TW201522641A - Plant regulatory genes promoting association with nitrogen fixing bacteria - Google Patents

Abstract

This disclosure concerns plant nitrogen responses. Embodiments concern regulatory factors that contribute to the functional association of plants (e.g., non-nodulating plants) with nitrogen-fixing bacteria.

Description

促進與固氮菌之關連性的植物調控基因 Plant regulatory genes that promote the association with nitrogen-fixing bacteria 優先權主張 Priority claim

本申請案主張皆於2013年11月18日提申之美國申請案編號14/083,193、與智利專利申請案編號2013-3314“PLANT REGULATORY GENES PROMOTING ASSOCIATION WITH NITROGEN FIXING BACTERIA”的申請日期之利益。 The application claims are based on the filing date of U.S. Application No. 14/083,193, filed on November 18, 2013, and the filing date of the patent application No. 2013-3314, "PLANT REGULATORY GENES PROMOTING ASSOCIATION WITH NITROGEN FIXING BACTERIA".

技術領域 Technical field

本揭示內容關於植物生化。具體例關於調控植物與固氮菌之關連性的遺傳因子。 This disclosure relates to plant biochemistry. Specific examples are genetic factors that regulate the association between plants and nitrogen-fixing bacteria.

背景 background

氮是植物生長和發展的基本巨量營養素，而且氮是對植物生長和作物生產力的主要限制因子。Marschner(1995)Mineral Nutrition of Higher Plants,Academic Press,Harcourt,San Diego,CA,p.889；Epstein(2005)Mineral Nutrition of Plants：Principles and Perspectives,2^nd Ed.,Sinauer Associates,Inc.,Sunderland,MA；Galloway and Cowling(2002)AMBIO 31：64。傳統農業係基於氮肥，以支援世界養分需求。然而，此類肥料在過度使用時會破壞環 境並破壞人類健康。 Nitrogen is a fundamentally large nutrient for plant growth and development, and nitrogen is a major limiting factor for plant growth and crop productivity. Marschner (1995) Mineral Nutrition of Higher Plants , Academic Press, Harcourt, San Diego, CA, p. 889; Epstein (2005) Mineral Nutrition of Plants: Principles and Perspectives , 2 ^nd Ed., Sinauer Associates, Inc., Sunderland, MA; Galloway and Cowling (2002) AMBIO 31:64. Traditional agriculture is based on nitrogen fertilizer to support the world's nutrient needs. However, such fertilizers can damage the environment and destroy human health when used excessively.

儘管大氣中豐含氮，但植物無法直接取得生物界的氮儲量。植物直接從土壤獲取氮，在一些情況可由固氮菌(NFB)經由生物固氮過程提供；將大氣N₂轉成生物可用之氮形式-例如自然生態系統中的銨-的主要過程(參閱譬如Olivares et al.(2013)Mol.Plant Microbe Interact.26：486)。由於土壤中的氮經常有限，一些植物物種演化出與固氮菌(NFB)關連的分子機制。Zehr et al.(2003)Env.Microbiol.5：539；Sprent and James(2007)Plant Physiol.144：575；Kraiser et al.(2011)J.Exp.Bot.62：1455。細菌和古細菌(archaea)是唯一能夠生物固氮的生物種類。將大氣N₂還原成銨係由固氮酶複合體-由二氮酶與二氮酶還原酶次單元構成-催化。Joerger et al.(1991)J.Bacteriol.173：4440。 Despite the abundance of nitrogen in the atmosphere, plants cannot directly access the nitrogen reserves of the biological world. Plants obtain nitrogen directly from the soil, in some cases by nitrogen-fixing bacteria (NFB) via biological nitrogen fixation processes; the main process of converting atmospheric N ₂ into biologically available nitrogen forms - such as ammonium in natural ecosystems (see, for example, Olivares et Al. (2013) Mol . Plant Microbe Interact. 26: 486). Due to the often limited nitrogen in the soil, some plant species evolved molecular mechanisms associated with nitrogen-fixing bacteria (NFB). Zehr et al. (2003) Env . Microbiol. 5: 539; Sprent and James (2007) Plant Physiol. 144: 575; Kraiser et al. (2011) J. Exp. Bot. 62: 1455. Bacteria and archaea are the only species that can biologically fix nitrogen. Reduction of atmospheric N ₂ to ammonium is catalyzed by a nitrogenase complex - consisting of a diazinase and a diazotase reductase subunit. Joerger et al. (1991) J. Bacteriol . 173: 4440.

與氮養分有關的植物-細菌交互作用係主要於豆科研究。豆科能夠和系統發育多樣化細菌群組-統稱為根瘤菌-以共生方式關連。Kistner and Parniske(2002)Trends Plant Sci.7：511。該等植物物種及其細菌夥伴的關連包括形成稱作“結瘤”的專門器官。結瘤包懷細菌並提供固氮發生的適當條件。Markmann and Parniske(2009)Trends Plant Sci.14：77。在該等結瘤中，植物提供碳源，交換細菌所固定的氮。Kistner and Parniske(2002)，見上文；Masson-Boivin et al.(2009)Trends Mirobiol.17：458。細菌與豆科之間的共生關連性係受到高度調控，僅在植物生長於限氮條件下時發生。低位準的硝酸鹽與銨刺激結瘤的形成，而高位準的該 等營養素亦抑制根部的感染位點數目與已存在結瘤的N-固定作用。Eaglesham(1989)Crop Sci.29：115；Zahran(1999)Microbiol.Mol.Biol.Rev.63：968；Bisseling et al.(1978)Bio.Biophys.Acta 539：1。 The plant-bacteria interactions associated with nitrogen nutrients are primarily in legume research. Leguminos can be associated with phylogenetically diverse bacterial groups - collectively referred to as Rhizobium - in a symbiotic manner. Kistner and Parniske (2002) Trends Plant Sci. 7: 511. The association of such plant species and their bacterial partners involves the formation of specialized organs known as "nodulation". Nodulation encompasses bacteria and provides appropriate conditions for nitrogen fixation. Markmann and Parniske (2009) Trends Plant Sci. 14:77. In these nodules, plants provide a source of carbon that exchanges nitrogen fixed by the bacteria. Kistner and Parniske (2002), supra; Masson-Boivin et al. (2009) Trends Mirobiol. 17:458. The symbiotic relationship between bacteria and legumes is highly regulated and occurs only when plants are grown under nitrogen-limited conditions. Low levels of nitrate and ammonium stimulate the formation of nodules, while high levels of these nutrients also inhibit the number of infection sites in the roots and the N-fixation of existing nodules. Eaglesham (1989) Crop Sci. 29: 115; Zahran (1999) Microbiol. Mol. Biol. Rev. 63: 968; Bisseling et al. (1978) Bio . Biophys. Acta 539: 1.

植物識得細菌所分泌的結瘤作用(Nod)因子，該因子活化包括初級轉錄因子1(NODULATION SIGNALING PATHWAY 1)(NSP1)與NSP2的信號傳導途徑。Smit et al.(2005)Science 308：1789；Kalo et al.(2005)Science 308：1786；Heckmann et al.(2006)Plant Physiol.142：1739。總之，該等轉錄因子調控結瘤起始(NIN)基因的表現，該基因是細菌感染與結瘤器官生成所必需的。Smit et al.(2005)，見上文；Hirsch et al.(2009)Plant Cell 21：545；Schauser et al.(1999)Nature 402：191。回應於細菌Nod因子的該等轉錄因子的活化係於植物處於限N條件時發生。Barbulova et al.(2007)Mol.Plant Microbe Interact.20：994。 The plant recognizes the Nod factor secreted by bacteria, which activates the signal transduction pathway of NODULATION SIGNALING PATHWAY 1 (NSP1) and NSP2. Smit et al. (2005) Science 308: 1789; Kalo et al. (2005) Science 308: 1786; Heckmann et al. (2006) Plant Physiol. 142: 1739. In summary, these transcription factors regulate the expression of the nodulation initiation ( NIN ) gene, which is required for bacterial infection and nodulation organogenesis. Smit et al. (2005), supra; Hirsch et al. (2009) Plant Cell 21: 545; Schauser et al. (1999) Nature 402: 191. Activation of these transcription factors in response to bacterial Nod factors occurs when the plant is in a N-limited condition. Barbulova et al. (2007) Mol . Plant Microbe Interact. 20:994.

除豆科外，來自不同分類單元的數量有限的其他植物物種已被報導和NFB有關連。儘管無法結瘤，但在限N條件下，一些植物(譬如小麥和甘蔗)可經由生物固氮作用吸納彼等顯著部分的氮需求。Iniguez et al.(2004)Mol.Plant Microbe Interact.17：1078；Boddey et al.(1991)Plant Soil 137：111。 In addition to legumes, a limited number of other plant species from different taxonomies have been reported to be associated with NFB. Although no tumors can be established, some plants (such as wheat and sugar cane) can absorb their significant nitrogen requirements through biological nitrogen fixation under N-limited conditions. Iniguez et al. (2004) Mol . Plant Microbe Interact. 17: 1078; Boddey et al. (1991) Plant Soil 137:111.

揭示內容 Reveal content

揭示了調控非結瘤植物物種與NFB(譬如苜蓿中 華根瘤菌(Sinorhizobium meliloti))之間N-養分交互作用的基因類別(譬如AtNSP1-樣、AtNLP4、與AtNLP9)。 Gene classes that regulate N-nutrient interactions between non-nodulated plant species and NFB (such as Sinorhizobium meliloti ) (eg, AtNSP1-like , AtNLP4 , and AtNLP9 ) are revealed .

在特定具體例揭示的是非天然核酸分子，其包含操作性地聯結至異源性促進子的聚核苷酸，其中該聚核苷酸為與SEQ ID NO：4至少80%一致的聚核苷酸；在嚴苛(譬如高度嚴苛)條件下雜交至由SEQ ID NO：4構成之核酸的聚核苷酸；與SEQ ID NO：5至少80%一致的聚核苷酸；在嚴苛(譬如高度嚴苛)條件下雜交至由SEQ ID NO：5構成之核酸的聚核苷酸；與SEQ ID NO：6至少80%一致的聚核苷酸；或在嚴苛(譬如高度嚴苛)條件下雜交至由SEQ ID NO：6構成之核酸的聚核苷酸。 Disclosed in a particular embodiment is a non-native nucleic acid molecule comprising a polynucleotide operably linked to a heterologous promoter, wherein the polynucleotide is at least 80% identical to SEQ ID NO: 4 An acid; a polynucleotide that hybridizes under stringent (eg, highly stringent) conditions to a nucleic acid consisting of SEQ ID NO: 4; a polynucleotide that is at least 80% identical to SEQ ID NO: 5; a polynucleotide that hybridizes to a nucleic acid consisting of SEQ ID NO: 5, for example, under highly stringent conditions; a polynucleotide that is at least 80% identical to SEQ ID NO: 6; or in harsh (eg, highly stringent) A polynucleotide that hybridizes to the nucleic acid consisting of SEQ ID NO: 6 under the conditions.

本案亦說明了增加植物(譬如非結瘤植物)氮效率的方法。在一些具體例中，該方法可包含將至少一異源性多肽引進植物，以製造基因轉殖植物，其中該異源性多肽為結瘤信號轉導途徑-樣(NSP)或NIN-樣蛋白(NLP)。在特定具體例中，該異源性多肽為NSP1、NLP4、或NLP9。此類異源性多肽可為，舉例來說，與SEQ ID NOs：1、3、與4當中一或多者至少約50%、約55%、約60%、約65%、約70%、約75%、約80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%96%、97%、98%、99%、或100%一致。 This case also illustrates methods for increasing the nitrogen efficiency of plants such as non-nodulated plants. In some embodiments, the method can comprise introducing at least one heterologous polypeptide into a plant to produce a gene transfer plant, wherein the heterologous polypeptide is a nodulation signal transduction pathway-like (NSP) or NIN-like protein (NLP). In a particular embodiment, the heterologous polypeptide is NSP1, NLP4, or NLP9. Such a heterologous polypeptide can be, for example, at least about 50%, about 55%, about 60%, about 65%, about 70%, with one or more of SEQ ID NOs: 1, 3, and 4. About 75%, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% consistent.

在一些具體例中，增加植物氮效率的方法可包含以編碼該異源性多肽的聚核苷酸使植物轉形。在特定具體例中，該聚核苷酸可實質上一致於SEQ ID NOs：4-6當中一 者，及/或為彼等的同源物或異種同源物。在特定具體例中，編碼該異源性多肽的聚核苷酸係於嚴苛(譬如高度嚴苛)條件下雜交至SEQ ID NOs：4-6當中至少一者。 In some embodiments, a method of increasing plant nitrogen efficiency can comprise transforming a plant with a polynucleotide encoding the heterologous polypeptide. In a particular embodiment, the polynucleotide can be substantially identical to one of SEQ ID NOs: 4-6 And/or homologs or heterologs of them. In a particular embodiment, the polynucleotide encoding the heterologous polypeptide is hybridized to at least one of SEQ ID NOs: 4-6 under stringent (e.g., highly stringent) conditions.

本案亦說明了增加植物(譬如非結瘤植物)氮效率的方法，該方法包含將促進與NFB之關連性的至少一至少一方式引進植物，舉例來說，以製造基因轉殖植物。促進與NFB之關連性的方式例子包括由SEQ ID NO：1構成之多肽；由SEQ ID NO：2構成之多肽；由SEQ ID NO：3構成之多肽；由SEQ ID NO：4構成之聚核苷酸；由SEQ ID NO：5構成之聚核苷酸；及由SEQ ID NO：6構成之聚核苷酸。 The present invention also illustrates a method of increasing the nitrogen efficiency of a plant, such as a non-nodulated plant, comprising introducing the plant into at least one of at least one mode that promotes its association with NFB, for example, to produce a genetically transformed plant. Examples of means for promoting the association with NFB include a polypeptide consisting of SEQ ID NO: 1; a polypeptide consisting of SEQ ID NO: 2; a polypeptide consisting of SEQ ID NO: 3; a polynucleus consisting of SEQ ID NO: a nucleotide; a polynucleotide consisting of SEQ ID NO: 5; and a polynucleotide consisting of SEQ ID NO: 6.

本案更說明了包含或穩定地轉形有任何前述多肽及/或核酸構築體的植物細胞、植物部分、植物材料、植物組織、植物種籽、及全株植物。相較於相同物種的野生型植物，此類基因轉殖植物在特定具體例展現在限氮生長條件下之增加的生長。 Further, the present invention describes plant cells, plant parts, plant material, plant tissues, plant seeds, and whole plants which comprise or stably transform any of the aforementioned polypeptides and/or nucleic acid constructs. Such genetically transgenic plants exhibit increased growth under nitrogen-limited growth conditions in specific embodiments compared to wild-type plants of the same species.

前述與其他特徵將由下列參照隨附圖式進行的數個具體例詳細說明而變得明顯。 The above and other features will be apparent from the following detailed description of the embodiments.

圖1A與1B包括在限N條件下NFB對植物生長的效應示意圖。植物生物質量(乾重；n=15)係於完全MS介質轉移至有N(40mM NO₃+20mM NH₄)或無N的MS介質並以不同細菌(NFB：越南伯克氏菌G4、噬異源伯克氏菌LB400、臺灣嗜銅菌LMG19424、菜豆根瘤菌CFN42、苜蓿中華根瘤菌RMP110；以及非-NFB：植物伯克氏菌PSJN、皮納突伯 嗜銅菌JMP134)(順序由左至右)接種或否的七日後測量。圖1A。植物乾重係於在含N介質生長七日並移植至限N條件且以活或死苜蓿中華根瘤菌接種、或未接種的植物測量。圖1B。每株植物的側根數量係於以上文圖1A所詳述的相同條件生長與處理的植物測量。圖1A(三個獨立生物重複的平均±SE；星號指示相較於無N生長的未接種植物明顯不同的平均(P<0.05))。 Figures 1A and 1B are schematic diagrams showing the effect of NFB on plant growth under N-limited conditions. Plant biomass (dry weight; n=15) was transferred to complete MS medium to MS medium with N (40 mM NO ₃ + 20 mM NH ₄ ) or no N and with different bacteria (NFB: Burk Bunge G4, phagocytosis) Heterologous Burkholderia LB400, Taiwan Copperophilus LMG19424, Rhizopus arrhizus CFN42, Rhizobium meliloti RMP110; and Non-NFB: Burkholderia PSJN, Pseudomonas aeruginosa JMP134) (sequence by left To the right) 7 days after inoculation or no measurement. Figure 1A. Plant dry weight was measured on plants grown in N-containing medium for 7 days and transplanted to N-limited conditions and inoculated with live or dead S. meliloti, or uninoculated. Figure 1B. The number of lateral roots per plant is measured by plants grown and treated under the same conditions as detailed above in Figure 1A. Figure 1A (mean ± SE of three independent biological replicates; asterisk indicates a significantly different average (P < 0.05) compared to uninoculated plants without N growth).

圖2A與2B包括在限N條件下的N固定作用增進植物生長的示意圖。生物質量係以生長在充足(2.5mM NH₄NO₃)或限N條件下、以苜蓿中華根瘤菌RMP110野生型接種或否或以無法固定N的成熟型接種的植物以乾重測量。圖2A。生物固氮作用係藉由¹⁵N稀釋技術測量。植物係生長在充足N(5% ¹⁵N)的介質。七日後，將植物移至不同處理的皿。使用質譜，在處理7日後於採收與乾燥植物測定¹⁴N與¹⁵N的份量。δ ¹⁵N表示相較於未接種條件的¹⁴N：¹⁵N同位素比率(三個獨立生物重複的平均±SE(P<0.05))。圖2A。 Figures 2A and 2B include schematic diagrams of N-immobilization enhancing plant growth under conditions of limited N. Bio-mass system to grow in a well (2.5mM NH ₄ NO ₃₎ or N limiting conditions, to a wild-type S. meliloti RMP110 inoculated plant or whether or not the mature N fixed inoculated on a dry weight measurement. Figure 2A. Biological nitrogen fixation was measured by a ¹⁵ N dilution technique. Plant lines are grown in a sufficient N (5% ¹⁵ N) medium. After seven days, the plants were moved to different treatment dishes. Using mass spectrometry, ¹⁴ N and ¹⁵ N portions were measured on harvested and dried plants after 7 days of treatment. δ ¹⁵ N represents the ¹⁴ N: ¹⁵ N isotope ratio compared to uninoculated conditions (mean ± SE of three independent biological replicates (P < 0.05)). Figure 2A.

圖3A與3B包括經由細菌接種引進某些***芥屬基因的示意圖。NSP1(圖3A)與NIN-樣轉錄因子(圖3A)基因表現係在處理第三或第七日後以即時定量逆轉錄PCR測量。植物係生長在充足(2.5mM NH₄NO₃)或限N條件、並以NFB接種或否。繪示值係相當於三個獨立生物重複的平均±SE(星號指示相較於未接種植物明顯不同的平均(P<0.05))。 Figures 3A and 3B include schematic representations of the introduction of certain Arabidopsis genes via bacterial vaccination. The NSP1 (Fig. 3A) and NIN-like transcription factor (Fig. 3A) gene expression lines were measured by real-time quantitative reverse transcription PCR after the third or seventh day of treatment. Plant lines were grown in sufficient (2.5 mM NH ₄ NO ₃ ) or N-limited conditions and inoculated with NFB or no. The values are equivalent to the mean ± SE of three independent biological replicates (the asterisk indicates a significantly different average (P < 0.05) compared to the uninoculated plants).

圖4包括***芥-就與苜蓿中華根瘤菌 RMP110之關連性而言至關重要的基因；AtNSP1-樣、AtNLP4、AtNLP8、與AtNLP9轉錄因子-的突變效應示意圖。生物質量係以生長在限氮條件下並以苜蓿中華根瘤菌接種或否的野生型與突變植物所測得的乾重表示(三個獨立生物重複的平均±SE；星號指示相較於未接種植物明顯不同的平均(P<0.05))。 Figure 4 includes Arabidopsis thaliana - a gene critical for the association with S. meliloti RMP110; a schematic representation of the mutational effects of AtNSP1-like , AtNLP4 , AtNLP8 , and AtNLP9 transcription factors. Biomass was expressed as the dry weight measured by wild-type and mutant plants grown under nitrogen-limited conditions and inoculated with N. faecalis (average ±SE of three independent biological replicates; asterisk indication compared to unvaccinated Plants were significantly different (P < 0.05)).

圖5包括展示NFB對根毛長度的效應的影像。在完全MS鹽介質生長達數日的植物係移植至無N的MS介質，並以NFB接種或否。在處理數日後拍照。 Figure 5 includes an image showing the effect of NFB on root hair length. Plant lines grown in complete MS salt medium for several days were transplanted to N-free MS medium and inoculated with NFB or not. Take a photo after a few days of processing.

圖6A與6B包括苜蓿中華根瘤菌對***芥屬根系建構的效應示意圖。側根密度(圖6A)與主要根長(圖6B)係於生長在充足/限N條件、以苜蓿中華根瘤菌RMP110野生型接種或否或以無法固定N的成熟型接種的植物測量。繪示值係相當於三個獨立生物重複的平均±SE。 Figures 6A and 6B are schematic diagrams showing the effect of Sinorhizobium meliloti on the Arabidopsis root architecture. The lateral root density (Fig. 6A) and the major root length (Fig. 6B) were measured on plants grown in a sufficient/limited N condition, inoculated with R. sphaeroides RMP110 wild type or no or inoculated with a mature type in which N could not be fixed. The plotted values are equivalent to the mean ± SE of three independent biological replicates.

圖7A與7B包括細菌接種AtNSP2-樣、AtNLP1、AtNLP2、與AtNLP5的回應示意圖。基因表現係在處理第三或第七日後以即時定量逆轉錄PCR測量(三個獨立生物重複的平均±SE；星號指示相較於未接種植物明顯不同的平均(P<0.05))。植物係生長在充足或限N條件、並以苜蓿中華根瘤菌接種或否。 Figures 7A and 7B include schematic representations of bacterial responses to AtNSP2-like , AtNLP1 , AtNLP2 , and AtNLP5 . Gene expression was measured by real-time quantitative reverse transcription PCR after the third or seventh day of treatment (mean ± SE of three independent biological replicates; asterisk indicates a significantly different average (P < 0.05) compared to uninoculated plants). Plant lines are grown in sufficient or limited N conditions and inoculated with S. meliloti or no.

序列表 Sequence table

在隨附序列表中列出的核酸序列係使用37 C.F.R.§ 1.822所定義的核苷酸鹼基標準字母縮寫顯示。僅顯示各核酸序列的一股，但可理解互補股係任意參照展示股而被 包括在內。在隨附序列表中：SEQ ID NO：1顯示NSP1-樣(SCL29；At3g13840)多肽： SEQ ID NO：2顯示NLP4(At1g20640)多肽： SEQ ID NO：3顯示NLP9(At3g59580)多肽： SEQ ID NO：4顯示NSP1-樣(SCL29；At3g13840)聚核苷酸編碼序列(CDS)： SEQ ID NO：5顯示NLP4(At1g20640)聚核苷酸編碼序列(CDS)： SEQ ID NO：6顯示NLP9(At3g59580)聚核苷酸編碼序列(CDS)： The nucleic acid sequences listed in the accompanying sequence listing are shown using the nucleotide base standard letter abbreviations as defined in 37 CFR § 1.822. Only one strand of each nucleic acid sequence is shown, but it is understood that the complementary strands are included with any reference to the display strand. In the accompanying sequence listing: SEQ ID NO: 1 shows the NSP1-like (SCL29; At3g13840) polypeptide: SEQ ID NO: 2 shows the NLP4 (At1g20640) polypeptide: SEQ ID NO: 3 shows the NLP9 (At3g59580) polypeptide: SEQ ID NO: 4 shows the NSP1-like (SCL29; At3g13840) polynucleotide coding sequence (CDS): SEQ ID NO: 5 shows the NLP4 (At1g20640) polynucleotide coding sequence (CDS): SEQ ID NO: 6 shows the NLP9 (At3g59580) polynucleotide coding sequence (CDS):

實行本發明之模式 Mode for carrying out the invention I.數個具體例的概觀I. Overview of several specific examples

在豆科中，植物與NFB之間的功能關連性係受到高度調控。分子調控系統阻止在氮存在下生長的植物形成結瘤。僅在限N條件且有NFB的存在下，共生性信號轉導途徑被激活而誘發植物中的結瘤發展。在Nod因子識別後，鈣振盪造成誘發初級轉錄因子NSP1與NSP2，其誘發NIN基因表現。共生性信號轉導途徑的一些組分在根瘤菌與叢枝菌根真菌關連性之間係共享。Oldroyd(2013)，見上文。然而且不像NSP2、NSP1與NIN基因係明確牽涉到與NFB之關連性。 In legumes, the functional association between plants and NFB is highly regulated. Molecular regulatory systems prevent the formation of nodules in plants grown in the presence of nitrogen. The symbiotic signal transduction pathway is activated to induce nodulation in plants only in the presence of N-limited conditions and in the presence of NFB. After recognition of the Nod factor, calcium oscillations induce the induction of the primary transcription factors NSP1 and NSP2, which induce NIN gene expression. Some components of the symbiotic signal transduction pathway are shared between Rhizobium and arbuscular mycorrhizal fungi. Oldroyd (2013), see above. However, unlike the NSP2 , NSP1 and NIN gene lines, it is explicitly related to NFB.

本案說明的是促進非結瘤植物物種與NFB之關 連性以增進N-養分的機制。在限N條件下，***芥屬係與苜蓿中華根瘤菌有關連，NFB能夠提供作為植物養分的還原氮，在限N條件下有助於植物生長。本案具體例運用就非結瘤植物與苜蓿中華根瘤菌之間的功能關連性而言至關重要的首次辨識植物轉錄因子。吾人已發現到專對豆科：NFB關連性的轉錄因子的同源性基因就介導在非結瘤植物中由苜蓿中華根瘤菌所誘發的植物生長促進作用而言係必要的。在具體例中，跨植物物種間的關連性機制的保留係容許***芥屬與苜蓿中華根瘤菌之間的關連性經由基因修飾在其他植物重現，以管理與NFB之有益N-養分交互作用，舉例來說，以加強耕作系統的氮使用效率。 This case illustrates the promotion of non-nodulated plant species and NFB Linkage to promote N-nutrients. Under the condition of N, Arabidopsis is associated with Sinorhizobium meliloti, and NFB can provide reducing nitrogen as plant nutrients, which is beneficial to plant growth under N-limited conditions. The specific case of this case uses the first identification of plant transcription factors that are critical for the functional relationship between non-nodulated plants and S. meliloti. We have found that homologous genes specific for leguminous: NFB-related transcription factors are essential for plant growth promoting effects induced by Sinorhizobium meliloti in non-nodulated plants. In a specific example, the retention of the cross-linkage mechanism between plant species allows the association between Arabidopsis and S. meliloti to be replicated in other plants via genetic modification to manage beneficial N-nutrient interactions with NFB For example, to enhance the efficiency of nitrogen use in farming systems.

如本案所述，***芥屬係與苜蓿中華根瘤菌-與豆科紫花苜蓿有關連的細菌-有功能關連性。Marsh et al.(2007)Plant Physiol.144：324；Peiter et al.(2007)Plant Physiol.145：192。在自由生活條件且與豆科關連時，苜蓿中華根瘤菌僅在限N條件下實行N-固定。Szeto et al.(1987)J.Bacteriol.169：1423。再如本案所述，苜蓿中華根瘤菌在限N條件下(譬如在沒有外加N-源的介質)係促進植物生長，於是適用於由於BNF的N-固定及植物生長促進。 As described in this case, the Arabidopsis genus has a functional association with S. meliloti - a bacterium associated with the leguminous alfalfa. Marsh et al. (2007) Plant Physiol. 144: 324; Peiter et al. (2007) Plant Physiol . 145: 192. In free-living conditions and related to legumes, Sinorhizobium meliloti is only N-fixed under N-limited conditions. Szeto et al. (1987) J. Bacteriol. 169: 1423. As described in the present case, Sinorhizobium meliloti is resistant to plant growth under conditions of limited N (for example, in the absence of an N-source medium), and is therefore suitable for N-immobilization of BNF and plant growth promotion.

II.縮寫II. Abbreviation

III.術語III. Terminology

在以下說明與表格中，使用許多術語。為了提供對說明書與請求項，包括此類術語所給定的範疇的清楚一致理解，提供了下列定義：回交(Backcrossing)：回交方法可用於將核酸序列引進植物。回交技術已被廣泛使用了數十年，以將新穎性狀引進植物。Jensen,N.,Ed.Plant Breeding Methodology,John Wiley & Sons,Inc.,1988。在典型回交流程中，感興趣的原始栽培品種(輪迴親代)係和攜帶欲轉移之感興趣基因的第二栽培品種(非輪迴親代)交配。從此交配得到的子代隨後和輪迴親代再次交配，重複該過程直到獲得植物為止，其中-除了來自非輪迴親代的轉移基因以外-在該轉換植物中回收了輪迴植物的基本上全部所欲形態與生理特質。 In the following descriptions and tables, many terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the categories given by such terms, the following definitions are provided: Backcrossing: Backcrossing methods can be used to introduce nucleic acid sequences into plants. Backcrossing techniques have been widely used for decades to introduce novel traits into plants. Jensen, N., Ed. Plant Breeding Methodology , John Wiley & Sons, Inc., 1988. In a typical backcrossing process, the original cultivar of interest (recurrent parent) is mated with a second cultivar (non-recurrent parent) carrying the gene of interest to be transferred. The progeny thus mated are then re-matured with the recurrent parent, and the process is repeated until the plant is obtained, wherein - in addition to the transfer gene from the non-recurrent parent - substantially all of the reincarnation of the plant is recovered in the transformed plant Morphological and physiological characteristics.

單離的：“單離的”生物組分(例如核酸或蛋白)已從天然存在該組分的生物體細胞內的其他生物組分(即其他染色體與染色體外DNA與RNA、及蛋白)實質上分離、製造、或純化出來，同時引起組分的化學與功能變化(譬如核酸可藉由斷開核酸連接至染色體內其餘DNA的化學鍵而從染色體單離)。已“單離的”核酸分子與蛋白包括藉由標準純化方法純化的核酸分子與蛋白，其中核酸或蛋白中已有化學與功能變化。該術語亦包含在宿主細胞中藉由重組表現製備的核酸與蛋白，還有化學合成的核酸分子、蛋白、與 肽。 Isolating: "Isolated" biological components (such as nucleic acids or proteins) have been derived from other biological components (ie, other chromosomes and extrachromosomal DNA and RNA, and proteins) within the organism's cells in which the component naturally exists. Isolation, manufacture, or purification, while causing chemical and functional changes in the components (eg, nucleic acids can be isolated from the chromosome by breaking the chemical bonds of the nucleic acid to the rest of the DNA in the chromosome). Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods in which chemical and functional changes have been made in the nucleic acid or protein. The term also encompasses nucleic acids and proteins produced by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules, proteins, and Peptide.

核酸分子：如本案所用，術語“核酸分子”可指稱核苷酸的聚合形式，可包括RNA、cDNA、基因組DNA的有義股與反義股，以及以上的合成形式與混合聚合物。核苷酸可指稱核糖核苷酸、去氧核糖核苷酸、或任一類核苷酸的修飾形式。本案使用的“核酸分子”係同義於“核酸”與“聚核苷酸”。核酸分子通常為至少10個鹼基長度，除非另有指明。術語包括DNA的單-與雙-股形式。核酸分子可包括藉由天然存在及/或非天然存在的核苷酸聯結而聯結在一起的天然存在與修飾核苷酸當中任一者或兩者。 Nucleic Acid Molecule: As used herein, the term "nucleic acid molecule" may refer to a polymeric form of a nucleotide, which may include the sense and antisense strands of RNA, cDNA, genomic DNA, and the above synthetic forms and mixed polymers. A nucleotide can refer to a modified form of a ribonucleotide, a deoxyribonucleotide, or any type of nucleotide. The "nucleic acid molecule" used in this context is synonymous with "nucleic acid" and "polynucleotide". Nucleic acid molecules are typically at least 10 bases in length unless otherwise indicated. The term includes both mono- and bi-strand forms of DNA. A nucleic acid molecule can include any one or both of the naturally occurring and modified nucleotides joined together by naturally occurring and/or non-naturally occurring nucleotide linkages.

核酸分子可藉由化學方式或生化方式修飾，或可含有非天然或衍化核苷酸鹼基，如熟習此藝者將輕易理解者。此類修飾包括，舉例來說，標記、甲基化、將天然存在核苷酸當中一或多者取代成類似物跨核苷酸修飾(譬如不帶電聯結：舉例來說，甲基膦酸酯、磷酸三酯、胺基磷酸酯、胺基甲酸酯等等；帶電聯結：舉例來說，硫代磷酸酯、二硫代磷酸酯等等；側基部分：舉例來說，肽；內嵌劑：舉例來說，吖啶、補骨脂素等等；螯合劑；烷化劑；以及修飾性聯結：舉例來說，α端基異構核酸等等)。術語“核酸分子”亦包括任何拓撲構形，包括單-股、雙-股、部分地雙螺旋、三螺旋、髮夾型、環型、以及鎖型構形。 Nucleic acid molecules may be modified by chemical or biochemical means, or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more of the naturally occurring nucleotides into an analog cross-nucleotide modification (eg, uncharged linkage: for example, methylphosphonate) , a phosphate triester, an amino phosphate, a urethane, etc.; a charged linkage: for example, a phosphorothioate, a phosphorodithioate, etc.; a pendant moiety: for example, a peptide; embedded Agents: for example, acridine, psoralen, etc.; chelating agents; alkylating agents; and modified linkages: for example, alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological configuration, including single-strand, double-strand, partially double helix, triple helix, hairpin, ring, and lock configurations.

寡核苷酸：寡核苷酸為短的核酸分子。寡核苷酸可藉由裂解較長的核酸分段、或藉由聚合個別核苷酸前驅物形成。自動化合成儀允許合成高達數百個鹼基對長度的 寡核苷酸。因為寡核苷酸可結合至互補核苷酸序列，所以彼等可用作偵測DNA或RNA的探針。由DNA構成的寡核苷酸(寡聚去氧核糖核苷酸)可用於PCR，一種用於擴增小型DNA序列的技術。在PCR中，寡核苷酸通常稱作“引子”，其容許DNA聚合酶延長寡核苷酸並複製互補股。 Oligonucleotide: An oligonucleotide is a short nucleic acid molecule. Oligonucleotides can be formed by cleavage of longer nucleic acid fragments, or by polymerization of individual nucleotide precursors. Automated synthesizers allow synthesis of up to hundreds of base pairs in length Oligonucleotides. Because oligonucleotides can bind to complementary nucleotide sequences, they can be used as probes for detecting DNA or RNA. Oligonucleotides (oligodeoxyribonucleotides) composed of DNA can be used for PCR, a technique for amplifying small DNA sequences. In PCR, oligonucleotides are often referred to as "primers" which allow the DNA polymerase to extend the oligonucleotide and replicate the complementary strand.

核酸分子可包括藉由天然存在及/或非天然存在的核苷酸聯結而聯結在一起的天然存在與修飾核苷酸當中任一者或兩者。核酸分子可藉由化學方式或生化方式修飾，或可含有非天然或衍化核苷酸鹼基，如熟習此藝者將輕易理解者。此類修飾包括，舉例來說，標記、甲基化、將天然存在核苷酸當中一或多者取代成類似物跨-核苷酸修飾(譬如不帶電聯結：舉例來說，甲基膦酸酯、磷酸三酯、胺基磷酸酯、胺基甲酸酯等等；帶電聯結：舉例來說，硫代磷酸酯、二硫代磷酸酯等等；側基部分：舉例來說，肽；內嵌劑：舉例來說，吖啶、補骨脂素等等；螯合劑；烷化劑；以及修飾性聯結：舉例來說，α端基異構核酸等等)。術語“核酸分子”亦包括任何拓撲構形，包括單-股、雙-股、部分地雙螺旋、三螺旋、髮夾型、環型、以及鎖型構形。 A nucleic acid molecule can include any one or both of the naturally occurring and modified nucleotides joined together by naturally occurring and/or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be modified by chemical or biochemical means, or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more of the naturally occurring nucleotides into an analog cross-nucleotide modification (eg, uncharged linkage: for example, methylphosphonic acid) Ester, phosphotriester, amino phosphate, urethane, etc.; charged linkage: for example, phosphorothioate, phosphorodithioate, etc.; pendant moiety: for example, peptide; Inserts: for example, acridine, psoralen, etc.; chelating agents; alkylating agents; and modified linkages: for example, alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological configuration, including single-strand, double-strand, partially double helix, triple helix, hairpin, ring, and lock configurations.

在本案針對DNA使用時，術語“編碼序列”係指稱在置於適當調控序列控制時轉錄成RNA的核苷酸序列。“蛋白質編碼序列”為經由轉錄與mRNA而最終轉譯成多肽的核苷酸序列(DNA或RNA)。就RNA而言，術語“編碼序列”係指稱轉譯成肽、多肽、或蛋白的核苷酸序列。編碼序列的邊界係由5'-端轉譯起始密碼子與3'-端轉譯停止密碼子決定。 編碼序列包括但不限於：基因組DNA；cDNA；EST；與重組核苷酸序列。 The term "coding sequence" when used in reference to DNA in this context refers to a nucleotide sequence that is transcribed into RNA upon administration of the appropriate regulatory sequences. A "protein coding sequence" is a nucleotide sequence (DNA or RNA) that is ultimately translated into a polypeptide via transcription and mRNA. In the case of RNA, the term "coding sequence" refers to a nucleotide sequence that is translated into a peptide, polypeptide, or protein. The boundaries of the coding sequence are determined by the 5'-end translation start codon and the 3'-end translation stop codon. Coding sequences include, but are not limited to, genomic DNA; cDNA; EST; and recombinant nucleotide sequences.

基因組：如本案所用，術語“基因組”係指稱在細胞核內發現的染色體DNA，亦係指稱在細胞的亞細胞組分內發現的胞器DNA。在本發明一些具體例中，DNA分子可被引進植物細胞，俾使DNA分子嵌入植物細胞的基因組。在該等與另外具體例中，DNA分子可嵌入植物細胞的細胞核DNA，或嵌入植物細胞的葉綠體或粒線體DNA。 Genome: As used herein, the term "genome" refers to chromosomal DNA found in the nucleus, and also refers to organelle DNA found within the subcellular components of a cell. In some embodiments of the invention, a DNA molecule can be introduced into a plant cell such that the DNA molecule is embedded in the genome of the plant cell. In these and other specific examples, the DNA molecule can be embedded in the nuclear DNA of the plant cell, or embedded in the chloroplast or mitochondrial DNA of the plant cell.

內源性：應用至本案核酸(譬如聚核苷酸、DNA、RNA、與基因)的術語“內源性”係指稱慣常(譬如在相同種類與物種的野生型細胞)出現在彼等特異性環境或背景內的一或多個(複數個)核酸。舉例來說，內源性基因為在論述的特定細胞且於相同背景(譬如關於調控序列)所慣常找到的基因。內源性核酸可和外源性及/或異源性區別-舉例而不限於-藉由偵測隨著細菌質體重組而得到的後者序列；識別非典型密碼子偏好性；以及在PCR反應中擴增從野生型細胞定徵的引子的非典型序列。 Endogenous: The term "endogenous" applied to nucleic acids (such as polynucleotides, DNA, RNA, and genes) in this case refers to the habitual (such as wild-type cells in the same species and species) appearing in their specificity. One or more (plural) nucleic acids within the environment or background. For example, an endogenous gene is a gene that is routinely found in a particular cell in question and in the same context, such as for regulatory sequences. Endogenous nucleic acids can be distinguished from exogenous and/or heterologous - by way of example and without limitation - by detecting the latter sequence obtained by bacterial plastid recombination; identifying atypical codon bias; and in PCR reactions Amplifying the atypical sequence of primers characterization from wild-type cells.

外源性：應用至本案核酸的術語“外源性”係指稱非慣常出現在彼等特異性環境或背景內的一或多個(複數個)核酸。舉例來說，假使宿主細胞被天然不存在於未轉形宿主細胞的核酸轉形，則該核酸對宿主細胞而言為外源性。術語外源性-如本案所用-亦係指稱和已存在於宿主細胞內的核酸序列一致、但位於和已存在於宿主細胞內具相同序列的核酸不同的細胞或基因組背景的一或多個(複數個) 核酸。舉例來說，嵌入和具相同序列的核酸慣常嵌入宿主細胞基因組的宿主細胞基因組不同位置的核酸對宿主細胞而言為外源性。再者，當具相同序列的核酸慣常僅存在於宿主細胞基因組時，存在於宿主細胞質體或載體的核酸(譬如DNA分子)對宿主細胞而言為外源性。 Exogenous: The term "exogenous" as applied to a nucleic acid of the present invention refers to one or more (plural) nucleic acids that are not normally found in their specific environment or context. For example, if the host cell is transformed by a nucleic acid that is not naturally found in the untransformed host cell, the nucleic acid is exogenous to the host cell. The term exogenous - as used herein - also refers to one or more of the cell or genomic backgrounds that are identical to the nucleic acid sequence already present in the host cell but that are different from the nucleic acid already present in the host cell with the same sequence ( Multiple) Nucleic acid. For example, nucleic acids embedded in and having the same sequence of nucleic acids that are conventionally embedded in the host cell genome at various locations in the host cell genome are exogenous to the host cell. Furthermore, nucleic acids (such as DNA molecules) present in the host cell plastid or vector are exogenous to the host cell when the nucleic acid having the same sequence is customarily present only in the host cell genome.

異源性：應用至本案核酸(譬如聚核苷酸、DNA、RNA、與基因)的術語“異源性”意指具不同來源。舉例來說，假使宿主細胞被天然不存在於未轉形宿主細胞的核酸轉形，則該核酸對宿主細胞而言為異源性(及外源性)。再者，轉形核酸的不同元件(譬如促進子、增強子、編碼序列、終止子等等)對另一者其他及/或對轉形宿主而言可為異源性。術語異源性-如本案所用-亦應用至和已存在於宿主細胞內的核酸序列一致、但現在聯結至不同額外序列及/或以不同複本數存在等等的一或多個(複數個)核酸。 Heterologous: The term "heterologous" as applied to a nucleic acid (such as a polynucleotide, DNA, RNA, and gene) of the present invention means having a different source. For example, a host cell is heterologous (and exogenous) to the host cell if the host cell is transformed by a nucleic acid that is not naturally found in the untransformed host cell. Furthermore, different elements of the transmorphic nucleic acid (such as promoters, enhancers, coding sequences, terminators, etc.) may be heterologous to the other and/or to the transpogic host. The term heterologous - as used herein - also applies to one or more (plural) of nucleic acid sequences already present in a host cell, but now joined to different additional sequences and/or present in different copies, and the like. Nucleic acid.

序列一致性：本案針對兩個核酸或多肽序列使用的術語“序列一致性”或“一致性”可指稱-當在特定比對窗口以最大對應性對齊時-兩序列內的相同殘基。 Sequence Consistency: The term "sequence identity" or "consistency" as used in this context for two nucleic acid or polypeptide sequences can be referred to - when aligned at a particular alignment window with maximum correspondence - the same residues within both sequences.

如本案所用，術語“序列一致性百分比”可指稱在比對窗口比對兩個最佳化對齊序列(譬如核酸序列、與胺基酸序列)所決定的值，其中在相比於參考序列(其不包含添加或刪除)以供兩個序列的最佳化對齊時，在比對窗口的序列部分可包含添加或刪除(即空隙)。百分比係藉由下列計算：決定兩序列中存在一致性核苷酸或胺基酸殘基的位置數目以生成配對位置數目、將配對位置數目除以比對窗口中的 全部位置數目、並將結果乘以100以生成序列一致性百分比。 As used herein, the term "percent sequence identity" can refer to a value determined by aligning two aligned alignment sequences (eg, a nucleic acid sequence, with an amino acid sequence) in an alignment window, wherein in comparison to a reference sequence ( When it does not include additions or deletions for optimal alignment of the two sequences, additions or deletions (ie, gaps) may be included in the sequence portion of the alignment window. The percentage is calculated by determining the number of positions in which the consensus nucleotide or amino acid residue is present in both sequences to generate the number of paired positions, dividing the number of paired positions by the alignment window The number of all positions, and the result is multiplied by 100 to generate a sequence consistency percentage.

對齊序列以供比對的方法在本領域係眾所周知。各式程式與對齊演算法係說明於，舉例來說：Smith and Waterman(1981)Adv.Appl.Math.2：482；Needleman and Wunsch(1970)J.Mol.Biol.48：443；Pearson and Lipman (1988)Proc.Natl.Acad.Sci.U.S.A.85：2444；Higgins and Sharp(1988)Gene 73：237-44；Higgins and Sharp(1989)CABIOS 5：151-3；Corpet et al.(1988)Nucleic Acids Res.16：10881-90；Huang et al.(1992)Comp.Appl.Biosci.8：155-65；Pearson et al.(1994)Methods Mol.Biol.24：307 31；Tatiana et al.(1999)FEMS Microbiol.Lett.174：247-50。序列對齊方法與同源性計算的詳細考量可在譬如Altschul et al.(1990)J.Mol.Biol.215：403-10找到。 Methods for aligning sequences for alignment are well known in the art. Various programs and alignment algorithms are described, for example: Smith and Waterman (1981) Adv. Appl. Math. 2: 482; Needleman and Wunsch (1970) J. Mol. Biol. 48: 443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444; Higgins and Sharp (1988) Gene 73: 237-44; Higgins and Sharp (1989) CABIOS 5: 151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8: 155-65; Pearson et al. (1994) Methods Mol . Biol. 24: 307 31; Tatiana et al. ) FEMS Microbiol. Lett. 174: 247-50. Detailed considerations for sequence alignment methods and homology calculations can be found, for example, in Altschul et al. (1990) J. Mol. Biol. 215:403-10.

國家生技資訊中心(NCBI)鹼基局部對齊搜尋工具(BLAST^TM；Altschul et al.(1990))可得自數個來源，包括國家生技資訊中心(Bethesda,MD)、與網路上，以連同數個序列分析程式使用。使用此程式如何決定序列一致性的說明可在網路上於BLAST^TM的“幫助(help)”章節取得。就核酸序列比對而言，可使用預設參數運用BLAST^TM(Blastn)程式的“比對雙序列(Blast 2 sequences)”功能。對參考序列具甚至更大相似度的核酸序列在藉此方法評估時將顯示增多之一致性百分比。 National Center for Biotechnology Information (NCBI) nucleotide local alignment search tool ^{(BLAST TM;. Altschul et al} (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and on the web to Used in conjunction with several sequence analysis programs. Use this program to illustrate how to determine sequence identity can be achieved in the BLAST ^TM chapter "Help (help)" on the web. To a nucleic acid sequence alignments, the default parameters can be used using BLAST ^TM (Blastn) of the program "Sequence alignment of bis (Blast 2 sequences)" function. Nucleic acid sequences with even greater similarity to the reference sequence will show an increased percent identity when evaluated by this method.

特異性地雜交/特異性地互補：如本案所用，術 語“特異性地雜交”與“特異性地互補”為指出足夠互補程度，俾使核酸分子與標靶核酸分子之間發生穩定的特異性結合的術語。兩核酸分子之間的雜交係涉及在兩核酸分子的核酸序列之間形成反向平行對齊。該兩分子隨後能夠和相對股上的對應鹼基形成氫鍵，而形成使用本領域眾所周知的方法可偵測到的雙螺旋分子，假使該分子足夠穩定。核酸分子不必100%互補於欲特異性地雜交之標靶序列。然而，必須存在以使雜交具特異性的序列互補性份量係為所使用雜交條件的函數。 Specific hybridization/specific complementation: as used in this case, surgery The terms "specifically hybridize" and "specifically complement" are terms that indicate a sufficient degree of complementarity to result in a stable specific binding between a nucleic acid molecule and a target nucleic acid molecule. Hybridization between two nucleic acid molecules involves the formation of an anti-parallel alignment between the nucleic acid sequences of the two nucleic acid molecules. The two molecules are then capable of forming hydrogen bonds with corresponding bases on the opposite strand to form a double helix molecule detectable using methods well known in the art, provided that the molecule is sufficiently stable. The nucleic acid molecule does not have to be 100% complementary to the target sequence to be specifically hybridized. However, there must be a function to make the sequence complementary portion of the hybrid specificity a function of the hybridization conditions used.

產生特定嚴苛度的雜交條件將取決於挑選的雜交方法本質和雜交核酸序列的組成物與長度而有所不同。一般而言，雜交溫度與雜交緩衝液的離子強度(尤其是Na⁺及/或Mg⁺⁺濃度)將決定雜交嚴苛性，儘管洗滌時間亦影響嚴苛性。關於實現特定嚴苛度所必需的雜交條件的計算係熟習此藝者所習知的，並在舉例來說Sambrook et al.(ed.)Molecular Cloning：A Laboratory Manual,2^nd ed.,vol.1-3,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,1989,chapters 9 and 11；與Hames and Higgins(eds.)Nucleic Acid Hybridization,IRL Press,Oxford,1985中有討論。關於核酸雜交的進一步詳細說明與指導可在，舉例來說，Tijssen,“Overview of principles of hybridization and the strategy of nucleic acid probe assays,”in Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes,Part I,Chapter 2,Elsevier,NY,1993；與 Ausubel et al.,Eds.,Current Protocols in Molecular Biology,Chapter 2,Greene Publishing and Wiley-Interscience,NY,1995中找到。 Hybridization conditions that produce a particular stringency will vary depending on the nature of the hybridization method selected and the composition and length of the hybrid nucleic acid sequence. In general, the hybridization temperature and the ionic strength of the hybridization buffer (especially the Na ⁺ and/or Mg ⁺⁺ concentration) will determine the stringency of the hybridization, although the wash time will also affect the severity. The calculation of the hybridization conditions necessary to achieve a particular severity is well known to those skilled in the art and is exemplified by Sambrook et al. (ed.) Molecular Cloning: A Laboratory Manual , 2 ^nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, chapters 9 and 11; discussed in Hames and Higgins (eds.) Nucleic Acid Hybridization , IRL Press, Oxford, 1985. Further details and guidance regarding nucleic acid hybridization can be, for example, Tijssen, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," in Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes , Part I, Chapter 2, Elsevier, NY, 1993; found in Ausubel et al. , Eds., Current Protocols in Molecular Biology , Chapter 2, Greene Publishing and Wiley-Interscience, NY, 1995.

如本案所用，“嚴苛條件”係涵蓋雜交僅會-在假使雜交分子與標靶核酸分子內的同源性序列之間有少於20%未配對-發生的條件。“嚴苛條件”包括另外的特別嚴苛性位準。於是，如本案所用，“中度嚴苛性”條件為該等具多於20%序列未配對之分子將不會雜交的條件；“高度嚴苛性”條件為該等具多於10%未配對之序列將不會雜交的條件；以及“極高度嚴苛性”條件為該等具多於5%未配對之序列將不會雜交的條件。 As used herein, "stringent conditions" encompasses hybridization only - in the case of a hypothesis that there is less than 20% unpaired-occurrence between the hybrid molecule and the homologous sequence within the target nucleic acid molecule. “Strict conditions” include additional levels of particular severity. Thus, as used in this context, "moderately harsh" conditions are those conditions in which more than 20% of the unpaired molecules will not hybridize; "highly stringent" conditions are such that they have more than 10% unpaired Conditions under which the sequence will not hybridize; and "very high severity" conditions are those conditions in which more than 5% of the unpaired sequences will not hybridize.

下列為代表性、非設限雜交條件。 The following are representative, non-limit hybridization conditions.

高度嚴苛條件(偵測共享至少90%序列一致性的序列)：在5x SSC緩衝液於65℃雜交16小時：在2x SSC緩衝液於室溫洗滌兩次，各為15分鐘；以及在0.5x SSC緩衝液於65℃洗滌兩次，各為20分鐘。 Highly stringent conditions (detecting sequences sharing at least 90% sequence identity): hybridization in 5x SSC buffer at 65 °C for 16 hours: wash twice in 2x SSC buffer at room temperature for 15 minutes each; and at 0.5 x SSC buffer was washed twice at 65 ° C for 20 minutes each.

中度嚴苛條件(偵測共享至少80%序列一致性的序列)：在5x-6x SSC緩衝液於65-70℃雜交16-20小時；在2x SSC緩衝液於室溫洗滌兩次，各為5-20分鐘；以及在1x SSC緩衝液於55-70℃洗滌兩次，各為30分鐘。 Moderately harsh conditions (detecting sequences sharing at least 80% sequence identity): hybridization in 5x-6x SSC buffer at 65-70 °C for 16-20 hours; washing twice in 2x SSC buffer at room temperature, each For 5-20 minutes; and wash twice in 1x SSC buffer at 55-70 ° C for 30 minutes each.

非嚴苛控制條件(共享至少50%序列一致性的序列將會雜交)：在6x SSC緩衝液於室溫至55℃雜交16-20小時；在2x-3x SSC緩衝液於室溫至55℃洗滌至少兩次，各為20-30分鐘。 Non-rigid control conditions (sequences that share at least 50% sequence identity will hybridize): 16-20 hours in 6x SSC buffer at room temperature to 55 °C; in room temperature to 55 °C in 2x-3x SSC buffer Wash at least twice, each for 20-30 minutes.

如本案所用，就聚核苷酸而言的術語“實質上同源”或“實質同源性”係指稱在嚴苛條件下雜交至參考核酸序列的聚核苷酸。舉例來說，實質上同源於參考DNA編碼序列的聚核苷酸為該等在嚴苛條件下(譬如列舉的中度嚴苛條件，見上文)雜交至參考DNA編碼序列的聚核苷酸。實質上同源性序列可具有至少80%序列一致性。舉例來說，實質上同源性序列可具有約80%至100%序列一致性，例如約81%；約82%；約83%；約84%；約85%；約86%；約87%；約88%；約89%；約90%；約91%；約92%；約93%；約94%；約95%；約96%；約97%；約98%；約98.5%；約99%；約99.5%；及約100%。實質同源性的特性係與特異性雜交密切相關。舉例來說，核酸分子係在有足夠互補程度時特異性地雜交，以避免在特異性結合係為所欲的條件下，舉例來說，在嚴苛雜交條件下，核酸非特異性結合至非標靶序列。 As used herein, the term "substantially homologous" or "substantially homologous" with respect to a polynucleotide refers to a polynucleotide that hybridizes under stringent conditions to a reference nucleic acid sequence. For example, a polynucleotide that is substantially homologous to a reference DNA coding sequence is a polynucleoside that hybridizes to a reference DNA coding sequence under stringent conditions (such as the list of moderately harsh conditions, see above). acid. Substantially homologous sequences can have at least 80% sequence identity. For example, a substantially homologous sequence can have from about 80% to 100% sequence identity, such as about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87% About 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94%; about 95%; about 96%; about 97%; about 98%; about 98.5%; 99%; about 99.5%; and about 100%. The properties of substantial homology are closely related to specific hybridization. For example, a nucleic acid molecule specifically hybridizes when there is sufficient complementarity to avoid non-specific binding of the nucleic acid to non-specific under stringent hybridization conditions under conditions where the specific binding system is desired. Target sequence.

如本案所用，術語“異種同源物”係指稱二或多個物種內從共同袓先核苷酸序列演化並在該二或多個物種內可保留相同功能的基因。 As used herein, the term "heterologous" refers to a gene that has evolved from a common prior nucleotide sequence within two or more species and that retains the same function within the two or more species.

如本案所用，當序列每一個核苷酸以5'至3'方向讀取時係互補於另一序列中的每一個核苷酸以3'至5'方向讀取時，兩個核酸序列分子被稱為展現“完全互補性”。互補於參考核苷酸序列的核苷酸序列將展現和參考核苷酸序列的反向互補序列一致的序列。該等術語與說明在本領域中係明確定義且為熟習此藝者所容易理解的。 As used herein, two nucleic acid sequence molecules are read when the nucleotide is read in the 5' to 3' direction and each nucleotide in the other sequence is read in the 3' to 5' direction. It is called to show "complete complementarity." A nucleotide sequence that is complementary to a reference nucleotide sequence will exhibit a sequence that is identical to the reverse complement of the reference nucleotide sequence. These terms and descriptions are expressly defined in the art and are readily understood by those skilled in the art.

如本案所用，術語“實質上一致”可指稱多於85% 一致的核苷酸序列。舉例來說，實質上一致的核苷酸序列可與參考序列至少85.5%；至少86%；至少87%；至少88%；至少89%；至少90%；至少91%；至少92%；至少93%；至少94%；至少95%；至少96%；至少97%；至少98%；至少99%；或至少99.5%一致。 As used in this case, the term "substantially consistent" may refer to more than 85%. Consistent nucleotide sequence. For example, a substantially identical nucleotide sequence can be at least 85.5%; at least 86%; at least 87%; at least 88%; at least 89%; at least 90%; at least 91%; at least 92%; at least 93 %; at least 94%; at least 95%; at least 96%; at least 97%; at least 98%; at least 99%; or at least 99.5% consistent.

表現：如本案所用，編碼序列(舉例來說，基因或轉殖基因)的“表現”係指稱核酸轉錄單元的編碼資訊(包括譬如基因組DNA或cDNA)轉換成細胞的操作、非操作、或結構部分(譬如蛋白質)的過程。基因表現可受到外在信號影響；舉例來說，使細胞、組織、或生物體暴露至增加或減少本案所包含之基因表現的藥劑。基因表現亦可在DNA至RNA至蛋白之途徑中的任何地方受到調控。基因表現的調控可經由下列發生，舉例來說，經由作用在轉錄、轉譯、RNA運送與加工、諸如mRNA之中介分子的降解的控制、及/或經由特定蛋白分子製成後的活化、失活、分室、或降解，或藉由前述任意組合。基因表現可藉由本領域習知方法於RNA位準或蛋白位準測量，該等方法包括而不限於北方墨點法、RT-PCR、西方墨點法、與(多個)體外、原位、或體內蛋白活性試驗。 Performance: As used in this context, the "expression" of a coding sequence (for example, a gene or a transgene) refers to the manipulation, non-operation, or structure of the coding information (including, for example, genomic DNA or cDNA) of a nucleic acid transcription unit into a cell. Part of the process (such as protein). Gene expression can be affected by extrinsic signals; for example, exposing cells, tissues, or organisms to agents that increase or decrease the gene expression contained in this case. Gene expression can also be regulated anywhere in the DNA to RNA to protein pathway. Regulation of gene expression can occur, for example, via activation, inactivation by transcription, translation, RNA delivery and processing, control of degradation of intervening molecules such as mRNA, and/or via specific protein molecules. , compartment, or degradation, or by any combination of the foregoing. Gene expression can be measured at RNA level or protein level by methods known in the art, including, but not limited to, Northern blotting, RT-PCR, Western blotting, and in vitro, in situ, Or in vivo protein activity test.

增加表現：如本案所用，術語“增加表現”係指稱啟始表現，也指稱模板構築體所製造的表現產物份量的數量增加。在一些具體例中，可提供至少一異源性基因至以其他方式包含相同基因之內源性複本的細胞或生物體，從而增加該基因所編碼的多肽的表現。在此類具體例中，表 現的增加可藉由比對包含異源性與內源性基因的細胞所製造的多肽份量和僅包含內源性基因細胞所製造的份量來測定。在一些具體例中，可提供影響轉錄的第一多肽(譬如NSP1-樣及/或NSP2-樣)至細胞或生物體，從而增加受到第一多肽控制的基因所編碼的第二多肽的表現。在此類具體例中，表現的增加可藉由比對在第一多肽存在下由該基因製造的多肽份量和無第一多肽存在下由該基因製造的份量來測定。在一些具體例中，調控序列可操作性地聯結至基因，從而增加基因的表現。在此類具體例中，表現的增加可藉由比對在調控序列操作性地聯結至基因之後由該基因所製造的多肽份量和操作性聯結或引進調控序列之前由該基因所製造的份量來測定。 Increased performance: As used in this case, the term "increase performance" refers to the initial performance and also refers to an increase in the number of performance product parts produced by the template structure. In some embodiments, at least one heterologous gene can be provided to a cell or organism that otherwise comprises an endogenous replica of the same gene, thereby increasing the performance of the polypeptide encoded by the gene. In such specific cases, the table The current increase can be determined by aligning the amount of polypeptide produced by cells containing the heterologous and endogenous genes and the amount of the part produced by only the endogenous gene cells. In some embodiments, a first polypeptide (eg, NSP1-like and/or NSP2-like) that affects transcription can be provided to a cell or organism, thereby increasing a second polypeptide encoded by a gene controlled by the first polypeptide. Performance. In such embodiments, the increase in performance can be determined by aligning the portion of the polypeptide made from the gene in the presence of the first polypeptide and the portion produced from the gene in the absence of the first polypeptide. In some embodiments, the regulatory sequences are operably linked to a gene, thereby increasing the performance of the gene. In such a specific example, the increase in expression can be determined by aligning the amount of the polypeptide produced by the gene after the regulatory sequence is operatively linked to the gene and the amount of the moiety produced by the gene prior to the operative linkage or introduction of the regulatory sequence. .

操作性地聯結：當第一核酸序列係與第二核酸序列呈功能性關聯時，第一核苷酸序列係和第二核酸序列操作性地聯結。當重組地製造時，以及，在有必要接合在相同讀取框(譬如在多順反子ORF)的兩蛋白-編碼區域時，操作性地聯結的核酸序列一般係相鄰。然而，核酸不需相鄰以操作性地聯結。 Operably linked: when the first nucleic acid sequence is functionally associated with the second nucleic acid sequence, the first nucleotide sequence and the second nucleic acid sequence are operatively linked. When recombinantly produced, and where it is necessary to join two protein-coding regions in the same reading frame (e.g., in a polycistronic ORF), the operably linked nucleic acid sequences are generally contiguous. However, nucleic acids do not need to be contiguous to be operatively linked.

在提及調控序列與編碼序列所使用的術語“操作性地聯結”意指調控序列影響所聯結編碼序列的表現。“調控序列”或“控制元”係指稱影響轉錄時點與位準/份量、RNA加工或穩定性、或相連編碼序列轉譯的核苷酸序列。調控序列可包括促進子；轉譯前導序列；內含子；增強子；主幹-迴圈結構；抑制子結合序列；終止序列；聚腺苷酸化識 別序列；等等。特別調控序列可位於操作性地聯結至彼等的編碼序列上游及/或下游。又，操作性地聯結至編碼序列的特別調控序列可位於雙股核酸分子的相連互補股上。 The term "operatively linked" as used in reference to a control sequence and a coding sequence means that the regulatory sequence affects the performance of the linked coding sequence. A "regulatory sequence" or "control element" refers to a nucleotide sequence that affects the timing and level of transcription, the processing or stability of RNA, or the translation of a contiguous coding sequence. Regulatory sequences may include promoters; translation leader sequences; introns; enhancers; backbone-loop structures; suppressor binding sequences; termination sequences; polyadenylation Don't sequence; wait. The particular regulatory sequences may be located upstream and/or downstream of the coding sequence operably linked to them. Further, a particular regulatory sequence operably linked to a coding sequence can be located on a contiguous complementary strand of a double stranded nucleic acid molecule.

促進子：如本案所用，術語“促進子”係指稱可在轉錄起點上游且可涉及啟始轉錄的RNA聚合酶與其他蛋白之識別與結合的DNA區域。促進子可操作性地聯結至編碼序列以在細胞中表現、或促進子可操作性地聯結至編碼信號序列的核苷酸序列，該信號序列可操作性地聯結至編碼序列以在細胞中表現。 Promoter: As used herein, the term "promoter" refers to a region of DNA that is upstream of the start of transcription and that may be involved in the recognition and binding of RNA polymerase and other proteins that initiate transcription. An promoter is operably linked to a coding sequence for expression in a cell, or a promoter is operably linked to a nucleotide sequence encoding a signal sequence operably linked to a coding sequence for expression in a cell .

本案一些具體例包括“植物促進子”。植物促進子為能夠在植物細胞內啟始轉錄的促進子。 Some specific examples of this case include "plant promoters". Plant promoters are promoters that initiate transcription in plant cells.

本案一些具體例包括“組織-偏好性促進子”。組織-偏好性促進子為能夠在發展控制之下啟始轉錄的促進子，包括，舉例而不限於：偏好啟始葉、花粉、穗、根、種籽、纖維、木質部導管、管胞、和厚壁組織的轉錄的促進子。基本上僅在某些組織啟始轉錄的促進子係稱作“組織-特異性”。“細胞類型-特異性”促進子主要驅動在一或多個器官中的某些細胞類型的表現，舉例來說，根或葉中的維管束細胞。“可誘發”促進子可為可受到環境控制的促進子。藉由可誘發促進子可啟始轉錄的環境條件例子包括厭氧條件與光照。組織-特異性、組織-偏好性、細胞類型-特異性、及可誘發進子構成“非-組成促進子類別。 Some specific examples of this case include the "organization-preference facilitator". Tissue-preference promoters are promoters that initiate transcription under developmental control, including, by way of example and not limitation: preferred initiation leaves, pollen, ears, roots, seeds, fibers, xylem vessels, tracheids, and Promoter of transcription of thick-walled tissues. Promoters that initiate transcription only in certain tissues are referred to as "tissue-specific". The "cell type-specific" promoter primarily drives the expression of certain cell types in one or more organs, for example, vascular cells in the root or leaf. An "inducible" promoter can be an environmentally motivated promoter. Examples of environmental conditions by which promoters can be induced to initiate transcription include anaerobic conditions and illumination. Tissue-specificity, tissue-preference, cell type-specificity, and inducible promoters constitute a "non-composition facilitator subclass.

任何可誘發促進子可用於本案一些具體例。參閱Ward et al.(1993)Plant Mol.Biol.22：361-366。藉由可誘發 促進子，回應誘發劑的轉錄速率增加了。例示的可誘發促進子包括但不限於：回應銅的來自ACEI系統的促進子；回應苯磺醯胺除草劑安全劑的來自玉米的In2基因；來自Tn10的Tet抑制子；與來自類固醇激素基因的可誘發促進子，其轉錄活性可被糖皮質類固醇激素誘發(Schena et al.(1991)Proc.Natl.Acad.Sci.USA 88：10421-5)。 Any inducible promoter can be used in some specific cases in this case. See Ward et al. (1993) Plant Mol. Biol. 22: 361-366. By inducing the promoter, the rate of transcription of the response elicitor is increased. Exemplary inducible promoters include, but are not limited to, promoters from the ACEI system that respond to copper; In2 genes from maize that respond to sulfonamide herbicide safeners ; Tet inhibitors from Tn10; and genes from steroid hormones Promoters can be induced whose transcriptional activity can be induced by glucocorticosteroids (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-5).

與非組成促進子相反，“組成”促進子為在大部分環境條件下活躍的促進子。例示的組成促進子包括但不限於：來自植物病毒的促進子，例如來自CaMV的35S促進子；來自水稻肌動蛋白的促進子；泛素促進子；pEMU；MAS；玉米H3組蛋白促進子；與ALS促進子，Xba1/NcoI分段5'至油菜ALS3結構基因(或類似於該Xba1/NcoI分段的核苷酸序列)(PCT國際專利公開案號WO 96/30530)。 In contrast to non-composed promoters, the "composition" promoter is a promoter that is active under most environmental conditions. Exemplary compositional promoters include, but are not limited to, promoters derived from plant viruses, such as the 35S promoter from CaMV; promoters from rice actin; ubiquitin promoter; pEMU; MAS; maize H3 histone promoter; With the ALS promoter, Xba1/NcoI is segmented 5' to the canola ALS3 structural gene (or a nucleotide sequence similar to the Xba1/NcoI segment) (PCT International Patent Publication No. WO 96/30530).

此外，任何組織-特異性或組織-偏好性促進子可利用在本發明的一些具體例。以包含操作性地聯結至組織特異性促進子之編碼序列的核酸分子轉形的植物可在特定組織中專一地、或偏好地製造所編碼序列的產物。例示的組織-特異性或組織-偏好性促進子包括但不限於：根部-偏好性促進子，例如來自菜豆基因者；葉特異性與光誘發促進子，例如來自cab或rubisco者；花粉囊特異性促進子，例如來自LAT52者；花粉特異性促進子，例如來自Zm13者；以及小孢子偏好性促進子，例如來自apg者。 Furthermore, any tissue-specific or tissue-preferential promoter can be utilized in some specific examples of the invention. A plant transformed with a nucleic acid molecule comprising a coding sequence operably linked to a tissue-specific promoter can specifically or preferentially produce a product of the encoded sequence in a particular tissue. Exemplary tissue-specific or tissue-preferential promoters include, but are not limited to, root-preference promoters, such as those from the bean gene; leaf-specific and light-inducible promoters, such as those from cab or rubisco ; of facilitator, for example those derived from LAT52; promote pollen-specific promoters, such as those derived from Zm13; and a microspore-preferred facilitator, for example, those derived from apg.

保守性取代：如本案所用，術語“保守性取代”係指稱胺基酸殘基被相同類別之其他胺基酸取代的取代作 用。非保守性胺基酸取代為殘基不落於相同類別的取代作用，舉例來說，鹼性胺基酸被中性或非極性胺基酸取代。可為實行保守性取代目的所定義的胺基酸類別為本領域所習知者。 Conservative substitution: As used herein, the term "conservative substitution" refers to a substitution of an amino acid residue by another amino acid of the same class. use. The non-conservative amino acid substitution is a substitution in which the residue does not fall within the same class, for example, the basic amino acid is substituted with a neutral or non-polar amino acid. The class of amino acids that can be defined for the purpose of conservative substitutions are well known in the art.

在一些具體例中，保守性取代包括第一脂族胺基酸被第二不同的脂族胺基酸取代。舉例來說，假使第一胺基酸為Gly；Ala；Pro；Ile；Leu；Val；與Met當中一者，則第一胺基酸可置換成選自Gly；Ala；Pro；Ile；Leu；Val；與Met的第二不同胺基酸。在特定例子中，假使第一胺基酸為Gly；Ala；Pro；Ile；Leu；與Val當中一者，則第一胺基酸可置換成選自Gly；Ala；Pro；Ile；Leu；與Val的第二不同胺基酸。在涉及取代疏水性脂族胺基酸的特定例子中，假使第一胺基酸為Ala；Pro；Ile；Leu；與Val當中一者，則第一胺基酸可置換成選自Ala；Pro；Ile；Leu；與Val的第二不同胺基酸。 In some embodiments, conservative substitutions include the substitution of a first aliphatic amino acid with a second, different aliphatic amino acid. For example, if the first amino acid is Gly; Ala; Pro; Ile; Leu; Val; and Met, the first amino acid can be substituted to be selected from Gly; Ala; Pro; Ile; Leu; Val; a second different amino acid from Met. In a specific example, the first amino acid is Gly; Ala; Pro; Ile; Leu; The second different amino acid of Val. In a specific example involving the substitution of a hydrophobic aliphatic amino acid, if the first amino acid is Ala; Pro; Ile; Leu; and one of Val, the first amino acid can be substituted to be selected from Ala; ; Ile; Leu; a second different amino acid from Val.

在其他具體例中，保守性取代包括第一芳香族胺基酸被第二、不同的芳香族胺基酸取代。舉例來說，假使第一胺基酸為His；Phe；Trp；與Tyr當中一者，則第一胺基酸可置換成選自His；Phe；Trp；與Tyr的第二不同胺基酸。在涉及取代不帶電芳香族胺基酸的特定例子中，假使第一胺基酸為Phe；Trp；與Tyr當中一者，則第一胺基酸可置換成選自Phe；Trp；與Tyr的第二不同胺基酸。 In other embodiments, conservative substitutions include the substitution of the first aromatic amino acid with a second, different aromatic amino acid. For example, if the first amino acid is His; Phe; Trp; and Tyr, the first amino acid can be substituted with a second different amino acid selected from the group consisting of His; Phe; Trp; and Tyr. In a specific example involving the substitution of an uncharged aromatic amino acid, if the first amino acid is Phe; Trp; and Tyr, the first amino acid can be substituted with a moiety selected from Phe; Trp; and Tyr. The second different amino acid.

在替換具體例中，保守性取代包括第一疏水性胺基酸被第二、不同的疏水性胺基酸取代。舉例來說，假使 第一胺基酸為Ala；Val；Ile；Leu；Met；Phe；Tyr；與Trp當中一者，則第一胺基酸可置換成選自Ala；Val；Ile；Leu；Met；Phe；Tyr；與Trp的第二不同胺基酸。在涉及取代非芳香族疏水性胺基酸的特定例子中，假使第一胺基酸為Ala；Val；Ile；Leu；與Met當中一者，則第一胺基酸可置換成選自Ala；Val；Ile；Leu；與Met的第二不同胺基酸。 In an alternative embodiment, the conservative substitution comprises the substitution of the first hydrophobic amino acid with a second, different hydrophobic amino acid. For example, if The first amino acid is Ala; Val; Ile; Leu; Met; Phe; Tyr; and Trp, the first amino acid can be substituted to be selected from Ala; Val; Ile; Leu; Met; Phe; Tyr a second different amino acid from Trp. In a specific example involving the substitution of a non-aromatic hydrophobic amino acid, if the first amino acid is Ala; Val; Ile; Leu; and Met, the first amino acid can be substituted to be selected from Ala; Val; Ile; Leu; a second different amino acid with Met.

在一些具體例中，保守性取代包括第一極性胺基酸被第二不同的極性胺基酸取代。舉例來說，假使第一胺基酸為Ser；Thr；Asn；Gln；Cys；Gly；Pro；Arg；His；Lys；Asp；與Glu當中一者，則第一胺基酸可置換成選自Ser；Thr；Asn；Gln；Cys；Gly；Pro；Arg；His；Lys；Asp；與Glu的第二不同胺基酸。在涉及取代不帶電極性胺基酸的特定例子中，假使第一胺基酸為Ser；Thr；Asn；Gln；Cys；Gly；與Pro當中一者，則第一胺基酸可置換成選自Ser；Thr；Asn；Gln；Cys；Gly；與Pro的第二不同胺基酸。在涉及取代帶電極性胺基酸的特定例子中，假使第一胺基酸為His；Arg；Lys；Asp；與Glu當中一者，則第一胺基酸可置換成選自His；Arg；Lys；Asp；與Glu的第二不同胺基酸。在涉及取代帶電極性胺基酸的另外例子中，假使第一胺基酸為Arg；Lys；Asp；與Glu當中一者，則第一胺基酸可置換成選自Arg；Lys；Asp；與Glu的第二不同胺基酸。在涉及取代帶正電(鹼性)極性胺基酸的特定例子中，假使第一胺基酸為His；Arg；與Lys當中一者，第一胺基酸可置換成選自His；Arg；與Lys的第二不同胺基酸。在涉及取代帶正電極性胺 基酸的另外例子中，假使第一胺基酸為Arg或Lys，則第一胺基酸可置換成Arg與Lys當中的另一胺基酸。在涉及取代帶負電(酸性)極性胺基酸的特定例子中，假使第一胺基酸為Asp或Glu，則第一胺基酸可置換成Asp與Glu當中的另一胺基酸。 In some embodiments, conservative substitutions include the substitution of a first polar amino acid by a second, different polar amino acid. For example, if the first amino acid is Ser; Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; and Glu, the first amino acid can be substituted to be selected from the group consisting of Ser; Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; second different amino acid with Glu. In a specific example involving the substitution of an unsubstituted amino acid, if the first amino acid is Ser; Thr; Asn; Gln; Cys; Gly; and Pro, the first amino acid can be replaced From Ser; Thr; Asn; Gln; Cys; Gly; a second different amino acid from Pro. In a specific example involving substitution of a charged amino acid, if the first amino acid is His; Arg; Lys; Asp; and Glu, the first amino acid can be substituted to select from His; Arg; Lys; Asp; a second different amino acid from Glu. In another example involving substitution of a charged amino acid, if the first amino acid is Arg; Lys; Asp; and Glu, the first amino acid can be substituted to be selected from Arg; Lys; Asp; A second amino acid different from Glu. In a specific example involving substitution of a positively charged (basic) polar amino acid, the first amino acid is His; Arg; and one of Lys, the first amino acid may be substituted to be selected from His; Arg; A second amino acid different from Lys. Involving a positive electrode with a positive electrode In another example of a base acid, if the first amino acid is Arg or Lys, the first amino acid can be substituted with another amino acid among Arg and Lys. In a specific example involving the substitution of a negatively charged (acidic) polar amino acid, if the first amino acid is Asp or Glu, the first amino acid can be substituted with another amino acid among Asp and Glu.

在額外具體例中，保守性取代包括第一電中性胺基酸被第二不同電中性胺基酸取代。舉例來說，假使第一胺基酸為Gly；Ser；Thr；Cys；Asn；Gln；與Tyr當中一者，則第一胺基酸可置換成選自Gly；Ser；Thr；Cys；Asn；Gln；與Tyr的第二不同胺基酸。 In additional embodiments, conservative substitutions include the substitution of a first electrically neutral amino acid by a second, different electrically neutral amino acid. For example, if the first amino acid is Gly; Ser; Thr; Cys; Asn; Gln; and Tyr, the first amino acid can be substituted to be selected from Gly; Ser; Thr; Cys; Asn; Gln; a second amino acid different from Tyr.

在一些具體例中，保守性取代包括第一非極性胺基酸被第二不同非極性胺基酸取代。舉例來說，假使第一胺基酸為Ala；Val；Leu；Ile；Phe；Trp；Pro；與Met當中一者，則第一胺基酸可置換成選自Ala；Val；Leu；Ile；Phe；Trp；Pro；與Met的第二不同胺基酸。 In some embodiments, conservative substitutions include the substitution of a first non-polar amino acid by a second, different, non-polar amino acid. For example, if the first amino acid is Ala; Val; Leu; Ile; Phe; Trp; Pro; and Met, the first amino acid can be substituted to be selected from Ala; Val; Leu; Phe; Trp; Pro; a second different amino acid with Met.

在眾多例子中，欲用於保守性取代以置換第一胺基酸的特定第二胺基酸可為了使第一與第二胺基酸均隸屬之前述類別數量最大化來進行選擇。於是，假使第一胺基酸為Ser(極性、非芳香族及電中性胺基酸)，則第二胺基酸可為另一極性胺基酸(即Thr；Asn；Gln；Cys；Gly；Pro；Arg；His；Lys；Asp；或Glu)；另一非芳香族胺基酸(即Thr；Asn；Gln；Cys；Gly；Pro；Arg；His；Lys；Asp；Glu；Ala；Ile；Leu；Val；或Met)；或另一電中性胺基酸(即Gly；Thr；Cys；Asn；Gln；或Tyr)。然而，較佳的是第二胺基 酸在此情況中可為Thr；Asn；Gln；Cys；與Gly當中一者，因為該等胺基酸共享根據極性、非芳香性及電中性的所有類別。在選擇欲用於保守性取代的特別第二胺基酸時可任擇地使用的附加要點為本領域所習知的。舉例來說，當Thr；Asn；Gln；Cys；與Gly皆可用於保守性取代Ser時，Cys可從選擇中排除，以避免形成非所欲的交叉聯結及/或雙硫鍵。同樣地，Gly可從選擇中排除，因為其缺少烷基側鏈。在此情況中，可選擇Thr，譬如以保留側鏈羥基的官能性。然而，欲用於保守性取代的特定第二胺基酸的選擇最終係依照熟練技術人員的判斷。 In many instances, a particular second amino acid to be used for conservative substitution to replace the first amino acid can be selected in order to maximize the number of the aforementioned classes to which both the first and second amino acids are attached. Thus, if the first amino acid is Ser (polar, non-aromatic, and electrically neutral amino acids), the second amino acid can be another polar amino acid (ie, Thr; Asn; Gln; Cys; Gly). ;Pro;Arg;His;Lys;Asp; or Glu); another non-aromatic amino acid (ie, Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; Glu; Ala; Ile ; Leu; Val; or Met); or another electrically neutral amino acid (ie, Gly; Thr; Cys; Asn; Gln; or Tyr). However, it is preferred that the second amine group The acid may in this case be one of Thr; Asn; Gln; Cys; and Gly, since the amino acids share all classes according to polarity, non-aromaticity and electrical neutrality. Additional points that may optionally be used in selecting a particular second amino acid to be used for conservative substitutions are well known in the art. For example, when Thr; Asn; Gln; Cys; and Gly are both useful for conservative substitution of Ser, Cys can be excluded from selection to avoid formation of undesired cross-linking and/or disulfide bonds. Likewise, Gly can be excluded from the selection because it lacks an alkyl side chain. In this case, Thr can be selected, for example, to retain the functionality of the side chain hydroxyl groups. However, the selection of a particular second amino acid to be used for conservative substitution is ultimately at the discretion of the skilled artisan.

限氮條件：如本案所用，術語“限氮條件”係指稱在土壤或培養基中的氮源(譬如硝酸鹽和銨鹽)份量有限的條件。“有限”份量在一些例子中為介在0.0至0.2mM；譬如0至0.1mM、0至0.03mM、及0至0.05mM之氮濃度的範圍。 Nitrogen-limited conditions: As used herein, the term "nitrogen-limited conditions" refers to conditions in which the nitrogen source (such as nitrates and ammonium salts) in the soil or medium is limited in part. The "limited" portion is in the range of 0.0 to 0.2 mM; such as 0 to 0.1 mM, 0 to 0.03 mM, and 0 to 0.05 mM nitrogen concentration in some examples.

性狀或表型：術語“性狀”與“表型”在本案係交替使用。就本揭示內容目的而言，特別感興趣的性狀包括，舉例來說，作物植物可表現的農藝上重要的性狀。 Traits or phenotypes: The terms "trait" and "phenotype" are used interchangeably in this case. For purposes of this disclosure, traits of particular interest include, for example, agronomically important traits that crop plants can exhibit.

轉形：如本案所用，術語“轉形”係指稱轉移一或多個(複數個)核酸分子進入細胞。當核酸分子變成穩定被細胞複製時，無論藉由核酸分子併入細胞基因組、或藉由游離基因複製，則細胞被引進細胞的核酸分子“轉形”。如本案所用，術語“轉形”涵蓋可藉以將核酸分子引進此類細胞的所有技術。例子包括但不限於：以病毒載體轉染；以質體載體轉形；電穿孔(Fromm et al.(1986)Nature 319：791-3)； 脂質轉染(Felgner et al.(1987)Proc.Natl.Acad.Sci.USA 84：7413-7)；顯微注射(Mueller et al.(1978)Cell 15：579 85)；農桿菌-介導的轉移(Fraley et al.(1983)Proc.Natl.Acad.Sci.USA 80：4803-7)；直接DNA攝取；以及微彈轟擊(Klein et al.(1987)Nature 327：70)。 Transformation: As used herein, the term "transformation" refers to the transfer of one or more (plural) nucleic acid molecules into a cell. When a nucleic acid molecule becomes stable to be replicated by a cell, whether it is incorporated into the genome of the cell by the nucleic acid molecule, or by replication of the free gene, the cell is "transformed" by the nucleic acid molecule introduced into the cell. As used in this context, the term "transformation" encompasses all techniques by which nucleic acid molecules can be introduced into such cells. Examples include, but are not limited to, transfection with viral vectors; transformation with plastid vectors; electroporation (Fromm et al. (1986) Nature 319:791-3); lipid transfection (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); Microinjection (Mueller et al. (1978) Cell 15:579 85); Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl .Acad.Sci. USA 80:4803-7); direct DNA uptake; and microprojectile bombardment (Klein et al. (1987) Nature 327:70).

轉殖基因：轉殖基因為外源性核酸序列。在一些例子中，轉殖基因可為包含與標靶核酸互補的核苷酸序列的dsRNA分子之一或雙(多)股的序列。在一些例子中，轉殖基因可為反義核酸序列，其表現抑制標靶核酸的表現。在再其他例子中，轉殖基因可為基因序列(譬如抗除草劑基因)、編碼工業上或藥學上有用化合物的基因、或編碼所欲農業性狀的基因。在該等與其他例子中，轉殖基因可含有操作性地聯結至轉殖基因的編碼序列的調控序列(譬如促進子)。 Transgenic gene: The transgenic gene is an exogenous nucleic acid sequence. In some examples, the transgenic gene can be one of a dsRNA molecule or a double (multiple) strand comprising a nucleotide sequence that is complementary to the target nucleic acid. In some examples, the transgenic gene can be an antisense nucleic acid sequence that exhibits inhibition of the performance of the target nucleic acid. In still other examples, the transgenic gene can be a genetic sequence (such as a herbicide resistant gene), a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding an desired agricultural trait. In these and other examples, the transgene may contain regulatory sequences (e.g., promoters) operably linked to the coding sequence of the transgene.

載體：載體係指稱引進細胞-舉例來說-以製造轉形細胞的核酸分子。載體可包括允許其在宿主細胞內複製的核酸序列，例如複製起點。載體的例子包括但不限於：攜帶外源性DNA進入細胞的質體；黏接質體；噬菌體；與病毒。載體亦可包括本領域習知的一或多個基因、反義分子、及/或可擇標記基因及其他遺傳元。載體可轉導、轉形、或轉染細胞，藉此致使細胞表現該載體所編碼的核酸分子及/或蛋白。載體任擇地包括幫助實現核酸分子進入細胞(譬如脂質體、蛋白質外衣等等)的材料。 Vector: A vector refers to a nucleic acid molecule that introduces a cell, for example, to produce a transforming cell. A vector can include a nucleic acid sequence that allows it to replicate within a host cell, such as an origin of replication. Examples of vectors include, but are not limited to, plastids that carry exogenous DNA into the cell; plastids; phage; and viruses. The vector may also include one or more genes, antisense molecules, and/or selectable marker genes and other genetic elements as are known in the art. The vector can transduce, transform, or transfect the cell, thereby causing the cell to express the nucleic acid molecule and/or protein encoded by the vector. The vector optionally includes materials that help achieve entry of the nucleic acid molecule into a cell, such as a liposome, a protein coat, and the like.

除非明確指示或暗示，術語“一(a)”、“一(an)”、 與“該(the)”在本案係用於象徵“至少一個”。 Unless explicitly indicated or implied, the terms "a", "an", And "the" is used in this case to symbolize "at least one."

除非另有明確解釋，否則本案使用的所有技術性與科學性術語係具有本揭示內容所屬領域中具有通常技藝者所一般理解的相同意義。分子生物學中的常用術語定義可在，舉例來說，Lewin B.,Genes V,Oxford University Press,1994(ISBN 0-19-854287-9)；Kendrew et al.(eds.),The Encyclopedia of Molecular Biology,Blackwell Science Ltd.,1994(ISBN 0-632-02182-9)；與Meyers R.A.(ed.),Molecular Biology and Biotechnology：A Comprehensive Desk Reference,VCH Publishers,Inc.,1995(ISBN 1-56081-569-8)找到。所有百分比係指重量且所有溶劑混合物比例係指體積，除非另有註記。所有溫度為攝氏度。 Unless otherwise expressly stated, all technical and scientific terms used in this context have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of commonly used terms in molecular biology can be found, for example, by Lewin B., Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology , Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Meyers RA (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference , VCH Publishers, Inc., 1995 (ISBN 1-56081 -569-8) Find it. All percentages refer to weight and all solvent mixture ratios refer to volume unless otherwise noted. All temperatures are in degrees Celsius.

IV.非結瘤植物的BNF基因 IV. BNF gene of non- noduce plants

本揭示內容提供開發轉錄因子-NSP1-樣與NSP2-樣、及NIN-樣基因(譬如NLPs1-9)-的新穎且非預期用途的組成物與方法。如本案所揭示，NSP1-樣、NLP4、及NLP9影響植物(譬如非結瘤植物，例如***芥屬)與NFB(譬如苜蓿中華根瘤菌，例如RMP110)之間的功能***互作用。於是，舉例來說，NSP1-樣、NLP4、及/或NLP9可用於調控NFB與作物植物的關連性。本案所述NSP1-樣、NLP4、及NLP9的特性可用於，舉例來說，提供具經調整BNF表型的基因轉殖植物。舉例來說，NSP1-樣(SCL29)、NLP4、及/或NLP9可在植物中表現或過度表現，以啟始及/或增加植物在限N條件下與NFB關連的能力，及/或增加植 物利用環境中的氮的效率。在某些例子中，NSP1-樣(SCL29)、NLP4、及/或NLP9可引進植物或在慣常包含該多肽的植物中過度表現(譬如藉由引進編碼該多肽之基因的額外複本、及/或改變慣常存在基因的調控控制)。 The present disclosure provides compositions and methods for the development of novel and unintended uses of transcription factor-NSP1-like and NSP2-like, and NIN-like genes (such as NLPs1-9). As disclosed herein, NSP1-like, NLP4, and NLP9 affect functional interactions between plants (such as non-noduce plants, such as Arabidopsis) and NFB (such as S. meliloti, such as RMP110). Thus, for example, NSP1-like, NLP4, and/or NLP9 can be used to modulate the association of NFB with crop plants. The characteristics of NSP1-like, NLP4, and NLP9 described herein can be used, for example, to provide genetically transgenic plants having an adjusted BNF phenotype. For example, NSP1-like (SCL29), NLP4, and/or NLP9 may behave or overexpress in plants to initiate and/or increase the ability of plants to associate with NFB under conditions of limited N, and/or to increase The efficiency of the use of nitrogen in the environment. In certain instances, NSP1-like (SCL29), NLP4, and/or NLP9 can be introduced into a plant or overexpressed in a plant that routinely comprises the polypeptide (eg, by introducing an additional copy of the gene encoding the polypeptide, and/or Change the regulatory control of genes that are routinely present).

一些具體例包括NSP1-樣轉錄因子多肽。根據特別具體例的NSP1-樣多肽包含在和SEQ ID NO：1(***芥NSP1-樣)對齊時顯示增多百分比一致性的胺基酸序列。在該等與其他具體例中的特定胺基酸序列可包含具有和SEQ ID NO：1，舉例來說，呈至少約50%、約55%、約60%、約65%、約70%、約75%、約80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%一致性的序列。 Some specific examples include NSP1-like transcription factor polypeptides. The NSP1-like polypeptide according to a particular embodiment comprises an amino acid sequence which shows an increased percent identity when aligned with SEQ ID NO: 1 (Arabid mustard NSP1-like). The particular amino acid sequence in these and other specific examples can comprise and SEQ ID NO: 1, for example, at least about 50%, about 55%, about 60%, about 65%, about 70%, About 75%, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Sequence of 95%, 96%, 97%, 98%, 99%, or 100% identity.

其他具體例包括NIN-樣蛋白多肽。特別具體例包括NLP4多肽、及/或NLP9多肽。 Other specific examples include NIN-like protein polypeptides. Particularly specific examples include NLP4 polypeptides, and/or NLP9 polypeptides.

根據一些例子的NLP4多肽包含在和SEQ ID NO：2(***芥NLP4)對齊時顯示增多百分比一致性的胺基酸序列。在該等與其他具體例中的特定胺基酸序列可包含具有和SEQ ID NO：2，舉例來說，呈至少約50%、約55%、約60%、約65%、約70%、約75%、約80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%一致性的序列。舉例來說，一些具體例包括AtNLP4異種同源物。 An NLP4 polypeptide according to some examples comprises an amino acid sequence that exhibits increased percent identity when aligned with SEQ ID NO: 2 (Arabid mustard NLP4). The particular amino acid sequence in these and other specific examples can comprise and SEQ ID NO: 2, for example, at least about 50%, about 55%, about 60%, about 65%, about 70%, About 75%, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Sequence of 95%, 96%, 97%, 98%, 99%, or 100% identity. For example, some specific examples include AtNLP4 xenogenic homologs.

根據一些例子的NLP9多肽包含在和SEQ ID NO：3(***芥NLP9)對齊時顯示增多百分比一致性的胺基酸序列。在該等與其他具體例中的特定胺基酸序列可包含具有和SEQ ID NO：9，舉例來說，呈至少約50%、約55%、約60%、約65%、約70%、約75%、約80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或100%一致性的序列。舉例來說，一些具體例包括AtNLP9異種同源物。 An NLP9 polypeptide according to some examples comprises an amino acid sequence that exhibits increased percent identity when aligned with SEQ ID NO: 3 (Arabid mustard NLP9). The particular amino acid sequence in these and other specific examples can comprise and SEQ ID NO: 9, for example, at least about 50%, about 55%, about 60%, about 65%, about 70%, About 75%, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Sequence of 95%, 96%, 97%, 98%, 99%, or 100% identity. For example, some specific examples include AtNLP9 xenogenes.

在一些具體例中，包含具有和SEQ ID NO：1(NSP1-樣多肽)或SEQ ID NO：2(NLP4多肽)及/或SEQ ID NO：3(NLP9多肽)對齊時的前述序列一致性之胺基酸序列的多肽係包含在就包含該蛋白的植物與NFB之關連性而言為必要的蛋白以內。NSP1-樣多肽可藉由，舉例來說，在序列資料庫搜尋和SEQ ID NO：1具有臨界序列一致性的多肽序列來辨識。NLP4多肽可藉由，舉例來說，在序列資料庫搜尋和SEQ ID NO：2具有某些序列一致性的多肽序列來辨識。NLP9多肽多肽可藉由，舉例來說，在序列資料庫搜尋和SEQ ID NO：3具有某些序列一致性的多肽序列來辨識。有用的序列資料庫可藉由熟習此藝者習知的任意眾多方法(譬如利用NCBI的BLAST^®工具)來搜尋。經由各種公共和私人商業來源，可取得眾多植物與其他生物體的其他資料庫。熟習此藝者將暸解到的是，NLP4與NLP9為同源性蛋白，於是，一被辨識包含與SEQ ID NO：2或SEQ ID NO：3共享序列 一致性之胺基酸序列的特定多肽亦可與SEQ ID NOs：2與4當中的另一者共享序列一致性。 In some embodiments, comprising the foregoing sequence identity when aligned with SEQ ID NO: 1 (NSP1-like polypeptide) or SEQ ID NO: 2 (NLP4 polypeptide) and/or SEQ ID NO: 3 (NLP9 polypeptide) The polypeptide of the amino acid sequence is contained within a protein essential for the association of the plant containing the protein with the NFB. An NSP1-like polypeptide can be identified by, for example, searching in a sequence library for a polypeptide sequence having a critical sequence identity to SEQ ID NO: 1. The NLP4 polypeptide can be identified by, for example, searching in a sequence library and a polypeptide sequence having certain sequence identity of SEQ ID NO: 2. A NLP9 polypeptide polypeptide can be identified by, for example, searching in a sequence library and a polypeptide sequence having certain sequence identity in SEQ ID NO:3. Useful sequence databases can be by any number of methods apparent to those skilled in the art having conventional (such as the use of the NCBI BLAST ^® tool) to search. Access to numerous databases of plants and other organisms is available through a variety of public and private commercial sources. It will be understood by those skilled in the art that NLP4 and NLP9 are homologous proteins, and thus, a specific polypeptide which is recognized to contain an amino acid sequence which shares sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3 is also recognized. Sequence identity can be shared with the other of SEQ ID NOs: 2 and 4.

其他具體例包括NSP1-樣多肽的結構性等效物及/或NLP4多肽及/或NLP9多肽的結構性等效物。結構性等效物包括但不限於本案NSP1-樣、NLP4、及NLP9多肽的胺基酸序列內的胺基酸殘基的保守性取代。如本案所用，“保守性取代”為胺基酸殘基被相同類別的其他胺基酸取代的取代作用。非保守性胺基酸取代為殘基不落於相同類別的取代作用，舉例來說，鹼性胺基酸被中性或非極性胺基酸取代。可為實行保守性取代目的所定義的胺基酸類別為本領域所習知者。 Other specific examples include structural equivalents of NSP1-like polypeptides and/or structural equivalents of NLP4 polypeptides and/or NLP9 polypeptides. Structural equivalents include, but are not limited to, conservative substitutions of amino acid residues within the amino acid sequence of the NSP1-like, NLP4, and NLP9 polypeptides of the present invention. As used herein, "conservative substitution" is the substitution of an amino acid residue by another amino acid of the same class. The non-conservative amino acid substitution is a substitution in which the residue does not fall within the same class, for example, the basic amino acid is substituted with a neutral or non-polar amino acid. The class of amino acids that can be defined for the purpose of conservative substitutions are well known in the art.

在替換具體例中，保守性取代包括第一脂族胺基酸被第二不同的脂族胺基酸取代。舉例來說，假使第一胺基酸為Gly；Ala；Pro；Ile；Leu；Val；與Met當中一者，則第一胺基酸可置換成選自Gly；Ala；Pro；Ile；Leu；Val；與Met的第二不同胺基酸。在特定例子中，假使第一胺基酸為Gly；Ala；Pro；Ile；Leu；與Val當中一者，則第一胺基酸可置換成選自Gly；Ala；Pro；Ile；Leu；與Val的第二不同胺基酸。在涉及取代疏水性脂族胺基酸的特定例子中，假使第一胺基酸為Ala；Pro；Ile；Leu；與Val當中一者，則第一胺基酸可置換成選自Ala；Pro；Ile；Leu；與Val的第二不同胺基酸。 In an alternate embodiment, conservative substitutions include the substitution of the first aliphatic amino acid with a second, different aliphatic amino acid. For example, if the first amino acid is Gly; Ala; Pro; Ile; Leu; Val; and Met, the first amino acid can be substituted to be selected from Gly; Ala; Pro; Ile; Leu; Val; a second different amino acid from Met. In a specific example, the first amino acid is Gly; Ala; Pro; Ile; Leu; The second different amino acid of Val. In a specific example involving the substitution of a hydrophobic aliphatic amino acid, if the first amino acid is Ala; Pro; Ile; Leu; and one of Val, the first amino acid can be substituted to be selected from Ala; ; Ile; Leu; a second different amino acid from Val.

在其他具體例中，保守性取代包括第一芳香族胺基酸被第二不同的芳香族胺基酸取代。舉例來說，假使第 一胺基酸為His；Phe；Trp；與Tyr當中一者，則第一胺基酸可置換成選自His；Phe；Trp；與Tyr的第二不同胺基酸。在涉及取代不帶電芳香族胺基酸的特定例子中，假使第一胺基酸為Phe；Trp；與Tyr當中一者，則第一胺基酸可置換成選自Phe；Trp；與Tyr的第二不同胺基酸。 In other embodiments, conservative substitutions include the substitution of the first aromatic amino acid with a second, different aromatic amino acid. For example, if you The monoamino acid is His; Phe; Trp; and one of Tyr, the first amino acid can be substituted with a second different amino acid selected from the group consisting of His; Phe; Trp; and Tyr. In a specific example involving the substitution of an uncharged aromatic amino acid, if the first amino acid is Phe; Trp; and Tyr, the first amino acid can be substituted with a moiety selected from Phe; Trp; and Tyr. The second different amino acid.

在一些具體例中，保守性取代包括第一疏水性胺基酸被第二不同的疏水性胺基酸取代。舉例來說，假使第一胺基酸為Ala；Val；Ile；Leu；Met；Phe；Tyr；與Trp當中一者，則第一胺基酸可置換成選自Ala；Val；Ile；Leu；Met；Phe；Tyr；與Trp的第二不同胺基酸。在涉及取代非芳香族疏水性胺基酸的特定例子中，假使第一胺基酸為Ala；Val；Ile；Leu；與Met當中一者，則第一胺基酸可置換成選自Ala；Val；Ile；Leu；與Met的第二不同胺基酸。 In some embodiments, conservative substitutions include the first hydrophobic amino acid being substituted with a second, different hydrophobic amino acid. For example, if the first amino acid is Ala; Val; Ile; Leu; Met; Phe; Tyr; and Trp, the first amino acid can be substituted to be selected from Ala; Val; Ile; Leu; Met; Phe; Tyr; a second different amino acid from Trp. In a specific example involving the substitution of a non-aromatic hydrophobic amino acid, if the first amino acid is Ala; Val; Ile; Leu; and Met, the first amino acid can be substituted to be selected from Ala; Val; Ile; Leu; a second different amino acid with Met.

在額外的具體例中，保守性取代包括將第一極性胺基酸取代成第二不同極性胺基酸。舉例來說，假使第一胺基酸為Ser；Thr；Asn；Gln；Cys；Gly；Pro；Arg；His；Lys；Asp；與Glu當中一者，則第一胺基酸可置換成選自Ser；Thr；Asn；Gln；Cys；Gly；Pro；Arg；His；Lys；Asp；與Glu的第二不同胺基酸。在涉及取代不帶電極性胺基酸的特定例子中，假使第一胺基酸為Ser；Thr；Asn；Gln；Cys；Gly；與Pro當中一者，則第一胺基酸可置換成選自Ser；Thr；Asn；Gln；Cys；Gly；與Pro的第二不同胺基酸。在涉及取代帶電極性胺基酸的特定例子中，假使第一胺基酸為His；Arg；Lys；Asp；與Glu當中一者，則第一胺基酸可置換成 選自His；Arg；Lys；Asp；與Glu的第二不同胺基酸。在涉及取代帶電極性胺基酸的另外例子中，假使第一胺基酸為Arg；Lys；Asp；與Glu當中一者，則第一胺基酸可置換成選自Arg；Lys；Asp；與Glu的第二不同胺基酸。在涉及取代帶正電(鹼性)極性胺基酸的特定例子中，假使第一胺基酸為His；Arg；與Lys當中一者，則第一胺基酸可置換成選自His；Arg；與Lys的第二不同胺基酸。在涉及取代帶正電極性胺基酸的另外例子中，假使第一胺基酸為Arg或Lys，則第一胺基酸可置換成Arg與Lys當中的另一胺基酸。在涉及取代帶負電(酸性)極性胺基酸的特定例子中，假使第一胺基酸為Asp或Glu，第一胺基酸可置換成Asp與Glu當中的另一胺基酸。 In additional embodiments, conservative substitutions include the substitution of a first polar amino acid for a second, differently polar amino acid. For example, if the first amino acid is Ser; Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; and Glu, the first amino acid can be substituted to be selected from the group consisting of Ser; Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; second different amino acid with Glu. In a specific example involving the substitution of an unsubstituted amino acid, if the first amino acid is Ser; Thr; Asn; Gln; Cys; Gly; and Pro, the first amino acid can be replaced From Ser; Thr; Asn; Gln; Cys; Gly; a second different amino acid from Pro. In a specific example involving substitution of a charged amino acid, if the first amino acid is His; Arg; Lys; Asp; and one of Glu, the first amino acid can be substituted It is selected from the group consisting of His; Arg; Lys; Asp; and a second different amino acid with Glu. In another example involving substitution of a charged amino acid, if the first amino acid is Arg; Lys; Asp; and Glu, the first amino acid can be substituted to be selected from Arg; Lys; Asp; A second amino acid different from Glu. In a specific example involving the substitution of a positively charged (basic) polar amino acid, if the first amino acid is His; Arg; and one of Lys, the first amino acid can be substituted to be selected from His; a second different amino acid from Lys. In another example involving the substitution of a positively charged amino acid, the first amino acid can be substituted with another amino acid among Arg and Lys, provided that the first amino acid is Arg or Lys. In a specific example involving the substitution of a negatively charged (acidic) polar amino acid, if the first amino acid is Asp or Glu, the first amino acid can be substituted with another amino acid among Asp and Glu.

在一些具體例中，保守性取代包括第一電中性胺基酸被第二不同電中性胺基酸取代。舉例來說，假使第一胺基酸為Gly；Ser；Thr；Cys；Asn；Gln；與Tyr當中一者，則第一胺基酸可置換成選自Gly；Ser；Thr；Cys；Asn；Gln；與Tyr的第二不同胺基酸。 In some embodiments, conservative substitutions include the substitution of a first electrically neutral amino acid by a second, different electrically neutral amino acid. For example, if the first amino acid is Gly; Ser; Thr; Cys; Asn; Gln; and Tyr, the first amino acid can be substituted to be selected from Gly; Ser; Thr; Cys; Asn; Gln; a second amino acid different from Tyr.

在另外的具體例中，保守性取代包括第一非極性胺基酸被第二不同非極性胺基酸。舉例來說，假使第一胺基酸為Ala；Val；Leu；Ile；Phe；Trp；Pro；與Met當中一者，則第一胺基酸可置換成選自Ala；Val；Leu；Ile；Phe；Trp；Pro；與Met的第二不同胺基酸。 In another embodiment, the conservative substitution comprises the first non-polar amino acid being the second different non-polar amino acid. For example, if the first amino acid is Ala; Val; Leu; Ile; Phe; Trp; Pro; and Met, the first amino acid can be substituted to be selected from Ala; Val; Leu; Phe; Trp; Pro; a second different amino acid with Met.

在眾多例子中，欲用於保守性取代以置換第一胺基酸的特定第二胺基酸可為了使第一與第二胺基酸均隸屬 之前述類別數量最大化來進行選擇。於是，假使第一胺基酸為Ser(極性、非芳香族及電中性胺基酸)，則第二胺基酸可為另一極性胺基酸(即Thr；Asn；Gln；Cys；Gly；Pro；Arg；His；Lys；Asp；或Glu)；其他非芳香族胺基酸(即Thr；Asn；Gln；Cys；Gly；Pro；Arg；His；Lys；Asp；Glu；Ala；Ile；Leu；Val；或Met)；或另一電中性胺基酸(即Gly；Thr；Cys；Asn；Gln；或Tyr)。然而，然而，較佳的是第二胺基酸在此情況中可為Thr；Asn；Gln；Cys；與Gly當中一者，因為該等胺基酸共享根據極性、非芳香性及電中性的所有類別。在選擇欲用於保守性取代的特別第二胺基酸時可任擇地使用的附加要件為本領域所習知的。舉例來說，當Thr；Asn；Gln；Cys；與Gly皆可用於保守性取代Ser時，Cys可從選擇中排除，以避免形成非所欲的交叉聯結及/或雙硫鍵。同樣地，Gly可從選擇中排除，因為其缺少烷基側鏈。在此情況中，可選擇Thr，譬如以保留側鏈羥基的官能性。然而，欲用於保守性取代的特定第二胺基酸的選擇最終係依照熟練技術人員的判斷。 In many instances, a particular second amino acid to be used for conservative substitution to replace the first amino acid may be for the first and second amino acids to be The number of the aforementioned categories is maximized to make a selection. Thus, if the first amino acid is Ser (polar, non-aromatic, and electrically neutral amino acids), the second amino acid can be another polar amino acid (ie, Thr; Asn; Gln; Cys; Gly). ;Pro;Arg;His;Lys;Asp; or Glu); other non-aromatic amino acids (ie, Thr; Asn; Gln; Cys; Gly; Pro; Arg; His; Lys; Asp; Glu; Ala; Ile; Leu; Val; or Met); or another electrically neutral amino acid (ie, Gly; Thr; Cys; Asn; Gln; or Tyr). However, it is preferred that the second amino acid may in this case be Thr; Asn; Gln; Cys; and Gly, since the amino acids are shared according to polarity, non-aromaticity and electrical neutrality. All categories. Additional elements that may optionally be used in selecting a particular second amino acid to be used for conservative substitution are well known in the art. For example, when Thr; Asn; Gln; Cys; and Gly are both useful for conservative substitution of Ser, Cys can be excluded from selection to avoid formation of undesired cross-linking and/or disulfide bonds. Likewise, Gly can be excluded from the selection because it lacks an alkyl side chain. In this case, Thr can be selected, for example, to retain the functionality of the side chain hydroxyl groups. However, the selection of a particular second amino acid to be used for conservative substitution is ultimately at the discretion of the skilled artisan.

可對植物NSP1-樣、NLP4、與NLP9編碼聚核苷酸執行其他突變，以生成更適用於，舉例來說，在包含該(多個)聚核苷酸的宿主細胞中表現與擴大規模的表現產物。舉例來說，半胱胺酸殘基可被刪除或被其他胺基酸取代，以排除雙硫橋鍵；N-聯結糖基化位點可經更動或消除，以實現，舉例來說，表現從習知過度糖基化N-聯結位點的酵母宿主更容易回收與純化的均質產物。為此，在細胞外區域 (ECD)發生於任何一或多個糖基化識別序列的第一或第三胺基酸位置當中一或兩者的各式各樣胺基酸取代(N-X-S或N-X-T)、及/或在ECD於任何一或多個此類識別序列第二位置的胺基酸刪除將阻止經修飾三肽序列之突變多肽的糖基化。 Other mutations can be performed on plant NSP1-like, NLP4, and NLP9 encoding polynucleotides to generate more suitable, for example, expression and scale-up in host cells comprising the polynucleotide(s). Performance product. For example, a cysteine residue can be deleted or replaced with another amino acid to exclude a disulfide bridge; the N-linked glycosylation site can be modified or eliminated to achieve, for example, performance Yeast hosts that are known to over-glycosylate N-linkage sites are more likely to recover and purify homogenous products. For this purpose, in the extracellular region (ECD) a wide variety of amino acid substitutions (NXS or NXT) occurring in one or both of the first or third amino acid positions of any one or more glycosylation recognition sequences, and/or at ECD Amino acid deletion at the second position of any one or more of such recognition sequences will prevent glycosylation of the mutant polypeptide of the modified tripeptide sequence.

親水性胺基酸一般包括且一般具有下列個別相對疏水度(於pH 7.0；kcal/mol)：天門冬胺酸(D)，-7.4；麩胺酸(E)-9.9；天門冬醯胺(N)，-0.2；麩醯胺(Q)，-0.3；離胺酸(K)，-4.2；精胺酸(R)，-11.2；絲胺酸(S)，-0.3；與半胱胺酸(C)，-2.8。疏水性胺基酸一般包括且一般具有下列個別相對疏水度：組胺酸(H)，0.5；蘇胺酸(T)，0.4；酪胺酸(Y)，2.3；色胺酸(W)，3.4；***酸(F)，2.5；白胺酸(L)，1.8；異白胺酸(I)，2.5；甲硫胺酸(M)，1.3；纈胺酸(V)，1.5；與丙胺酸(A)，0.5。甘胺酸具有疏水度0並可被視為親水或疏水。 Hydrophilic amino acids generally include and generally have the following individual relative hydrophobicities (at pH 7.0; kcal/mol): aspartic acid (D), -7.4; glutamic acid (E)-9.9; aspartame ( N), -0.2; branamine (Q), -0.3; lysine (K), -4.2; arginine (R), -11.2; serine (S), -0.3; and cysteamine Acid (C), -2.8. Hydrophobic amino acids generally include and generally have the following individual relative degrees of hydrophobicity: histidine (H), 0.5; threonine (T), 0.4; tyrosine (Y), 2.3; tryptophan (W), 3.4; phenylalanine (F), 2.5; leucine (L), 1.8; isoleucine (I), 2.5; methionine (M), 1.3; valine (V), 1.5; with propylamine Acid (A), 0.5. Glycine has a degree of hydrophobicity of 0 and can be considered hydrophilic or hydrophobic.

如本領域所習知的，肽的胺基酸同源性可藉由比對其胺基酸序列輕易地測定。同樣地，肽的兩性同源性可藉由比對胺基酸序列的親水性和疏水性測定。 As is known in the art, the amino acid homology of a peptide can be readily determined by comparison to its amino acid sequence. Likewise, the amphoteric homology of the peptide can be determined by comparing the hydrophilicity and hydrophobicity of the amino acid sequence.

一些具體例包括了包含編碼NSP1-樣(“NSP1-樣聚核苷酸”)、NLP4(“NLP4聚核苷酸”)、及/或NLP9多肽(“NLP9聚核苷酸”)的核苷酸序列的核酸，例如上文所說明者。舉例來說，一些具體例中的核酸序列與SEQ ID NO：4(***芥NSP1-樣)或SEQ ID NO：5(***芥NLP4)及/或SEQ ID NO：6(***芥NLP9)對齊時展現增多的百分比一致性。 在該等與其他具體例中的特定核酸序列可包含具有，舉例而不限於：至少約50%、約55%、約60%、約65%、約70%、約75%、約80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%，或100%一致性SEQ ID NO：4或SEQ ID NO：5及/或SEQ ID NO：6的序列。 Some specific examples include nucleosides comprising NSP1-like ("NSP1-like polynucleotides"), NLP4 ("NLP4 polynucleotides"), and/or NLP9 polypeptides ("NLP9 polynucleotides"). A nucleic acid of an acid sequence, such as those described above. For example, when the nucleic acid sequence in some specific examples is aligned with SEQ ID NO: 4 ( Arabid mustard NSP1-like ) or SEQ ID NO: 5 ( Arabid mustard NLP4 ) and/or SEQ ID NO: 6 ( Arabid mustard NLP9 ) Shows increased percentage consistency. The particular nucleic acid sequences in these and other specific examples can include, by way of example and not limitation, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5 and/or SEQ ID NO: 6.

熟習此藝者可輕易地辨識出包含編碼NSP1-樣、NLP4、及/或NLP9多肽的核苷酸序列的許多核酸。舉例來說，可修飾核酸分子，而無實質上改變編碼多肽的胺基酸序列，舉例來說，藉由引進根據密碼子簡併性的可容許核苷酸取代。於是，將可理解到既定胺基酸序列的任何NSP1-樣、NLP4、及/或NLP9多肽可立即反向改造成任何眾多冗餘核苷酸序列。進一步舉例，編碼NSP1-樣、NLP4、及/或NLP9多肽的基因可選自任何眾多可得植物基因組庫、cDNA庫、EST庫、等等(譬如藉由對SEQ ID NOs：4-6當中一者的同源性)，或藉由所編碼多肽與SEQ ID NO：1或SEQ ID NOs：2及/或3的序列類似性，或此類基因可根據分子生物學的可靠與眾所周知技術選殖自生物體。 A number of nucleic acids comprising nucleotide sequences encoding NSP1-like, NLP4, and/or NLP9 polypeptides can be readily identified by those skilled in the art. For example, a nucleic acid molecule can be modified without substantially altering the amino acid sequence encoding the polypeptide, for example, by introducing a permissible nucleotide substitution based on codon degeneracy. Thus, it will be appreciated that any NSP1-like, NLP4, and/or NLP9 polypeptide of a given amino acid sequence can be immediately reverse engineered into any of a number of redundant nucleotide sequences. By way of further example, the gene encoding the NSP1-like, NLP4, and/or NLP9 polypeptide can be selected from any of a number of available plant genomic libraries, cDNA libraries, EST libraries, and the like (eg, by SEQ ID NOs: 4-6 Homology, or by sequence similarity of the encoded polypeptide to SEQ ID NO: 1 or SEQ ID NOs: 2 and/or 3, or such genes can be selected according to reliable and well-known techniques of molecular biology Since the organism.

任何與所有NSP1-樣、NLP4、與NLP9多肽、以及編碼彼等的核酸分子可利用於本發明的某些具體例。 Any and all of the NSP1-like, NLP4, and NLP9 polypeptides, as well as nucleic acid molecules encoding the same, can be utilized in certain embodiments of the invention.

在本案一些具體例中，包含編碼NSP1-樣、NLP4、及/或NLP9多肽之核苷酸序列的核酸包含基因調控元(譬如促進子)。促進子可基於欲***載體構築體的細胞類型來選擇。作用於細菌、酵母、與植物的促進子為本領域眾所周 知。促進子亦可基於彼等的調控特徵來選擇。此類特徵的例子包括轉錄活性的增強、誘發能力、組織-特異性、與發展階段-特異性。在植物中，已說明了可誘發、具病毒或合成來源、具組成性活性、以時間方式調控、及以空間方式調控的促進子。參閱譬如Poszkowski et al.(1989)EMBO J.3：2719；Odell et al.(1985)Nature 313：810；與Chau et al.(1989)Science 244：174-81)。 In some embodiments of the present invention, a nucleic acid comprising a nucleotide sequence encoding an NSP1-like, NLP4, and/or NLP9 polypeptide comprises a gene regulatory element (such as a promoter). Promoters can be selected based on the type of cell into which the vector construct is to be inserted. Promoters acting on bacteria, yeast, and plants are well known in the art. Promoters can also be selected based on their regulatory characteristics. Examples of such features include increased transcriptional activity, inducing ability, tissue-specificity, and developmental phase-specificity. In plants, promoters that can be induced, have a viral or synthetic origin, are constitutively active, are regulated in a temporal manner, and are spatially regulated are described. See, for example, Poszkowski et al. (1989) EMBO J. 3:2719; Odell et al. (1985) Nature 313:810; and Chau et al. (1989) Science 244: 174-81).

為獲得(多個)異源性基因的較高度表現，可較佳的是再改造(多個)基因，俾使更有效率地在表現宿主細胞(譬如植物細胞，舉例來說，油菜、水稻、煙草、玉米、棉花、與大豆)中表現。因此，在設計編碼NSP1-樣或NLP4及/或NLP9多肽的基因以供植物表現時的任擇額外步驟(即除了提供一或多個基因調控元以外)為再改造異源性基因蛋白編碼區域，以使表現最佳化。特別例子包括已最佳化以增加來自第二植物物種之基因轉殖植物細胞中的表現位準(即製造更多蛋白)的再設計***芥屬基因，其係相比於來自第二植物物種、轉形有原始(即未修飾)***芥屬基因序列的植物細胞。 In order to obtain a higher degree of expression of the heterologous gene(s), it may be preferred to reprogram the gene(s) to render the host cell more efficiently (eg, plant cells, for example, rapeseed, rice) , tobacco, corn, cotton, and soybeans). Thus, an additional step in designing a gene encoding a NSP1-like or NLP4 and/or NLP9 polypeptide for expression in a plant (ie, in addition to providing one or more gene regulatory elements) is a reprogramming of the heterologous gene protein coding region To optimize performance. Specific examples include redesigned Arabidopsis genes that have been optimized to increase performance levels in gene transfer plant cells from a second plant species (ie, to make more protein) compared to the second plant species Plant cells transformed with the original (ie unmodified) Arabidopsis gene sequence.

由於基因密碼冗餘性/簡併性(即一些胺基酸係由不止一個密碼子所指定)所給予的可塑性，在不同生物體或生物體類別的基因組演化已造成同義密碼子的使用差異。此“密碼子偏見”反映在蛋白編碼區域的平均鹼基組成。舉例來說，具有相對低G+C含量的基因組的生物體係利用較多在同義密碼子第三位置具有A或T的密碼子，而該等具有 相對高G+C含量者則利用較多在第三位置具有G或C的密碼子。再者，據認為在mRNA內“微量”密碼子的存在可降低該mRNA的絕對轉譯率，尤其是當對應於微量密碼子的帶電tRNA相對冗餘性極低的時候。此論述的延伸是因個別微量密碼子而減少的轉譯率就多個微量密碼子而言將至少加成。因此，在特別表現宿主中具有高相對含量的微量密碼子的mRNAs係具有相應低的轉譯率。此比率可由所編碼蛋白的相應低位準反映。 Genomic evolution in different organisms or organism classes has resulted in differences in the use of synonymous codons due to the plasticity conferred by gene redundancy/degenerate (ie, some amino acids are specified by more than one codon). This "codon bias" reflects the average base composition of the protein coding region. For example, a biological system with a relatively low G+C content of the genome utilizes more codons with A or T at the third position of the synonymous codon, and such Relatively high G+C content utilizes more codons with G or C at the third position. Furthermore, it is believed that the presence of "trace" codons in the mRNA reduces the absolute translational rate of the mRNA, especially when the relative redundancy of charged tRNAs corresponding to the microcodons is extremely low. An extension of this discussion is that the reduced translation rate due to individual microcodons will add at least for multiple microcodons. Thus, mRNAs with particularly high levels of microcodons in a particular expression host have a correspondingly low translation rate. This ratio can be reflected by the corresponding low level of the encoded protein.

在改造編碼NSP1-樣或NLP4及/或NLP9多肽以在植物細胞(譬如水稻、煙草、玉米、棉花、與大豆)中表現的最佳化基因時，假使已測定(多個)預期宿主植物的密碼子偏見是有幫助的。存在多個公開可用的DNA序列資料庫，其中吾人可找到關於植物基因組密碼子分佈情形的資訊或各種植物基因的蛋白編碼區域。 In engineering an optimized gene encoding an NSP1-like or NLP4 and/or NLP9 polypeptide for expression in plant cells (eg, rice, tobacco, corn, cotton, and soybean), if the desired host plant(s) have been determined Codon bias is helpful. There are a number of publicly available databases of DNA sequences in which we can find information about the distribution of plant genome codons or protein coding regions of various plant genes.

密碼子偏見是表現宿主用來編碼其蛋白的胺基酸的密碼子統計分佈。密碼子偏見可計算成相對於所有胺基酸的密碼子使用單一密碼子的頻率。或者，密碼子偏見可計算成編碼特定胺基酸的單一密碼子相對於該胺基酸所有其餘密碼子(同義密碼子)的使用頻率。 Codon bias is a codon statistical distribution that represents the amino acid that the host uses to encode its protein. Codon bias can be calculated as the frequency with which a single codon is used relative to the codons of all amino acids. Alternatively, codon bias can be calculated as the frequency of use of a single codon encoding a particular amino acid relative to all remaining codons (synonymous codons) of the amino acid.

在設計供植物表現NSP1-樣或NLP4及/或NLP9多肽的最佳化編碼區域時，當存在多重選項時，應測定植物所偏好的主要密碼子(“第一選項”)，還有第二、第三、第四等等選項的偏好密碼子。隨後可設計編碼NSP1-樣或NLP4及/或NLP9多肽的胺基序列的新穎DNA序列，其中該 新穎DNA序列係異於原生DNA序列(編碼該多肽)，藉由取代成表現宿主-所偏好(第一偏好、第二偏好、第三偏好、或第四偏好等等)密碼子，以指定胺基酸序列內各位置的胺基酸。隨後分析新穎序列中藉由修飾可能創建的限制酶位點。所辨識的推定限制位點再藉由以下一偏好密碼子置換該等密碼子來修飾，以移除限制位點。在序列中可能影響異源性序列轉錄或轉譯的其他位點為外顯子：內含子接點(5'或3')、聚-A添加信號、及/或RNA聚合酶終止信號。可再分析與修飾序列，以降低TA或CG雙聯體的頻率。除該等雙聯體以外，具有超過約六個相同的G或C核苷酸的序列嵌段亦可不利地影響序列轉錄或轉譯。因此，該等嵌段係有利地藉由將第一或第二等等密碼子選項置換成下一偏好密碼子選項來修飾。 When designing an optimized coding region for a plant to express NSP1-like or NLP4 and/or NLP9 polypeptides, when multiple options exist, the plant's preferred major codon ("first option") should be determined, and a second Preferred codons for options such as third, fourth, etc. A novel DNA sequence encoding an amino sequence of an NSP1-like or NLP4 and/or NLP9 polypeptide can then be designed, wherein A novel DNA sequence differs from a native DNA sequence (encoding the polypeptide) by the substitution of a host-preferred (first preference, second preference, third preference, or fourth preference, etc.) codon to specify the amine Amino acids at various positions within the base acid sequence. The restriction enzyme sites that may be created by modification in the novel sequences are then analyzed. The identified putative restriction sites are then modified by replacing the codons with a preferred codon to remove the restriction sites. Other sites in the sequence that may affect transcription or translation of the heterologous sequence are exons: intron junctions (5' or 3'), poly-A addition signals, and/or RNA polymerase termination signals. The sequence can be reanalyzed and modified to reduce the frequency of TA or CG doublets. In addition to the duplexes, sequence blocks having more than about six identical G or C nucleotides can also adversely affect sequence transcription or translation. Thus, the blocks are advantageously modified by replacing the first or second codons of the codons with the next preferred codon option.

諸如上文所說明的方法能使熟習此藝者修飾就特定植物而言為外來的(多個)基因，俾使該基因最佳化地表現於植物。該方法另例示於PCT國際專利公開案號WO 97/13402 A1。於是，一些具體例的功能上等效於NSP1-樣、NLP4、及/或NLP9聚核苷酸的最佳化合成基因可用於轉形宿主，包括植物與植物細胞。再者，亦可在電腦上(in silico)由初始胺基酸序列生成NSP1-樣、NLP4、與NLP9聚核苷酸。論及製造合成基因的額外指導可在，舉例來說，U.S.專利5,380,831找到。 A method such as that described above enables a person skilled in the art to modify a gene(s) that is foreign to a particular plant, such that the gene is optimally expressed in the plant. This method is further exemplified in PCT International Patent Publication No. WO 97/13402 A1. Thus, some specific examples of optimized synthetic genes that are functionally equivalent to NSP1-like , NLP4 , and/or NLP9 polynucleotides can be used in transformed hosts, including plant and plant cells. Furthermore, also generated in the computer (in silico) from the initial amino acid sequence of NSP1- like, nLP4, and NLP9 polynucleotide. Additional guidance for the manufacture of synthetic genes can be found, for example, in U.S. Patent 5,380,831.

一旦NSP1-樣、NLP4、或NLP9聚核苷酸序列已在紙上或電腦上設計，則包含該聚核苷酸序列的實際核酸 分子可在實驗室合成，以使序列精確對應所設計的序列。此類合成DNA分子可選殖或以其他方式操作，正如彼等係衍生自天然或原生來源一般。 Once the NSP1-like , NLP4 , or NLP9 polynucleotide sequence has been designed on paper or on a computer, the actual nucleic acid molecule comprising the polynucleotide sequence can be synthesized in the laboratory such that the sequence corresponds exactly to the designed sequence. Such synthetic DNA molecules are selected or otherwise manipulated, as are derived from natural or native sources.

V.在限N條件以NSP1-樣、NLP4、及/或NLP9調整植物生長 V. Adjusting plant growth with NSP1-like , NLP4 , and/or NLP9 under N-limited conditions

一些具體例係採用NSP1-樣、NLP4、與NLP9就植物在限N生長條件下經由與NFB之關連性維持及/或增加生長而言係為必需的發現。舉例來說，NSP1-樣、NLP4、及/或NLP9多肽可引進或異源性表現植物細胞，舉例而不限於：增加包含植物細胞的植物與NFB之關連性；增加限N條件下的植物生長；並增加植物利用環境氮的效率。 Some specific examples use NSP1-like, NLP4, and NLP9 to be necessary for plants to maintain and/or increase growth via association with NFB under N-limited growth conditions. For example, NSP1-like, NLP4, and/or NLP9 polypeptides can introduce or heterologously express plant cells, by way of example and without limitation: increasing the association of plants comprising plant cells with NFB; increasing plant growth under conditions of limited N And increase the efficiency of plants using environmental nitrogen.

在特定具體例中，NSP1-樣、NLP4、及/或NLP9多肽可在細胞或生物體內表現或過度表現，舉例而不限於：藉由將NSP1-樣、NLP4、及/或NLP9聚核苷酸引進細胞或生物體；藉由將NSP1-樣、NLP4、及/或NLP9多肽引進細胞或生物體；及/或藉由提供在細胞或生物體內經由(多個)信號和操作性地聯結至NSP1-樣、NLP4、及/或NLP9聚核苷酸的調控元交互作用而足以促進NSP1-樣、NLP4、及/或NLP9多肽表現的正向或負向信號。在另外的具體例中，NSP1-樣、NLP4、及/或NLP9多肽在細胞或生物體內可被剔除或低度表現，舉例而不限於：藉由破壞、突變、或失活NSP1-樣、NLP4、及/或NLP9聚核苷酸；將反義核酸引進靶向NSP1-樣、NLP4、及/或NLP9聚核苷酸的細胞或生物體；藉由以抗體或其他特異性結合蛋白結合NSP1-樣、NLP4、及/或NLP9多肽從細胞或生物體的細胞機構以物理方式移除 NSP1-樣、NLP4、及/或NLP9多肽；及/或藉由提供在細胞或生物體內經由(多個)信號和操作性地聯結至NSP1-樣、NLP4、及/或NLP9聚核苷酸的調控元交互作用而足以減少或消除NSP1-樣、NLP4、及/或NLP9多肽表現的正向或負向信號。 In a particular embodiment, the NSP1-like, NLP4, and/or NLP9 polypeptides can be expressed or overexpressed in a cell or organism, by way of example and without limitation: by NSP1-like , NLP4 , and/or NLP9 polynucleotides Introducing a cell or organism; introducing a NSP1-like, NLP4, and/or NLP9 polypeptide into a cell or organism; and/or by providing a signal or operatively linked to NSP1 via the signal(s) in the cell or organism The regulatory elemental interaction of the -like , NLP4 , and/or NLP9 polynucleotides is sufficient to promote positive or negative signaling of NSP1-like, NLP4, and/or NLP9 polypeptide expression. In another embodiment, the NSP1-like, NLP4, and/or NLP9 polypeptides can be eliminated or rendered low in a cell or organism, by way of example and without limitation: by disrupting, mutating, or inactivating NSP1-like , NLP4 And/or NLP9 polynucleotide; introducing an antisense nucleic acid into a cell or organism that targets an NSP1-like , NLP4 , and/or NLP9 polynucleotide; binding NSP1- by an antibody or other specific binding protein , NLP4, and/or NLP9 polypeptides physically remove NSP1-like, NLP4, and/or NLP9 polypeptides from cellular machinery or cells of a living organism; and/or via (s) provided in a cell or organism Signaling and operably linked to a regulatory elemental interaction of NSP1-like , NLP4 , and/or NLP9 polynucleotides sufficient to reduce or eliminate positive or negative signaling of NSP1-like, NLP4, and/or NLP9 polypeptide expression .

在一些具體例中，NSP1-樣多肽可在細胞或生物體內表現或過度表現，從而促進至少一NIN(多個)基因的表現；舉例而不限於NLP4與NLP9。在另外的具體例中，NSP1-樣多肽在細胞或生物體內可被移除或低度表現，從而減少或消除至少一NIN(多個)基因的表現，舉例來說，以研究在非結瘤植物中的NFB關連性的機制。在一些具體例中，NLP4及/或NLP9多肽可在細胞或生物體內表現或過度表現，從而直接增加植物細胞與NFB關連的能力。在另外的具體例中，NLP4及/或NLP9多肽在細胞或生物體內可被移除或低度表現，從而減少或消除植物細胞與NFB關連的能力。在特定具體例中，NSP1-樣、NLP4、及/或NLP9多肽係由操作性地聯結至在該等氮為生長限制以外的條件中引導該(多個)多肽表現的調控元的聚核苷酸表現，藉此增加植物在該等其他條件下利用環境氮的效率。 In some embodiments, the NSP1-like polypeptide can be expressed or overexpressed in a cell or organism, thereby promoting the expression of at least one NIN (s) gene; by way of example and without limitation, NLP4 and NLP9 . In another embodiment, the NSP1-like polypeptide can be removed or expressed in a low degree in a cell or organism, thereby reducing or eliminating the performance of at least one NIN (s) gene, for example, to study in a non-junction The mechanism of NFB association in plants. In some embodiments, the NLP4 and/or NLP9 polypeptide can be expressed or overexpressed in a cell or organism, thereby directly increasing the ability of the plant cell to be associated with NFB. In additional embodiments, the NLP4 and/or NLP9 polypeptides can be removed or understated in cells or organisms, thereby reducing or eliminating the ability of plant cells to be associated with NFB. In a particular embodiment, the NSP1-like, NLP4, and/or NLP9 polypeptide is a nucleoside operatively linked to a regulatory element that directs expression of the polypeptide(s) in conditions other than growth restriction of the nitrogen. The acid is expressed thereby increasing the efficiency of the plant in utilizing environmental nitrogen under these other conditions.

在特定具體例中，跨不同植物物種與不同植物物種之間的NFB關連性機制的保守性係被放大，以經由NSP1-樣、NLP4、及/或NLP9多肽的異源性表現將新穎NFB關連性表型引進植物。舉例來說，NSP1-樣、NLP4、及/或NLP9多肽可在通常不表現NSP1-樣、NLP4、及/或NLP9多肽的非 結瘤植物中表現，從而賦予該植物與NFB關連之增多能力。 In a particular embodiment, the conservation of the NFB-related mechanism between different plant species and different plant species is amplified to correlate novel NFB via heterologous expression of NSP1-like, NLP4, and/or NLP9 polypeptides. Sexual phenotype introduces plants. For example, NSP1-like, NLP4, and/or NLP9 polypeptides may be non-expressing non-NSP1-like, NLP4, and/or NLP9 polypeptides. Expressed in nodulation plants, thereby conferring increased ability to bind the plant to NFB.

在本案替換具體例中，植物細胞、植物部分、及/或植物可被基因改造，以藉由本領域所習知引進異源性分子的任何數個方法包含至少一NSP1-樣、NLP4、及/或NLP9聚核苷酸，藉此製造非天然基因轉殖植物細胞、植物部分、或植物。在本案特定具體例中，異源性分子係藉由選自-舉例而不限於-轉形與選擇性育種(譬如回交育種)的方法引進植物細胞、植物部分、及/或植物。 In an alternative embodiment of the present invention, plant cells, plant parts, and/or plants can be genetically engineered to include at least one NSP1-like , NLP4 , and/ by any number of methods known in the art to introduce heterologous molecules. Or a NLP9 polynucleotide whereby a non-native gene transfer plant cell, plant part, or plant is made. In a particular embodiment of the present invention, the heterologous molecule is introduced into a plant cell, a plant part, and/or a plant by a method selected from the group consisting of, but not limited to, -transformation and selective breeding, such as backcross breeding.

在一些具體例中，NSP1-樣、NLP4、及/或NLP9聚核苷酸被引進，俾使操作性地聯結至組成促進子，從而引導基因產物在彼等通常不表現(譬如氮非有限)的條件下表現。在特定具體例中，NSP1-樣、NLP4、及/或NLP9聚核苷酸被引進，俾使操作性地聯結至非-組成促進子，從而引導基因產物以組織-偏好性(譬如在根部組織)或組織特異性方式表現。在特定具體例中，NSP1-樣、NLP4、及/或NLP9聚核苷酸被引進，俾使操作性地聯結至可誘發促進子，從而以調控方式引導基因產物的表現。 In some embodiments, NSP1-like , NLP4 , and/or NLP9 polynucleotides are introduced and operably linked to a compositional promoter, thereby directing the gene product to generally not exhibit (such as nitrogen non-limited) Performance under the conditions. In a specific embodiment, NSP1-like , NLP4 , and/or NLP9 polynucleotides are introduced and operably linked to a non-constituting promoter to direct gene product to tissue-preference (eg, in root tissue) ) or tissue-specific performance. In a particular embodiment, NSP1-like , NLP4 , and/or NLP9 polynucleotides are introduced and operably linked to an elicitible promoter to direct the expression of the gene product in a regulated manner.

任何植物物種或植物細胞可被基因改造成包含本案異源性核酸。在一些具體例中，依此基因改造的植物細胞能夠再生，以製造植物。在一些具體例中，被基因改造的植物細胞(譬如宿主植物細胞)包括來自下列的細胞，舉例而不限於：高等植物、雙子葉植物、單子葉植物、耗材植物、作物植物、油用植物(譬如油籽植物)、以及非結瘤植 物。此類植物包括，舉例而不限於：苜蓿；大豆；棉花；油菜籽(油菜)；亞麻籽；玉米；稻米；臂形草；小麥；紅花；高粱；甜菜；向日葵；煙草；以及草類(譬如草坪草)。在特定例子中，本案的基因改造植物細胞或植物包括，舉例而不限於：油菜(Brassica napus)；印度芥菜(Brassica juncea)；衣索比亞芥(Brassica carinata)；白蘿蔔(Brassica rapa)；花椰菜(Brassica oleracea)；大豆(Glycine max)；亞麻(Linum usitatissimum)；玉米(Zea mays)；紅花(Carthamus tinctorius)；向日葵(Helianthus annuus)；煙草(Nicotiana tabacum)；***芥、巴西堅果(Betholettia excelsa)；蓖麻籽(Ricinus communis)；椰子(Cocus nucifera)；芫荽(Coriandrum sativum)；棉花屬(Gossypium spp.)；花生(Arachis hypogaea)；荷荷芭(Simmondsia chinensis)；油棕(Elaeis guineeis)；橄欖(Olea eurpaea)；水稻(Oryza sativa)；西葫蘆(Cucurbita maxima)；大麥(Hordeum vulgare)；甘蔗(Saccharum officinarum)；小麥屬(包括杜蘭小麥(Triticum durum)與小麥(Triticum aestivum))；與紫萍屬(Lemnaceae sp.)。在一些具體例中，植物可尤其具有基因背景，作為優良栽培種、野生型栽培種、及商業可區別物種。 Any plant species or plant cell can be genetically engineered to comprise a heterologous nucleic acid of the present invention. In some embodiments, the genetically engineered plant cells are capable of being regenerated to produce plants. In some embodiments, genetically engineered plant cells (eg, host plant cells) include cells from, for example, without limitation: higher plants, dicots, monocots, consumable plants, crop plants, oil plants ( Such as oilseed plants, and non-nodulated plants. Such plants include, by way of example and not limitation:: soy; soybean; cotton; rapeseed (rapeseed); flaxseed; corn; rice; arm-shaped grass; wheat; safflower; sorghum; beet; sunflower; tobacco; Lawn grass). In a specific example, the genetically modified plant cell or plant of the present invention includes, by way of example and not limitation: Brassica napus ; Brassica juncea ; Brassica carinata ; Brassica rapa ; Cauliflower ( Brssica oleracea ); soybean ( Glycine max ); flax ( Linum usitatissimum ); corn ( Zea mays ); safflower ( Carthamus tinctorius ); sunflower ( Helianthus annuus ); tobacco ( Nicotiana tabacum ); arabian mustard, brazil nut ( Betholettia excelsa ); Ricinus communis ; Cocus nucifera ; Coriandrum sativum ; Gossypium spp .; Arachis hypogaea ; Simmondsia chinensis ; Elaeis guineeis Olea eurpaea ; Oryza sativa ; Cucurbita maxima ; Hordeum vulgare ; Saccharum officinarum ; Triticum durum and Triticum aestivum ; With the genus Lemnaceae ( Lemnaceae sp. ). In some embodiments, plants may have in particular a genetic background as an excellent cultivar, a wild-type cultivar, and a commercially distinguishable species.

根據本領域習知方法，核酸可引進基本上任何植物。本案具體例可運用本領域所習知用於植物轉形(及製造基因改造植物)的任意眾多方法。此類方法包括，舉例而不限於用於雙子葉植物-還有單子葉植物-的生物與物理轉形流程。參閱譬如Goto-Fumiyuki et al.(1999)Nat. Biotechnol.17：282；Miki et al.(1993)Methods in Plant Molecular Biology and Biotechnology(Glick,B.R.and Thompson,J.E.,Eds.),CRC Press,Inc.,Boca Raton,FL,pp.67-88。此外，用於植物細胞與組織轉形及再生植物的載體與體外培養方法係說明於，舉例來說，Gruber and Crosby(1993)Methods in Plant Molecular Biology and Biotechnology，見上文，於pp.89-119。 Nucleic acids can be introduced into essentially any plant according to methods known in the art. Specific examples of the present invention can employ any of a wide variety of methods known in the art for plant transformation (and for the manufacture of genetically modified plants). Such methods include, by way of example and not limitation, biological and physical transformation procedures for dicotyledonous plants, as well as monocotyledons. See, for example, Goto-Fumiyuki et al. (1999) Nat. Biotechnol . 17:282; Miki et al. (1993) Methods in Plant Molecular Biology and Biotechnology (Glick, BRand Thompson, JE, Eds.), CRC Press, Inc. , Boca Raton, FL, pp. 67-88. In addition, vectors and in vitro culture methods for plant cell and tissue transformation and regenerative plants are described, for example, in Gruber and Crosby (1993) Methods in Plant Molecular Biology and Biotechnology , supra, at pp. 89- 119.

將核酸引進植物宿主細胞的植物轉形技術包括，舉例而不限於：使用根瘤農桿菌(Agrobacterium tumefaciens)或毛根農桿菌(A.rhizogenes)作為轉形劑以卸防T-DNA轉形；磷酸鈣轉染；聚凝胺轉形；原生質體融合；電穿孔(D'Halluin et al.(1992)Plant Cell 4：1495)；超音波方法(譬如超音波穿孔)；脂質體轉形；顯微注射；接觸裸DNA；接觸質體載體；接觸病毒載體；生物彈道學(譬如DNA粒子轟擊(參閱譬如Klein et al.(1987)Nature 327：70)與微粒子轟擊(Sanford et al.(1987)Part.Sci.Technol.5：27；Sanford(1988)Trends Biotech.6：299,Sanford(1990)Physiol.Plant 79：206；與Klein et al.(1992)Biotechnology 10：268)；碳化矽WHISKERS-介導轉形(Kaeppler et al.(1990)Plant Cell Rep.9：415)；奈米粒子轉形(參閱譬如U.S.專利公開案號US2009/0104700A1)；氣溶膠發射；與聚乙二醇(PEG)-介導的攝取。在特別例子中，異源性核酸可直接引進植物細胞的基因組DNA。 Plant transformation techniques for introducing nucleic acids into plant host cells include, by way of example and not limitation, the use of Agrobacterium tumefaciens or A. rhizogenes as a transforming agent to de-trace T-DNA transformation; calcium phosphate Transfection; polyamine transduction; protoplast fusion; electroporation (D'Halluin et al. (1992) Plant Cell 4: 1495); ultrasonic methods (such as ultrasonic perforation); liposome transformation; microinjection Contact with naked DNA; contact with plastid vectors; contact with viral vectors; biological ballistics (such as DNA particle bombardment (see, for example, Klein et al. (1987) Nature 327:70) and microprojectile bombardment (Sanford et al. (1987) Part. Sci. Technol. 5:27; Sanford (1988) Trends Biotech. 6:299, Sanford (1990) Physiol. Plant 79:206; and Klein et al. (1992) Biotechnology 10:268); 碳WHISKERS-mediated 碳Transformation (Kaeppler et al. (1990) Plant Cell Rep. 9: 415); nanoparticle transformation (see, for example, US Patent Publication No. US 2009/0104700 A1); aerosol emission; and polyethylene glycol (PEG)- Mediated uptake. In a particular example, a heterologous nucleic acid can be introduced directly into the base of a plant cell. Group DNA.

用於將表現載體引進植物的廣泛利用方法係以 農桿菌的天然轉形系統為基礎。Horsch et al.(1985)Science 227：1229。根瘤農桿菌與毛根農桿菌為習知可用於以基因方式轉形植物細胞的植物致病性土壤細菌。根瘤農桿菌與毛根農桿菌的Ti與Ri質體分別攜帶負責植物基因轉形的基因。Kado(1991)Crit.Rev.Plant.Sci.10：1。論及農桿菌載體系統的細節與用於農桿菌-介導基因轉移的方法亦可得自舉例來說Gruber et al.，見上文；Miki et al.，見上文；Moloney et al.(1989)Plant Cell Reports 8：238，以及U.S.專利號4,940,838與5,464,763。 The widely used method for introducing expression vectors into plants is based on the natural transformation system of Agrobacterium. Horsch et al. (1985) Science 227:1229. Agrobacterium tumefaciens and Agrobacterium rhizogenes are phytopathogenic soil bacteria which are conventionally used for genetically transforming plant cells. The Agrobacterium tumefaciens and the Ti and Ri plastids of Agrobacterium tumefaciens carry genes responsible for plant gene transformation, respectively. Kado (1991) Crit. Rev. Plant. Sci. 10:1. Details of the Agrobacterium vector system and methods for Agrobacterium-mediated gene transfer can also be obtained, for example, from Gruber et al. , supra; Miki et al. , supra; Moloney et al. 1989) Plant Cell Reports 8: 238, and US Patent Nos. 4,940,838 and 5,464,763.

假使農桿菌係用作轉形，欲***的DNA通常被選殖到特別的質體內；進入中間載體或二元載體。中間載體無法在農桿菌內自行複製。中間載體可藉由輔助質體(共軛)的方式轉移至根瘤農桿菌。日本煙草超級二元系統(Japan Tobacco Superbinary System)是此類系統的例子(回顧於Komari et al.(2006)Methods in Molecular Biology(K.Wang,ed.)No.343；Agrobacterium Protocols,2^nd Edition,Vol.1,Humana Press Inc.,Totowa,NJ,pp.15-41；與Komori et al.(2007)Plant Physiol.145：1155)。二元載體在大腸桿菌與農桿菌皆能自行複製。二元載體包含選擇標記基因以及被右側與左側T-DNA邊界區域包夾的聯接子或聚聯接子。彼等可直接轉形至農桿菌(Holsters,1978)。農桿菌包含攜帶vir區域的質體。Ti或Ri質體亦包含T-DNA轉移所必需的vir區域。vir區域是T-DNA轉移至植物細胞所必需的。可含有額外的T-DNA。 In case the Agrobacterium is used as a transformation, the DNA to be inserted is usually selected into a special plastid; into an intermediate vector or a binary vector. The intermediate vector cannot replicate itself in Agrobacterium. The intermediate vector can be transferred to Agrobacterium tumefaciens by means of an auxiliary plastid (conjugate). The Japan Tobacco Superbinary System is an example of such a system (reviewed in Komari et al. (2006) Methods in Molecular Biology (K. Wang, ed.) No. 343; Agrobacterium Protocols , ^2nd Edition , Vol. 1, Humana Press Inc., Totowa, NJ, pp. 15-41; and Komori et al. (2007) Plant Physiol . 145: 1155). The binary vector can replicate itself in both E. coli and Agrobacterium. The binary vector comprises a selectable marker gene and a linker or polylinker that is sandwiched by the right and left T-DNA border regions. They can be directly transformed into Agrobacterium (Holsters, 1978). Agrobacterium contains a plastid carrying the vir region. The Ti or Ri plastid also contains the vir region necessary for T-DNA transfer. The vir region is required for the transfer of T-DNA to plant cells. May contain additional T-DNA.

根瘤農桿菌宿主的病毒功能將引導含構築體與毗鄰標記的T-股***植物細胞DNA，當該細胞被使用二元T DNA載體的細菌感染時(Bevan(1984)Nuc.Acid Res.12：8711)或共培養程序(Horsch et al.(1985)Science 227：1229)。一般而言，農桿菌轉形系統係用於改造雙子葉植物。Bevan et al.(1982)Ann.Rev.Genet 16：357；Rogers et al.(1986)Methods Enzymol.118：627。農桿菌轉形系統亦可用於轉形，還有轉移核酸至單子葉植物與植物細胞。參閱U.S.專利號5,591,616；Hernalsteen et al.(1984)EMBO J 3：3039；Hooykass-Van Slogteren et al.(1984)Nature 311：763；Grimsley et al.(1987)Nature 325：1677；Boulton et al.(1989)Plant Mol.Biol.12：31；與Gould et al.(1991)Plant Physiol.95：426。 The viral function of the Agrobacterium tumefaciens host will direct the inclusion of the construct and the adjacent marker T-strand into the plant cell DNA when the cell is infected with a binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12: 8711) or co-culture program (Horsch et al. (1985) Science 227: 1229). In general, Agrobacterium transformation systems are used to engineer dicots. Bevan et al. (1982) Ann. Rev. Genet 16:357; Rogers et al. (1986) Methods Enzymol. 118:627. Agrobacterium transformation systems can also be used for transformation, as well as transfer of nucleic acids to monocots and plant cells. See US Patent No. 5,591,616; Hernalsteen et al. (1984) EMBO J 3:3039; Hooykass-Van Slogteren et al. (1984) Nature 311:763; Grimsley et al. (1987) Nature 325:1677; Boulton et al. (1989) Plant Mol. Biol. 12:31; and Gould et al. (1991) Plant Physiol. 95:426.

本案重組宿主的基因操縱可使用標準基因技術進行。並可在適用於基因操縱的任何宿主細胞實行。在一些具體例中，重組宿主細胞可為適用於基因操縱及/或重組基因表現的任何生物體或微生物體宿主。在一些具體例中，重組宿主可為植物。本案使用的標準重組DNA與分子選殖技術為本領域眾所周知並說明於，舉例而不限於：Sambrook et al.(1989)，見上文；Silhavy et al.(1984)Experiments with Gene Fusions,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY；與Ausubel et al.(1987)Current Protocols in Molecular Biology,Greene Publishing Assoc.and Wiley-Interscience,New York,NY。 Genetic manipulation of recombinant hosts in this case can be performed using standard genetic techniques. It can be carried out in any host cell suitable for gene manipulation. In some embodiments, the recombinant host cell can be any organism or microbial host suitable for use in gene manipulation and/or recombinant gene expression. In some embodiments, the recombinant host can be a plant. The standard recombinant DNA and molecular selection techniques used in this case are well known and described in the art, by way of example and not limitation: Sambrook et al. (1989), supra; Silhavy et al. (1984) Experiments with Gene Fusions , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel et al. (1987) Current Protocols in Molecular Biology , Greene Publishing Assoc. and Wiley-Interscience, New York, NY.

在將核酸引進植物細胞之後，植物細胞可生長，並待諸如芽與根之分生組織出現後，可產生成熟植物。在一些具體例中，可生成複數株植物。再生植物的方法論為熟習此藝者所習知且可在，舉例來說：Plant Cell and Tissue Culture(Vasil and Thorpe,Eds.),Kluwer Academic Publishers,1994找到。本案說明的基因改造植物可培養在發酵介質或生長在適宜介質，例如土壤。在一些具體例中，高等植物的適宜生長介質可為用於植物的任何生長介質，包括，舉例而不限於：土壤、沙、支持根部生長(譬如蛭石、珍珠岩等等)或水耕栽培的任何其他特定介質，還有適宜的光、水與幫助高等植物生長的營養補充劑。 After introduction of the nucleic acid into the plant cell, the plant cell can grow and, after emergence of meristems such as buds and roots, can produce mature plants. In some embodiments, a plurality of plants can be produced. The methodology for regenerating plants is well known to those skilled in the art and can be found, for example, in Plant Cell and Tissue Culture (Vasil and Thorpe, Eds.), Kluwer Academic Publishers, 1994. The genetically modified plants described in this case can be cultured in a fermentation medium or grown in a suitable medium, such as soil. In some embodiments, a suitable growth medium for higher plants may be any growth medium for the plant, including, by way of example and not limitation: soil, sand, supporting root growth (such as vermiculite, perlite, etc.) or hydroponic cultivation Any other specific medium, as well as suitable light, water and nutrient supplements that help the growth of higher plants.

藉由任何上述轉形技術製造的轉形植物細胞可培養再生擁有轉形表型且於是所欲表型的全植物。此類再生技術係依靠操縱培養組織生長介質中的某些植物激素，通常依靠已和所欲核苷酸序列一起引進的殺生物劑及/或除草劑標記。由培養的原生質體再生植物係說明於Evans et al.(1983)“Protoplasts Isolation and Culture,”in Handbook of Plant Cell Culture,Macmillian Publishing Company,New York,pp.124-176；與Binding(1985)Regeneration of Plants,Plant Protoplasts,CRC Press,Boca Raton,pp.21-73。再生亦可由植物痂、外植體、器官、花粉、胚胎或其之一部分進行。此類再生技術係大致上說明於Klee et al.(1987)Ann.Rev.Plant Phys.38：467。 The transformed plant cells produced by any of the above-described transformation techniques can be cultured to regenerate whole plants having a transformed phenotype and thus a desired phenotype. Such regeneration techniques rely on manipulation of certain plant hormones in tissue growth media, typically relying on biocides and/or herbicide markers that have been introduced with the desired nucleotide sequence. Regeneration of plant lines from cultured protoplasts is described in Evans et al. (1983) "Protoplasts Isolation and Culture," in Handbook of Plant Cell Culture, Macmillian Publishing Company, New York, pp. 124-176; and Binding (1985) Regeneration Of Plants, Plant Protoplasts , CRC Press, Boca Raton, pp. 21-73. Regeneration can also be carried out by plant mites, explants, organs, pollen, embryos or a part thereof. Such regeneration techniques are generally described in Klee et al. (1987) Ann. Rev. Plant Phys. 38:467.

在其他具體例中，轉形的植物細胞不能再生以製 造植物。此類細胞可運用，舉例來說，在發展具有相關表型-舉例來說，NFB關連性-的植物細胞系。 In other specific examples, the transformed plant cells cannot be regenerated to make Planting. Such cells can be used, for example, in the development of plant cell lines with associated phenotypes - for example, NFB-related properties.

轉形的植物細胞、痂、組織或植物可藉由以存在於轉形DNA上的標記基因所編碼的性狀選擇或篩揀改造植物材料來辨識與單離。舉例而言，選擇可藉由使改造的植物材料生長在含有轉形基因構築體賦予抗性的抗生素或除草劑抑制量的介質上來進行。再者，轉形的植物與植物細胞亦可藉由篩揀可存在於重組核酸構築體上的任何可視標記基因(譬如β-葡萄糖醛酸酶、螢光素酶、或gfp基因)的活性來辨識。此類選擇與篩揀方法對熟習此藝者而言係眾所周知。 The transformed plant cell, sputum, tissue or plant can be identified and isolated by selecting or sieving the plant material by the trait encoded by the marker gene present on the transmorphic DNA. For example, selection can be carried out by growing the engineered plant material on a medium containing an antibiotic or herbicide inhibiting amount that confers resistance to the transforming gene construct. Furthermore, transformed plants and plant cells can also be screened for the activity of any visible marker gene (such as beta-glucuronidase , luciferase , or gfp gene) that can be present on the recombinant nucleic acid construct. Identification. Such selection and screening methods are well known to those skilled in the art.

含有本案異源性分子的基因轉殖植物可經由選擇性育種製造，舉例來說，使包含該分子的第一親代植物與第二親代植物有性雜交，藉此製造複數個第一子代植物。隨後可選擇抵抗選擇標記(譬如草甘膦，對草甘膦的抗性可由本案異源性分子賦予子代植物)的第一子代植物。第一子代植物隨後可自交，藉此製造複數個第二子代植物。隨後，可選擇抵抗選擇標記的第二子代植物。該等步驟可再包括第一子代植物或第二子代植物回交至第二親代植物或第三親代植物。 A genetically transgenic plant comprising a heterologous molecule of the present invention can be produced by selective breeding, for example, by sexually crossing a first parental plant comprising the molecule with a second parental plant, thereby producing a plurality of first sub-species Generation of plants. A first progeny plant can then be selected that is resistant to a selection marker (such as glyphosate, which is conferred to the progeny plant by the heterologous molecule in this case). The first progeny plant can then be selfed, thereby producing a plurality of second progeny plants. Subsequently, a second progeny plant resistant to the selection marker can be selected. The steps may further comprise backcrossing the first progeny plant or the second progeny plant to the second parental plant or the third parental plant.

亦欲被理解的是兩個不同基因轉殖植物亦可交配，以製造含有兩個獨立地分隔、外加外源性基因的後代。適當後代的自交可製造兩個外加外源性基因純合子的植物。亦設想到親代植物的回交以及與非基因轉殖植物的異交， 無性繁殖亦然。普遍用於不同性狀與作物的其他育種方法係本領域習知的。回交育種已用於將簡單遺傳的高度遺傳性性狀的基因轉移進入所欲純合栽培品種或自交系，其為輪迴親代。所得植物係預期具有輪迴親代(譬如栽培品種)的屬性及從給方親代轉移的所欲性狀。在初始交配後，選出擁有給方親代表型的個體並重複交配(回交)至輪迴親代。所得親代係預期具有輪迴親代(譬如栽培品種)的屬性及從給方親代轉移的所欲性狀。 It is also understood that two different genetically transformed plants can also be mated to produce offspring containing two independently segregating, plus exogenous genes. Selfing of appropriate offspring can produce two plants that are homozygous for the exogenous gene. Backcrossing of parental plants and outcrossing with non-genetically transformed plants are also envisaged. Asexual reproduction is also true. Other breeding methods commonly used for different traits and crops are well known in the art. Backcross breeding has been used to transfer genes that are simply inherited highly heritable traits into the desired homozygous cultivar or inbred line, which is a recurrent parent. The resulting plant line is expected to have the attributes of a recurrent parent (such as a cultivar) and the desired trait transferred from the parental parent. After the initial mating, the individuals who have the representative type are given and the mating (backcrossing) is repeated to the recurrent parent. The resulting parental line is expected to have the attributes of a recurrent parent (such as a cultivar) and the desired trait transferred from the parental parent.

核酸亦可經由同源性重組引進植物基因組的預定區塊。經由同源性重組在植物細胞的特異性染色體位點內穩定整合聚核苷酸序列的方法已說明於本領域。舉例而言，說明於美國專利公開案號2009/0111188 A1的位點特異性整合係涉及使用重組酶或整合酶，以介導給方聚核苷酸序列引進染色體標靶。此外，PCT國際專利公開案號WO 2008/021207說明鋅指結構介導的同源性重組，以在基因組特異位置內穩定整合一或多個給方聚核苷酸序列。重組酶的使用，例如美國專利6,720,475說明的FLP/FRT、或美國專利5,658,772說明的CRE/LOX可用來使聚核苷酸序列穩定整合至特異性染色體位點。最後，使用巨核酸酶使給方聚核苷酸靶向進入特異性染色體位置係說明於Puchta et al.(1996)Proc.Natl.Acad.Sci.USA 93：5055。 Nucleic acids can also be introduced into predetermined blocks of the plant genome via homologous recombination. Methods for the stable integration of polynucleotide sequences within specific chromosomal sites of plant cells via homologous recombination have been described in the art. For example, the site-specific integration described in U.S. Patent Publication No. 2009/0111188 A1 relates to the use of a recombinase or integrase to mediate the introduction of a chromosomal target into a polynucleotide sequence. In addition, PCT International Patent Publication No. WO 2008/021207 describes zinc finger structure-mediated homologous recombination to stably integrate one or more donor polynucleotide sequences within a genome-specific location. The use of a recombinase, such as the FLP/FRT described in U.S. Patent No. 6,720,475, or the CRE/LOX described in U.S. Patent No. 5,658,772, can be used to stably integrate a polynucleotide sequence into a specific chromosomal site. Finally, the use of meganucleases to target a polynucleotide to a specific chromosomal location is described in Puchta et al. (1996) Proc. Natl. Acad. Sci. USA 93:5055.

用於植物細胞內的位點特異性整合的其他各種方法係一般習知並可應用。Kumar et al.(2001)Trends Plant Sci.6(4)：155。再者，已在數個原核與低等真核生物體辨識 到的位點特異性重組系統可應用於植物。此類系統的例子包括但不限於：得自魯氏酵母(Zygosaccharomyces rouxii)的pSR1質體的R/RS重組酶系統(Araki et al.(1985)J.Mol.Biol.182：191)、與噬菌體Mu的Gin/gix系統(Maeser and Kahlmann(1991)Mol.Gen.Genet.230：170)。 Other various methods for site-specific integration within plant cells are generally known and applicable. Kumar et al. (2001) Trends Plant Sci. 6(4): 155. Furthermore, site-specific recombination systems that have been identified in several prokaryotic and lower eukaryotes can be applied to plants. Examples of such systems include, but are not limited to, the R/RS recombinase system derived from the pSR1 plastid of Zygosaccharomyces rouxii (Araki et al. (1985) J. Mol . Biol . 182: 191), and Gin/gix system of phage Mu (Maeser and Kahlmann (1991) Mol. Gen. Genet. 230: 170).

各種試驗可連同本案某些具體例的核酸分子運用。除表型觀察外，下列技術可用於偵測植物細胞中核酸分子的存在。舉例來說，分子的存在可藉由使用序列的引子或探針、偵測編碼蛋白的ELISA試驗、偵測蛋白的西方墨點法、或偵測RNA或DNA的北方或南方墨點法來測定。額外的技術，例如原位雜交、酶染色、和免疫染色亦可用於偵測在特定植物器官與組織以內的重組構築體的存在或表現。 Various assays can be used in conjunction with nucleic acid molecules of certain embodiments of the present invention. In addition to phenotypic observation, the following techniques can be used to detect the presence of nucleic acid molecules in plant cells. For example, the presence of a molecule can be determined by using a primer or probe of the sequence, an ELISA assay to detect the encoded protein, a Western blotting method for detecting the protein, or a northern or southern dot method for detecting RNA or DNA. . Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, can also be used to detect the presence or expression of recombinant constructs within specific plant organs and tissues.

南方分析是常用的偵測方法，其中DNA以限制內切酶切割並在瓊脂凝膠上分級，以藉由分子量分離DNA且隨後轉移到尼龍膜。隨後和以³²P(或其他探針標記)放射性標記的探針片段雜交並於SDS溶液洗滌。 Southern analysis is a commonly used detection method in which DNA is cleaved by restriction endonucleases and fractionated on an agar gel to separate DNA by molecular weight and then transferred to a nylon membrane. The radiolabeled probe fragment is then hybridized with ³² P (or other probe label) and washed in the SDS solution.

同樣地，北方分析採用類似流程，其中RNA以限制內切酶切割並在瓊脂凝膠上分級，以藉由分子量分離RNA且隨後轉移到尼龍膜。隨後和以³²P(或其他探針標記)放射性標記的探針片段雜交並於SDS溶液洗滌。分析從感興趣組織單離的RNA(譬如mRNA)可指示相關表現位準。通常，假使mRNA出現或mRNA量增加了，可推論對應轉殖基因正在表現。北方分析、或其他mRNA分析流程可用於 測定所引入轉殖基因或原生基因的表現位準。 Similarly, Northern analysis employed a similar procedure in which RNA was cleaved by restriction endonucleases and fractionated on an agar gel to isolate RNA by molecular weight and subsequently transferred to a nylon membrane. The radiolabeled probe fragment is then hybridized with ³² P (or other probe label) and washed in the SDS solution. Analysis of RNA isolated from tissue of interest (eg, mRNA) can indicate relevant performance levels. In general, if mRNA is present or the amount of mRNA is increased, it can be inferred that the corresponding transgene is being expressed. Northern analysis, or other mRNA analysis procedures, can be used to determine the level of expression of the introduced transgene or native gene.

本案的核酸、或其片段可用於設計PCR擴增的引子。在實行PCR擴增時，在引子與模板之間可忍受某些程度的未配對。藉由熟習此藝者習知的方法在既定引子中可製造突變、***、與刪除。 The nucleic acids of this case, or fragments thereof, can be used to design primers for PCR amplification. When performing PCR amplification, some degree of unpairing can be tolerated between the primer and the template. Mutations, insertions, and deletions can be made in a given primer by a method known to those skilled in the art.

水解探針試驗-或習知為TAQMAN^®(Life Technologies,Foster City,Calif.)-為偵測與量化DNA序列存在的另一方法。簡言之，FRET寡核苷酸探針被設計成一寡聚物位於轉殖基因內且一位於旁側基因組序列內，以供事件特異性偵測。FRET探針與PCR引子(一引子位於***DNA序列且一位於旁側基因組序列)在熱穩定聚合酶與dNTPs的存在下循環。FRET探針的雜交使得FRET探針上的螢光部分從淬熄部分斷裂與釋放。螢光信號指示因成功擴增與雜交而存在的旁側/轉殖基因***序列。 Hydrolysis probe assay - or conventionally known as TAQMAN ^® (Life Technologies, Foster City, Calif.) - is another method for detecting and quantifying the presence of DNA sequences. Briefly, FRET oligonucleotide probes are designed such that an oligo is located within the transgene and one is located in the flanking genomic sequence for event specific detection. The FRET probe and the PCR primer (one primer located in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe causes the fluorescent moiety on the FRET probe to cleave and release from the quenching moiety. Fluorescent signals indicate flanking/transgenic gene insertion sequences that are present due to successful amplification and hybridization.

VII.包含NSP1-樣、NLP4、與NLP9的植物、植物部分、及植物材料 VII. Plants, plant parts , and plant materials containing NSP1-like , NLP4 , and NLP9

本案一些具體例提包含至少一異源性NSP1-樣、NLP4、及/或NLP9聚核苷酸的植物，例如可由穩定轉形的植物細胞或組織生成，或可藉由來自給方品系的此類核酸的遺傳漸滲(introgression)製造。此類植物可以任何方式使用或培養，其中存在(多個)感興趣的轉形聚核苷酸係所欲的。據此，基因轉殖植物尤其可藉由轉形改造成具有一或多個所欲性狀(譬如NFB關連性)且隨後可藉由熟習此藝者習知 的任何方法種植與培養。本案的特別具體例提供此類基因轉殖植物的部分、細胞、及/或組織。植物部分不設限地包括種籽、胚乳、胚珠與花粉。在一些具體例中，植物部分為種籽。 Some specific examples of the present invention include plants comprising at least one heterologous NSP1-like , NLP4 , and/or NLP9 polynucleotide, such as may be produced from stably transformed plant cells or tissues, or may be derived from a donor line Genetic introgression of nucleic acids. Such plants can be used or cultured in any manner in which the desired polynucleotide(s) of interest are present. Accordingly, the genetically transformed plant can be engineered and cultured, inter alia, by transformation into one or more desired traits (e.g., NFB affinity) and then by any method known to those skilled in the art. Particular embodiments of the present invention provide portions, cells, and/or tissues of such genetically transformed plants. Plant parts include seeds, endosperms, ovules and pollen without limitation. In some embodiments, the plant parts are seeds.

代表性、非設限植物包括非結瘤植物；***芥屬；田間作物(譬如苜蓿、大麥、豆類、三葉草、玉蜀黍、棉花、亞麻、扁豆、玉米、豌豆、油菜/油菜籽、水稻、黑麥、紅花、高粱、大豆、向日葵、煙草、與小麥)；蔬菜作物(譬如蘆筍、甜菜、油菜、青花菜、抱子甘藍、白菜、胡蘿蔔、花椰菜、芹菜、黃瓜(瓜類作物)、茄子、萵苣、芥菜、洋蔥、甜椒、馬鈴薯、南瓜、白蘿蔔、菠菜、南瓜、芋頭、蕃茄、與節瓜)；水果與堅果作物(譬如杏仁、蘋果、杏子、香蕉、黑莓、藍莓、可可、木薯、櫻桃、柑桔、椰子、蔓越莓、棗子、榛果、葡萄、葡萄柚、番石榴、奇異果、檸檬、萊姆、芒果、甜瓜、油桃、橘子、木瓜、百香果、桃子、花生、梨、鳳梨、開心果、李子、覆盆子、草莓、蜜桔、胡桃、與西瓜)；樹木與觀賞植物(譬如赤楊、水曲柳、白楊、杜鵑、樺樹、黃楊、山茶、康乃馨、菊花、榆樹、冷杉、常春藤、茉莉、杜松、橡木、棕櫚、楊柳、松、紅木、杜鵑、玫瑰、與橡膠)。 Representative, non-limited plants include non-nodulated plants; Arabidopsis; field crops (such as alfalfa, barley, beans, clover, maize, cotton, flax, lentils, corn, peas, rapeseed/rapeseed, rice, rye) , safflower, sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops (such as asparagus, beets, canola, broccoli, Brussels sprouts, cabbage, carrots, broccoli, celery, cucumber (melon crops), eggplant, lettuce) , mustard, onions, sweet peppers, potatoes, pumpkins, turnips, spinach, squash, taro, tomatoes, and zucchini; fruit and nut crops (such as almonds, apples, apricots, bananas, blackberries, blueberries, cocoa, cassava, Cherry, citrus, coconut, cranberry, date, hazelnut, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, Pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut, and watermelon); trees and ornamental plants (such as alder, ash, white poplar, rhododendron, birch Tree, boxwood, camellia, carnation, chrysanthemum, eucalyptus, fir, ivy, jasmine, juniper, oak, palm, willow, pine, rosewood, azalea, rose, and rubber).

為確認用以再生植物的(多個)感興趣轉形聚核苷酸的存在，可實行各式各樣試驗。此類試驗包括，舉例而不限於：生化試驗，例如偵測蛋白產物的存在，譬如藉 由免疫方式(ELISA及/或西方墨點法)或藉由酶的功能；植物部分試驗(譬如葉或根試驗)；以及分析植物的表型。 To confirm the presence of the polynucleotide of interest(s) used to regenerate the plant, a variety of assays can be performed. Such tests include, by way of example and not limitation, biochemical tests, such as detecting the presence of a protein product, such as By immunological means (ELISA and / or Western blotting method) or by the function of enzymes; plant part tests (such as leaf or root test); and analysis of plant phenotype.

在發展任何新穎的所欲植物種源時，有眾多步驟，其可以生成基因轉殖作物植物開始。在一些具體例中，包含至少一NSP1-樣、NLP4、及/或NLP9聚核苷酸的基因轉殖植物可用於植物育種及/或種源發展計劃。 In the development of any novel source of desired plant, there are numerous steps that can be initiated to produce a genetically transformed crop plant. In some embodiments, a genetically transgenic plant comprising at least one NSP1-like , NLP4 , and/or NLP9 polynucleotide can be used in a plant breeding and/or provenance development program.

植物育種開始於分析與定義當前種源的問題與弱點、建立計劃目標，並定義特定育種目的。下一步驟為選出擁有符合計劃目標之性狀的種源。目標是在單一變種中合併來自親代種源之所欲性狀的改良組合。該等重要性狀可包括較高種籽產率、抵抗疾病與昆蟲、較佳的莖與根、耐旱與熱、及較佳的農藝品質。 Plant breeding begins with the analysis and definition of current sources of problems and weaknesses, the establishment of planning goals, and the definition of specific breeding purposes. The next step is to select the source that has the traits that meet the planned goals. The goal is to combine improved combinations of desirable traits from the parental source in a single variant. Such traits may include higher seed yield, resistance to disease and insects, preferred stem and root, drought and heat tolerance, and better agronomic qualities.

育種或選擇方法的挑選係取決於植物生殖模式、欲改良(多個)性狀的遺傳力、商用栽培品種類型(譬如F₁雜交栽培品種、純系栽培品種等等)。就高度遺傳的性狀而言，在單一場所評估的優良個體植物選項將會有效，但就具低遺傳力的性狀而言，選擇應基於對相關植物家族的重複評估所獲得的平均值。受歡迎的選擇方法包括譜系選擇、修飾譜系選擇、質量選擇、與輪迴選擇。 The selection of breeding or selection methods depends on the reproductive mode of the plant, the heritability of the trait to be improved, and the type of commercial cultivar (such as F ₁ hybrid cultivar, pure cultivar, etc.). In the case of highly heritable traits, superior individual plant options evaluated at a single location will be effective, but for low heritability traits, the selection should be based on an average obtained from repeated assessments of the relevant plant family. Popular selection methods include pedigree selection, modified lineage selection, quality selection, and recurrent selection.

遺傳複雜性影響育種方法的選擇。回交育種係用於將高度遺傳性狀的一或數個合意基因轉移進入栽培品種。此方式已被廣泛地用於抗病栽培品種的育種。各式輪迴選擇技術係用於改良由眾多基因調控的量化遺傳性狀。在自 花授粉作物中使用輪迴選擇係取決於授粉容易度、各次授粉的成功雜交頻率、以及各次成功交配的雜交後代數量。 Genetic complexity affects the choice of breeding methods. Backcross breeding lines are used to transfer one or several desirable genes of highly hereditary traits into cultivars. This method has been widely used for breeding of disease resistant cultivars. Various recurrent selection techniques are used to improve the quantitative genetic traits regulated by numerous genes. In self The use of recurrent selection in flower pollination crops depends on the ease of pollination, the frequency of successful hybridizations for each pollination, and the number of hybrid progeny that have been successfully mated.

各個育種計劃應包括育種程序效率的定期、客觀評估。評估要點係視目標與目的而有所更動，但應包括基於比對先進育種品系的適當標準總值的選擇增益、以及每份輸入單位(譬如每年、每元支出等等)的成功栽培品種數量。 Each breeding program should include a regular, objective assessment of the efficiency of the breeding program. Assessment points are subject to change by purpose and purpose, but should include selection gains based on the appropriate standard value of the comparison of advanced breeding lines, and the number of successful cultivars per input unit (eg, annual, per dollar, etc.) .

有前途的先進育種品系係徹底測試並在代表(多個)商業目標區域的環境中比對適當標準達三或更多年。最好的品系是新穎商業栽培品種的候選者；該等仍缺乏數個性狀者可用作親代，以製造新的種群而供進一步選擇。 Promising advanced breeding lines are thoroughly tested and compared to appropriate standards for three or more years in an environment representing the business target area(s). The best strains are candidates for novel commercial cultivars; those who still lack a number of traits can be used as parents to make new populations for further selection.

從進行首次交配起算，該等過程-引至銷售與配送的最終步驟-通常需時八至十二年。因此，發展新穎栽培品種是需要精確的前瞻性規劃、有效使用資源、且甚少改變方向的耗時過程。 From the time of the first mating, these processes - the final steps leading to sales and distribution - usually take eight to twelve years. Therefore, the development of novel cultivars is a time-consuming process that requires precise forward-looking planning, efficient use of resources, and little change of direction.

譜系育種與輪迴選擇育種方法係用於發展來自育種種群的栽培品種。育種計劃係藉由自交與選擇所欲表型將來自二或多個栽培品種的所欲性狀或基礎廣泛的各種資源併至發展栽培品種的育種池中。評估新穎栽培品種，以測定何者具有商業潛力。 Pedigree breeding and recurrent selection breeding methods are used to develop cultivars from breeding populations. The breeding program integrates the desired traits or broad-based resources from two or more cultivars into the breeding pool of the cultivar by self-intersection and selection of desired phenotypes. Evaluate novel cultivars to determine which has commercial potential.

譜系育種係普遍用於改良自花授粉作物。使擁有合意互補性狀的兩親代交配，以製造F₁。F₂種群係使一或數個F₁s自交而製得。最佳個體的選擇可於F₂種群開始；隨 後，於F₃開始，選出最佳家族的最佳個體。重複的家族測試可於F₄世***始，以改善具低遺傳力之性狀的選擇有效性。在育種的後期階段(即F₆與F₇)，測試最佳品系或表型相似品系的混合物發佈為新穎栽培品種的潛力。 Lineage breeding systems are commonly used to improve self-pollination crops. So has the desirable traits of two complementary parental mating to produce F _1. The F ₂ population is made by selfing one or several F ₁ s. The best choice may be subject to a F ₂ population started; then, begins at F _3, select the best individual optimal family. These tests can be repeated beginning family generations F _4, to improve with low heritability of the trait selection effectiveness. In the later stages of breeding (ie F ₆ and F ₇ ), testing the mixture of the best line or phenotypically similar lines is released as a potential for novel cultivars.

質量與輪迴選擇可用於改良自花授粉作物或交叉授粉作物的種群。異型接合個體的基因多變種群係為識得或藉由數個不同親代的交叉交配創建。最佳植物係基於個體優勢、傑出後代、或傑出結合能力來選擇。使選出植物交叉交配以製造新穎種群，其中持續著進一步的選擇循環。 Quality and recurrent selection can be used to improve the population of self-pollinated crops or cross-pollinated crops. The genetically variable population of a heterozygous individual is either recognized or created by cross-mating of several different parents. The best plant lines are selected based on individual strengths, outstanding offspring, or outstanding binding abilities. The selected plants are cross-matured to create a novel population in which a further selection cycle continues.

回交育種已用於將簡單遺傳的高度遺傳性性狀的基因轉移進入所欲純合栽培品種或自交系，其為輪迴親代。欲轉移性狀的來源係稱作給方親代。所得植物係預期具有輪迴親代(譬如栽培品種)的屬性及從給方親代轉移的所欲性狀。在初始交配後，選出擁有給方親代表型的個體並重複交配(回交)至輪迴親代。所得親代係預期具有輪迴親代(譬如栽培品種)的屬性及從給方親代轉移的所欲性狀。 Backcross breeding has been used to transfer genes that are simply inherited highly heritable traits into the desired homozygous cultivar or inbred line, which is a recurrent parent. The source of the trait to be transferred is called the parental parent. The resulting plant line is expected to have the attributes of a recurrent parent (such as a cultivar) and the desired trait transferred from the parental parent. After the initial mating, the individuals who have the representative type are given and the mating (backcrossing) is repeated to the recurrent parent. The resulting parental line is expected to have the attributes of a recurrent parent (such as a cultivar) and the desired trait transferred from the parental parent.

單一種籽後裔程序在嚴格意義上係指稱種植分離種群、每株植物採收一顆種籽樣本、並使用該一顆-種籽樣本種植下一世代。當種群已從F₂演進至所欲的近交位準時，衍生出品系的植物將各追溯至不同的F₂個體。在種群中的植物數量係隨著各世代衰減，因為若干種籽無法發芽或若干植物無法製造至少一種籽。結果，當世代演進完成 時，種群中並非所有最初取樣的F₂植物將由子代表示。 A single seed descendant program refers in the strict sense to planting isolated populations, harvesting one seed sample per plant, and using the one seed sample to plant the next generation. When the evolution of the population from the F ₂ to the desired time position inbred derived plants produced based traced back to each different F ₂ individuals. The number of plants in the population is attenuated with generations because several seeds are unable to germinate or several plants are unable to produce at least one seed. As a result, when the completion of the evolution of generations, not all of the initial sample population of F ₂ progeny plants will be represented.

在本案具體例，NSP1-樣、NLP4、及/或NLP9聚核苷酸可引進植物種源，舉例來說，以發展特徵在於-在操作性地聯結至該(多個)聚核苷酸的調控元調控下-與NFB之增多關連性的新穎自交系。此類發展計劃的特別優點可在於，舉例來說，在非結瘤植物中表現NFB表型產生增加的氮利用率及/或生長。 In the specific case of the present invention, NSP1-like , NLP4 , and/or NLP9 polynucleotides can be introduced into a plant provenance, for example, by development characterized by - operatively linked to the polynucleotide(s) A novel inbred line that regulates meta-regulation and is associated with increased NFB. A particular advantage of such development programs may be, for example, the expression of the NFB phenotype in non-nodulated plants to produce increased nitrogen utilization and/or growth.

提供下列實施例係例示某些特別特徵及/或具體例。實施例不應解讀成揭示內容限於所例示的特別特徵或具體例。 The following examples are provided to illustrate certain specific features and/or specific examples. The examples should not be construed as limited to the particular features or specific examples illustrated.

實施例 Example 實施例1：材料與方法 Example 1: Materials and Methods

***芥哥倫比亞0生態型係用於所有實驗，除非另有指明。 Arabidopsis Columbia 0 Ecotype was used for all experiments unless otherwise indicated.

生長與處理條件.***芥幼苗係以16：8光：暗光週期(hrs.)與22℃恒溫生長在帶有穆-斯氏(Murashige and Skoog)(MS)鹽介質或MS鹽介質、無所指之5mM KNO₃氮(N)補充的0.8%瓊脂皿上。七日後，將植物移至完全、或無N、補充有或沒有2.5mM NH₄NO₃的MS鹽介質。 Growth and treatment conditions. Arabidopsis seedlings were grown at a constant temperature of 16:8 light: dark light cycle (hrs.) and 22 °C in a medium with MS or salt of Murashige and Skoog (MS), no The 5 mM KNO ₃ nitrogen (N) was added to a 0.8% agar dish. After seven days, the plants were moved to the full, or no N, supplemented with or without 2.5mM NH MS medium salts of ₄ NO _3.

視處理而定，植物係以NFB或非-NFB接種或否。先讓所有細菌在稀釋869介質中生長，達到於600nm之光密度0.4的20mL過夜培養物係用於接種。在接種前，細菌以無菌水洗滌並再懸浮於5mL。20mL植物的瓊脂介質係接種 5mL細菌，或者以無菌水呈現未接種。 Depending on the treatment, the plant is inoculated with NFB or non-NFB or not. All bacteria were first grown in diluted 869 medium and 20 mL overnight cultures at an optical density of 0.4 at 600 nm were used for inoculation. Prior to inoculation, the bacteria were washed with sterile water and resuspended in 5 mL. 20mL plant agar medium inoculation 5 mL of bacteria, or uninoculated as sterile water.

實施例2：非結瘤植物與NFB之間的功能關連性 Example 2: Functional association between non-nodulated plants and NFB

為測定***芥是否建立和NFB的有益N-養分交互作用，吾人評估不同NFB物種在限N條件下對植物生長的效應。吾人選擇在與植物關連時顯示固定N的五個不同NFB物種：苜蓿中華根瘤菌RMP110(Pichon et al.(1992)植物細胞4：1199)，菜豆根瘤菌CFN42(Poupot et al.(1995)J.Biol.Chem.270：6050)，臺灣嗜銅菌LMG 19424(Marchetti et al.(2011)Appl.Env.Microbiol.77：2161)，噬異源伯克氏菌LB400(Perin et al.(2006)Appl.Env.Microbiol.72：3103)，以及越南伯克氏菌G4(Perin et al.(2006)，見上文)。 To determine whether Arabidopsis has established a beneficial N-nutrient interaction with NFB, we evaluated the effects of different NFB species on plant growth under N-limited conditions. We have chosen to display five different NFB species that are fixed N when associated with plants: Rhizobium meliloti RMP110 (Pichon et al. (1992) Plant Cell 4: 1199), Rhizopus genus CFN42 (Poupot et al. (1995) J .Biol. Chem. 270: 6050), Taiwan's copper bacterium LMG 19424 (Marchetti et al. (2011) Appl . Env . Microbiol. 77: 2161), B. plasmophilus LB400 (Perin et al. (2006) ) Appl. Env. Microbiol. 72: 3103), and B. vietii G4 (Perin et al. (2006), supra).

吾人亦測試不能實行BFN的兩株額外細菌作為控制組：植物伯克氏菌PsJN，習知加強***芥屬生長(Zuniga et al.(2013)Mol.Plant Microbe Interact.26：546)，及皮納突伯嗜銅菌JMP134，能夠和植物關連的土壤細菌，但在吾等實驗條件下對植物生長無正向影響(Ledger et al.(2012)Antonie Van Leeuwenhoek 101：713)。 We also tested two additional bacteria that could not be used as BFN as a control group: Burkholderia psJN, which is known to enhance Arabidopsis growth (Zuniga et al. (2013) Mol . Plant Microbe Interact . 26: 546), and Neisseria pallidum JMP134, a soil bacterium that is associated with plants, but has no positive effects on plant growth under our experimental conditions (Ledger et al. (2012) Antonie Van Leeuwenhoek 101: 713).

使植物生長在帶有MS介質的垂直皿上達七日， 並移至帶有相同MS介質但無N(MS-N)、或以104菌落形成單位(cfu)/mL不同細菌接種的MS-N皿。吾人在移至MS-N的七日後評估植物乾重。 Plant the plants on vertical dishes with MS media for seven days. And moved to an MS-N dish with the same MS medium but no N (MS-N), or inoculated with 104 colony forming units (cfu) / mL of different bacteria. We evaluated the dry weight of the plants seven days after moving to MS-N.

在限N條件下，相較於未接種介質，植物乾重在苜蓿中華根瘤菌RMP110的存在下明顯較高。圖1。而且，在吾等實驗條件下，植物的生長係相仿於完整MS介質所達到者。圖1。該等結果指出苜蓿中華根瘤菌RMP110在無N源時可促進植物生長。所利用的其他細菌物種皆不能增加***芥屬乾重，指出僅有中性或有益細菌的存在無法解釋此觀察。***芥屬與不同細菌之間的有效交互作用係藉由回應於在所有情況下的細菌接種所觀察到的根毛長度的非特異性增加來確認。圖5。 Under the condition of limited N, the dry weight of the plant was significantly higher in the presence of R. sphaeroides RMP110 than the uninoculated medium. figure 1. Moreover, under our experimental conditions, the growth of plants is similar to that achieved with intact MS media. figure 1. These results indicate that R. sphaeroides RMP110 promotes plant growth in the absence of N source. None of the other bacterial species utilized could increase the dry weight of Arabidopsis, indicating that the presence of only neutral or beneficial bacteria could not explain this observation. The effective interaction between Arabidopsis and different bacteria was confirmed by a non-specific increase in the length of the root hair observed in response to bacterial inoculation in all cases. Figure 5.

BNF係經由¹⁵N稀釋技術在限N條件下比對以野生型與△nifH突變種接種的植物來印證。吾人的結果指出苜蓿中華根瘤菌能夠固定大氣的氮並使其可用作植物養分。 BNF was confirmed by the ¹⁵ N dilution technique under plants in the N-limited condition with plants inoculated with wild-type and Δ nifH mutants. Our results indicate that S. meliloti can fix atmospheric nitrogen and make it useful as plant nutrients.

為測定N固定作用在限N條件下是否為植物生長所必需的，吾人使用不能固定N的苜蓿中華根瘤菌RMP110突變株(△nifH)。Bobik et al.(2006)J.Bacteriol.188：4890。如圖2A所示，在限N條件下，相較於野生型細菌，苜蓿中華根瘤菌RMP110△nifH對植物生長具有顯著降低的影響。 In order to determine whether N fixation is necessary for plant growth under N-limited conditions, we used a Rhizobium meliloti RMP110 mutant (ΔnifH) that could not fix N. Bobik et al. (2006) J. Bacteriol. 188: 4890. As shown in Fig. 2A, under the condition of N, the Rhizobium meliloti RMP110ΔnifH had a significant effect on plant growth compared to the wild type bacteria.

為證實苜蓿中華根瘤菌RMP110的生長促進作用係部分地歸因於所固定的N係有助於***芥屬養分，吾人使植物生長在補充有5mM KNO₃的MS-N達七日，該MS-N 係以5% ¹⁵N同位素標記。隨後使植物暴露至MS-N介質中的野生型苜蓿中華根瘤菌RMP110或△nifH突變種。相較於以△nifH接種的植物或未接種植物，在野生型苜蓿中華根瘤菌RMP110的存在下以補充有經標記KNO₃的MS-N介質處理的植物顯示減少的¹⁵N同位素比例。圖2B。此結果指出***芥屬植物需要大氣的N，其中¹⁴N同位素係佔絕大多數。野生型與△nifH突變種細菌株兩者對側根密度與根毛延長具有類似效應，指出兩株均能與植物交互作用。圖6A。 In order to confirm that the growth promoting effect of R. sphaeroides RMP110 is partly due to the fact that the immobilized N line contributes to Arabidopsis nutrients, we have grown plants on MS-N supplemented with 5 mM KNO ₃ for seven days. The -N is labeled with a 5% ¹⁵ N isotope. The plants are then exposed to wild-type S. meliloti RMP110 or ΔnifH mutants in MS-N medium. Compared to △ nifH inoculated plants were inoculated plant or, in the presence of wild-type S. meliloti to RMP110 supplemented with ¹⁵ N isotope ratio KNO medium processing plant MS-N labeled ₃ exhibit reduced. Figure 2B. This result indicates that Arabidopsis requires atmospheric N, with the ¹⁴ N isotope being the majority. Both wild-type and ΔnifH mutant strains had similar effects on lateral root density and root hair elongation, indicating that both plants could interact with plants. Figure 6A.

該等結果顯示***芥可與苜蓿中華根瘤菌RMP110功能性地關連，以增強在限N條件下的植物生長。此植物：細菌交互作用代表了所論述非豆科植物機制的傑出模式系統，以促進與細菌物種之關連性而引致營養相關的生物N固定作用。 These results indicate that Arabidopsis can be functionally associated with S. meliloti RMP110 to enhance plant growth under N-limited conditions. This plant: bacterial interaction represents an outstanding model system for the non-legume mechanisms discussed to promote the association with bacterial species to cause nutrient-related biological N fixation.

實施例3：調控***芥屬與苜蓿中華根瘤菌之交互作用的基因 Example 3: Genes regulating the interaction between Arabidopsis and Sinorhizobium meliloti

在***芥屬中，NSP1與NIN基因的同源物僅在植物生長於限N條件下時受到細菌存在的調控。此暗示N的可得性為調控植物回應細菌之易感性的主要因素。藉由利用植物攜帶位於NSP1-樣與選定NLPs基因中的***突變的功能基因組方式，吾人發現該等轉錄因子對***芥屬與苜蓿中華根瘤菌之間的功能關連性而言係至關重要。 In Arabidopsis, homologs of the NSP1 and NIN genes are regulated by the presence of bacteria only when the plants are grown under N-limited conditions. This suggests that the availability of N is a major factor in regulating plant susceptibility to bacterial susceptibility. By utilizing a functional genomic approach in which plants carry insertional mutations in NSP1-like and selected NLPs genes, we have found that these transcription factors are critical for functional connectivity between Arabidopsis and S. meliloti.

豆科物種中的氮固定作用係取決於調控何時及如何與根瘤菌建立共生關連性的精密分子機制。Oldroyd (2013)Nat.Rev.Microbiol.11：252)。為測定調控吾等新近發現-其中***芥屬與苜蓿中華根瘤菌RMP110交互作用-的模式系統中的交互作用的機制是否可存在於***芥屬，吾人分別分析NSP1-樣與NSP2-樣基因、At3g13840(AtNSP1-樣)與At4g08250(AtNSP2-樣)的功能。該等基因在***芥並不具有已知功能。 Nitrogen fixation in legume species depends on the precise molecular mechanisms governing when and how to establish symbiotic association with Rhizobium. Oldroyd (2013) Nat. Rev. Microbiol. 11: 252). To determine whether the mechanism of interaction in the model system that regulates our recent discovery - the interaction between Arabidopsis and R. sphaeroides RMP110 - can exist in Arabidopsis, we separately analyzed NSP1-like and NSP2-like genes, At3g13840 ( AtNSP1-like ) and At4g08250 ( AtNSP2-like ). These genes do not have a known function in Arabidopsis.

AtNSP1-樣與AtNSP2-樣基因的表現係以生長在N-充足條件(5mM KNO₃)的植物評估且隨後移至2.5mM NH₄NO₃或移至MS-N介質，有或無苜蓿中華根瘤菌RMP110的存在。植物係於轉移的3與7日後採收。製備總RNA，並使用即時定量逆轉錄聚合酶鏈反應(qRT-PCR)測量感興趣基因的轉錄位準。 The expression of AtNSP1-like and AtNSP2-like genes was assessed by plants grown in N-sufficient conditions (5 mM KNO ₃ ) and subsequently transferred to 2.5 mM NH ₄ NO ₃ or moved to MS-N medium with or without sputum Chinese root nodule The presence of the bacterium RMP110. Plants were harvested 3 and 7 days after transfer. Total RNA was prepared and the transcriptional level of the gene of interest was measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR).

吾等結果顯示AtNSP1-樣基因表現在植物移至帶有細菌的MS-N介質時被誘發，但在其他實驗條件則否。圖3A。此結果暗示AtNSP1-樣功能在限N條件下、苜蓿中華根瘤菌存在時是必需的。反之，AtNSP2-樣在受測實驗條件下係不受調控。圖7A。 Our results show that the AtNSP1-like gene expression is induced when the plants are moved to the MS-N medium with bacteria, but not under other experimental conditions. Figure 3A. This result suggests that AtNSP1-like function is required under the condition of N and in the presence of S. meliloti. Conversely, AtNSP2-like samples were not regulated under the experimental conditions tested. Figure 7A.

在豆科中，NSP1與NSP2調控NIN基因的表現。***芥屬基因組編碼九個NIN-樣基因(NLPs)。作為評估NLPs在***芥屬與苜蓿中華根瘤菌交互作用的角色的第一步驟，吾人在圖3A所述相同實驗條件下分析該等9個NLPs(NLP1(At2g17150)；NLP2(At4g35270)；NLP3(At4g38340)；NLP4(At1g20640)；NLP5(At1g76350)；NLP8 (At2g43500)；與NLP9(At3g59580))當中七者的表現。 In Leguminosae, NSP1 and NSP2 regulate the performance of the NIN gene. The Arabidopsis genome encodes nine NIN -like genes ( NLPs ). As a first step in assessing the role of NLPs in the interaction between Arabidopsis and S. meliloti, we analyzed these 9 NLPs ( NLP1 ( At2g17150 ); NLP2 ( At4g35270 ); NLP3 ( NLP3 ( At4g38340 ); NLP4 ( At1g20640 ); NLP5 ( At1g76350 ); NLP8 ( At2g43500 ); and NLP9 ( At3g59580 )).

吾等結果顯示AtNLP4、AtNLP8、與AtNLP9的基因表現在限N條件與苜蓿中華根瘤菌RMP110的存在下(圖3B)被誘發，類似於AtNSP1-樣。反之，AtNLP3顯示不同表現樣貌，其在N-充足條件下但僅於細菌存在時被誘發。圖3B。AtNLP1、AtNLP2、與AtNLP5的基因表現在所評估的實驗條件下並無顯著改變。圖7B。 Our results showed that the gene expression of AtNLP4 , AtNLP8 , and AtNLP9 was induced in the presence of N-limited conditions and Rhizobium meliloti RMP110 (Fig. 3B), similar to AtNSP1-like . Conversely, AtNLP3 showed a different appearance, which was induced under N-sufficient conditions but only in the presence of bacteria. Figure 3B. The gene expression of AtNLP1 , AtNLP2 , and AtNLP5 did not change significantly under the experimental conditions evaluated. Figure 7B.

為論述AtNSP1-樣、AtNLP4、AtNLP8、與AtNLP9基因針對***芥屬與苜蓿中華根瘤菌RMP110交互作用的功能，具AtNSP1-樣(salk_036071C-salk_023595C)、AtNLP4(salk_100786C-salk_063595C)、AtNLP8(salk_140298-salk_031064C)與AtNLP9(salk_025839C-salk_042082C)的兩個獨立純合突變品系***芥係獲自***芥屬生物資源中心(ABRC)。 In order to discuss the interaction of AtNSP1-like , AtNLP4 , AtNLP8 , and AtNLP9 genes against Arabidopsis and R. sphaeroides RMP110, AtNSP1-like (salk_036071C-salk_023595C), AtNLP4 (salk_100786C-salk_063595C), AtNLP8 (salk_140298-salk_031064C Two independent homozygous mutant lines of AtNLP9 (salk_025839C-salk_042082C) are obtained from the Arabidopsis Bioresource Center (ABRC).

生長在限N條件下的突變與野生型植物係以野生型苜蓿中華根瘤菌RMP110接種(或假仿接種作為控制組)。吾等結果顯示，在限N條件下，藉由苜蓿中華根瘤菌RMP110的植物生長促進作用在atnsp1突變種中丟失了。圖4A。同樣地，AtNLP4與AtNLP9基因的突變植物在苜蓿中華根瘤菌RMP110的存在下並未展現增加的生長。圖4B。反之，atnlp8突變植物並未顯示關於苜蓿中華根瘤菌RMP110在限N條件下所誘發的植物生長促進效應。該等結果指出AtNSP1-樣、AtNLP4、與AtNLP9對於***芥屬與苜蓿中華根瘤菌 RMP110之間在限N條件下增強生長的功能***互作用是必需的。該等結果亦指出保守性機制係存在於植物，以介導就N養分而言的植物：細菌有益交互作用，其可運用於將BNF提供至非結瘤植物物種。 Mutations that were grown under N-limited conditions were inoculated with wild-type S. meliloti RMP110 (or mock imitation as a control group). Our results showed that under the condition of N, the plant growth promoting effect of R. sphaeroides RMP110 was lost in the atnsp1 mutant. Figure 4A. Similarly, mutant plants of AtNLP4 and AtNLP9 genes did not exhibit increased growth in the presence of R. sphaeroides RMP110. Figure 4B. Conversely, the atnlp8 mutant plant did not show a plant growth promoting effect induced by R. sphaeroides RMP110 under N-limited conditions. These results indicate that AtNSP1-like , AtNLP4 , and AtNLP9 are required for functional interaction between Arabidopsis and S. meliloti RMP110 to enhance growth under N-limited conditions. These results also indicate that conserved mechanisms are present in plants to mediate plant-to-bacterial beneficial interactions with respect to N-nutrients, which can be applied to provide BNF to non-nodulated plant species.

<110> 智利天主教大學(Pontificia Universidad Catolica de Chile) Kraiser,Tatiana Gonzalez,Bernardo Guitierrez,Rodrigo <110> Pontificia Universidad Catolica de Chile Kraiser, Tatiana Gonzalez, Bernardo Guitierrez, Rodrigo

<120> 促進與固氮菌之關連性的植物調控基因 <120> Plant regulatory genes that promote the association with nitrogen-fixing bacteria

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Claims (22)

一種用於增加非結瘤植物氮效率的方法，該方法包含將至少一異源性多肽引進植物，以製造基因轉殖植物，其中該異源性多肽係選自由結瘤信號轉導途徑-樣(NSPs)與NIN-樣蛋白(NLPs)構成之群組。 A method for increasing nitrogen efficiency of a non-nodulated plant, the method comprising introducing at least one heterologous polypeptide into a plant to produce a genetically transformed plant, wherein the heterologous polypeptide is selected from a nodulation signal transduction pathway Groups of (NSPs) and NIN-like proteins (NLPs). 如請求項1之方法，其中該異源性多肽係選自由NSP1-樣、NLP4、與NLP9構成之群組。 The method of claim 1, wherein the heterologous polypeptide is selected from the group consisting of NSP1-like, NLP4, and NLP9. 如請求項2之方法，其中該異源性多肽係與SEQ ID NOs：1-4當中一或多者至少90%一致。 The method of claim 2, wherein the heterologous polypeptide is at least 90% identical to one or more of SEQ ID NOs: 1-4. 如請求項2之方法，其中將異源性多肽引進植物包含以編碼異源性多肽的聚核苷酸轉形植物。 The method of claim 2, wherein the heterologous polypeptide is introduced into a plant comprising a polynucleotide-transformed plant encoding a heterologous polypeptide. 如請求項4之方法，其中該聚核苷酸係與SEQ ID NOs：4-6當中一或多者至少80%一致。 The method of claim 4, wherein the polynucleotide is at least 80% identical to one or more of SEQ ID NOs: 4-6. 如請求項4之方法，其中該聚核苷酸在嚴苛條件下係雜交至SEQ ID NOs：4-6當中一或多者。 The method of claim 4, wherein the polynucleotide hybridizes under stringent conditions to one or more of SEQ ID NOs: 4-6. 如請求項4之方法，其中該聚核苷酸係經密碼子-最佳化，以在植物中表現異源性多肽。 The method of claim 4, wherein the polynucleotide is codon-optimized to display a heterologous polypeptide in the plant. 如請求項4之方法，其中該聚核苷酸係操作性地聯結至選自下列所構成群組的植物促進子：組成促進子、組織偏好性促進子、組織特異性促進子、與可誘發促進子。 The method of claim 4, wherein the polynucleotide is operatively linked to a plant promoter selected from the group consisting of: a composition promoter, a tissue-preferred promoter, a tissue-specific promoter, and an inducer Promoter. 如請求項1之方法，其中該增加的氮效率係包含基因轉殖植物在限氮條件下增加的生長。 The method of claim 1, wherein the increased nitrogen efficiency comprises increased growth of the genetically transformed plant under nitrogen-limited conditions. 如請求項1之方法，其中該異源性多肽係引進植物的根 部組織。 The method of claim 1, wherein the heterologous polypeptide is introduced into the root of the plant Department organization. 如請求項1之方法，其中該方法包含使基因轉殖植物在限氮條件生長。 The method of claim 1, wherein the method comprises growing the genetically transformed plant under nitrogen-limited conditions. 一種基因轉殖植物，其係藉由如請求項1之方法製造。 A genetically modified plant produced by the method of claim 1. 一種細胞、組織、種籽、或材料，其係獲自如請求項12之基因轉殖植物。 A cell, tissue, seed, or material obtained by the genetically transgenic plant of claim 12. 一種核酸分子，其包含具有農藝功能的聚核苷酸，其中該聚核苷酸係選自由下列構成之群組：與SEQ ID NO：4至少80%一致的聚核苷酸；在嚴苛條件下雜交至由SEQ ID NO：4構成之核酸的聚核苷酸；與SEQ ID NO：5至少80%一致的聚核苷酸；在嚴苛條件下雜交至由SEQ ID NO：5構成之核酸的聚核苷酸；與SEQ ID NO：6至少80%一致的聚核苷酸；以及在嚴苛條件下雜交至由SEQ ID NO：6構成之核酸的聚核苷酸；其中該核酸序列係操作性地聯結至異源性促進子。 A nucleic acid molecule comprising an agronomically functional polynucleotide, wherein the polynucleotide is selected from the group consisting of: a polynucleotide that is at least 80% identical to SEQ ID NO: 4; in harsh conditions a polynucleotide that hybridizes to a nucleic acid consisting of SEQ ID NO: 4; a polynucleotide that is at least 80% identical to SEQ ID NO: 5; hybridizes under stringent conditions to the nucleic acid consisting of SEQ ID NO: Polynucleotide; a polynucleotide that is at least 80% identical to SEQ ID NO: 6; and a polynucleotide that hybridizes under stringent conditions to the nucleic acid consisting of SEQ ID NO: 6; wherein the nucleic acid sequence is Operatively linked to a heterologous promoter. 一種基因轉殖植物細胞，其係以如請求項14之核酸分子穩定轉形。 A gene transfer plant cell which is stably transformed with the nucleic acid molecule of claim 14. 一種用於製造基因轉殖植物的方法，該方法包含將核酸引進植物細胞，其中該核酸包含編碼異源性結瘤信號轉導途徑1-樣(NSP1-樣)、NIN-樣蛋白-4(NLP4)、或NIN-樣蛋白-9(NLP9)多肽的核苷酸序列，藉此製造基因轉殖植物。 A method for producing a genetically transgenic plant, the method comprising introducing a nucleic acid into a plant cell, wherein the nucleic acid comprises a heterologous nodulation signal transduction pathway 1-like (NSP1-like), NIN-like protein-4 ( The nucleotide sequence of NLP4), or NIN-like protein-9 (NLP9) polypeptide, thereby producing a genetically transformed plant. 如請求項16之方法，其中該植物為非結瘤植物。 The method of claim 16, wherein the plant is a non-nodulated plant. 一種基因轉殖植物，其係藉由如請求項16之方法製造。 A genetically modified plant produced by the method of claim 16. 一種細胞、組織、種籽、或材料，其係獲自如請求項17之基因轉殖植物。 A cell, tissue, seed, or material obtained by the genetically transgenic plant of claim 17. 如請求項17之基因轉殖植物，其中該基因轉殖植物包含基因轉殖植物在限氮條件下相較於相同物種的野生型植物所增加的生長。 The gene transfer plant of claim 17, wherein the gene transfer plant comprises increased growth of the genetically transformed plant under nitrogen-limited conditions compared to wild-type plants of the same species. 一種用於增加非結瘤植物氮效率的方法，該方法包含將促進與固氮菌(NFB)之關連性的至少一方式引進植物，其中該植物的氮效率係經由生物固氮作用(BNF)增加。 A method for increasing the nitrogen efficiency of a non-nodulated plant, the method comprising introducing into the plant at least one mode that promotes the association with a nitrogen-fixing bacteria (NFB), wherein the nitrogen efficiency of the plant is increased via biological nitrogen fixation (BNF). 如請求項21之方法，其中該促進與固氮菌NFB之關連性的方式係選自由下列構成之群組：由SEQ ID NOs：1、3、與4當中一者構成的多肽、以及由SEQ ID NOs：4-6當中一者構成的聚核苷酸。 The method of claim 21, wherein the means for promoting the association with the nitrogen-fixing bacteria NFB is selected from the group consisting of: a polypeptide consisting of one of SEQ ID NOs: 1, 3, and 4, and SEQ ID NOs: A polynucleotide consisting of one of 4-6.