TWI686477B - Cloning vector, kit, and method for specifically inducing mutagenesis in chloroplast genes, and transgenic plant cells and agrobacterium generated by the same - Google Patents

Abstract

The present application provides a method for generating chloroplast gene modified plant cells, comprising: constructing a pair of nuclear gene modifying vectors; transforming the pair of nuclear gene modifying vectors into plant cells via Agrobacterium; and selecting and culturing the plant cells. The nuclear gene modifying vectors comprises a promoter, a recombinant gene conferred the chloroplast transit peptide and TALEN protein domain, a terminator as well as right border and left border of T-DNA regions. Thus, by using nuclear transformation to mutate chloroplast gene, the problems such as low transformation efficiency, long selection time, labor-intensive and high cost encountering in chloroplast transformation would be avoided.

Description

特異性造成植物葉綠體基因變異的轉殖載體、套組、方法及利用其產生之轉殖植物細胞與農桿菌Transplantation vector, set and method for specifically causing plant chloroplast gene variation, and transgenic plant cells and Agrobacterium produced using the same

本發明關於一種植物基因轉殖載體及方法，特別是關於一種植物細胞核基因轉殖載體及方法，其可用來造成葉綠體基因的變異。The invention relates to a plant gene transfer vector and method, in particular to a plant cell nuclear gene transfer vector and method, which can be used to cause chloroplast gene mutation.

傳統上要進行植物葉綠體基因的靜默、修飾與編輯，需要先行構築含有欲修飾的基因序列之葉綠體表現載體，接著利用基因槍轟擊法，將表現載體導入葉綠體內，再經由同源重組的方式取代原有的基因序列（Bock, 2015）。但是基因槍轟擊法的技術門檻高，需要訓練有素、經驗豐富的技術人員和昂貴的儀器去操作執行，而且基因槍耗材需求的成本高，於葉綠體的轉殖效率低。此外，由於一個植物細胞含有數十至數百個葉綠體，一個葉綠體也含有數十或上百個基因組，因此基因槍轟擊法所需篩選過程的時間冗長。Traditionally, to silence, modify and edit plant chloroplast genes, it is necessary to construct a chloroplast expression vector containing the gene sequence to be modified first, and then use the gene gun bombardment method to introduce the expression vector into the chloroplast and replace it by homologous recombination. The original gene sequence (Bock, 2015). However, the technical threshold of the gene gun bombardment method is high, which requires trained, experienced technicians and expensive instruments to operate and execute, and the cost of the gene gun consumables is high, and the efficiency of chloroplast transformation is low. In addition, since a plant cell contains tens to hundreds of chloroplasts, and a chloroplast also contains tens or hundreds of genomes, the time required for the screening process by the gene gun bombardment method is lengthy.

除了基因槍轟擊法，也可利用PEG（polyethylene glycol）媒介法進行葉綠體基因轉殖，亦有報導可以成功地將表現載體經由原生質體（protoplasts）導入少數植物的葉綠體中（Bock, 2015）。此方法雖然不需要昂貴的儀器，但是需要製備原生質體，而原生質體脆弱、操作不易，而且轉殖後又需自原生質體再生成植株。雖然耗材成本較低，但原生質體的再生技術門檻高，且再生過程所需的時間很長，大部分植物尚未建立此技術，現行只有少數植物可行。此外，PEG媒介法也有可能會產生出多倍體的細胞、轉殖效率低、篩選過程時間冗長等問題。In addition to the gene gun bombardment method, PEG (polyethylene glycol) vector method can also be used for chloroplast gene transformation. It has also been reported that expression vectors can be successfully introduced into chloroplasts of a few plants via protoplasts (Bock, 2015). Although this method does not require expensive equipment, it requires the preparation of protoplasts, and the protoplasts are fragile and difficult to handle, and plants need to be regenerated from the protoplasts after transplantation. Although the cost of consumables is low, the threshold of protoplast regeneration technology is high, and the time required for the regeneration process is very long. Most plants have not yet established this technology, and currently only a few plants are feasible. In addition, the PEG-mediated method may also produce polyploid cells, low colonization efficiency, and lengthy screening process.

總而言之，現有葉綠體基因轉殖技術仍有許多缺點、問題有待克服和改進，包括門檻高、轉殖效率低、篩選過程的時間冗長、需投入甚多人力和物力等。此外，由於葉綠體基因組較小且基因密度高，一般皆是利用同源重組的方式將外源基因***基因組中，相較於細胞核轉殖的隨機地***細胞核基因組中的方式，葉綠體轉殖效率低很多。All in all, the existing chloroplast gene transfer technology still has many shortcomings and problems to be overcome and improved, including high threshold, low transfer efficiency, lengthy screening process, and a lot of manpower and material resources. In addition, because the chloroplast genome is small and the gene density is high, the foreign genes are generally inserted into the genome by homologous recombination. Compared with the method of randomly inserting the nuclear genome into the nuclear genome, the efficiency of chloroplast transformation is low. a lot of.

有鑒於上述先前技術的問題，本發明的目的就是在提供一種細胞核基因轉殖載體、套組及方法，來造成葉綠體基因的變異，能夠有效改善傳統葉綠體基因轉殖效率低、篩選時間長、耗費人力及物力等問題。In view of the above problems of the prior art, the object of the present invention is to provide a nuclear gene transfer vector, set and method to cause chloroplast gene mutation, which can effectively improve the traditional chloroplast gene transfer efficiency, low screening time, and cost Human and material issues.

根據本發明之一目的，提出一種細胞核基因轉殖載體，包括：啟動子；葉綠體訊息蛋白基因，其位於啟動子下游；重組TALEN蛋白域，包含如SEQ ID NO: 21或SEQ ID NO: 22所示之序列；以及第一邊界區及第二邊界區，例如T-DNA的左邊界區及右邊界區。其中啟動子、葉綠體訊息蛋白基因、重組TALEN蛋白域及依序位於第一邊界區與第二邊界區之間。According to an object of the present invention, a nuclear gene transfer vector is proposed, which includes: a promoter; a chloroplast signaling protein gene, which is located downstream of the promoter; and a recombinant TALEN protein domain, which includes SEQ ID NO: 21 or SEQ ID NO: 22 The sequence shown; and the first and second border regions, such as the left and right border regions of T-DNA. The promoter, the chloroplast signaling protein gene, and the recombinant TALEN protein domain are sequentially located between the first border region and the second border region.

較佳地，啟動子可為 rbcS啟動子。 Preferably, the promoter may be the rbcS promoter.

較佳地，重組TALEN蛋白域可包括編碼DNA辨識蛋白及核酸內切酶的DNA序列。更佳地，核酸內切酶可為Fok I，而該DNA辨識蛋白可辨識葉綠體之 rpoB基因之片段。最佳地，編碼DNA辨識蛋白之DNA序列可包括如SEQ ID NO: 3或SEQ ID NO: 4所示之序列。 Preferably, the recombinant TALEN protein domain may include DNA sequences encoding DNA recognition proteins and endonuclease. More preferably, the endonuclease can be Fok I, and the DNA recognition protein can recognize a fragment of the chloroplast rpoB gene. Most preferably, the DNA sequence encoding the DNA recognition protein may include the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

較佳地，轉殖載體可更包括篩選基因，篩選基因可為抗藥性篩選基因、非抗藥性篩選基因、或其組合。更佳地，篩選基因可包括卡那黴素（kanamycin）抗性基因或甲硫胺酸磺醯亞胺（methionine sulfoximine, MSO）殺草劑抗性基因。最佳地，卡那黴素抗性基因包括 nptII或該甲硫胺酸磺醯亞胺殺草劑抗性基因包括 bar。 Preferably, the transfection vector may further include a screening gene, and the screening gene may be a drug resistance screening gene, a non-drug resistance screening gene, or a combination thereof. More preferably, the screening gene may include a kanamycin (kanamycin) resistance gene or a methionine sulfoximine (MSO) herbicide resistance gene. Most preferably, the kanamycin resistance gene includes nptII or the methionine sulfonimide herbicide resistance gene includes bar .

較佳地，轉殖載體可更包括編碼標誌物之序列。更佳地，標誌物可包含選自由發光分子、化學發光分子、螢光染料、螢光猝滅劑、脂質、有色分子、放射性同位素、閃爍劑、生物素、抗生物素蛋白、鏈黴親和素、蛋白質A、蛋白質G、抗體或其片段、聚組胺酸、Ni ²⁺、Flag標籤、myc標籤、HA標籤、和酶所組成的群組中的可檢測標記物。 Preferably, the transfer vector may further include a sequence encoding a marker. More preferably, the marker may comprise a molecule selected from the group consisting of luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorescent quenchers, lipids, colored molecules, radioisotopes, scintillators, biotin, avidin, streptavidin , Protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni ²⁺ , Flag tag, myc tag, HA tag, and detectable markers in the group consisting of enzymes.

較佳地，轉殖載體可包含如SEQ ID NO: 1或SEQ ID NO: 2所示之序列。Preferably, the transfer vector may comprise the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

較佳地，轉殖載體可更包括 rbcS基因的3端非轉錄區，其位於重組TALEN蛋白域下游。 Preferably, the transfection vector may further include a 3-terminal non-transcribed region of the rbcS gene, which is located downstream of the recombinant TALEN protein domain.

根據本發明之又一目的，提出一種特異性造成植物葉綠體基因變異的套組，包括：一對前述之細胞核基因轉殖載體。較佳地，套組可包括第一轉殖載體及第二轉殖載體。第一轉殖載體包括：第一啟動子；第一葉綠體訊息蛋白基因，其位於第一啟動子下游；第一重組TALEN蛋白域，包含如SEQ ID NO: 21所示之序列；以及第一邊界區及第二邊界區，其中第一啟動子、第一葉綠體訊息蛋白基因及第一重組TALEN蛋白域依序位於第一邊界區與第二邊界區之間。第二轉殖載體包括：第二啟動子；第二葉綠體訊息蛋白基因，其位於第二啟動子下游；第二重組TALEN蛋白域，包含如SEQ ID NO: 22所示之序列；以及第三邊界區及第四邊界區，其中第二啟動子、第二葉綠體訊息蛋白基因及第二重組TALEN蛋白域依序位於第三邊界區與第四邊界區之間。According to another object of the present invention, a kit for specifically causing plant chloroplast gene variation is proposed, which includes: a pair of the aforementioned nuclear gene transfer vectors. Preferably, the kit may include a first transgenic vector and a second transgenic vector. The first transfer vector includes: a first promoter; a first chloroplast information protein gene, which is located downstream of the first promoter; a first recombinant TALEN protein domain, including the sequence shown in SEQ ID NO: 21; and a first border Region and the second boundary region, in which the first promoter, the first chloroplast signaling protein gene and the first recombinant TALEN protein domain are sequentially located between the first boundary region and the second boundary region. The second transfer vector includes: a second promoter; a second chloroplast information protein gene, which is located downstream of the second promoter; a second recombinant TALEN protein domain, including the sequence shown in SEQ ID NO: 22; and a third border Region and the fourth border region, in which the second promoter, the second chloroplast signaling protein gene and the second recombinant TALEN protein domain are sequentially located between the third border region and the fourth border region.

根據本發明之再一目的，提出一種產生細胞核基因轉殖植物細胞並造成葉綠體基因變異的方法，包括：建構一對前述之細胞核基因轉殖載體；透過農桿菌將所述一對細胞核基因轉殖載體併入植物細胞；以及篩選及培養植物細胞。According to yet another object of the present invention, a method for generating nuclear gene transplantation of plant cells and causing chloroplast gene mutations is provided, comprising: constructing a pair of nuclear gene transfection vectors as described above; transfecting the pair of nuclear genes through Agrobacterium The vector is incorporated into the plant cell; and the plant cell is selected and cultured.

較佳地，併入可包含同源性導向的修復機制或非同源性末端接合導向的修復機制。Preferably, the incorporation may include homology-directed repair mechanisms or non-homologous end-joint-directed repair mechanisms.

較佳地，方法可更包括轉殖細胞核基因轉殖載體至農桿菌。Preferably, the method may further include transferring the nuclear gene transfer vector to Agrobacterium.

較佳地，方法可更包括分析植物細胞並確認細胞核基因轉殖載體併入該植物細胞。Preferably, the method may further include analyzing the plant cell and confirming that the nuclear gene transfer vector is incorporated into the plant cell.

根據本發明之再一目的，提出一種細胞核基因轉殖植物細胞，其係以前述之方法所建構。According to another object of the present invention, a nuclear gene transgenic plant cell is proposed, which is constructed by the aforementioned method.

根據本發明之再一目的，提出一種農桿菌，包括前述之細胞核基因轉殖載體。According to another object of the present invention, an Agrobacterium is proposed, which includes the aforementioned nuclear gene transfer vector.

總的來說，本案透過於細胞核表現重組TALEN蛋白的方式來造成葉綠體基因變異的方式，至少可解決前述傳統葉綠體基因轉殖的問題，並至少提供以下優點：In summary, the method of causing chloroplast gene mutation by expressing recombinant TALEN protein in the cell nucleus can at least solve the aforementioned problem of traditional chloroplast gene transfer and provide at least the following advantages:

（1）提昇轉殖效率並解決傳統葉綠體基因轉殖技術門檻高之問題。(1) Improve the efficiency of transformation and solve the problem of high threshold of traditional chloroplast gene transformation technology.

（2）由於利用細胞核基因轉殖，篩選的時間較短。(2) Due to the use of nuclear gene transfer, the screening time is shorter.

（3）利用農桿菌媒介法進行細胞核基因轉殖的成本低，故無須昂貴的儀器及製備原生質體。(3) The cost of using the Agrobacterium-mediated method for nuclear gene transfer is low, so there is no need for expensive equipment and preparation of protoplasts.

本案所請發明的上述以及其它目的、特徵與優點，在參照以下的詳細說明與較佳實施例和隨文檢附的圖式後，將變得明顯。The above and other objects, features, and advantages of the invention claimed in this case will become apparent after referring to the following detailed description and preferred embodiments and accompanying drawings.

本發明之各個具體實例的細節說明如後。本發明之其他特徵將會經由以下各個具體實例中的詳細說明及申請專利範圍而清楚呈現。The detailed description of each specific example of the present invention is as follows. Other features of the present invention will be clearly presented through the detailed description and patent application scope in the following specific examples.

無須進一步的闡述，咸相信本發明所屬技術領域中具有通常知識者基於前述說明即可利用本發明至最廣的程度。因此，可以理解以下的說明僅僅是作為例示說明之用，而非以任何方式限制其餘的揭露內容。Without further elaboration, Xian believes that those with ordinary knowledge in the technical field to which the present invention belongs can use the present invention to the fullest extent based on the foregoing description. Therefore, it can be understood that the following description is only for illustrative purposes and does not limit the remaining disclosure in any way.

除非另有說明，否則此處使用之全部技術和科學名詞與本發明所屬技術領域中具有通常知識者通常所瞭解的意義相同。Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those with ordinary knowledge in the technical field to which the present invention belongs.

除非另有說明，所有百分比、部分、比例等皆以重量計。Unless otherwise stated, all percentages, parts, ratios, etc. are by weight.

如本文中所用者，術語「由……製成」（produced from）與「包含」（comprising）同義。如本文中所用者，術語「包含」（includes, including）、「包括」（comprises, comprising）、「具有」（has, having）、「含有」（contains, containing）、或其任何其他變型，係意欲涵蓋非排他性的涵括。例如，含有清單列出的複數元素的一組合物、製程、方法、製品或裝置不一定僅限於清單上所列出的這些元素而已，而是可以包括未明確列出但卻是該組合物、製程、方法、製品或裝置固有的其他元素。術語「包含」一般係以包括之意思被使用，此即係允許一或數種其他特徵或組成份之存在。As used herein, the term "produced from" is synonymous with "comprising". As used herein, the terms "includes", "includes", "comprises", "has, having", "contains", or any other variants of It is intended to cover non-exclusive inclusions. For example, a composition, process, method, product, or device containing a plurality of elements listed in the list is not necessarily limited to those elements listed on the list, but may include the composition not specifically listed, but Other elements inherent in the process, method, product or device. The term "comprising" is generally used to mean including, which means that one or several other features or components are allowed to exist.

本申請案的目的在於提供一種細胞核基因轉殖載體，其在透過農桿菌或其他本發明所屬技術領域中具有通常知識者習知的方法轉殖至植物細胞核並經過細胞核的轉錄後，可於細胞質轉譯合成出重組TALEN蛋白。重組TALEN蛋白可以藉由位於N端的訊息蛋白之導引，經由葉綠體膜的傳送通道（Toc-Tic translocon）到達葉綠體基質內，而後該重組TALEN蛋白可特異性地結合於目標葉綠體DNA的特定位置。重組TALEN蛋白再由核酸內切酵素（例如Fok I）作用，造成雙股葉綠體DNA的斷裂，進而啟動葉綠體DNA之修復機制，包括為同源性導向的修復機制（homologous recombination, HR）或非同源性末端接合導向的修復機制（non-homologous end joining, NHEJ）。當利用葉綠體DNA的非同源性重組之修復機制，常導致目標基因變異，將進而導致目標基因失去活性。The purpose of the present application is to provide a nuclear gene transfer vector which can be transferred to the plant cell nucleus through Agrobacterium or other methods known to those of ordinary skill in the technical field of the present invention and transcribed by the cell nucleus, and can be transferred to the cytoplasm Translation and synthesis of recombinant TALEN protein. The recombinant TALEN protein can be guided into the chloroplast matrix through the transmission channel (Toc-Tic translocon) of the chloroplast membrane through the guidance of the N-terminal signaling protein, and then the recombinant TALEN protein can specifically bind to the specific location of the target chloroplast DNA. The recombinant TALEN protein is then endonuclease (such as Fok I) to cause the double-stranded chloroplast DNA to break, and then start the repair mechanism of chloroplast DNA, including homologous-oriented repair mechanism (homologous recombination, HR) or different Non-homologous end joining (NHEJ). When the repair mechanism of non-homologous recombination of chloroplast DNA is used, the target gene is often mutated, which will in turn cause the target gene to lose activity.

在一實施例中，如圖1所示，提供一種細胞核基因轉殖載體，包括：啟動子（promoter）；葉綠體訊息蛋白基因（TP），其位於啟動子下游；重組TALEN蛋白域（TALEN），其位於葉綠體訊息蛋白基因下游；以及T-DNA左邊界區（LB-T border）及T-DNA右邊界區（RB-T border），其中啟動子、葉綠體訊息蛋白基因及重組TALEN蛋白域依序位於T-DNA左邊界區與T-DNA右邊界區之間。In one embodiment, as shown in FIG. 1, a nuclear gene transfer vector is provided, including: a promoter; a chloroplast signaling protein gene (TP), which is located downstream of the promoter; a recombinant TALEN protein domain (TALEN), It is located downstream of the chloroplast signaling protein gene; and T-DNA left border region (LB-T border) and T-DNA right border region (RB-T border), in which the promoter, chloroplast signaling protein gene and recombinant TALEN protein domain are in order Located between the left border area of T-DNA and the right border area of T-DNA.

在一實施態樣中，該細胞核基因轉殖載體可具有如SEQ ID NO: 1或SEQ ID NO: 2所示之序列，並將該細胞核基因轉殖載體命名為pTpTalen-L或pTpTalen-R。In one embodiment, the nuclear gene transfer vector may have the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the nuclear gene transfer vector is named pTpTalen-L or pTpTalen-R.

值得注意的是，本發明所屬技術領域中具有通常知識者，可根據所欲重組TALEN蛋白辨識或切割的區域，而設計重組TALEN蛋白域的序列。轉殖載體中的其他元件，例如啟動子、篩選標記、終結子等，本發明所屬技術領域中具有通常知識者可依據實驗需求而自行選擇適合的元件進行替換。It is worth noting that those with ordinary knowledge in the technical field of the present invention can design the sequence of the recombinant TALEN protein domain according to the region to be recognized or cleaved by the desired TALEN protein. For other elements in the transfection vector, such as promoters, selection markers, terminators, etc., those with ordinary knowledge in the technical field to which the present invention belongs may choose suitable elements to replace according to experimental needs.

在一實施例中，提供一種產生細胞核基因轉殖植物細胞的方法，包括：建構一對前述之細胞核基因轉殖載體；透過農桿菌將所述一對細胞核基因轉殖載體併入植物細胞；以及篩選及培養植物細胞。In one embodiment, a method for producing nuclear gene transgenic plant cells is provided, comprising: constructing a pair of the aforementioned nuclear gene transfection vectors; incorporating the pair of nuclear gene transduction vectors into plant cells through Agrobacterium; and Screen and cultivate plant cells.

在又一實施例中，透過前述的方法，可提供一種細胞核基因轉殖植物細胞或細胞核基因轉殖植物，或可提供一種包含前述細胞核基因轉殖載體的農桿菌。In yet another embodiment, through the aforementioned method, a nuclear gene transgenic plant cell or a nuclear gene transgenic plant can be provided, or an Agrobacterium comprising the aforementioned nuclear gene transgenic vector can be provided.

本文中所稱「植物」、「植物細胞」及「重組植物細胞」等術語於本文中可彼此相互交替使用，其可表示雙子葉植物、單子葉植物、裸子植物等的植物細胞，例如菸草、***芥、馬鈴薯、大豆、番茄、蘭花、水稻、玉米、小麥、大麥、高粱、甘蔗等。可於該植物細胞中引入一或數種本案所述之載體或經基因工程方法***欲表現基因的所述載體。如本發明所屬技術領域中通常知識者所瞭解，上述術語不僅意指特定之細胞，而且亦指該種細胞之後代或潛在可能之後代。因為基因突變或環境之影響，後繼之世代中可能會出現改變，故該等細胞之後代實質上不會與親代之細胞相同，然而卻仍包括於本文中所使用之該術語之範圍內。The terms "plant", "plant cell" and "recombinant plant cell" referred to herein may be used interchangeably with each other herein, which may refer to plant cells of dicotyledonous plants, monocotyledonous plants, gymnosperms, etc., such as tobacco, Arabidopsis, potato, soybean, tomato, orchid, rice, corn, wheat, barley, sorghum, sugarcane, etc. One or several vectors described in this case can be introduced into the plant cell or the vector to be inserted into the gene to be expressed by genetic engineering methods. As those of ordinary knowledge in the technical field to which the present invention is understood, the above term means not only a specific cell, but also a progeny or a potential progeny of the cell. Because of genetic mutations or environmental influences, there may be changes in subsequent generations, so the descendants of these cells will not be substantially the same as the cells of the parent, but they are still included in the scope of the term used herein.

本文中所稱「葉綠體訊息蛋白基因」係指一段核苷酸序列，在經過細胞核的轉錄、細胞質的轉譯後可合成出一訊息蛋白。此訊息蛋白會表現在蛋白質的一端，例如N端，此訊息蛋白包含了將蛋白質運送至葉綠體所需的導引訊息。如同「地址標籤」，此訊息蛋白能將蛋白質指引到葉綠體表面。而在葉綠體表面有一組特殊的傳送通道（Toc-Tic translocon），可辨識這個「地址標籤」，經辨識後，接受器會將含正確「地址標籤」的蛋白質轉運入葉綠體基質。在進入葉綠體基質後，這多出來的一段訊息蛋白便會被切除。The term "chloroplast signaling protein gene" as used herein refers to a nucleotide sequence that can be synthesized by transcription of the nucleus and translation of the cytoplasm. This information protein will appear at one end of the protein, such as the N-terminus. This information protein contains the guiding information needed to transport the protein to the chloroplast. Like the "address tag", this information protein can direct the protein to the surface of the chloroplast. On the surface of the chloroplast, there is a special transmission channel (Toc-Tic translocon), which can recognize this "address label". After recognition, the receiver will transfer the protein containing the correct "address label" into the chloroplast matrix. After entering the chloroplast matrix, this extra piece of signal protein will be excised.

本文中所稱「篩選基因」係用來確認前述載體是否有順利轉形（transform）進入宿主中。前述篩選基因可為但不限於：抗藥性篩選基因、非抗藥性篩選基因、或其組合。在一可行實施態樣中，前述篩選基因可為抗藥性篩選基因。舉例來說，前述抗藥性篩選基因可為卡那黴素或新黴素抗性基因。在此可行實施態樣中，順利轉形有前述載體的植物細胞（如，菸草或***芥）便可以產生對卡那黴素或新黴素的抗性，而得以存活於含有卡那黴素或新黴素的環境。The "screening gene" referred to herein is used to confirm whether the aforementioned vector has successfully transformed into the host. The aforementioned screening gene may be, but not limited to, a drug resistance screening gene, a non-drug resistance screening gene, or a combination thereof. In a feasible embodiment, the aforementioned screening gene may be a drug resistance screening gene. For example, the aforementioned drug resistance screening gene may be a kanamycin or neomycin resistance gene. In this feasible embodiment, a plant cell (for example, tobacco or Arabidopsis) that has been transformed with the aforementioned vector can develop resistance to kanamycin or neomycin, and can survive in the presence of kanamycin. Or neomycin environment.

本文中所稱「非抗藥性篩選基因」係指非以對抗生素之抗性與否來確認轉形的基因。前述非抗藥性篩選基因例如但不限於：Phosphinothricin N‐acetyltransferase基因（ bar或 pat），其做為篩選基因可將含有殺草劑的成分草銨膦（glufosinate）或甲硫胺酸磺醯亞胺（methionine sulfoximine; MSO）等化合物進行乙醯基化而去掉其毒性，因此可以利用殺草劑譬如草銨膦等類化合物來進行轉基因植物的篩選。另外，突變或過量表現5‐enolpyruvoyl‐shikimate‐3‐phosphate synthetase (EPSPS) 基因可以不受到含有嘉賽磷（Glyphosate; N‐(phosphonomethyl)glycine）殺草劑的抑制，因此也可以做為篩選基因，利用殺草劑譬如嘉賽磷等來進行轉基因植物的篩選。 The term "non-drug resistance screening gene" as used herein refers to a gene that has not been confirmed by the resistance to antibiotics. The aforementioned non-drug resistance screening genes are, for example but not limited to, Phosphhinothricin N-acetyltransferase gene ( bar or pat ), which can be used as a screening gene to include the herbicide-containing components glufosinate or methionine sulfonamide (Methionine sulfoximine; MSO) and other compounds are acetylated to remove their toxicity, so herbicides such as glufosinate and other compounds can be used to screen transgenic plants. In addition, mutations or excessive performance of the 5‐enolpyruvoyl‐shikimate‐3‐phosphate synthetase (EPSPS) gene may not be inhibited by herbicides containing Glyphosate; N‐(phosphonomethyl)glycine), so it can also be used as a screening gene Utilizing herbicides such as glufosinate to screen transgenic plants.

在一較佳實施態樣中，前述非抗藥性篩選基因為營養缺乏篩選基因（如，cytosine deaminase ( codA)）、醣類代謝基因（如，phosphomannose isomerase (manA); xylose isomerase ( xylA)）、植物賀爾蒙相關基因（如，isopentenyltransferase (ipt)）、重金屬抗性基因（如，鎘抗性基因）、D-胺基酸消旋酶（如，alanine racemase）基因、熱休克蛋白質（heat shock protein）基因或報導基因 （如，β‐glucuronidase( GUS);luciferase( LUC) ;fluorescent proteins( FPs)）。 In a preferred embodiment, the aforementioned non-drug resistance screening genes are nutritional deficiency screening genes (eg, cytosine deaminase ( codA )), sugar metabolism genes (eg, phosphomannose isomerase (manA) ; xylose isomerase ( xylA )), Plant hormone related genes (eg, isopentenyltransferase (ipt) ), heavy metal resistance genes (eg, cadmium resistance genes), D-amino acid racemase (eg, alanine racemase) genes, heat shock proteins (heat shock) protein) gene or reporter gene (eg, β-glucuronidase ( GUS ); luciferase ( LUC ) ; fluorescent proteins ( FPs) ).

本文中所稱「併入」、「經併入」或「將核酸送入細胞中」等術語係表示利用任何之方法，使細胞外之核酸（如載體）於有或無伴隨物質（例如另一載體或寡核苷酸）之下進入宿主細胞。「細胞併入」或「經併入之細胞」等術語係表示於該植物細胞或其子代細胞中送入細胞外之核酸，因此植物細胞含該細胞外之核酸。核酸送入細胞中，而使該核酸可與染色體結合成一體之片段形式或以染色體外之元件之形式進行複製，或是該核酸破壞細胞中內源基因的表現。The terms "incorporated", "incorporated", or "sending nucleic acid into a cell" as used herein mean using any method to make the extracellular nucleic acid (such as a carrier) with or without accompanying substances (such as another A vector or oligonucleotide) into the host cell. Terms such as "cell incorporation" or "incorporated cells" refer to nucleic acids that are delivered outside the cell in the plant cell or its progeny cells, so the plant cell contains the nucleic acid outside the cell. The nucleic acid is fed into the cell, so that the nucleic acid can be combined with the chromosome in the form of fragments or replicated in the form of extrachromosomal elements, or the nucleic acid disrupts the expression of endogenous genes in the cell.

在一實施例中，迫使含有外源基因的載體進入農桿菌體內有兩種方式，即電穿孔法及三親雜交法。電穿孔法就是透過電擊農桿菌的方式，使其產生暫時性通透孔使載體進入農桿菌中。三親雜交法就是將含有特殊質體的大腸桿菌（稱為幫助者菌系）與含有外源基因的大腸桿菌，及含有幫助者質體的農桿菌三種菌類一起培養，含有外源基因的載體即會進入含有幫助者質體的農桿菌中。在一實施態樣中，進一步利用該含有外源基因載體的農桿菌，將外源基因送入目標細胞中，例如植物細胞。In one embodiment, there are 兩 ways to force the vector containing the foreign gene into Agrobacterium, namely electroporation and three-parent hybridization. Electroporation is the method of electroporation of Agrobacterium to produce a temporary through hole for the carrier to enter Agrobacterium. The three-parent hybridization method is to cultivate E. coli containing special plastids (referred to as helper strains) together with E. coli containing exogenous genes, and three strains of Agrobacterium containing helper plastids 類, a carrier containing exogenous genes Will enter the Agrobacterium containing helper plastids. In one embodiment, the Agrobacterium containing the foreign gene vector is further used to deliver the foreign gene into a target cell, such as a plant cell.

位於本文中之元素或成份之前的不定冠詞「一」及「一個」旨在非限制性地說明該元素或成份的實例數目（即出現數）。因此「一」或「一個」應理解為包括一個或至少一個，且該元素或成分的單數詞形也包括複數，除非該數目顯然是指單數。The indefinite articles "a" and "an" before the element or component in this article are intended to illustrate without limitation the number of instances (ie the number of occurrences) of the element or component. Therefore, "a" or "an" should be understood as including one or at least one, and the singular form of the element or component also includes the plural unless the number obviously refers to the singular.

本文中之材料、方法及實例僅為說明性質，除非特別說明，否則非意欲為限制本發明。儘管可用類似或等效於本文所述的方法或材料來實施或測試本發明，然本文所述者為較適當的方法及材料。The materials, methods, and examples herein are illustrative only, and unless specifically stated otherwise, are not intended to limit the invention. Although methods or materials similar or equivalent to those described herein can be used to practice or test the present invention, those described herein are more suitable methods and materials.

建構似轉錄活化因子核酸酶（Construction of transcription-like activator nuclease ( Transcription activator-like effector nuclease; TALENTranscription activator-like effector nuclease; TALEN ）)

TALEN是經由人為重組似轉錄活化因子（Transcription activator-like effector, TALE）的DNA辨識結合域和核酸內切酶活性區域而形成。TALE是由植物黃單胞菌（ Xanthomonasbacteria）分泌的蛋白質，此蛋白質含有一個進核訊號（NLS）以及轉錄活化子，能幫助TALE蛋白從細胞質進入細胞核，然後專一性結合到宿主基因的啟動子，進而活化轉錄作用之進行。 TALEN is formed by the DNA recognition binding domain and endonuclease active region of artificial recombination transcription activator-like effector (TALE). TALE is a protein secreted by the plant Xanthomonas bacteria. This protein contains a nuclear entry signal (NLS) and transcription activator, which can help the TALE protein enter the nucleus from the cytoplasm and then specifically bind to the promoter of the host gene. , Which in turn activates transcription.

TALE的中間區域為多個串聯重複單元組成的串聯重複區，每個串聯重複單元一般含有34個胺基酸，其中第12和13位置的胺基酸之變異性高，被稱為重複可變區（repeat-variable diresidue，RVD）。每個串聯重複單元中的RVD可以辨認DNA的不同鹼基，並有簡單的一對一之對應關係。例如，RVD如果是NI（Asn-Ile）可辨認A，HD（His-Asp）可辨認C，NG（Asn-Gly）可辨認T，而NN（Asn-Asn）可辨認G/A。The middle area of TALE is a tandem repeating area composed of multiple tandem repeating units. Each tandem repeating unit generally contains 34 amino acids, of which the amino acids at positions 12 and 13 have high variability and are called repeatable variable District (repeat-variable diresidue, RVD). The RVD in each tandem repeat unit can identify different bases of DNA, and there is a simple one-to-one correspondence. For example, if RVD is NI (Asn-Ile) can recognize A, HD (His-Asp) can recognize C, NG (Asn-Gly) can recognize T, and NN (Asn-Asn) can recognize G/A.

另一方面，核酸內切酶活性區域可為Fok I核酸內切酶，而Fok I核酸內切酶需形成二元體才可切割DNA。因此，目前可利用合成一對TALEN，以配對上任何所期望結合的DNA序列且特異性地用來切割其雙股DNA （Cermak et al., 2011）。On the other hand, the active region of the endonuclease can be Fok I endonuclease, and Fok I endonuclease needs to form a binary body to cut DNA. Therefore, it is currently possible to synthesize a pair of TALENs to match any desired DNA sequence and specifically use it to cut its double-stranded DNA (Cermak et al., 2011).

在一實施例中，使用者可根據其目標基因設計重組TALEN蛋白域，使重組TALEN蛋白域中之重複可變區可辨識目標核酸序列。舉例而言，重組TALEN蛋白域可包含如SEQ ID NO: 21或SEQ ID NO: 22所示之序列，其中重複可變區以「NNNNNN」表示，亦即N可選自於A、T、C及G中任一個，以使所表現出的重複可變區為可辨認A之NI（Asn-Ile），可辨認C之HD（His-Asp），可辨認T之NG（Asn-Gly），或可辨認G或A之NN（Asn-Asn）。在一實施例中，設計一對細胞核基因轉殖載體，如圖1所示，pTpTalen-L和pTpTalen-R中所編碼的Talen-L與Talen-R為一對TALEN基因。在一實施例中，設計pTpTalen-L和pTpTalen-R中Talen-L與Talen-R的序列，使其轉譯後的蛋白質可辨識***芥或菸草的葉綠體 rpoB基因序列。 In one embodiment, the user can design the recombinant TALEN protein domain according to his target gene, so that the repeated variable region in the recombinant TALEN protein domain can recognize the target nucleic acid sequence. For example, the recombinant TALEN protein domain may include the sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22, wherein the repeating variable region is represented by "NNNNNN", that is, N may be selected from A, T, C And any one of G, so that the repeated variable regions shown are NI (Asn-Ile) recognizable A, HD (His-Asp) recognizable C, and NG (Asn-Gly) recognizable T, Or the NN (Asn-Asn) of G or A can be recognized. In one embodiment, a pair of nuclear gene transfer vectors are designed. As shown in FIG. 1, Talen-L and Talen-R encoded in pTpTalen-L and pTpTalen-R are a pair of TALEN genes. In one embodiment, the sequences of Talen-L and Talen-R in pTpTalen-L and pTpTalen-R are designed so that the translated protein can recognize the chloroplast rpoB gene sequence of Arabidopsis or tobacco.

設計design TALENTALEN 欲作用於葉綠體基因之目標序列Target sequence to act on chloroplast gene

在本實施例中，以菸草葉綠體 rpoB基因為例子，根據其序列設計Talen-L與Talen-R。 rpoB基因位於葉綠體 DNA 的 rpoB-rpoCl-rpoC2的操作基因組 (Operon) (Little et al., 1988；Inada et al., 1996；Hajdukiewicz et al., 1997)。而根據先前研究顯示，若將 rpoB基因自菸草的葉綠體 DNA 中剔除，雖仍有細胞核編碼的 RNA 合成酶維持部分葉綠體的功能，不會造成植株死亡，但會促使菸草形成白色的外表性狀，且生長發育也比正常的菸草植株來得緩慢 （Allison et al., 1996）。由於其較易於觀察，因此選擇 rpoB基因作為 TALENs進行葉綠體基因編輯的目標基因。 In this example, taking the tobacco chloroplast rpoB gene as an example, Talen-L and Talen-R are designed according to their sequences. The rpoB gene is located in the operational genome (Operon) of rpoB-rpoCl-rpoC2 of chloroplast DNA (Little et al., 1988; Inada et al., 1996; Hajdukiewicz et al., 1997). According to previous research, if the rpoB gene is removed from the chloroplast DNA of tobacco, although RNA synthetase encoded by the nucleus still maintains part of the chloroplast function, it will not cause plant death, but it will promote tobacco to form a white appearance. Growth and development are also slower than normal tobacco plants (Allison et al., 1996). Because it is easier to observe, the rpoB gene was selected as the target gene for chloroplast gene editing of TALENs.

首先，自NCBI資料庫下載***芥與菸草的 rpoB基因序列 （Accession number 分別是 NC_000932.1 和 NC_001879.2） 進行序列比對，擷取一段具有共同保留性較高的序列，並藉由TAL Effector Nucleotide Targeter 2.0 (https://talent.cac.corne-ll.edu/node/add/talen) 網頁設計 TALENs 的目標位。在一實施態樣中，選擇Talen-L與Talen-R所辨識的目標DNA序列分別為 5’-TCCAAAAATTGAA-3’（SEQ ID NO: 11） 與 5’- TAATTGAAATTCA -3’（SEQ ID NO: 12）。 First, download the rpoB gene sequences (Accession numbers NC_000932.1 and NC_001879.2, respectively) from Arabidopsis and Tobacco from the NCBI database, and perform sequence alignment to extract a sequence with high common retention and use TAL Effector Nucleotide Targeter 2.0 (https://talent.cac.corne-ll.edu/node/add/talen) The goal of TALENs for web design. In one embodiment, the target DNA sequences identified by Talen-L and Talen-R are 5'-TCCAAAAATTGAA-3' (SEQ ID NO: 11) and 5'-TAATTGAAATTCA-3' (SEQ ID NO: 12).

構築細胞核重組Constructing nuclear reorganization TALENTALEN 基因的表現載體Gene expression vector

根據前述目標DNA序列，分別針對位於葉綠體 rpoB基因的目標DNA序列SEQ ID NO: 11與SEQ ID NO: 12，使用 Golden Gate TALEN assembly kit (Cermak et al., 2011) 人工合成並構築合成出一對TALEN基因，使其所轉譯出的重複可變區的RVD 序列分別為N’-HD-HD-NI-NI-NI-NI-NI-NG-NG-NN-NI-NI-C’ 與N’-NI-NI-NG-NG-NN-NI-NI-NI-NG-NG-HD-NI-C’，其可特異性辨識結合於上述目標DNA序列SEQ ID NO: 11與SEQ ID NO: 12。在一實施例中，依照 Golden Gate TALEN assembly kit的使用說明，構築前述RVD 序列時需分成前段與後段進行，將其分別命名為 pTALEN-L1、pTALEN-L2、pTALEN-R1 和 pTALEN-R2，且其 RVD 序列分別為N’-HD-HD-NI-NI-NI-NI-C’、N’-NI-NG-NG-NN-NI-C’、N’-NI-NI-NG-NG-NN-NI-C’ 和 N’-NI-NI-NG-NG-HD-C’。 Based on the aforementioned target DNA sequence, a pair of target DNA sequences SEQ ID NO: 11 and SEQ ID NO: 12 located in the chloroplast rpoB gene were synthesized and constructed using the Golden Gate TALEN assembly kit (Cermak et al., 2011). TALEN gene, so that the RVD sequences of the repeated variable regions that are translated are N'-HD-HD-NI-NI-NI-NI-NI-NG-NG-NN-NI-NI-C' and N'-NI-NI-NG-NG-NN-NI-NI-NI-NG-NG-HD-NI-C', which can specifically recognize and bind to the above target DNA sequences SEQ ID NO: 11 and SEQ ID NO: 12 . In one embodiment, according to the instructions of the Golden Gate TALEN assembly kit, the construction of the aforementioned RVD sequence needs to be divided into the front section and the rear section, which are named pTALEN-L1, pTALEN-L2, pTALEN-R1, and pTALEN-R2, and The RVD sequences are N'-HD-HD-NI-NI-NI-NI-NI-C', N'-NI-NG-NG-NG-NN-NI-C', N'-NI-NI-NG-NG- NN-NI-C' and N'-NI-NI-NG-NG-HD-C'.

接著，利用前述pTALEN-L1、pTALEN-L2、pTALEN-R1 和 pTALEN-R2構築重組TALEN基因的細胞核表現載體。簡言之，將合成出的TALEN 基因片段重組於植物表現載體pImpactVector 4-1，基因表現卡匣最後的組成為 rbcS的葉綠體訊息蛋白和HA 標籤或 Flag 標籤重組於TALEN的N端，並由 rbcS基因啟動子來調控重組TALEN的表現。再將基因表現卡匣分別重組於農桿菌雙偶載體pBinPlus （含 nptII基因做為篩選標誌）或pZGB9R （含 bar基因做為篩選標誌），而分別得到 pTpTalen-R與pTpTalen-L的細胞核表現載體，如圖1所示。以下詳細說明實驗步驟： Next, the nuclear expression vector of the recombinant TALEN gene was constructed using the aforementioned pTALEN-L1, pTALEN-L2, pTALEN-R1, and pTALEN-R2. In short, the synthesized TALEN gene fragment was recombined into the plant expression vector pImpactVector 4-1, and the final composition of the gene expression cassette was rbcS chloroplast signaling protein and HA tag or Flag tag were recombined at the N-terminus of TALEN, and then rbcS Gene promoter to regulate the performance of recombinant TALEN. Then, the gene expression cassettes were recombined into the Agrobacterium double-coupled vector pBinPlus (containing the nptII gene as a selection marker) or pZGB9R (containing the bar gene as a selection marker), and pTpTalen-R and pTpTalen-L nuclear expression vectors were obtained respectively ,As shown in Figure 1. The experimental steps are described in detail below:

pTALEN-L1 和 pTALEN-R1 載體之構築：Construction of pTALEN-L1 and pTALEN-R1 vectors:

利用引子對 pFUSA-M6-R（AGCGGCGCCGGTCTCGATAGCC；SEQ ID NO: 13） 和 pFUS-A-Del-F （AGCAAGCGGGTCTCACGGTGCAGCGGCTGTTG；SEQ ID NO: 14） 以 pFUS-A 載體為模板進行 PCR 擴增放大，得到2,573 bp 的 DNA 片段。將 pFUS-A 之 PCR 放大片段  用限制酶 BsaI切割，再以1%的瓊膠電泳分離，並取2,560 bp DNA 片段進行純化。再利用引子對 pTC14-F3 （TCTGATGTTACATTGCACAAGA；SEQ ID NO: 15）和 pTC14-R3 （TGAACGCTCTCCTGAGTAGG；SEQ ID NO: 16）以 Module plasmid 載體（pTALEN-L1：pHD1、pHD2、pNI3、pNI4、pNI5、pNI6；pTALEN-R1：pNI1、pNI2、pNG3、pNG4、pNN5、pNI6） 為模板各自進行 PCR 擴增放大並定量。將 Module plasmid 之 PCR 放大片段 混合後，用限制酶 BsaI 切割，再以2%的瓊膠電泳分離，並取79～100 bp DNA 片段進行純化。將 Module plasmid 的 PCR 產物經 BsaI 切割後之 DNA 片段與 pFUS-A 的 PCR 產物經 BsaI 切割後之 DNA 片段，進行黏合反應。經轉形大腸桿菌後，再經100mg/L Spectinomycin 篩選，分別得到3,087 bp 的 pTALEN-L1 和3,087 bp的 pTALEN-R1載體。Using primers to pFUSA-M6-R (AGCGGCGCCGGTCTCGATAGCC; SEQ ID NO: 13) and pFUS-A-Del-F (AGCAAGCGGGTCTCACGGTGCAGCGGCTGTTG; SEQ ID NO: 14) PCR amplification using pFUS-A vector as a template, resulting in 2,573 bp DNA fragment. The PCR amplified fragment of pFUS-A was cut with restriction enzyme BsaI, and then separated by 1% agar electrophoresis, and a 2,560 bp DNA fragment was purified. Reuse primer pairs pTC14-F3 (TCTGATGTTACATTGCACAAGA; SEQ ID NO: 15) and pTC14-R3 (TGAACGCTCTCCTGAGTAGG; SEQ ID NO: 16) with Module plasmid vectors (pTALEN-L1: pHD1, pHD1, pHD2, pNI3, pNI4, pNI5, pNI6; pTALEN-R1: pNI1, pNI2, pNG3, pNG4, pNN5, pNI6) PCR amplification and quantification for each template. After mixing the PCR amplified fragments of Module plasmid, it is cut with restriction enzyme BsaI, and then separated by 2% agarose electrophoresis, and 79～100 bp DNA fragments are purified. The PCR product of Module plasmid was cut by BsaI DNA fragment and the PCR product of pFUS-A was cut by BsaI DNA fragment, and then the adhesion reaction was carried out. After transforming E. coli, and then screening with 100mg/L Spectinomycin, 3,087 bp pTALEN-L1 and 3,087 bp pTALEN-R1 vectors were obtained.

pTALEN-L2 和 pTALEN-R2 載體之構築：Construction of pTALEN-L2 and pTALEN-R2 vectors:

將 pFUS-B5 用限制酶 BsaI 切割，再以1%的瓊膠電泳分離，並取2,557 bp DNA 片段進行純化。利用引子對pTC14-F3 （TCTGATGTTACATTGCACAAGA；SEQ ID NO: 15）和 pTC14-R3 （TGAACGCTCTCCTGAGTAGG；SEQ ID NO: 16）以 Module plasmid 載體 （pTALENL2：pNI1、pNG2、pNG3、pNN4、pNI5；pTALEN-R2：pNI1、pNI2、pNG3、pNG4、pHD5） 為模板各自進行 PCR 擴增放大。將 Module plasmid 之 PCR 放大片段混合後，用限制酶BsaI 切割，再以2%的瓊膠電泳分離，並取79～100 bpDNA 片段進行純化。將 Module plasmid 的 PCR 產物經 BsaI 切割後之DNA 片段與 pFUS-B5 經 BsaI 切割之片段，進行黏合反應。經轉形大腸桿菌後再經100 mg/L Spectinomycin 篩選，分別得到3,007 bp 的 pTALEN-L2 和3,007 bp 的 pTALEN-R2載體。PFUS-B5 was cut with restriction enzyme BsaI, then separated by 1% agar electrophoresis, and 2,557 bp DNA fragments were purified. Using primer pairs pTC14-F3 (TCTGATGTTACATTGCACAAGA; SEQ ID NO: 15) and pTC14-R3 (TGAACGCTCTCCTGAGTAGG; SEQ ID NO: 16) with Module plasmid vectors (pTALENL2: pNI1, pNG2, pNG3, pNN4, pNI5; pTALEN-R2: pNI1 , PNI2, pNG3, pNG4, pHD5) are the templates for PCR amplification. After mixing the amplified PCR fragments of the Module plasmid, it is cut with the restriction enzyme BsaI, and then separated by 2% agar electrophoresis, and 79 to 100 bp DNA fragments are purified. The PCR product of Module plasmid was cleaved by BsaI and the fragment of pFUS-B5 cleaved by BsaI to carry out the adhesion reaction. After transforming E.coli and then screening with 100 mg/L Spectinomycin, 3,007 bp pTALEN-L2 and 3,007 bp pTALEN-R2 vectors were obtained.

pTALEN-L 載體之構築：Construction of pTALEN-L carrier:

將 pCS2TAL3-DD、pTALEN-L1 與 pTALEN-L2 載體用限制酶Esp3I 切割，再以1%的瓊膠電泳分離，並分別取5,309 bp、616 bp 和536 bp DNA 片段進行純化。再將 pLR-NI 載體用限制酶Esp3I 切割，以2%的瓊膠電泳分離，並取89 bp DNA 片段進行純化。將 pCS2TAL3-DD、TALEN-L1、TALEN-L2 和 pLR-NI 經Esp3I 切割之片段進行黏合反應。經轉形大腸桿菌後，再經100 mg/L Ampicillin 篩選，得到6,550 bp 的 pTALEN-L載體。The pCS2TAL3-DD, pTALEN-L1 and pTALEN-L2 vectors were cut with the restriction enzyme Esp3I, and then separated by 1% agar electrophoresis, and 5,309 bp, 616 bp and 536 bp DNA fragments were respectively purified. The pLR-NI vector was then cut with restriction enzyme Esp3I, separated by 2% agar electrophoresis, and 89 bp DNA fragments were purified. The fragments of pCS2TAL3-DD, TALEN-L1, TALEN-L2, and pLR-NI cleaved by Esp3I were subjected to bonding reaction. After transforming Escherichia coli, it was screened with 100 mg/L Ampicillin to obtain 6,550 bp pTALEN-L vector.

pTALEN-R 載體之構築：Construction of pTALEN-R carrier:

將 pCS2TAL3-RR、pTALEN-R1 與 pTALEN-R2 載體用限制酶Esp3I 切割，再以1%的瓊膠電泳分離，並分別取5,339 bp、616 bp 和536 bp DNA 片段進行純化。再將 pLR-NI 載體用限制酶Esp3I 切割，以2%的瓊膠電泳分離，並取89 bp DNA 片段進行純化。將 pCS2TAL3-DD、TALEN-L1、TALEN-L2 和 pLR-NI 經Esp3I 切割之片段進行黏合反應。經轉形大腸桿菌後，再經100 mg/L Ampicillin 篩選，得到6,580 bp 的 pTALEN-R載體。The pCS2TAL3-RR, pTALEN-R1 and pTALEN-R2 vectors were cut with restriction enzyme Esp3I, and then separated by 1% agar electrophoresis, and 5,339 bp, 616 bp and 536 bp DNA fragments were purified respectively. The pLR-NI vector was then cut with restriction enzyme Esp3I, separated by 2% agar electrophoresis, and 89 bp DNA fragments were purified. The fragments of pCS2TAL3-DD, TALEN-L1, TALEN-L2, and pLR-NI cleaved by Esp3I were subjected to bonding reaction. After transforming Escherichia coli, it was screened with 100 mg/L Ampicillin to obtain 6,580 bp pTALEN-R vector.

構築植物組織特異性表現載體： 利用來自菊花 (Chrysanthemum spp.) 的 rbcS（Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit） 基因啟動子 ( Outchkourov et al., 2003) 來調控基因表現，其可在行光合作用的組織中啟動下游基因的表現。因此構築含有 rbcS啟動子之載體，包含 pTpTalen-L和pTpTalen-R與構築此二載體過程中的其他過渡載體 。 Construction of plant tissue-specific expression vectors: the use of the rbcS (Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit) gene promoter from Chrysanthemum spp. (Outchkourov et al., 2003) to regulate gene expression, which can Initiate the expression of downstream genes in tissues that engage in photosynthesis. Therefore, constructing a vector containing the rbcS promoter includes pTpTalen-L and pTpTalen-R and other transition vectors in the process of constructing these two vectors.

pIV4-TL 載體之構築：Construction of pIV4-TL vector:

利用引子對 TAL-BamHI-F（CATGGCTCCAAAGAAGGATCCTAAGGTA；SEQ ID NO: 17） 和 TAL-NsiI-R （GCACCCGTCAGTGCATTGC；SEQ ID NO: 18），以 pTALEN-L 為模板進行 PCR 擴增放大，再利用限制酶 NsiI 與 BamHI 切割 PCR 片段，並以1%瓊膠電泳分離，取460 bp DNA 片段進行純化。同時，將 pTALEN-L 與 ImpactVector ^TM1.4 分別利用限制酶 NotI/ NsiI 與 NotI/ BamHI 切割，並以1%瓊膠電泳分離，分別取2,233 bp 與4,906 bp DNA 片段進行純化。再將經限制酶切割後之 ImpactVector ^TM1.4 (4,906 bp)、pTALEN-L (2,233 bp) 和 pTALEN-L 之 PCR 產物 (460 bp) 的DNA 片段進行黏合反應，經轉形大腸桿菌後，再經 100 mg/L Ampicillin 篩選，得到7,598 bp 的 pIV4-TL 載體。 Using primer pairs TAL-BamHI-F (CATGGCTCCAAAGAAGGATCCTAAGGTA; SEQ ID NO: 17) and TAL-NsiI-R (GCACCCGTCAGTGCATTGC; SEQ ID NO: 18), using pTALEN-L as a template for PCR amplification, and then use the restriction enzyme Nsi I and BamH I cut the PCR fragment and separated it with 1% agarose electrophoresis. Take 460 bp DNA fragment for purification. At the same time, pTALEN-L and ImpactVector ^TM 1.4 were cut with restriction enzymes Not I/ Nsi I and Not I/ BamH I, respectively, and separated by 1% agar electrophoresis, and 2,233 bp and 4,906 bp DNA fragments were purified respectively. Then the DNA fragments of ImpactVector ^TM 1.4 (4,906 bp), pTALEN-L (2,233 bp) and pTALEN-L PCR product (460 bp) after restriction enzyme cleavage were subjected to the binding reaction. Screened with 100 mg/L Ampicillin, a 7,598 bp pIV4-TL vector was obtained.

pIV4-TR 載體之構築：Construction of pIV4-TR carrier:

利用引子對 TAL-BamHI-F （CATGGCTCCAAAGAAGGATCCTAAGGTA；SEQ ID NO: 17）和 TAL-NsiI-R （GCACCCGTCAGTGCATTGC；SEQ ID NO: 18），以 pTALEN-R 為模板進行 PCR 擴增放大，再利用限制酶 NsiI 與 BamHI 切割 PCR 擴增片段，並以1%瓊膠電泳分離，取490 bp DNA 片段進行純化。同時，將 pTALEN-R 與 ImpactVector ^TM1.4 分別利用限制酶 NotI/ NsiI 與 NotI/ BamHI 切割，並以1%瓊膠電泳分離，分別取2,233 bp 與4,906 bp DNA 片段進行純化。再將經限制酶切割後之 ImpactVector ^TM1.4 (4,906 bp)、pTALEN-R (2,233 bp) 和 pTALEN-R 之 PCR 產物 (490 bp) DNA片段進行黏合反應，經轉形大腸桿菌後，再經100 mg/L Ampicillin 篩選，得到7,628 bp 的 pIV4-TR 載體。 Use primer pairs TAL-BamHI-F (CATGGCTCCAAAGAAGGATCCTAAGGTA; SEQ ID NO: 17) and TAL-NsiI-R (GCACCCGTCAGTGCATTGC; SEQ ID NO: 18), using pTALEN-R as a template for PCR amplification and then use the restriction enzyme Nsi I and BamH I cut the PCR amplified fragments and separated them by 1% agarose electrophoresis, and took 490 bp DNA fragments for purification. At the same time, pTALEN-R and ImpactVector ^TM 1.4 were cut with restriction enzymes Not I/ Nsi I and Not I/ BamH I, respectively, and separated by 1% agar electrophoresis, and 2,233 bp and 4,906 bp DNA fragments were purified respectively. Restriction enzyme cleaved ImpactVector ^TM 1.4 (4,906 bp), pTALEN-R (2,233 bp) and pTALEN-R PCR product (490 bp) DNA fragments were subjected to a binding reaction, after transforming E. coli, and then through 100 Screened with mg/L Ampicillin and obtained 7,628 bp pIV4-TR vector.

pZGB-IV4 載體之構築Construction of pZGB-IV4 carrier

利用引子對 AscI-IV-F （TGGCGCGCCAGACAAACACCCCTTGTTATAC；SEQ ID NO: 19）與 H3-PacI-IV-R （CATAAGCTTTTAATTAATGCGTCCACTATAGAC；SEQ ID NO: 20） 以 ImpactVector ^TM1.4 為模板進行 PCR 擴增放大，再利用限制酶 HindIII 和 AscI 切割PCR 擴增片段，並以1%瓊膠電泳分離，取2,293 bp DNA 片段進行純化。同時，將 pZGB9R 利用限制酶 HindIII 和 AscI 切割，並以1%瓊膠電泳分離，取8,479 bp DNA 片段進行純化。再將經限制酶切割後之pZGB9R (8,479 bp) 和 ImpactVector ^TM1.4 之 PCR 產物 (2,293 bp) DNA片段進行黏合反應，經轉形大腸桿菌後，再經100 mg/L Kanamycin 篩選，得到10,747 bp 的 pZGB-IV4 載體。 Using primer pairs AscI-IV-F (TGGCGCGCCAGACAAACACCCCTTGTTATAC; SEQ ID NO: 19) and H3-PacI-IV-R (CATAAGCTTTTAATTAATGCGTCCACTATAGAC; SEQ ID NO: 20) using ImpactVector ^TM 1.4 as a template for PCR amplification and then using restriction enzymes HindIII and AscI cut the PCR amplified fragments and separated them by 1% agarose electrophoresis. The 2,293 bp DNA fragments were purified. At the same time, pZGB9R was cleaved with restriction enzymes Hind III and Asc I, and separated by 1% agarose electrophoresis, and 8,479 bp DNA fragments were purified. Then, the restriction enzyme cleaved pZGB9R (8,479 bp) and ImpactVector ^TM 1.4 PCR product (2,293 bp) DNA fragments were subjected to a binding reaction, after transforming Escherichia coli, and then screened with 100 mg/L Kanamycin to obtain 10,747 bp. pZGB-IV4 vector.

pTpTalen-L載體之構築：Construction of pTpTalen-L vector:

將 pIV4-TL 與 pZGB-IV4 分別利用限制酶 AscI 與 PacI 切割，並以1%瓊膠電泳分離，分別取4,953 bp 與8,478 bp DNA 片段進行純化。再將經限制酶切割後之 pZGB-IV4 (8,478 bp) 和 pIV4-TL (4,953 bp) DNA片段進行黏合反應，經轉形大腸桿菌後，再經100 mg/L Kanamycin 篩選，得到13,435 bp 的 pTpTalen-L 載體。其中，pTpTalen-L 載體之重組TALEN蛋白域中的重複可變區序列係包括如SEQ ID NO: 3所示的核苷酸序列。 PIV4-TL and pZGB-IV4 were cut with restriction enzymes Asc I and Pac I, respectively, and separated by 1% agar electrophoresis, and 4,953 bp and 8,478 bp DNA fragments were purified respectively. Then pZGB-IV4 (8,478 bp) and pIV4-TL (4,953 bp) DNA fragments cleaved by restriction enzymes were subjected to a binding reaction. After transforming E. coli, they were screened with 100 mg/L Kanamycin to obtain pTpTalen of 13,435 bp. -L carrier. The repeat variable region sequence in the recombinant TALEN protein domain of the pTpTalen-L vector includes the nucleotide sequence shown in SEQ ID NO: 3.

pTpTalen-R 載體之構築：Construction of pTpTalen-R vector:

將 pIV4-TR 與 pBinPLUS 分別利用限制酶 AscI 與 PacI 切割，並以1%瓊膠電泳分離，分別取4,983 bp 與12,328 bp DNA 片段進行純化。再將 經限制酶切割後之 pBinPLUS (12,328 bp) 和 pIV4-TR (4,983 bp) DNA 片段進行黏合反應，經轉形大腸桿菌後，再經100 mg/L Kanamycin 篩選，得到17,311 bp 的 pTpTalen-R 載體。其中，pTpTalen-R載體之重組TALEN蛋白域中的重複可變區序列係包括如SEQ ID NO: 4所示的核苷酸序列 The pIV4-TR and pBinPLUS were cut with restriction enzymes Asc I and Pac I, respectively, and separated by 1% agarose electrophoresis, and 4,983 bp and 12,328 bp DNA fragments were purified. Then, the restriction enzyme cleaved pBinPLUS (12,328 bp) and pIV4-TR (4,983 bp) DNA fragments were subjected to a binding reaction, and after transformation into E. coli, and then screened with 100 mg/L Kanamycin, the 17,311 bp pTpTalen-R was obtained. Carrier. The repeat variable region sequence in the recombinant TALEN protein domain of the pTpTalen-R vector includes the nucleotide sequence shown in SEQ ID NO: 4

表現載體的轉殖Expression vector transfer

轉殖表現載體至菸草：Transplant the expression vector to tobacco:

簡言之，分別將前述構築的細胞核重組TALEN表現載體，利用電穿孔法分別轉入農桿菌中，經適當抗生素（kanamycin或hygromycin）篩選後，分別得到含有不同載體的轉殖農桿菌。將轉入pTpTalen-L與轉入pTpTalen-R的農桿菌培養液依相同菌液濃度比例混合後，再放入菸草葉圓盤組織，進行共轉殖(Chang et al., 2007)。In short, the nuclear recombinant TALEN expression vectors constructed as described above were transferred into Agrobacterium by electroporation, respectively, and screened with appropriate antibiotics (kanamycin or hygromycin) to obtain transformed Agrobacterium containing different vectors. The pTpTalen-L and pTpTalen-R-transformed Agrobacterium culture solution was mixed according to the same bacterial solution concentration ratio, and then put into tobacco leaf disc tissue for co-transplantation (Chang et al., 2007).

詳言之，從凍菌管中抽取2 μL農桿菌菌液 （GV3101） 加入含有10 ml LB (含100 mg/L Rifomycin 和100 mg/L Gentamycin) 的試管中，於28 °C下，150 rpm 震盪培養1 天。從培養1 天的菌液中抽取200 μL加入含有4 mL LB（含100 mg/L Rifomycin 和100 mg/L Gentamycin）的試管中，於28 °C下，150 rpm 震盪培養4 小時。取1.5 mL 倒入微量離心管內，以轉速6,000 rpm，於4 °C下，離心3 分鐘，移除上清液，並重複此步驟直到所有菌體沉澱於微量離心管內。加入200 μL 在冰上預冷的無菌10%甘油，將沉澱後菌體回溶，再以轉速6,000 rpm，於4 °C下，離心3 分鐘，將上清液倒於廢液瓶中，重複此步驟一次。加入100 μL 在冰上預冷的無菌10%甘油，將菌體回溶，置於冰上5 分鐘。加入1.5 μg 質體。電擊前先將電擊管置於冰上。再將菌液移至電擊管中，以2,500 伏特、100 歐姆加以電擊 (BTX ECM399 Electroporation Generator，USA)。將菌液移至新的微量離心管，並加入1 mL LB，於28 °C下，150 rpm 震盪培養1 小時。將菌液以5,000 rpm 離心1 分鐘，移除約950 μL 上清液，剩下的上清液與菌體混合，並移至 LB 固態培養基上均勻塗抹。置於室溫培養，約2～3 天即可在培養盤上觀察轉形的農桿菌菌落出現。In detail, take 2 μL of Agrobacterium solution (GV3101) from the frozen bacteria tube and add it to a test tube containing 10 ml LB (containing 100 mg/L Rifomycin and 100 mg/L Gentamycin) at 28 °C, 150 rpm Shake culture for 1 day. Extract 200 μL from the bacterial solution cultured for 1 day and add it to a test tube containing 4 mL of LB (containing 100 mg/L Rifomycin and 100 mg/L Gentamycin), and incubate at 28 °C with shaking at 150 rpm for 4 hours. Pour 1.5 mL into a microcentrifuge tube, spin at 6,000 rpm, 4 °C, centrifuge for 3 minutes, remove the supernatant, and repeat this step until all bacterial cells settle in the microcentrifuge tube. Add 200 μL of sterile 10% glycerol pre-chilled on ice, re-dissolve the bacteria after precipitation, and then centrifuge at 6,000 rpm at 4 °C for 3 minutes. Pour the supernatant into the waste bottle and repeat This step once. Add 100 μL of sterile 10% glycerol pre-chilled on ice, re-dissolve the bacteria and place on ice for 5 minutes. Add 1.5 μg plastid. Place the shock tube on ice before shock. The bacterial solution was then transferred to an electric shock tube and shocked at 2,500 volts and 100 ohms (BTX ECM399 Electroporation Generator, USA). Transfer the bacterial solution to a new microcentrifuge tube, add 1 mL of LB, and incubate at 28 °C with shaking at 150 rpm for 1 hour. Centrifuge the bacterial solution at 5,000 rpm for 1 minute, remove about 950 μL of the supernatant, and mix the remaining supernatant with the bacterial cells, and move to LB solid medium to spread evenly. Incubate at room temperature and observe the appearance of transformed Agrobacterium colonies on the culture plate in about 2 to 3 days.

轉殖菸草組織的篩選和植物的再生：Screening of transgenic tobacco tissues and plant regeneration:

簡言之，將共同轉殖二個表現載體的植物組織，依照表現載體上帶有的篩選標誌基因，結合兩種不同的藥物（kanamycin、methionine sulfoximine (MSO)）進行篩選。轉殖菸草組織經由再生過程分化成植株，由自花授粉得到轉殖菸草種子(T ₁子代)。T ₁子代可經自花授粉得到轉殖T ₂子代，詳細的菸草核基因轉殖過程如下所述： In short, the plant tissues of the two expression vectors are co-transformed, and combined with two different drugs (kanamycin, methionine sulfoximine (MSO)) for screening according to the screening marker gene carried on the expression vector. Colonization turn differentiate into tobacco plant tissue by regeneration process by the self-pollinated to give seed transfected colonization tobacco (T ₁ progeny). The T ₁ progeny can be transformed into T ₂ progeny by self-pollination. The detailed tobacco nuclear gene transfer process is as follows:

以白金接種環沾取含重組質體的農桿菌單一菌落，在5 mL 含有抗生素（含150 μg/L Rifampicin 和150 μg/L Kanamycin）的 LB 中混勻，於28 °C，150 rpm 震盪培養18～24小時。隔天將菌液放大培養至50 mL含適當抗生素（150 μg/L Rifampicin 和150 μg/L Kanamycin）、1 mL的500 mM MES及5 μL 的0.2M Acetosyringone的LB培養液中，震盪培養18～24小時。將培養完成之菌液以轉速1,700 xg，於室溫下，離心10 分鐘，並移除上清液。加入5 mL的infiltration buffer （10 mM MES 1 mL、10 mM MgCl ₂），震盪均勻回溶菌體。測量菌液的OD ₆₀₀，並調整菌液至OD ₆₀₀為1。將菌液與0.2 M Acetosyringone solution （1000：1的比例）混勻，以150 rpm搖晃培養3～4小時。接著以無菌針頭輕刮菸草葉背微管束組織。以針筒吸取0.1～0.3 mL的菌液，持注射針筒將其出口處靠在葉背傷口處，另一手以指腹輕壓葉面。開始緩慢注射菌液進入菸草葉片內部，可見液體在葉片中擴散且充滿內部。注射完後標記注射區域。 Take a platinum inoculation ring to take a single colony of Agrobacterium containing recombinant plastids, mix in 5 mL of LB containing antibiotics (containing 150 μg/L Rifampicin and 150 μg/L Kanamycin), and culture at 28 °C with shaking at 150 rpm 18 to 24 hours. The next day, the bacteria solution was enlarged and cultured to 50 mL of LB culture medium containing appropriate antibiotics (150 μg/L Rifampicin and 150 μg/L Kanamycin), 1 mL of 500 mM MES, and 5 μL of 0.2M Acetosyringone, shaking culture 18～ 24 hours. The cultured bacterial solution was centrifuged at 1,700 xg at room temperature for 10 minutes, and the supernatant was removed. Add 5 mL of infiltration buffer (10 mM MES 1 mL, 10 mM MgCl ₂ ), and shake back to the lysate evenly. Measure the OD _{600 of the} bacterial solution and adjust the bacterial solution to OD ₆₀₀ of 1. Mix the bacterial solution with 0.2 M Acetosyringone solution (1000:1 ratio), and shake at 150 rpm for 3 to 4 hours. Then the microtubule bundle tissue of tobacco leaf back was gently scraped with a sterile needle. Aspirate 0.1-0.3 mL of bacterial solution with a syringe, hold the injection syringe to place its exit against the wound on the back of the leaf, and gently press the leaf surface with the fingertip of the other hand. Begin to slowly inject the bacterial liquid into the tobacco leaf, and it can be seen that the liquid diffuses in the leaf and fills the interior. Mark the injection area after injection.

將經注射後的煙草植株放入紙箱中避光黑暗培養2天，再光照培養3天後，將葉片注射範圍內的組織切割下來，以 1.2～2 % 漂白水，搖晃殺菌10分鐘，殺菌完成後。利用手術刀在葉片外圍切出傷口，將切成適當大小之葉片，葉背朝上，置於含適當抗生素之 Tobacco shooting medium（TSM）培養基，以透氣膠帶封好後置於25℃植物生長箱，以日照時間16小時培養，待其生長出癒傷組織和莖葉後，進行發根培養，最後移至土壤中。Put the injected tobacco plants in a carton in the dark for 2 days, and then culture for 3 days under light. Cut the tissues within the injection area of the leaves, sterilize with 1.2-2% bleach, shake for 10 minutes, and complete the sterilization. Rear. Use a scalpel to cut the wound around the blade, cut the blade into the appropriate size, with the back of the leaf facing up, place it in Tobacco shooting medium (TSM) medium containing the appropriate antibiotic, seal it with a breathable tape, and place it in a 25℃ plant growth box After cultivating for 16 hours of sunshine, callus, stems and leaves are grown, then hair root culture is carried out, and finally transferred to the soil.

分析轉殖植物Analysis of transgenic plants

a. PCR分子檢測：a. PCR molecular detection:

根據前述實施例，培養獲得許多菸草轉殖品系，選擇其中6個轉殖品系M0、M11、M14、M17、M29及M42進行PCR擴增放大，藉此檢測外源基因（TpTalen-L與TpTalen-R）是否同時***菸草DNA中。According to the foregoing embodiment, many tobacco transgenic lines were obtained through cultivation, and six transgenic lines M0, M11, M14, M17, M29, and M42 were selected for PCR amplification and amplification, thereby detecting foreign genes (TpTalen-L and TpTalen- R) Whether it is inserted into tobacco DNA at the same time.

在一實施態樣中，利用Plant genomic DNA mini kit （Geneaid，Taiwan）萃取植物葉片組織DNA。首先，秤取0.1 g 植物葉片組織，並以液態氮研磨成粉末。加入400 μL GP1 buffer 與5 μL RNase A 混合後移至1.5 mL 微量離心管中，並以60 °C加熱10 分鐘，並於此時預熱 Elution buffer (200 μL /sample) 備用。再加入100 μL GP2 buffer 混合均勻後，置於冰上3 分鐘。將植物組織液移到 Filter column 中並以轉速1,000 xg 離心1 分鐘。將濾液移至新的1.5 mL 微量離心管，再加入1.5 倍體積的 GP3 buffer，並震盪5 秒。將700 μL 植物組織混合液移至 GD column 中並以轉速14,000 xg 離心2 分鐘後，移除濾液，重複此步驟，直至把植物組織混合液離完。加入400 μL W1 buffer 至 GD column 中並以轉速14,000 xg 離心30 秒後，移除濾液。加入600 μL Wash buffer 至 GD column 中並以轉速14,000 xg 離心30 秒後，移除濾液。再以14,000 xg 離心3 分鐘，取出管柱置於新的1.5 mL 微量離心管。於管柱膜上加入50～100 μL 之 Elution buffer，靜置5 分鐘，以14,000 xg 離心30秒，使 DNA 溶液自管柱中濾出，並收集至1.5 ml 微量離心管中，置於-20 °C保存。In one embodiment, Plant genomic DNA mini kit (Geneaid, Taiwan) is used to extract DNA from plant leaf tissue. First, weigh 0.1 g of plant leaf tissue and grind it into powder with liquid nitrogen. Add 400 μL GP1 buffer and 5 μL RNase A, mix and move to a 1.5 mL microcentrifuge tube, and heat at 60 °C for 10 minutes. At this time, preheat the Elution buffer (200 μL /sample) for use. Add 100 μL of GP2 buffer and mix well, then place on ice for 3 minutes. Transfer the plant tissue fluid to the Filter column and centrifuge at 1,000 xg for 1 minute. Move the filtrate to a new 1.5 mL microcentrifuge tube, add 1.5 times the volume of GP3 buffer, and shake for 5 seconds. Transfer 700 μL of plant tissue mixture to the GD column and centrifuge at 14,000 xg for 2 minutes. Remove the filtrate and repeat this step until the plant tissue mixture is separated. Add 400 μL W1 buffer to the GD column and centrifuge at 14,000 xg for 30 seconds, then remove the filtrate. Add 600 μL Wash buffer to the GD column and centrifuge at 14,000 xg for 30 seconds, then remove the filtrate. Centrifuge at 14,000 xg for 3 minutes, remove the column and place in a new 1.5 mL microcentrifuge tube. Add 50～100 μL of Elution buffer to the column membrane, let it stand for 5 minutes, centrifuge at 14,000 xg for 30 seconds to filter the DNA solution from the column, and collect it in a 1.5 ml microcentrifuge tube and place it at -20 Store at °C.

接著，將對照組（WT）與轉殖菸草的DNA（150 ng）以一對引子Check-F （SEQ ID NO: 5；AATTCACTCATTGGATTCATAGAAG）與Check-R （SEQ ID NO: 6；CTGTAGCCGAGCGTGCGTAG）進行PCR，以確認外源基因（TpTalen-L與TpTalen-R）是否同時***菸草DNA中。PCR 的反應溫度與時間的三階段條件為，第一階段以95 °C反應2 分鐘。第二階段分成三部分，先以95 °C反應30 秒進行 Denaturation，再以61 °C反應30 秒進行 Annealing，再以72 °C反應反應45 秒進行 Elongation。而此階段約重複45 次。第三階段則以72 °C反應5分鐘。再使用2.2%的瓊膠，以100 伏特40 分鐘進行電泳分離。Next, PCR was performed on the control group (WT) and the DNA (150 ng) of the transferred tobacco with a pair of primers Check-F (SEQ ID NO: 5; AATTCACTCATTGGATTCATAGAAG) and Check-R (SEQ ID NO: 6; CTGTAGCCGAGCGTGCGTAG). To confirm whether the foreign genes (TpTalen-L and TpTalen-R) are inserted into tobacco DNA at the same time. The three-stage condition of PCR reaction temperature and time is that the first stage reacts at 95 °C for 2 minutes. The second stage is divided into three parts, first react at 95 °C for 30 seconds for Denaturation, then at 61 °C for 30 seconds for Annealing, then at 72 °C for 45 seconds for Elongation. This stage is repeated about 45 times. In the third stage, the reaction was carried out at 72 °C for 5 minutes. Then use 2.2% agar and perform electrophoretic separation at 100 volts for 40 minutes.

電泳結果如圖2 B部分所顯示，在這6個不同轉殖品系的電泳結果中都可以同時偵測到452 bp及482bp的PCR產物，即Talen-L和Talen-R基因皆同時表現於6個轉殖品系中，而WT中則無偵測到Talen-L和Talen-R基因。這就表示已成功轉殖Talen-L和Talen-R基因至菸草中。The electrophoresis results are shown in Part B of Figure 2. In the electrophoresis results of these six different transgenic lines, PCR products of 452 bp and 482 bp can be simultaneously detected, that is, the Talen-L and Talen-R genes are simultaneously expressed in 6 Among the transgenic lines, Talen-L and Talen-R genes were not detected in WT. This means that the Talen-L and Talen-R genes have been successfully transformed into tobacco.

b. 觀察轉殖植物之外表性狀：b. Observe the external traits of the transformed plants:

觀察不同轉殖植物品系與對照組間之外表性狀與生長發育情況之差異。結果如圖3顯示，轉殖植物的外表性狀和生長發育呈現多樣性。部分品系轉殖植物無任何外表性狀，部分品系轉殖植物的葉片呈現斑點狀白化或局部區域白化（如M29），或整個葉片白(黃)化（如M11、M17），而有些轉殖植物為全株白化(淡黃色)的植株（如M0）。此外，轉殖植物之生長發育也有不同程度的遲緩。如先前研究報導，當菸草葉綠體 rpoB基因發生變異，會導致植物產生白化的外表性狀 (Allison et al., 1996)。因此，推測轉殖植物白化程度的差異可能與重組TALEN蛋白表現在轉殖植物葉片的量有關，而造成葉綠體 rpoB基因受到不同程度的破壞而導致葉片產生這些不同程度的外表性狀。 Observe the difference in appearance and growth between different transgenic plant lines and the control group. The results are shown in Figure 3, the appearance and growth of the transgenic plants are diverse. Some lines of transgenic plants do not have any appearance traits. The leaves of some lines of transgenic plants are spotted or partially whitened (such as M29), or the entire leaves are white (yellow) (such as M11, M17), while some of the transformed plants It is an all-albino (light yellow) plant (such as M0). In addition, the growth and development of transgenic plants are also delayed to varying degrees. As reported in previous studies, when the rpoB gene of the tobacco chloroplast changes, it will cause the plant to produce albino appearance (Allison et al., 1996). Therefore, it is speculated that the difference in the degree of albinism of the transgenic plants may be related to the amount of recombinant TALEN protein expressed in the leaves of the transgenic plants, which caused the chloroplast rpoB gene to be damaged to varying degrees and caused the leaves to produce these different degrees of appearance traits.

c. 高解析度解離分析葉綠體 rpoB基因的突變： c. High-resolution dissociation analysis of chloroplast rpoB gene mutations:

為研究造成植物白化性狀的產生是否與葉綠體 rpoB基因的變異有關。因此選擇2個不同轉殖品系（M11和M17）的白化葉片組織，萃取DNA後，利用一對引子（SEQ ID NO: 7； GGATTTAATCAGATACAATTTGAAG）和 （SEQ ID NO: 8；TTCCAAATTAATCCCGCGGATAC）進行定量PCR(Q-PCR, Fast Evagreen ^®qPCR Master Mixer, Biotium），以擴增放大重組TELEN蛋白作用於 rpoB基因的附近DNA序列（預期PCR產物大小為229 bp），並將PCR產物進行高解析度解離分析（high resolution melting; HRM）分析後，與對照組（正常菸草）比較，觀察DNA序列是否有所變異。 To investigate whether the generation of plant albino traits is related to the variation of chloroplast rpoB gene. Therefore, the albino leaf tissues of two different transgenic lines (M11 and M17) were selected. After DNA extraction, a pair of primers (SEQ ID NO: 7; GGATTTAATCAGATACAATTTGAAG) and (SEQ ID NO: 8; TTCCAAATTAATCCCGCGGATAC) were used for quantitative PCR (Q -PCR, Fast Evagreen ^® qPCR Master Mixer, Biotium) to amplify and amplify the recombinant TELEN protein in the vicinity of the rpoB gene DNA sequence (the expected PCR product size is 229 bp), and the PCR product is subjected to high resolution dissociation analysis (high After resolution melting (HRM) analysis, compare with the control group (normal tobacco) to observe whether the DNA sequence has changed.

詳言之，將含有反應總體積為20 μL的2X Fast Evagreen ^®qPCR Master Mixer (Biotium)、2.5 mM MgCl ₂、0.2 μM前述引子及20 ng 轉殖菸草DNA的混合物進行Q-PCR，預期產物為229 bp。Q-PCR及HRM皆是利用Roche Light Cycler ^®Nano 進行操作。首先，利用95 °C進行180秒的處理，接著進行兩階段的循環步驟，每次循環進行95 °C的denature 10秒及60 °C的annealing/extension 50秒，進行45次循環後，接著以95 °C進行60秒使雙股DNA完全解開，接著以40 °C進行60秒處理，使其形成異合子（heterozygous）的結構出現，最後以每秒0.05由65 °C逐漸升溫至95 °C進行HRM分析。 In detail, containing a total reaction volume of 20 μL of ^{2X Fast Evagreen ® qPCR Master Mixer (} Biotium), 2.5 mM MgCl 2, a mixture of 0.2 μM of the preceding primer and 20 ng transfected colonization tobacco DNA is Q-PCR, the expected product is 229 bp. Both Q-PCR and HRM are operated using Roche Light Cycler ^® Nano. First, use 95 °C for 180 seconds, followed by a two-step cycle step, each cycle of 95 °C denature 10 seconds and 60 °C annexing/extension 50 seconds, after 45 cycles, and then 95°C for 60 seconds to completely unwind the double-stranded DNA, followed by treatment at 40°C for 60 seconds to form a heterozygous structure, and finally gradually heated from 65°C to 95° at 0.05 per second C Perform HRM analysis.

結果如圖4顯示，轉殖植物的白色葉片組織（M11-W和M17-W）與對照組相比，有明顯的熔解曲線之差異，這就表示在這 2個轉殖品系的 rpoB基因有發生核苷酸之變異。 The results are shown in Figure 4. Compared with the control group, the white leaf tissues (M11-W and M17-W) of the transgenic plants have obvious melting curve differences, which means that the rpoB genes in these two transgenic lines have Nucleotide variation occurs.

d. 葉綠體 rpoB基因的定序分析： d. Sequencing analysis of chloroplast rpoB gene:

為研究重組TALEN蛋白在轉殖植物的表現是否最後造成葉綠體rpoB序列的突變，因此萃取對照組（WT）和4個不同轉殖品系（M0, M11, M17 和M29）的葉片組織，其中M11和M17品系植物的DNA又分別萃取自白色（M11-W和M17-W）和綠色（M11-G和M17-G）的葉片組織。以一對引子（SEQ ID NO: 9；AGCAGAAGTCTGTTTCTAGGGATGT）和（SEQ ID NO: 10；TAGCTGATGATAGAACTAGAATA）利用PCR去擴增放大重組TALEN蛋白作用於 rpoB基因的附近序列，並將PCR產物進行定序，將定序結果利用CLC Genomics Workbench 9.5.2 軟體進行多重序列比對。 In order to investigate whether the performance of the recombinant TALEN protein in the transgenic plants finally caused the mutation of the chloroplast rpoB sequence, the leaf tissues of the control group (WT) and 4 different transgenic lines (M0, M11, M17 and M29) were extracted, among which M11 and The DNA of the M17 strain plants is extracted from the leaf tissues of white (M11-W and M17-W) and green (M11-G and M17-G), respectively. Use a pair of primers (SEQ ID NO: 9; AGCAGAAGTCTGTTTCTAGGGATGT) and (SEQ ID NO: 10; TAGCTGATGATAGAACTAGAATA) to amplify and amplify the sequence around the recombinant TALEN protein acting on the rpoB gene by PCR and sequence the PCR products. The sequence results were compared using CLC Genomics Workbench 9.5.2 software.

結果如圖5顯示，在轉殖品系M0，M11，M17和M29的白色或黃化葉片組織（M0, M11-W, M17-W, M29）的 rpoB-295位置明顯有發生A突變至T的情形，如箭頭所指處。但是在M11和M17的綠色葉片組織（M11-G，M17-G）的 rpoB基因序列則與對照組相同。這就表示，透過pTpTalen-L及pTpTalen-R對菸草進行轉殖，可針對特定葉綠體基因進行突變，例如葉綠體 rpoB基因的突變，進而導致植物外表性狀的改變。 The results shown in Figure 5 show that the mutation of A to T is apparent in the rpoB- 295 position of the white or yellow leaf tissues (M0, M11-W, M17-W, M29) of the transgenic lines M0, M11, M17 and M29. The situation, as indicated by the arrow. However, the rpoB gene sequence of green leaf tissues (M11-G, M17-G) in M11 and M17 was the same as that in the control group. This means that the transformation of tobacco through pTpTalen-L and pTpTalen-R can mutate specific chloroplast genes, such as the mutation of the chloroplast rpoB gene, which in turn leads to changes in plant appearance.

以上所述僅為示例性，而非為限制性。本發明所屬發明技術領域中具有通常知識者能依需求及所針對的核苷酸序列，設計重組TALEN蛋白域中之重複可變區，使重組TALEN蛋白可專一辨識目標核酸序列。任何未脫離本發明的精神與範疇，而對其進行的等效修改或變更，均應包含於申請專利範圍所界定的範圍中。The above is only exemplary and not restrictive. Those with ordinary knowledge in the technical field of the invention to which the invention belongs can design the repeating variable region in the recombinant TALEN protein domain according to the needs and the targeted nucleotide sequence, so that the recombinant TALEN protein can specifically identify the target nucleic acid sequence. Any equivalent modifications or changes made without departing from the spirit and scope of the present invention shall be included in the scope defined by the scope of the patent application.

參考文獻： Allison, L. A., Simon, L. D. and Maliga, P. Deletion of rpoBreveals a second distinct transcription system in plastids of higher plants. The European Molecular Biology Organization Journal15, 2802-2809, 1996 . Bock, R.  Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology. Annual Review of Plant Biology, 66, 211-241, 2015. Cermak, T., Doyle, E. L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J. A., Somia, N. V., Bogdanove, A. J. and Voytas, D. F. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research39, e28, 2011. Chang, C. C., Huang, P. S., Lin, H. R., & Lu, C. H.. Transactivation of protein expression by rice HSP101 in planta and using Hsp101 as a selection marker for transformation. Plant and cell physiology, 48(8), 1098-1107, 2007 Hajdukiewicz, P. T., Allison, L. A. and Maliga, P. The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids. The European Molecular Biology Organization Journal16, 4041-4048, 1997. Inada, H., Kusumi, K., Nishimura, M. and Iba, K. Specific expression of the chloroplast gene for RNA polymerase ( rpoB) at an early stage of leaf development in rice. Plant and Cell Physiology37, 229-232, 1996. Little, M. C. and Hallick, R. B. Chloroplast rpoA, rpoB, and rpoCgenes specify at least three components of a chloroplast DNA-dependent RNA polymerase active in tRNA and mRNA transcription. The Journal of Biological Chemistry263, 14302-14307, 1988. Outchkourov, N. S., Peters, J., de Jong, J., Rademakers, W. and Jongsma, M. A. The promoter-terminator of chrysanthemum rbcS1directs very high expression levels in plants. Planta6, 1003-1012, 2003. References: Allison, LA, Simon, LD and Maliga, P. Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants. The European Molecular Biology Organization Journal 15, 2802-2809, 1996. Bock, R. Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology. Annual Review of Plant Biology , 66 , 211-241, 2015. Cermak, T., Doyle, EL, Christian, M., Wang, L., Zhang, Y ., Schmidt, C., Baller, JA, Somia, NV, Bogdanove, AJ and Voytas, DF Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research 39, e28, 2011. Chang , CC, Huang, PS, Lin, HR, & Lu, CH. Transactivation of protein expression by rice HSP101 in planta and using Hsp101 as a selection marker for transformation. Plant and cell physiology , 48 (8), 1098-1107, 2007 Hajdukiewicz, PT, Allison, LA and Maliga, P. The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct grou ps of genes in tobacco plastids. The European Molecular Biology Organization Journal 16, 4041-4048, 1997. Inada, H., Kusumi, K., Nishimura, M. and Iba, K. Specific expression of the chloroplast gene for RNA polymerase ( rpoB ) at an early stage of leaf development in rice. Plant and Cell Physiology 37, 229-232, 1996. Little, MC and Hallick, RB Chloroplast rpoA , rpoB , and rpoC genes specify at least three components of a chloroplast DNA-dependent RNA polymerase active in tRNA and mRNA transcription. The Journal of Biological Chemistry 263, 14302-14307, 1988. Outchkourov, NS, Peters, J., de Jong, J., Rademakers, W. and Jongsma, MA The promoter-terminator of chrysanthemum rbcS1 directs very high expression levels in plants. Planta 6, 1003-1012, 2003.

無no

圖 1係根據本案所請發明一實施例的細胞核基因轉殖載體的圖譜。其中，A部分及B部分分別為pTpTalen-L與pTpTalen-R，皆為細胞核基因表現的農桿菌雙偶載體，並且為具葉片組織特異性的表現載體。pTpTalen-L的骨架為來自pZGB9R，具有KanR （ npt II）基因做為篩選標誌；而pTpTalen-R的骨架為來自pBinPlus，其具有 bar基因做為篩選標誌。各縮寫代表之涵義為：LB-T Border與RB-T Border分別為農桿菌T-DNA的左右邊界。CaMV 35S為Califlower mosaic virus 35S 轉錄子的啟動子。35S polyA為Califlower mosaic virus 35S 轉錄子的3’UTR。RbcS promoter為 rbcS基因的啟動子。RbcS terminator為 rbcS基因的3’UTR。KanR （ npt II）為抗卡那黴素（kanamycin）的篩選標誌基因，而 bar為抗殺草劑（Methionine sulfoximine, MSO）的篩選標誌基因。TP為葉綠體訊息蛋白的序列。Flag為Flag標籤，而HA為HA標籤。 FIG. 1 is a map of a nuclear gene transfer vector according to an embodiment of the invention claimed in this case. Among them, part A and part B are pTpTalen-L and pTpTalen-R, respectively, which are both Agrobacterium double-coupled vectors expressed by nuclear genes, and are expression vectors with leaf tissue specificity. The skeleton of pTpTalen-L is from pZGB9R, with KanR ( npt II ) gene as a selection marker; and the skeleton of pTpTalen-R is from pBinPlus, which has bar gene as a selection marker. The meaning of each abbreviation is: LB-T Border and RB-T Border are the left and right borders of Agrobacterium T-DNA, respectively. CaMV 35S is the promoter of Califlower mosaic virus 35S transcript. 35S polyA is the 3'UTR of Califlower mosaic virus 35S transcript. RbcS promoter is the promoter of rbcS gene. RbcS terminator is the 3'UTR of rbcS gene. KanR ( npt II ) is a selection marker gene for kanamycin resistance, and bar is a selection marker gene for herbicide resistance (Methionine sulfoximine, MSO). TP is the sequence of chloroplast signaling protein. Flag is the Flag tag, and HA is the HA tag.

圖 2係於本案所請發明一實施例中利用PCR確認共轉殖 pTpTalen-L 與 pTpTalen-R後基因是否嵌入菸草基因組中。(A) 所使用之PCR引子對 Check-F （SEQ ID NO: 5；AATTCACTCATTGGATTCATAGAAG）與Check-R （SEQ ID NO: 6；CTGTAGCCGAGCGTGCGTAG）位於 pTpTalen-L 和 pTpTalen-R載體上的位置。(B) 將6個共轉殖菸草品系（M0、M11、M14、M17、M39、M42）、對照組（Wt為未轉殖菸草）、pTpTalen-L （Lp）和pTpTalen-Rp （Rp） 載體等的DNA，使用特異性引子對 （Check-F 與 Check-R）進行 PCR 擴增放大，再將 PCR 產物以2.2%的瓊膠電泳進行分離。圖中，M 為100 bp DNA 標誌。箭頭處為預期PCR產物大小分別為482 bp 和452 bp。Fig. 2 is an example of the invention claimed in this case, using PCR to confirm whether the genes after co-transfection of pTpTalen-L and pTpTalen-R are embedded in the tobacco genome. (A) The PCR primer pairs Check-F (SEQ ID NO: 5; AATTCACTCATTGGATTCATAGAAG) and Check-R (SEQ ID NO: 6; CTGTAGCCGAGCGTGCGTAG) are located on the pTpTalen-L and pTpTalen-R vectors. (B) 6 co-transgenic tobacco lines (M0, M11, M14, M17, M39, M42), control group (Wt is untransformed tobacco), pTpTalen-L (Lp) and pTpTalen-Rp (Rp) vectors The DNA of such products is amplified by PCR using specific primer pairs (Check-F and Check-R), and then the PCR products are separated by 2.2% agar electrophoresis. In the figure, M is a 100 bp DNA marker. The arrows indicate the expected PCR product sizes of 482 bp and 452 bp, respectively.

圖 3係為根據本案發明一實施例之經細胞核基因轉殖的菸草品系之植株外表型態。如圖所示，經細胞核基因轉殖之4個不同轉殖品系呈現不同程度葉片白化或黃化的外表性狀。WT為對照組（未轉殖菸草）。M0為全株白化的植物品系。M11為2個分別生長在培養基或泥土的不同個體。M17為2個分別生長在培養基或泥土的不同個體。M29為2個分別生長在泥土中的不同個體。FIG. 3 is a plant phenotype of a tobacco line transfected with nuclear genes according to an embodiment of the present invention. As shown in the figure, the four different transgenic lines transfected with nuclear genes exhibited the appearance of leaf albino or yellowing to varying degrees. WT is the control group (untransformed tobacco). M0 is an all-albino plant line. M11 is two different individuals that grow on medium or soil. M17 is two different individuals that grow on medium or soil, respectively. M29 is two different individuals growing in the soil.

圖 4係利用高解析度解離（High Resolution Melting, HRM）分析根據本案發明一實施例之菸草葉綠體 rpoB基因的突變。自WT對照組（未轉殖菸草）的葉片組織和2個不同轉殖品系（M11和M17）的白色葉片組織萃取DNA，接著利用一對引子（SEQ ID NO: 7； GGATTTAATCAGATACAATTTGAAG）和 （SEQ ID NO: 8；TTCCAAATTAATCCCGCGGATAC）進行Q-PCR （Fast Evagreen ^®qPCR Master Mix, Biotium），以擴增放大重組TELEN蛋白作用區域附近的DNA（229 bp），將PCR產物進行高解析度解離分析（HRM, LightCycler ^®Nano, Roche）。結果顯示，轉殖植物的白色葉片組織（M11-W和M17-W）與WT對照組有明顯的熔解曲線之差異，因此推測 rpoB基因發生核苷酸之變異。 4 is a high-resolution dissociation (High Resolution Melting, HRM) analysis of tobacco chloroplast rpoB gene mutation according to an embodiment of the present invention. DNA was extracted from the leaf tissue of the WT control group (untransformed tobacco) and the white leaf tissue of two different transgenic lines (M11 and M17), followed by a pair of primers (SEQ ID NO: 7; GGATTTAATCAGATACAATTTGAAG) and (SEQ ID NO: 8; TTCCAAATTAATCCCGCGGATAC) Q-PCR (Fast Evagreen ^® qPCR Master Mix, Biotium) to amplify the DNA (229 bp) near the area of action of the recombinant TELEN protein, and the PCR products are subjected to high-resolution dissociation analysis (HRM, LightCycler ^® Nano, Roche). The results showed that the white leaf tissues (M11-W and M17-W) of the transformed plants had a significant difference in melting curve from the WT control group, so it was speculated that there was a nucleotide variation in the rpoB gene.

圖 5係顯示重組TALEN蛋白造成根據本案發明一實施例之菸草葉片之葉綠體 rpoB基因序列的變異。萃取對照組（WT）和4個不同轉殖品系（M0, M11, M17 和M29）的葉片組織，其中M11和M17品系植物的DNA又分別萃取自白色（M11-W和M17-W）和綠色（M11-G和M17-G）葉片組織。以一對引子（SEQ ID NO: 9；AGCAGAAGTCTGTTTCTAGGGATGT）和（SEQ ID NO: 10；TAGCTGATGATAGAACTAGAATA）利用PCR去擴增放大DNA，並將PCR產物進行定序，將定序結果利用CLC Genomics Workbench 9.5.2 軟體進行多重序列比對。結果顯示，在轉殖品系M0，M11，M17和M29的白色或黃化葉片組織（M0-W; M11-W, M17-W, M29）的 rpoB-295位置明顯有發生A突變至T的情形。但是在M11和M17的綠色葉片組織（M11-G、M17-G）的 rpoB基因序列則與對照組相同。箭頭處為發生變異之核苷酸。 FIG. 5 shows that the recombinant TALEN protein causes variation in the chloroplast rpoB gene sequence of tobacco leaves according to an embodiment of the present invention. Extract the leaf tissues of the control group (WT) and 4 different transgenic lines (M0, M11, M17 and M29), where the DNA of the plants of the M11 and M17 lines are extracted from white (M11-W and M17-W) and green respectively (M11-G and M17-G) Leaf tissue. Use a pair of primers (SEQ ID NO: 9; AGCAGAAGTCTGTTTCTAGGGATGT) and (SEQ ID NO: 10; TAGCTGATGATAGAACTAGAATA) to amplify the amplified DNA using PCR, and sequence the PCR products, using the CLC Genomics Workbench 9.5.2 The software performs multiple sequence alignments. The results showed that in the transgenic lines M0, M11, M17 and M29, the white or yellowed leaf tissues (M0-W; M11-W, M17-W, M29) had a mutation from A to T in the rpoB- 295 position. . However, the rpoB gene sequence in the green leaf tissues (M11-G, M17-G) of M11 and M17 was the same as the control group. The arrows show the nucleotides that have mutated.

在以下的詳細描述中，為了解釋本發明，提供了許多具體細節，以便能徹底理解所揭露的實施方式。然而，顯而易見的是，一個或多個的實施方式可以在沒有所述具體細節的情況下實現。在其它情況中，為了簡化附圖，習知的結構和流程將以示意性的方式顯示。In the following detailed description, in order to explain the present invention, many specific details are provided in order to thoroughly understand the disclosed embodiments. However, it is obvious that one or more embodiments can be implemented without the specific details. In other cases, in order to simplify the drawings, the conventional structure and flow will be shown in a schematic manner.

Claims (20)

一種細胞核基因轉殖載體，包括：一啟動子；一葉綠體訊息蛋白基因，其位於該啟動子下游；一重組TALEN蛋白域，包含如SEQ ID NO：21或SEQ ID NO：22所示之序列；以及一第一邊界區及一第二邊界區，其中該啟動子、該葉綠體訊息蛋白基因及該重組TALEN蛋白域依序位於該第一邊界區與該第二邊界區之間；其中該重組TALEN蛋白域包括編碼一DNA辨識蛋白的DNA序列；其中該DNA辨識蛋白辨識葉綠體之rpoB基因之片段。 A nuclear gene transfer vector, including: a promoter; a chloroplast information protein gene, which is located downstream of the promoter; a recombinant TALEN protein domain, including the sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22; And a first border region and a second border region, wherein the promoter, the chloroplast signal protein gene and the recombinant TALEN protein domain are sequentially located between the first border region and the second border region; wherein the recombinant TALEN The protein domain includes a DNA sequence encoding a DNA recognition protein; wherein the DNA recognition protein recognizes a fragment of the chloroplast rpoB gene. 如請求項1所述之轉殖載體，其中該啟動子為rbcS啟動子。 The transfer vector according to claim 1, wherein the promoter is an rbcS promoter. 如請求項1所述之轉殖載體，其中該重組TALEN蛋白域包括一核酸內切酶的DNA序列。 The transfer vector according to claim 1, wherein the recombinant TALEN protein domain includes a DNA sequence of an endonuclease. 如請求項3所述之轉殖載體，其中該核酸內切酶為Fok I。 The transfer vector according to claim 3, wherein the endonuclease is Fok I. 如請求項1所述之轉殖載體，其中編碼該DNA辨識蛋白之DNA序列具有如SEQ ID NO：3或SEQ ID NO：4所示之序列。 The transfer vector according to claim 1, wherein the DNA sequence encoding the DNA recognition protein has the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4. 如請求項1所述之轉殖載體，更包括一篩選基因。 The transfer vector according to claim 1, further comprising a selection gene. 如請求項6所述之轉殖載體，其中該篩選基因為一抗藥性篩選基因、一非抗藥性篩選基因、或其組合。 The transfer vector according to claim 6, wherein the screening gene is a drug resistance screening gene, a non-drug resistance screening gene, or a combination thereof. 如請求項6所述之轉殖載體，其中該篩選基因包括一卡那黴素(kanamycin)抗性基因或一甲硫胺酸磺醯亞胺(methionine sulfoximine,MSO)殺草劑抗性基因。 The transfer vector according to claim 6, wherein the screening gene comprises a kanamycin (kanamycin) resistance gene or a methionine sulfoximine (MSO) herbicide resistance gene. 如請求項8所述之轉殖載體，其中該卡那黴素抗性基因包括nptII或該甲硫胺酸磺醯亞胺殺草劑抗性基因包括bar。 The transfer vector according to claim 8, wherein the kanamycin resistance gene includes nptII or the methionine sulfonimide herbicide resistance gene includes bar . 如請求項1所述之轉殖載體，更包括編碼一標誌物之序列。 The transfer vector as described in claim 1, further comprising a sequence encoding a marker. 如請求項10所述之轉殖載體，其中該標誌物包含選自由發光分子、化學發光分子、螢光染料、螢光猝滅劑、脂質、有色分子、放射性同位素、閃爍劑、生物素、抗生物素蛋白、鏈黴親和素、蛋白質A、蛋白質G、抗體或其片段、聚組胺酸、Ni²⁺、Flag標籤、myc標籤、HA標籤、和酶所組成的群組中的可檢測標記物。 The transfection carrier according to claim 10, wherein the marker comprises a luminescent molecule, a chemiluminescent molecule, a fluorescent dye, a fluorescent quencher, a lipid, a colored molecule, a radioactive isotope, a scintillant, biotin, an anti-cancer Detectable labels in the group consisting of biotin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni ²⁺ , Flag tags, myc tags, HA tags, and enzymes Thing. 如請求項1所述之轉殖載體，其包含如SEQ ID NO：1或SEQ ID NO：2所示之序列。 The transfer vector according to claim 1, which comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. 如請求項1所述之轉殖載體，更包括一rbcS基因的3端非轉錄區，其位於該重組TALEN蛋白域下游。 The transfection vector according to claim 1, further comprising a 3-terminal non-transcribed region of the rbcS gene, which is located downstream of the recombinant TALEN protein domain. 一種特異性造成植物葉綠體基因變異的套組，包括：一第一轉殖載體，包括：一第一啟動子；一第一葉綠體訊息蛋白基因，其位於該第一啟動子下游；一第一重組TALEN蛋白域，包含如SEQ ID NO：21所示之序列；以及一第一邊界區及一第二邊界區，其中該第一啟動子、該第一葉綠體訊息蛋白基因及該第一重組TALEN蛋白域依序位於該第一邊界區與該第二邊界區之間；以及一第二轉殖載體，包括：一第二啟動子；一第二葉綠體訊息蛋白基因，其位於該第二啟動子基因下游； 一第二重組TALEN蛋白域，包含如SEQ ID NO：22所示之序列；以及一第三邊界區及一第四邊界區，其中該第二啟動子、該第二葉綠體訊息蛋白基因及該第二重組TALEN蛋白域依序位於該第三邊界區與該第四邊界區之間；其中該第一重組TALEN蛋白域和該第二重組TALEN蛋白域包括編碼一DNA辨識蛋白的DNA序列；其中該DNA辨識蛋白辨識葉綠體之rpoB基因之片段。 A kit for specific variation of plant chloroplast genes, including: a first transgenic vector, including: a first promoter; a first chloroplast signaling protein gene, which is located downstream of the first promoter; a first recombination TALEN protein domain, including the sequence shown in SEQ ID NO: 21; and a first border region and a second border region, wherein the first promoter, the first chloroplast signaling protein gene and the first recombinant TALEN protein The domain is located between the first border region and the second border region in sequence; and a second transfection vector, including: a second promoter; a second chloroplast signaling protein gene, which is located in the second promoter gene Downstream; a second recombinant TALEN protein domain, including the sequence shown in SEQ ID NO: 22; and a third border region and a fourth border region, wherein the second promoter, the second chloroplast signaling protein gene and The second recombinant TALEN protein domain is sequentially located between the third border region and the fourth border region; wherein the first recombinant TALEN protein domain and the second recombinant TALEN protein domain include a DNA sequence encoding a DNA recognition protein; The DNA recognition protein recognizes a fragment of the rpoB gene of the chloroplast. 一種產生細胞核基因轉殖植物細胞的方法，包括：建構一對如請求項1所述之細胞核基因轉殖載體；透過一農桿菌將該細胞核基因轉殖載體併入一植物細胞；以及篩選及培養該植物細胞。 A method for producing nuclear gene transgenic plant cells, comprising: constructing a pair of nuclear gene transfection vectors as described in claim 1; incorporating the nuclear gene transduction vectors into a plant cell through an Agrobacterium; and screening and culturing The plant cell. 如請求項15所述之方法，其中該併入包含一同源性導向的修復機制或一非同源性末端接合導向的修復機制。 The method of claim 15, wherein the incorporation includes a homology-directed repair mechanism or a non-homologous end-joint-directed repair mechanism. 如請求項15所述之方法，更包括轉殖該細胞核基因轉殖載體至該農桿菌。 The method according to claim 15, further comprising transferring the nuclear gene transfer vector to the Agrobacterium. 如請求項15所述之方法，更包括分析該植物細胞並確認該細胞核基因轉殖載體併入該植物細胞。 The method of claim 15, further comprising analyzing the plant cell and confirming that the nuclear gene transfer vector is incorporated into the plant cell. 一種細胞核基因轉殖植物細胞，係以請求項15所述之方法所建構。 A nuclear gene transgenic plant cell is constructed by the method described in claim 15. 一種農桿菌，包括如請求項1所述之細胞核基因轉殖載體。An Agrobacterium comprising the nuclear gene transfer vector as described in claim 1.

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