CN110343157A - Cotton verticillium wilt related gene GhBONI and its coding albumen and application - Google Patents

Abstract

The invention discloses cotton verticillium wilt related gene GhBONI and its coding albumen and applications.The present invention provides amino acid sequences to connect the fusion protein formed after label for SEQ ID No.1 or by the substitution and/or deletion and/or addition of one or several amino acid residues or with 99%, 95%, 90%, 85% or 80% or more identity and protein with the same function or its N-terminal and/or C-terminal.Upland cotton TM-1 is converted using GhBONI gene order building VIGS plant expression vector, the transgene cotton of acquisition, the resistance to verticillium wilt is shown after being inoculated with verticillium dahliae V991, illustrates that the albumen of GhBONI gene and its coding may participate in verticillium wilt resistance of cotton by same mechanism.The present invention is of great significance to resisting verticillium transgene cotton is cultivated.

Description

Cotton verticillium wilt related gene GhBONI and its coding albumen and application

Technical field

The present invention relates to genetic engineering fields, and in particular to cotton verticillium wilt related gene GhBONI and its coding albumen with Using.

Background technique

Cotton is industrial crops important in the world, is the maximum crop of fiber production scale in the world.Gossypium (Gossypium) upland cotton (Gossypium hirsutum L.) in is since its fiber quality is good, yield is high, growth cycle It is short, it is to plant most commonly used Main Cultivation cotton seed at present, yield accounts for about the 95% of World cotton total output.However, cotton yellow Disease of withering (Verticillium wilt) exists as a kind of soil-borne disease fungal pathogens verticillium dahliae (Verticillium dahliae) There are destructive vascular bundle diseases, the serious growth and development for restricting cotton becomes the of worldwide cotton in Cotton Production One disease, " cancer " referred to as cotton.In the late three decades, which has a generation in each cotton region in China, and in the part time and Region population outbreak.So far, cotton verticillium wilt can't be controlled effectively in upland cotton, the land of high resistance to verticillium wilt Cotton variety is still difficult to obtain at present.Many years it was verified that excavate disease-resistant gene, understanding cotton to the reaction mechanism of verticillium wilt, Accelerate verticillium wilt resistance of cotton by same breeding process, and then improve the disease resistance of cotton, it has also become the master of verticillium wilt resistance of cotton by same breeding work Want approach.

With the fast development of molecular biology, people gradually add the understanding of plant biological Stress responses molecular mechanism Deep, discovery disease-resistant gene (Disease Resistance, R) is the key factor that defense response difference regulates and controls between mediating kind. Disease-resistant gene R can directly or indirectly identify pathogen nontoxic gene product, and then induce a series of defense response in downstream. Plant containing R gene, which can resist, carries infecting for corresponding AVR gene germ, and the plant without R gene is then to having Corresponding AVR gene germ shows as susceptible.The NB-LRR albumen of most of plant R genes coding is animal immune receptor (NOD- Like receptor, NLR) homologous protein, research find R gene plant transcription level in exist closely regulation, this Regulation plays an important role in plant defense and balance plant defense and growth.The aseptically table of R gene General relatively low up to level, in the case where germ infects induction, the expression of R gene can just be activated, the up-regulated expression or mistake of composition The normal growth that amount expression R gene will affect plant even results in Plant death.In addition, also being sent out in plant disease-resistant defense response Existing active oxygen (ROS) as the signals such as signaling molecule and calcium signal, protein phosphorylation and hormone regulating and controlling by way of interaction, Change the disease resistance of plant.The transgenic research process of cotton is more time-consuming, virus induced gene silencing technology (Virus-induced gene silencing, VIGS) is a kind of inherent immunity mechanism generally existing in plant, is belonged to Posttranscriptional gene silencing makes plant generate silencing phenotype by silencing target gene, thus judge the function of target gene, and Avoid Genetic Transformation in Higher Plants and gene function redundancy.VIGS technology can in a short time invade Agrobacterium in plant, A large amount of inoculated plant seedling or root system complete the function that genetic transformation carrys out goal in research gene.

Summary of the invention

The object of the present invention is to provide cotton verticillium wilt related gene GhBONI and its coding albumen and applications.

In a first aspect, a kind of claimed protein.

Present invention protein claimed specifically may be used from cotton (Gossypium hirsutum L.) is obtained from It is following any:

(A1) amino acid sequence is the protein of SEQ ID No.1；

(A2) by amino acid sequence shown in SEQ ID No.1 by one or several amino acid residues substitution and/or Deletion and/or addition and protein with the same function；

(A3) with (A1)-(A2) in it is any defined by amino acid sequence have 99% or more, 95% or more, 90% with Above, 85% or more or 80% or more identity and protein with the same function；

(A4) it is obtained after N-terminal and/or C-terminal the connection protein tag of protein defined by any in (A1)-(A3) Fusion protein.

In above-mentioned protein, the protein tag (protein-tag) refers to using DNA extracorporeal recombination, with purpose A kind of polypeptide or albumen of albumen amalgamation and expression together, in order to the expression of destination protein, detection, tracer and/or purifying.Institute Stating protein tag can be Flag label, His label, MBP label, HA label, myc label, GST label and/or SUMO label etc..

Second aspect, the nucleic acid molecules of claimed code for said proteins.

Claimed nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid Molecule is also possible to RNA, such as mRNA, rRNA or tRNA.

Further, the nucleic acid molecules are the gene of code for said proteins, and the gene can be following any described DNA molecular:

(B1) DNA molecular shown in SEQ ID No.2；

(B2) hybridize and the DNA molecular of code for said proteins with (B1) DNA molecular limited under strict conditions；

(B3) DNA sequence dna limited with (B1) or (B2) has 99% or more, 95% or more, 90% or more, 85% or more Or 80% or more identity and code for said proteins DNA molecular.

Above-mentioned stringent condition can for 6 × SSC, the solution of 0.5%SDS hybridizes at 65 DEG C, then with 2 × SSC, It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.

The third aspect, the claimed recombinant vector containing above-mentioned nucleic acid molecules, expression cassette, transgenic cell line or Recombinant bacterium.

Claimed recombinant vector can be recombinant expression carrier, can also be recombinant cloning vector.

The recombinant expression carrier can use existing plant expression vector construction.The plant expression vector includes double base agriculture Bacillus carrier and the carrier etc. that can be used for plant micropellet bombardment, as pCAMBIA1300, pCUbi1390, pCHF3, PGreen0029, pCAMBIA3301, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other are derivative to plant Object expression vector.The plant expression vector also may include 3 ' end untranslated regions of foreign gene, i.e., believes comprising polyadenylic acid Number and it is any other participation mRNA processing or gene expression DNA fragmentation.The bootable polyadenylic acid of polyadenylation signals adds Enter the 3 ' ends to mRNA precursor.When using the gene constructed recombinant expression carrier, it can be added before its transcription initiation nucleotide Any enhanced, composing type, organizing specific type or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S are opened Mover, ubiquitin gene Ubiquitin promoter (pUbi), stress induced promoter rd29A etc., they can be used alone or with Other plant promoters are used in combination；In addition, enhancing also can be used when using gene constructed recombinant expression carrier of the invention Son, including translational enhancer or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region starting Codon etc., but must be identical as the reading frame of coded sequence, to guarantee the correct translation of entire sequence.The translation control letter Number and initiation codon source be it is extensive, can be natural, be also possible to synthesis.Translation initiation region can come from Transcription initiation region or structural gene.It, can be to used for the ease of transgenic plant cells or plant are identified and screened Recombinant expression carrier is processed, and can produce the enzyme or luminophor of color change as the coding that can be expressed in plant is added Gene, resistant antibiotic marker or anti-chemical reagent marker gene etc..Any selected marker can also be not added Gene directly screens transformed plant with adverse circumstance.

Claimed expression cassette is by that can start the promoter of the gene expression, the gene, Yi Jizhuan Record termination sequence composition.

Fourth aspect, the claimed protein or the nucleic acid molecules or the recombinant vector, expression cassette, Transgenic cell line or recombinant bacterium it is following it is any in application:

(a) regulate and control disease resistance of plant；

(b) plant variety breeding disease resistance enhancing or weakened；

(c) plant breeding.

In (a), the regulation disease resistance of plant can specifically embody are as follows: in the plant, the protein (or it is described Gene) expression quantity reduce, the resistance disease of the plant improves.

In (b), the method for the plant variety of breeding disease resistance enhancing, specifically may include by the protein (or The gene) the relatively low plant of expression quantity the step of hybridizing as parent.The plant that the breeding disease resistance weakens The method of article kind specifically may include using the relatively high plant of the expression quantity of the protein (or described gene) as parent The step of being hybridized.

In (c), the plant variety of disease resistance enhancing is concretely cultivated in the plant breeding.

5th aspect, a kind of claimed method for cultivating plant variety.

The method of claimed cultivation plant variety can be method A or method B:

Method A: a method of plant variety that cultivating disease resistance enhancing, including making protein described in recipient plant The step of expression quantity and/or activity reduce；

Method B: a method of the plant variety that disease resistance weakens being cultivated, including making protein described in recipient plant The step of expression quantity and/or activity improve.

6th aspect, a kind of claimed method for cultivating genetically modified plants.

The method of claimed cultivation genetically modified plants, can be method C or method D:

Method C: a method of genetically modified plants that cultivating disease resistance enhancing include the following steps: to inhibit recipient plant Described in protein encoding gene expression, obtain genetically modified plants；The genetically modified plants are compared with the recipient plant Disease resistance enhancing；

Method D: a method of the genetically modified plants that disease resistance weakens are cultivated, are included the following steps: into recipient plant The nucleic acid molecules that can express the protein are imported, genetically modified plants are obtained；The genetically modified plants and the recipient plant Weaken compared to disease resistance.

In method C, inhibit the expression of the encoding gene of protein described in the recipient plant specifically can be by as follows It realizes: importing pTRV2-GhCLA1 carrier and pTRV1 carrier in Xiang Suoshu recipient plant；The pTRV2-GhCLA1 carrier is will After DNA fragmentation shown in 1-417 of SEQ ID No.2 is inserted between restriction enzyme site EcoR I and the Kpn I of pTRV2 carrier Obtained recombinant vector.

In method D, the gene can specifically be imported in the recipient plant by the recombinant expression carrier, obtain institute State genetically modified plants.The recombinant expression carrier is to obtain after the gene is inserted into the multiple cloning sites of plant expression vector The recombinant vector for capableing of expressing said gene.

In above-mentioned various aspects, the disease can be verticillium wilt.

In above-mentioned various aspects, the disease can be the plant disease as caused by verticillium dahliae.

Further, the verticillium wilt can be cotton verticillium wilt.

Further, the verticillium dahliae can be verticillium dahliae bacterial strain V991 (i.e. verticillium wilt pathogen V991).

In above-mentioned various aspects, the plant can be dicotyledon.

Further, the dicotyledon can be cotton.

Further, the cotton can be cotton.

The present invention provides cotton calcium to rely on cardiolipin binding protein BONI gene and its coding albumen, utilizes GhBONI gene Sequence information expands the gene, and constructs VIGS plant expression vector conversion upland cotton TM-1, and the transgene cotton of acquisition is connecing The resistance to verticillium wilt is shown after kind verticillium dahliae V991, illustrates that the albumen of GhBONI gene and its coding may participate in Verticillium wilt resistance of cotton by same mechanism.The present invention is of great significance to resisting verticillium transgene cotton is cultivated.

Detailed description of the invention

Fig. 1 is the VIGS system and VIGS cotton plants inoculation after two weeks that TRV mediation is successfully established in upland cotton TM-1 Growing state after verticillium wilt pathogen V991 12 days compares.A is the VIGS system that TRV mediation is successfully established in upland cotton TM-1. TRV:00 (control): injection zero load pTRV2；TRV:GhCLA1: injection albefaction gene.B is that cotton plants are inoculated with VIGS after two weeks Growing state after verticillium wilt pathogen V991 12 days compares.TRV:00 (control): injection zero load pTRV2；TRV:BONI: injection GhBONI gene.

Fig. 2 is the expression quantity that fluorescent quantitation Real time-PCR detects GhBONI gene.

Fig. 3 is that disease incidence and disease index statistically analyze.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Upland cotton TM-1: it receives in the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute.Bibliography: Sun Zhengwen, Kuang Meng, Ma Zhi English, Wang Shengfen using CottonSNP63K chip building cotton variety finger-print Scientia Agricultura Sinica, 2017,50 (24): the 4692-4704. public can obtain from applicant, can only be used to repeat the present invention experiment use, must not he use.

Plant VIGS silencing expression vector:

PTRV1: Changsha Yingrun Biological Technology Co., Ltd., article No. VRW0365；

PTRV2: Changsha Yingrun Biological Technology Co., Ltd., article No. VRW0366.

PTRV2-GhCLA1, bibliography: Liu Hui, Yu Xiuli, Huang Xianzhong .TRV virus-mediated gene silencing system exists Cotton Science Cotton Science 2016,28 (5): 485-492 are established in Xinjiang Upland Cotton and Asiatic cotton.The public can Obtained from applicant, can only be used to repeat the present invention experiment use, must not he use.

Agrobacterium GV3101: Urumchi section, new and high-tech development zone is farsighted to reach firm, AC1001.

Verticillium wilt pathogen V991: this laboratory saves, and the public can be from Xinjiang Agricultural Sciences institute nuclear technology biotechnology research Institute (Xinjiang Uygur Autonomous Regions biotech research center) obtains.Bibliography: Zhang Hui, Tian Xinquan, Gao Wei, Cai Yingfan, dragon The identification of fine jade upland cotton PPO gene full-length genome and the response analysis Cotton Science Cotton Science to verticillium wilt pathogen 2017,29 (5): 428-436.The public can obtain from applicant, can only be used to repeat the present invention experiment use, must not he use.

Embodiment 1, cotton verticillium wilt related gene GhBONI and its acquisition for encoding albumen

The total serum IgE of cotton variety upland cotton TM-1 is mentioned, and reverse transcription obtains cDNA, then using gained cDNA as template, adopted PCR amplification is carried out with primer GhBONIORF-F and primer GhBONIORF-R.

Primer GhBONIORFF:5 '-ATGGGAAATTGCTGCTCCGA-3 '；

Primer GhBONIORFR:5 '-TCAAATCCTGGGTTTAATAT-3 '.

Amplified production is sequenced, it is known that gained cDNA nucleotides sequence is classified as SEQ ID No.2, by gene shown in it It is named as GhBONI, the amino acid sequence of the protein of coding is SEQ ID No.1.

Above-mentioned cDNA (SEQ ID No.2) can also pass through artificial synthesized acquisition.

Embodiment 2, cotton verticillium wilt related gene GhBONI and its application for encoding albumen

One, the building of recombinant expression carrier

1, the total serum IgE of cotton (upland cotton standard system TM-1) is extracted, and reverse transcription is cDNA, using cDNA as template, used The primer pair of primer GhBONI-1F and primer GhBONI-1R composition carries out PCR amplification, obtains pcr amplification product.

Primer GhBONI-VIGS-F:5 '-GAATTCATGGGAAATTGCTGCTCCGA-3'；

Primer GhBONI-VIGS-R:5 '-GGGGTACCCCACCAAGAGATTGCTGCTCCT-3’。

In primer GhBONI, underscore is restriction enzyme EcoRI and KpnI sequence, and subsequent connection plant silencing is facilitated to express Carrier pTRV2 carries out Cotton Transformation.

2, the pcr amplification product and pEASY-Blunt Zero Cloning Kit (cloning vector) obtained step 1 is even It connects, send sequencing with positive transformant of the GhBONI-1F/R primer after PCR is identified.Obtaining sequence is " 5 '-GAATTC+SEQ ID 1-417 of No.2+GGGGTACCCC-3’”。

3, step 2 is obtained sequence fragment to connect with pTRV2 carrier by EcoRI and KpnI double digestion, obtains VIGS plant Object expression vector recombinant plasmid, is named as pTRV2-GhBONI.PTRV2-GhBONI structure description: in the digestion of pTRV2 carrier Resulting recombinant plasmid after DNA fragmentation shown in 1-417 of insertion SEQ ID No.2 between site EcoRI and KpnI.

4, the plant expression vector recombinant plasmid pTRV2-GhBONI for obtaining step 3 converts Agrobacterium GV3101, uses GhBONI-1F/R primer obtains Agrobacterium positive colony after PCR screening and identification.

Two, the acquisition of transgene cotton

1, by the sun plant expression vector recombinant plasmid pTRV2-GhBONI Agrobacterium of acquisition in (50 μ containing kanamycins G/ml it) and on the LB solid medium of gentamicin (20 μ g/ml) resistance crosses, 28 DEG C are cultivated 2 days.

2, the monoclonal access 5ml that picking step 1 obtains contains kanamycins (50 μ g/ml) and gentamicin (20 μ g/ml) In the LB liquid medium of resistance, 28 DEG C of 180 turns of min^-1Culture is for 24 hours；In 50m L LB liquid medium of transferring, 28 DEG C 180 Turn min^-1Cultivate 12h, 4000 turns of min^-1Be centrifuged 5min and collect somatic cells, with the re-suspension liquid of proper volume (formula: 10mmol·L^–1MgCl₂, 10mmolL^–1MES and 200 μm of olL^–1Acetosyringone) it is resuspended to final concentration of 1.5 (OD₆₀₀).Re-suspension liquid is stood into 3h or more at room temperature.Obtain the re-suspension liquid containing recombinant plasmid pTRV2-GhBONI.Similar side Method is prepared the re-suspension liquid containing pTRV1 carrier, the re-suspension liquid of the pTRV2-GhCLA1 carrier containing CLA1 genetic fragment, contains There is the re-suspension liquid of pTRV2 empty carrier.

The re-suspension liquid of re-suspension liquid containing pTRV1 carrier and the recombinant plasmid pTRV2-GhBONI containing target gene fragment Injection cotton cotyledon is mixed at 1: 1 by volume, for obtaining gene silencing transformant；Re-suspension liquid containing pTRV1 carrier and contain There is the re-suspension liquid of the pTRV2-GhCLA1 carrier of CLA1 genetic fragment to mix injection cotton cotyledon at 1: 1 by volume, for detecting Whether gene silencing system is correct；Re-suspension liquid containing pTRV1 carrier and the re-suspension liquid containing pTRV2 empty carrier by volume 1: 1 mixes injection cotton cotyledon, for doing genetic transformation control strain.

3, TM-1 cotton seeds are seeded in Nutrition Soil, and 12h illumination/12h is dark, and temperature is 26-28 DEG C of illumination cultivation.It is wet Degree is maintained at 60% or more, 4-5d and pours a water, opens and when true leaf is not yet developed can be used to VIGS when two panels cotyledon is open and flat Operation.

4, the cotyledon back side gently first being punctured with syringe needle and causing microtrauma mouth, the syringe for spending syringe needle is infused from wound Enter the ready re-suspension liquid mixed according to volume ratio 1: 1 in step 2, obtains cotton GhBONI gene silencing transformant.After 2 weeks Observe the phenotype of different disposal cotton, and the expression of testing goal gene.To 30 single plants of every kind of material processing.

Agrobacterium VIGS specific method bibliography: Gao, X., Shan, L.Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.Methods Mol Biol,2013,975:157-165.

Three, the detection of VIGS genetic conversion system

1, upland cotton CLA1 gene (cloroplastos alterados 1gene) is used to carry out VIGS for marker gene System detection.The gene participates in Development of Chloroplasts process, encodes 1-deoxyxylulose 5-phosphate synthase egg White, it is readily identified mark property that highly conserved in evolution, cotton plant, which has apparent albefaction phenotype, after CLA1 gene silencing.Knot Fruit sees A in Fig. 1.After VIGS infects 2 weeks, the plant true leaf almost albefaction of pTRV1 and pTRV2-GhCLA1 is injected, and is injected Empty carrier pTRV1 and pTRV2 are that the blade for compareing (TRV:00) does not have any variation.Illustrate to be successfully established in upland cotton TM-1 The VIGS system that TRV is mediated.

2, fluorescent quantitation Real time-PCR is detected

Cotton plants blade after VIGS infects 2 weeks is extracted using Plant Total RNA Extraction Kit kit Total serum IgE.It is reference gene with cotton Histon 3, passes through GhBONI base after fluorescent quantitation Real time-PCR detection silencing The expression of the disturbed silencing of cause.

Quantitative fluorescent PCR instrument is Applied Biosystems StepOne (Applied Biosystems, beauty State), agents useful for same is SYBR Premix Ex Taq^TMThe SYBR Premix Ex Taq of kit (TransGene, Beijing)^TMExamination Agent box.Using reverse transcription cDNA as template, initial amount is 150ng, 3 repetitions of each processing.Response procedures are as follows: denaturation (95 DEG C, 30s)；(95 DEG C, 5s；58 DEG C, 15s；72 DEG C, 31s) 40 circulations of amplification；Dissolution (95 DEG C, 15s；60 DEG C, 1min；95 DEG C, 15s)。

Primer Histon3-RT-F:5 '-GCCAAGCGTGTCACAATTATGC-3 '；

Primer Histon3-RT-R:5 '-ACATCACATTGAACCTACCACTACC-3 '.

Primer GhBONI-RT-F:5 '-CATCACTGGCAACCAAAGCG-3 '；

Primer GhBONI-RT-R:5 '-CCACCAGCATCGGATCACTC-3 '.

As a result see Fig. 2.Compared with empty carrier (TRV:00) control, infected in the two GhBONI gene VIGS randomly selected In plant (BONI-1, BONI-2), the expression quantity of GhBONI gene significantly declines, and silencing efficiency is obvious.

Four, verticillium wilt resistance of cotton by same inoculation and Resistance Identification

The verticillium wilt pathogenic bacteria verticillium dahliae bacterial strain V991 of preservation (is recorded in " Zhang Hui, Tian Xinquan, Gao Wei, Cai Ying It is numerous, the identification of Long Lu upland cotton PPO gene full-length genome and the response analysis Cotton Science Cotton to verticillium wilt pathogen Science 2017,29 (5): 428-436 " " the verticillium wilt pathogen V991 " in a text) it is activated in PDA culture medium.Picking Thallus in Czapek`s culture solution, 25 DEG C, 200r/min, cultivate 3~5d.By 4 layers of filtered through gauze of Bacteria culturing liquid, Cause of disease bacteria concentration is counted using blood counting chamber, adjusts final concentration to 1.0 × 10 with sterilizing distilled water⁷A spore/mL, and be added Tween-20 is to final concentration 0.001% (volumn concentration).To the transformant after obtained VIGS gene silencing 2 weeks, with leaching Root method is inoculated with verticillium wilt pathogen V991 spore liquid, investigation statistics verticillium wilt incidence after 12d is inoculated with, using 0~4 grade of method statistic Sick grade.To 30 single plants of every kind of material processing.If 3 biology repeat.

Sick grade index statistical-reference document: Xu Li, Zhu Longfu, Zhang Xianlong verticillium wilt resistance of cotton by same Recent Advances in Mechanism crop Journal, 2012,38:1553-1560；Xu L,Zhu L F,Zhang X L.Research on resistance mechanism of cotton to Verticillium wilt.Acta Agron Sin,2012,38:1553–1560.

Disease index=[(diseased plant numbers at different levels × corresponding sick grade)/investigation total strain number × highest disease grade (4)] × 100

As a result as shown in figure 1 shown in B.The silencing plant of pTRV2-GhBONI after being infected 2 weeks to VIGS and pair of empty carrier According to (TRV:00) plant, it is inoculated with the incidence of verticillium wilt after verticillium dahliae bacterial strain V991,12d.By B in Fig. 1 as it can be seen that control (TRV:00) plant blade compared with GhBONI gene silencing strain occurs that light yellow patch is more and area is bigger, and leaf margin is to last volume Qu Chengdu becomes apparent from.It is statisticallyd analyze by 3 secondary pollutant repeated observations, as a result such as Fig. 3.The adjoining tree of empty carrier is injected, Average disease index is up to 41.72, and the plant disease resistance after GhBONI gene silencing significantly increases, and average disease index is 32.18.Show that GhBONI gene takes part in the allergic reaction of verticillium wilt induction.

Above-mentioned experimental result sufficiently shows that the expression quantity that rear GhBONI gene is infected by VIGS significantly declines, disease-resistant Enhancing.

<110>Institute of Nuclear Technology and Biotechnology, Xinjiang Academy of Agricultu is (in Xinjiang Uygur Autonomous Regions's biotechnology research The heart)

<120>cotton verticillium wilt related gene GhBONI and its coding albumen and application

<130> GNCLN191655

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<170> PatentIn version 3.5

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acatggaagc cagtattttt gaatattcaa caagtaggaa gcaaggatag tccattagtg 780

atagagtgct ttaacttcaa tagcaatggg aagcatgatc tgattggaaa agtccagaag 840

tcactagcag atttggaaaa gattcattct ggaagggaag gagaaaattt atttttgcca 900

actctggttg ggcatgattg cgagaacaag atattaaaaa gcaagctttt cgtggaaaat 960

ttctctgaga ctatccaaca taccttcctg gattacctgg ctgggggagt tgaacttaac 1020

tttatggtgg ctattgattt tacggcttca aatggaaatc cccgtcttcc tgattccttg 1080

cattacattg atccatctgg acggcagaat gcataccaga aagcaatcta tgaggttgga 1140

gaagtattgc agttctatga tacagataag tgttttcctg catggggatt tggagcacgg 1200

cccattgatg gtccagtttc tcattgtttc aacttgaatg gaagcaataa ctactgtaag 1260

gttgaaggaa tccgaggcat tatgatggcc tatacaagcg cgcttttcaa tgtttcgctt 1320

gcaggaccaa cactatttgg gcatgtggtt aacaaagctg cactaattgc cagccaatct 1380

cttgccgatg aagctcaaaa atactttgtt ttgttgatta tcacggatgg agttgtgacc 1440

gatctccaag aaaccaaaga tgcactggta aaagcatcag atctcccact gtcgatcctc 1500

attgttggag ttggaggagc agacttcaaa gaaatggaga ttttagatgc agacaaaggg 1560

gaaagacttg aaagctcgac cggacgtgtt gcttcacgtg atattgttca atttgttcca 1620

tttagggatg tacaaggtgg tgaagtatct attgttcaag cgcttttggc tgaattaccg 1680

acacaatttt taacctatat gcgaagcaga gatattaaac ccaggatttg a 1731

Claims (10)

1. protein is following any:

(A1) amino acid sequence is the protein of SEQ ID No.1；

(A2) amino acid sequence shown in SEQ ID No.1 is passed through to the substitution and/or missing of one or several amino acid residues And/or addition and protein with the same function；

(A3) with (A1)-(A2) in it is any defined by amino acid sequence have 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity and protein with the same function；

(A4) fusion obtained after N-terminal and/or C-terminal the connection protein tag of protein defined by any in (A1)-(A3) Albumen.

2. encoding the nucleic acid molecules of protein described in claim 1.

3. nucleic acid molecules according to claim 2, it is characterised in that: the nucleic acid molecules are described in coding claim 1 The gene of protein, the gene are following any DNA molecular:

(B1) DNA molecular shown in SEQ ID No.2；

(B2) hybridize and encode DNA points of protein described in claim 1 with (B1) DNA molecular limited under strict conditions Son；

(B3) with (B1) or (B2) limit DNA sequence dna have 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity and the DNA molecular for encoding protein described in claim 1.

4. recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing nucleic acid molecules described in Claims 2 or 3.

5. recombinant vector according to claim 4, it is characterised in that: the recombinant vector is recombinant expression carrier or recombination Cloning vector.

6. nucleic acid molecules described in protein or Claims 2 or 3 described in claim 1 or recombination described in claim 4 or 5 carry Body, expression cassette, transgenic cell line or recombinant bacterium it is following it is any in application:

(a) regulate and control disease resistance of plant；

(b) plant variety breeding disease resistance enhancing or weakened；

(c) plant breeding.

7. a kind of method for cultivating plant variety, is method A or method B:

Method A: a method of the plant variety of disease resistance enhancing is cultivated, including makes egg described in claim 1 in recipient plant The step of expression quantity and/or activity of white matter reduce；

Method B: a method of the plant variety that disease resistance weakens is cultivated, including makes egg described in claim 1 in recipient plant The step of expression quantity and/or activity of white matter improve.

8. a kind of method for cultivating genetically modified plants, is method C or method D:

Method C: a method of genetically modified plants that cultivating disease resistance enhancing include the following steps: to inhibit to weigh in recipient plant Benefit requires the expression of the encoding gene of 1 protein, obtains genetically modified plants；The genetically modified plants and the recipient plant Enhance compared to disease resistance；

Method D: a method of the genetically modified plants that disease resistance weakens are cultivated, include the following steps: to import into recipient plant The nucleic acid molecules that protein described in claim 1 can be expressed, obtain genetically modified plants；The genetically modified plants and the receptor Plant weakens compared to disease resistance.

9. according to the application or method any in claim 6-8, it is characterised in that: the disease is verticillium wilt；Or

The disease is the plant disease as caused by verticillium dahliae；

Further, the verticillium wilt is cotton verticillium wilt.

10. according to the application or method any in claim 6-9, it is characterised in that: the plant is dicotyledon；

Further, the dicotyledon is cotton；

Further, the cotton is cotton.