CN106754963B - NtRRS3 gene and its application in tobacco resistance to bacterial wilt - Google Patents

Abstract

The invention discloses tobacco CC-NBS-LRR class disease-resistant genesNtRRS3And its application in tobacco resistance to bacterial wilt, belong to plant genetic engineering field.Present invention firstly provides from the tobacco resistance to bacterial wilt kind rock cigarette 97 clone obtainNtRRS3Gene, nucleotides sequence are classified as shown in SEQ ID No.1.The present invention further will be above-mentionedNtRRS3Gene, which is transferred in tobacco, carries out functional verification.The result shows that overexpressionNtRRS3Gene can significantly increase susceptible variety to the resistance of Ralstonia solanacearum.What the present invention separatedNtRRS3Gene is to regulate and control tobacco to the important gene of Ralstonia solanacearum resistance, and tobacco can be improved to the resistance of bacterial wilt, have important application prospect in terms of tobacco resistance to bacterial wilt.

Description

NtRRS3 gene and its application in tobacco resistance to bacterial wilt

Technical field

The present invention relates toNtRRS3Gene and its application in tobacco resistance to bacterial wilt belong to plant gene engineering technology neck Domain.

Background technique

Tobacco is the important industrial crops in China.Tobacco bacterial wilt is one of important disease of tobacco, which passes Property bacterial disease, invaded from root or wound, lead to plant withered death, be commonly called as " cigarette pest " in China.The disease is distributed more Extensively, the torrid zone and subtropical zone of high temperature and humidity are distributed mainly on.It shows in recent years gradually to high latitude, High aititude frigid zone The trend of area extension.The disease seriously endangers China Yangtze river basin and southern tobacco leaf producing region, causes huge damage to tobacco grower It loses, causes the cigarette district for having multiple provinces such as Fujian, Guangdong, Guangxi of serious production loss.There has been no cost-effective anti-at present Tobacco bacterial wilt means are controlled, and the effective way fundamentally solved is to cultivate disease-resistant variety, compared with traditional breeding way, benefit Tobacco bacterial wilt can be more effectively solved the problems, such as with the resistance to bacterial wilt kind that genetic engineering breeding means cultivate good quality and high output.

China has carried out the examining order of tobacco gene group at present, and the genome for completing two wild original species is surveyed Sequence, but the genome sequence of cultivar is still in an assembling process.This is beneficial to the clone of tobacco Bacterial wilt resistance gene and identification, Promote tobacco resistance to bacterial wilt molecular breeding.The bacterial wilt host range that causes harm is very wide, but in addition to arabidopsis, not yet from forward direction Science of heredity means obtain Bacterial wilt resistance gene.The anti-green cumyl of the TIR-NB-LRR class obtained on arabidopsis at present becauseRRS1-RWith The anti-green cumyl of LRR-RLK class becauseERECTA.And NBS-LRR class resistant gene often includes nucleotide binding site (NBS) He Fuliang Propylhomoserin repeats (LRR) structural domain.The conserved domain of NBS, which contains, multiple to play a crucial role during plant signaling transduction Conservative functional areas, such as P-Loop, Kinase-2, Kinase-3a and GLPL etc..LRR structural domain then mediates pathogen-associated molecular mould In the immune response (PTI) that formula causes.After plant is invaded by pathogen, it will usually anti-by generating allergy infecting position It answers, to resist the diffusion and breeding of pathogen, and then activates a series of related defense response in downstream.The defense reaction mistake of plant Journey is that the signal transduction regulated and control network of complexity acts on as a result, the regulated and control network is by resistance channel or SA, the plant hormones such as ET, JA The signal transduction pathway of participation intersects to form.At present in tobacco the anti-green cumyl of NBS-LRR class because report it is very few, the anti-blueness of tobacco is withered Molecule mechanism and its regulated and control network research it is very few, this seriously constrains the molecular breeding process of tobacco resistance to bacterial wilt.

The present invention is directed to background above technology, study the tobacco gene chip data analysis based on laboratory early period as a result, Have found one after Ralstonia solanacearum induces in disease-resistant variety up-regulated expression, the CC-NBS-LRR of expression is lowered in susceptible variety Class resistant gene, is named asNtRRS3, as resistance to bacterial wilt key candidate gene.Using Race technology from tobacco disease resistance kind rock The full length sequence of the gene has been cloned in cigarette 97, includes complete open reading frame, it is bright comprising CC motif, NB-ARC and multiple richnesses Propylhomoserin motif (LRR-RI) conserved domain, the characteristics of meeting the disease-resistant gene family of CC-NBS-LRR class.Utilize Gateway skill Art constructs overexpression vector, and digestion connection method constructs RNAi carrier, is transferred to susceptible variety Hongda tobacco and disease-resistant respectively In kind rock cigarette 97, genetic complement functional verification is carried out.Ralstonia solanacearum inoculated identification is carried out to transgenic tobacco plant, as a result, it has been found thatNtRRS3Overexpression transgenic plant makes susceptible variety show slightly resistance, and NtRRS3-RNAi interference of transgene plant makes Susceptible variety shows apparent susceptible.After being inoculated with Ralstonia solanacearum, overexpresses defense-related gene in transgenic plant and induced Up-regulated expression makes it generate Defense response reaction；And it is significant after defense-related gene is induced in RNAi transgenic plant under Mileometer adjustment reaches, and increases it by the susceptibility to bacterial wilt.This illustratesNtRRS3Gene may participate in rock cigarette 97 and resist to Ralstonia solanacearum Property channel, may be non-allelic genes in anti-sense kind, signals-modulating network has different resistance access and SA, and JA etc. is more Bars transduction pathway intersects to form, and discloses the diversity and complexity of tobacco resistance to bacterial wilt molecule mechanism.The present invention is benefit Resistance to bacterial wilt new product of tobacco, which is cultivated, with genetic engineering means provides genetic resources, it is with important application prospects.

Summary of the invention

The present invention providesNtRRS3Gene and its application in tobacco resistance to bacterial wilt.Its purpose is to provide tobaccos CC-NBS-LRR class disease-resistant geneNtRRS3The coded sequence of gene and its encoded albumen, are provided containing above-mentionedNtRRS3Base The over-express vector and RNAi interference carrier of cause, and it is applied to improve tobacco to the resistance of Ralstonia solanacearum, and then it is withered to cultivate anti-blueness Sick new product of tobacco (being).Genetic resources are provided to cultivate resistance to bacterial wilt new product of tobacco using genetic engineering means, are had Important application prospect.

To achieve the above object, the present invention adopts the following technical scheme:

The present invention is cloned from resistance to bacterial wilt tobacco bred rock cigarette 97 by design primerNtRRS3Gene, nucleotide Sequence is shown in SEQ ID NO.1, and amino acid sequence is shown in SEQ ID NO.2.

The present invention provides contain the tobacco CC-NBS-LRR class disease-resistant geneNtRRS3The over-express vector of gene with And RNAi carrier.It, will using Gateway technologyNtRRS3Gene is introduced into over-express vector pEarleyGate 205, by increasing Strong type promoter 35S driving, carrier are named as p35S:: NtRRS3-TAP；It, will using digestion connection methodNtRRS3Gene Forward and reverse Insert Fragment is inserted into RNAi expression vector pGSA2285, is named asNtRRS3-RNAi.Above-mentioned carrier is turned respectively Change EHA105 Agrobacterium, is conducted into sense bacterial wilt kind Hongda tobacco and resistance to bacterial wilt kind rock respectively by leaf disk method In cigarette 97.Ralstonia solanacearum inoculated identification is carried out to transgenic progeny plant, the results showed thatNtRRS3The overexpression of gene can be significant Enhance tobacco plant to the resistance of Ralstonia solanacearum, which can reduce tobacco plant to the resistance of Ralstonia solanacearum.

Beneficial effect

The tobacco CC-NBS-LRR class disease-resistant gene that the present invention clonesNtRRS3Gene can be improved tobacco and resist to Ralstonia solanacearum Property, genetic improvement and cultivation resistance to bacterial wilt new product of tobacco (being) for tobacco resistance to bacterial wilt apply valence with important Value.

Detailed description of the invention

Fig. 1 RACE is obtainedNtRRS33 ' the unknown nucleotide sequences and 5 ' unknown nucleotide sequences and ORF frame electrophoretogram of gene.A:5 ' RACE target fragment；B:3 ' RACE target fragment；C:cDNA target fragment.M1: DL2,000 DNA Marker; M2: DL15,000 DNA Marker。

The Multiple Sequence Alignment of Fig. 2-1 NtRRS3 protein structure domain.

The Multiple Sequence Alignment of Fig. 2-2 NtRRS3 protein structure domain.

The Multiple Sequence Alignment of Fig. 2-3 NtRRS3 protein structure domain.

Fig. 3NtRRS3The building schematic diagram of overexpression vector (A) and RNAi carrier (B).

Fig. 4NtRRS3Overexpress transgene tobacco T0 generation identification.Identification in A.DNA level；B. on rna level Identification；M:DL2,000 DNA Marker.1: negative control；2: positive control；Remaining swimming lane is with T0 for the DNA of individual plants Or sscDNA is the amplified production of template.

Fig. 5NtRRS3-RNAiTransgene tobacco T0 generation identification.A. the identification in DNA level；B. on rna level Identification；M:DL2,000 DNA Marker；1: negative control；2: positive control；Remaining swimming lane is with T0 for the DNA of individual plants Or sscDNA is the amplified production of template.

Fig. 6NtRRS3Overexpress the phenotype and its disease index of transgene tobacco inoculation Ralstonia solanacearum.

Fig. 7 RNAi interferes silencingNtRRS3The phenotype and its disease index of transgene tobacco inoculation Ralstonia solanacearum.

Specific embodiment

[embodiment 1] RACE is obtainedNtRRS33 ' unknown nucleotide sequence of gene and 5 ' unknown nucleotide sequences

Tobacco gene chip data analysis result of this research based on laboratory early period, it was found that one induces through Ralstonia solanacearum The up-regulated expression in disease-resistant variety afterwards is lowered the CC-NBS-LRR class resistant gene of expression in susceptible variety, is named asNtRRS3.According to the genetic fragment, design primer NtRRS3-3 ' race-F(5 '-AGATTAGGCACTGGAGAGCCAGTTG- 3 ') and NtRRS3-5 ' race-R(5 '-CTGTGCTGCATGCTAGCTCTATCAC-3 '), according to template joint sequence design one To primer NtLib45-F(5 '-GATTCTGTGGATAACCGTATTACCGCCTTACGCGTGTAAAACGAC-3 ') and NtLib39-R(5 '-ACCAGGATCTCCTAGGGAAACAGCTATGACCATGTTCAC-3 '), drawn with NtRRS3-5 ' race-R Object and the pairing of NtLib45-F primer and NtRRS3-3 ' race-F primer and NtLib39-R primer carry out 5 ' and 3 '-respectively RACE reaction, 5 '-RACE reaction conditions are 95 DEG C, 5 min → (95 DEG C, 30 s → 72 DEG C, 2 min) 5 cycles → (95 DEG C, 30 s → 55 DEG C, 30 s → 72 DEG C, 30 s) 30 cycles → 72 DEG C, 10 min；3 '-RACE reaction conditions It is 95 DEG C, 5 min → (95 DEG C, 30 s → 72 DEG C, 2 min) 5 cycles → (95 DEG C, 30 s → 55 DEG C, 30 s → 72 DEG C, 30 s) 30 cycles → 72 DEG C, 10 min.

Pcr amplification product is separated by electrophoresis with the Ago-Gel that concentration is 2%, according to the mesh of Bioinformatics Prediction Stripe size recycling purposive to product and be connected to carrier pMD18-T, then transformed competence colibacillus Escherichia coliDH5ɑ, choose Monoclonal is taken, PCR detection is carried out, selects positive colony that Hua Da gene Co., Ltd is sent to be sequenced.By the 5 ' of acquisition and 3 ' unknown sequences Column obtain its full length cDNA sequence after splicing, and the special primer of amplification ORF frame is designed according to full length cDNA sequenceNtRRS3- ORF-F(5 '-ATGGCTTATGCTGCTATTACTTCCCTCATG-3 ') andNtRRS3- ORF-R(5 '- CTAATGGATATGGACCTCAATAGAAATTG-3 '), the ORF for obtaining target gene is cloned as template using 97 leaf cDNA of rock cigarette Frame sequence.Reaction condition is 95 DEG C, 5 min → (95 DEG C, 30 s → 68 DEG C, 2 min) 5 cycles → (95 DEG C, 30 s → 55 DEG C, 30 s → 72 DEG C, 30 s) 30 cycles → 72 DEG C, 10 min；Pcr amplification product concentration Ago-Gel for 1.5 % is separated by electrophoresis, purposeful to product according to the purpose band size of Bioinformatics Prediction Recycling and be connected to carrier pDONR207, then transformed competence colibacillus Escherichia coliDH5ɑ, picking monoclonal, progress PCR detection, Selection positive colony send Hua Da gene Co., Ltd to be sequenced.

NtRRS33 ' the unknown nucleotide sequences and 5 ' unknown nucleotide sequences of gene and the electrophoretogram of ORF frame sequence are as shown in Figure 1；Its is complete Whole ORF sequence is as shown in SEQ ID No.1.The cDNA sequence overall length 3016bp of the gene contains 902 amino acid residues of coding ORF frame, length is 83bp 5 ' end non-translational regions, 3 ' the end non-translational regions and 26 bp that length is 224 bp Poly (A) tail.Utilizing the CDD(Conserved Domain Database on NCBI) software carries out conservative domain to NtRRS3 albumen Prediction, the sequence include CC motif, NB-ARC and multiple leucine rich motifs (LRR-RI) conserved domain, meet CC-NBS- The characteristics of disease-resistant gene family of LRR class.Tobacco is compared by BlastPNtRRS3The albumen of coding and the resistance of other crops Homology between albumen, itself and the homology of potato, tomato, black nightshade are respectively 61%, 60%, 56% as the result is shown.With ClustalW software, to the CC-NBS-LRR class resistance of the Four Plants such as NtRRS-3 and arabidopsis, potato, barley and wheat The result that protein amino acid sequence compares is as shown in Fig. 2-1 ~ 2-3, the CC-NBS-LRR domain amino acid sequence ratio of NtRRS3 More conservative, containing the region P-Loop, Kinase-2a, Kinase-3a and GLPL, and the amino acid sequence except conservative region is poor It is different bigger, thus deducibility tobaccoNtRRS3Gene belongs to CC-NBS-LRR genoid family.

[embodiment 2]NtRRS3Overexpression vector and RNAi carrier building and verifying

Using Gateway technology, pass through primer pairNtRRS3- OE-F(5 '-GGGGACAAGTTTGTACAAAAAAGCAGG CTTCACCATGGCTTATGCTGC-3 ') andNtRRS3- OE-R(5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTA ATGGATATGGACCTC-3 ') amplification obtain do not include termination codonNtRRS3The cDNA coding domain segment of gene, it is anti-through BP The target fragment should be connected in entry vector pDONR207, then by LR reaction by the segment from entry vector pDONR207 In be transferred in over-express vector pEarleyGate 205, construct p35S::NtRRS3- TAP Overexpression vector (Fig. 3 A) is used In genetic transformation tobacco Hongda tobacco.

Using digestion connection method, pass through the primer pair of forward direction insertionNtRRS3- RNAi-Xho I-F(5 '-ATTACTCGAG TCTAATTGATATATGGAGGTGTG-3 '),NtRRS3- RNAi-NcoI-R(5 '-ATTACCATGGACAGAGGATTGTTAA TGTTAGAG-3 ') and the primer pair be reversely inserted intoNtRRS3- RNAi-PacI-F(5 '-ACCTTAATTAATCTAATTGATATA TGGAGGTGTG-3 ') andNtRRS3- RNAi-BamHI-R(5 '-ATTAGGATCCACAGAGGATTGTTAATGTTAGAG-3 ') It expands and obtains from 97 leaf cDNA template of rock cigarette respectivelyNtRRS3Forward and reverse Insert Fragment of gene, after inserting it into digestion RNAi expression vector pGSA2285 in, buildingNtRRS3-RNAi expression vector (Fig. 3 B) is used for genetic transformation tobacco rock cigarette 97.The carrier built converts Agrobacterium EHA105 in case Transgenic Tobacco is used by frozen-thawed method.

The PCR identification of the genetic transformation and transgene tobacco of [embodiment 3] tobacco

Plasmid p35S: will be respectively provided with:NtRRS3- TAP Overexpression vector andNtRRS3-The agriculture bar of RNAi interference carrier Bacterium is by the way that in leaf disk method transformation of tobacco kind Hongda tobacco and rock cigarette 97, T0 passes through Basta and card for transgene tobacco respectively The screening of that mycin.The kanamycin screening leaf bud and root growth of 100mg/mL and 75mg/L are used respectively, during the cultivation process Inhibit the growth of Agrobacterium with 500 mg/L(Cef), 25 DEG C ± 2 DEG C of condition of culture temperature, the daily 14h daytime/10h of illumination is black Night.Precultivation medium :+1.5 mg/L 6-BA+0.1 mg/L IAA of 1/2 MS culture medium, co-culture medium: 1/2 MS training + 1.5 mg/L 6-BA+0.1 mg/L IAA+1 mg/L AS of base is supported, screening and culturing medium :+1.5 mg/L 6- of MS culture medium is induced + 1.5 mg/L 6-BA+0.1 mg/L IAA of BA+0.1 mg/L IAA+500 mgL cef+2.5 mg/L PPt, MS culture medium + 500 mg/L cef+25 mg/L Km, screening and culturing medium :+1.5 mg/L 6-BA+0.1 mg/L IAA+500 of MS culture medium + 500 mg/L cef+50 mg/L of+1.5 mg/L 6-BA+0.1 mg/L IAA of mg/L cef+4 mg/L PPt, MS culture medium Km, root media :+0.5 mg/L of+0.5 mg/L IAA+500 mg/L cef+4 mg/L PPt, MS culture medium of MS culture medium IAA+500 mg/L cef+75 mg/L Km。

Overexpress the PCR detection of transgene tobacco: according to 35S promoter andNtRRS3Gene order design pair of primers draws Object 35S-F(5 '-TGATGTGATATCTCCACTGACGTAAG-3 ') andNtRRS3- R(5 '-GACCCGACTTCGACTCCTCG- 3 '), CTAB method extracts transgenosis and non-transgenic tobacco DNA, and using the DNA of transgenic plant as template, unconverted tobacco DNA is Control, with 35S-F andNtRRS3- R primer carries out PCR amplification.PCR reaction system are as follows: 10 × buffer, 2.0 μ l, dNTP 1.5 0.5 μ l of μ l, 35S-F,NtRRS30.5 μ l of-R, Taq enzyme 0.1 μ l, ddH₂14.4 μ l of O, 1.0 μ l of template DNA, total volume are 20µl.Response procedures are as follows: 94 DEG C of 5 min → 40 cycles → 72 (94 DEG C of 30 s → 58 DEG C, 30 s → 72 DEG C, 60 s) ℃ 10min→ hold at 4℃.The results show that positive plant PCR amplification obtains the band (Fig. 4 A) of a 1650bp, 73 plants of positive plants are obtained from 84 transgenic plants.To understand whether positive plant transgenosis is expressed, CTAB method is extracted The total serum IgE of positive plant is single-stranded cDNA through PrimeScript reverse transcriptase reverse transcription, according toNtRRS3Gene order is set Count primer NtRRS3-F(5 '-TGCAGTGTCTGTCTGGATGGAATC-3 ') and NtRRS3-R(5 '- ACTGGCTCTCCAGTGCCTAATCT-3 '), RT-PCR analysis is carried out, all item is expressed in display to the 73 plants of positive plants analyzed Band, positive rate are 87%(Fig. 4 B).

RNAi interference carrier transgenic plant detection: design primer NptII-F(5 '- AGATGGATTGCACGCAGGTTCTC-3 ') and NptII-R(5 '-ATCGGGAGCGGCGATACCGTA-3 '), CTAB method is extracted Transgenosis and non-transgenic tobacco DNA, using the DNA of transgenic plant as template, unconverted tobacco DNA is control, with NptII-F It is that primer carries out PCR amplification with NptII-R.PCR reaction system are as follows: 10 × buffer 2.0 μ l, dNTP 1.5 μ l, NptII-F 0.5 0.5 μ l of μ l, NptII-R, 0.1 14.4 μ l of μ l, ddH2O of Taq enzyme, 1.0 μ l of template DNA, total volume is 20 μ l.Reaction interval Sequence are as follows: 94 DEG C of 5 min → (94 DEG C of 30 s → 55 DEG C, 30 s → 72 DEG C, 30 s) 40 cycles → 72 DEG C 10min → hold at 4℃.The results show that positive plant PCR amplification obtains the band (Fig. 5 A) of a 500bp, from 79 transgenosis 60 positive plants are obtained in plant；To understand whether positive plant transgenosis inhibits to express, it is extracted the total of sun plant RNA, with primer pair NtRRS3-RNAi-Xho I-F(5 '-ATTACTCGAGTCTAATTGATATATGGAGGTGTG-3 ') and NtRRS3-RNAi-Nco I-R(5 '-ATTACCATGGACAGAGGATTGTTAATGTTAGAG-3 ') carry out RT-PCR analysis, institute All displays band of expression (Fig. 5 B) of 60 positive plants of analysis.60 plants of T0 are obtained for transgenic positive plant by identification, Positive rate about 79%.

The identification of [embodiment 4] transgenic plant resistance to bacterial wilt

To the wild-type tobacco plants of 6 leaf phases, overexpression transgenic tobacco plant and RNAi transgenic tobacco plant 2,3 functional leafs that fall carry out inoculation processing, inject tobacco Ralstonia solanacearum bacterium solution along vein with disposable 1 mL syringe, carry out resistance Identification.Every kind of processing combination sets three biology and repeats.The disease resistance of inoculation plant entirety is specifically evaluated by disease index.

It is inoculated with transgenic plant after 20 dNtRRS3- OE growing way is still normal, and apparent necrosis occurs in blade inoculation position Spot, but stem does not occur infecting or only slight infection occurs in minority, and there is dead symptom in control group Hongda tobacco, Transgenic plantNtRRS3The Resistant expression of-OE is obviously stronger (Fig. 6 A) than wild type Hongda tobacco.63 plantsNtRRS3Transgenosis In plant identification, there are 16 plants absolutely not to fall ill, the morbidity of other showed differents, disease index is 37.67(figure 6B).

It is inoculated with transgenosis after 30 dNtRRS3There is apparent necrotic plaque and infects stem in-RNAi plant leaf inoculation position There is the phenomenon of plant death in portion, and only blade necrotic plaque occurs and do not infect downwards control group rock cigarette 97, and growing way does not go out normally Existing dead symptom, transgenosisNtRRS3The Resistant expression of-RNAi plant is obviously (Fig. 7 A) more susceptible than control group rock cigarette 97.48 StrainNtRRS3In the identification of-RNAi transgenic plant, there are 2 plants absolutely not to fall ill, others have different degrees of morbidity, disease Feelings index is 59.72(Fig. 7 B).

The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

SEQUENCE LISTING

<110>University Of Agriculture and Forestry In Fujian

<120>NtRRS3 gene and its application in tobacco resistance to bacterial wilt

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Met Ala Tyr Ala Ala Ile Thr Ser Leu Met Ser Thr Ile Gln Gln Ser

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Met Gln Val Thr Gly Leu Asn Leu Gln Ser Phe Tyr Glu Lys Leu Glu

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Ser Leu Ile Ala Ile Thr Gly Lys Pro Cys Asn Lys Ile Gly Asn Leu

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Gly Ala Leu Thr Arg Leu Glu Ala Glu Val Ile Glu Leu Ala Cys Ser

50 55 60

Thr Glu Asp Met Val Asp Ser Glu Ser Arg Asn Val Met Val Asp Leu

65 70 75 80

Lys Ser Arg Asn Val Ile Asn Ile Ile Ser Arg Thr Val Thr Phe Gly

85 90 95

Lys Leu Arg Ser Leu Leu Asn Gln Ala Ile Arg Asp Ile Asp Ser Thr

100 105 110

Thr Lys Lys Trp Ile Gly Thr Gln Thr Ser Leu Asp Leu Lys Ala Gln

115 120 125

Asn Thr Ile Leu Ala Ser Thr Ser Glu Arg Ala Leu Glu Pro Glu Asn

130 135 140

Met Met Val Gly His Glu Asn Glu Phe Glu Met Met Gln Val Gln Leu

145 150 155 160

Ala Arg Gly Ala Ser Glu Leu Glu Val Val Ser Ile Val Gly Met Gly

165 170 175

Gly Ile Gly Lys Thr Thr Leu Ala Asn Lys Ile Phe Ser Asp Pro Leu

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Ile Met Tyr His Phe Asp Ile Arg Ala Lys Val Thr Ile Ser Gln Glu

195 200 205

Tyr Cys Ala Arg Asn Gly Leu Leu Gly Leu Leu Ser Ser Ile Thr Gly

210 215 220

Lys Thr Asp Lys Pro Tyr Glu Gln Gln Asp Asp Gly Gln Leu Lys Asp

225 230 235 240

Gln Leu Gln Lys Leu Leu Lys Gly Arg Arg Tyr Leu Ile Val Ile Asp

245 250 255

Asp Ile Trp Asn Glu Ala Ala Trp Asp Asp Ile Lys Leu Cys Phe Pro

260 265 270

Asp Cys Asn Asn Arg Ser Arg Ile Leu Leu Thr Thr Arg Asn Val Lys

275 280 285

Val Ala Glu Tyr Ala Ser Ser Gly Lys Pro Pro Tyr Gln Met Arg Leu

290 295 300

Leu Asn Cys Asn Glu Ser Trp Asp Leu Leu His Lys Arg Val Phe Leu

305 310 315 320

Asn Glu Cys Phe Pro Pro Glu Phe Glu Gln Leu Gly Lys Gln Ile Ala

325 330 335

Leu Asn Cys Arg Gly Leu Pro Leu Ala Ile Ile Val Ile Ala Gly Leu

340 345 350

Leu Ser Lys Ile Gly Lys Ala Leu Asp Glu Trp Gln Met Val Val Glu

355 360 365

Asn Val Ser Ser Leu Val Ser Thr Asp Val Asp Val Gln Cys Met Arg

370 375 380

Val Leu Ala Leu Ser Tyr His His Leu Pro His Arg Leu Lys Ser Cys

385 390 395 400

Phe Leu Tyr Phe Ala Ile Phe Pro Glu Asp Glu Gln Ile Phe Val Asp

405 410 415

Lys Leu Val Glu Leu Trp Ala Val Glu Gly Phe Phe Lys Val Glu Asp

420 425 430

Met Lys Asn Ile Glu Lys Val Gly Gly Glu Phe Leu Lys Glu Leu Ile

435 440 445

Asp Arg Ser Leu Ile Ser Ile Gln Asn Leu Ser Phe Asp Gly Glu Ile

450 455 460

Glu Ser Cys Gly Met His Asp Leu Ile Arg Glu Leu Cys Leu Arg Glu

465 470 475 480

Ala His Asn Met Asn Phe Val Asn Val Ile Gly Glu Glu Asn Asp Arg

485 490 495

Asn Pro Cys Ala Gln Leu Met His Phe Ser Arg Ser Arg Ser Arg Val

500 505 510

Ser Ile Gln Leu Asn Asn Pro Asp Asp Ser Ile Glu Ala Lys Leu Ala

515 520 525

Ile Tyr Pro Glu Lys Asp Ala Arg Ser Ile Ile Cys Phe Arg Gly His

530 535 540

Trp Phe Val Gln Lys Ser Leu Arg Phe Lys Leu Val Arg Val Leu Asp

545 550 555 560

Leu Ala Leu Val Arg Cys Ser Thr Phe Pro Arg Gly Ile Leu Asp Leu

565 570 575

Ile His Leu Arg Tyr Leu Ala Leu Thr Leu Tyr Pro His Leu Glu Ser

580 585 590

Gly Lys Glu Ile Pro Ser Thr Thr Val Ile Pro Pro Leu Ile Ser Ser

595 600 605

Leu Cys Tyr Leu Gln Thr Phe Lys Leu Tyr Leu Pro Phe Ser Lys Asp

610 615 620

Leu Val Phe Pro Phe Ile Leu Pro Ser Glu Ile Leu Ala Ile Pro Gln

625 630 635 640

Leu Arg His Leu Phe Leu Asp Trp Asn Tyr Leu Gln Ser Tyr Lys Pro

645 650 655

Thr Lys Lys Ser Leu Val Leu Lys Asn Leu Gln Cys Leu Ser Gly Trp

660 665 670

Asn Pro Trp Tyr Cys Thr Gly Ser Val Phe Arg Leu Phe Pro Asn Leu

675 680 685

Lys Lys Leu Gln Ile Arg Gly Ile Pro Lys Asp Phe Ile Asn His Tyr

690 695 700

Ala Leu Phe Asp Phe Cys Asn Leu Asp Gln Leu Glu Glu Leu Glu Phe

705 710 715 720

Cys Val Thr Tyr Pro Arg Phe Gly Cys Phe Leu Asp Arg Thr Thr His

725 730 735

Gln Glu Gly Arg Leu Arg Leu His Thr Pro Pro Leu Phe Asn Thr Glu

740 745 750

Asp Ala Phe Ala Pro Phe Arg Leu Pro His Pro Asn Asp Phe Pro Gln

755 760 765

Asn Leu Lys Asn Leu Ala Phe Ser Gly Thr Phe Phe Arg Trp Lys Asp

770 775 780

Leu Ser Ile Phe Gly Lys Leu Pro Lys Leu Glu Ser Leu Lys Leu Gly

785 790 795 800

Tyr Asp Ala Phe Leu Asp Lys Glu Trp Glu Val Phe Asp Glu Val Phe

805 810 815

Pro Arg Leu Lys Phe Leu Leu Leu Glu Asp Leu Glu Ile Arg His Trp

820 825 830

Arg Ala Ser Cys Asp Asn Phe Pro Phe Leu Glu Arg Leu Phe Leu Lys

835 840 845

Gly Cys Trp Tyr Leu Asp Ser Ile Pro Gln Asn Phe Ala Asp Ile Thr

850 855 860

Thr Leu Ala Leu Ile Asp Ile Arg Arg Cys Ala Lys Ser Val Glu Asn

865 870 875 880

Ser Ala Lys Gln Ile Gln Gln Asp Met Gln Asp Asn Tyr Ala Ile Ser

885 890 895

Ile Glu Val His Ile His

900

<210> 3

<211> 25

<212> DNA

<213>artificial sequence

<400> 3

agattaggca ctggagagcc agttg 25

<210> 4

<211> 25

<212> DNA

<213>artificial sequence

<400> 4

ctgtgctgca tgctagctct atcac 25

<210> 5

<211> 45

<212> DNA

<213>artificial sequence

<400> 5

gattctgtgg ataaccgtat taccgcctta cgcgtgtaaa acgac 45

<210> 6

<211> 39

<212> DNA

<213>artificial sequence

<400> 6

accaggatct cctagggaaa cagctatgac catgttcac 39

<210> 7

<211> 30

<212> DNA

<213>artificial sequence

<400> 7

atggcttatg ctgctattac ttccctcatg 30

<210> 8

<211> 29

<212> DNA

<213>artificial sequence

<400> 8

ctaatggata tggacctcaa tagaaattg 29

<210> 9

<211> 48

<212> DNA

<213>artificial sequence

<400> 9

ggggacaagt ttgtacaaaa aagcaggctt caccatggct tatgctgc 48

<210> 10

<211> 48

<212> DNA

<213>artificial sequence

<400> 10

ggggaccact ttgtacaaga aagctgggtc ctaatggata tggacctc 48

<210> 11

<211> 33

<212> DNA

<213>artificial sequence

<400> 11

attactcgag tctaattgat atatggaggt gtg 33

<210> 12

<211> 33

<212> DNA

<213>artificial sequence

<400> 12

attaccatgg acagaggatt gttaatgtta gag 33

<210> 13

<211> 34

<212> DNA

<213>artificial sequence

<400> 13

accttaatta atctaattga tatatggagg tgtg 34

<210> 14

<211> 33

<212> DNA

<213>artificial sequence

<400> 14

attaggatcc acagaggatt gttaatgtta gag 33

<210> 15

<211> 26

<212> DNA

<213>artificial sequence

<400> 15

tgatgtgata tctccactga cgtaag 26

<210> 16

<211> 20

<212> DNA

<213>artificial sequence

<400> 16

gacccgactt cgactcctcg 20

<210> 17

<211> 24

<212> DNA

<213>artificial sequence

<400> 17

tgcagtgtct gtctggatgg aatc 24

<210> 18

<211> 23

<212> DNA

<213>artificial sequence

<400> 18

actggctctc cagtgcctaa tct 23

<210> 19

<211> 23

<212> DNA

<213>artificial sequence

<400> 19

agatggattg cacgcaggtt ctc 23

<210> 20

<211> 21

<212> DNA

<213>artificial sequence

<400> 20

atcgggagcg gcgataccgt a 21

<210> 21

<211> 33

<212> DNA

<213>artificial sequence

<400> 21

attactcgag tctaattgat atatggaggt gtg 33

<210> 22

<211> 33

<212> DNA

<213>artificial sequence

<400> 22

attaccatgg acagaggatt gttaatgtta gag 33

Claims (6)

1. tobacco CC-NBS-LRR class disease-resistant geneNtRRS3, it is characterised in that: its nucleotides sequence is classified as SEQ ID NO.1 institute Show.

2. tobacco CC-NBS-LRR class disease-resistant gene described in claim 1NtRRS3Encoded albumen, it is characterised in that: its institute The amino acid sequence of coding is shown in SEQ ID NO.2.

3. tobacco CC-NBS-LRR class disease-resistant gene described in claim 1NtRRS3Overexpression carrier, the overexpression The construction method of carrier is: using Gateway technology, passes through primer pairNtRRS3- OE-F:5 '-GGGGACAAGTTTGTAC AAAAAAGCAGGCTTCACCATGGCTTATGCTGC-3 ' andNtRRS3- OE-R:5 '-GGGGACCACTTTGTACAAGAAAG It does not include termination codon that CTGGGTCCTAATGGATATGGACCTC-3 ' amplification, which obtains,NtRRS3The code area the cDNA piece of gene Section reacts through BP and the target fragment is connected in entry vector pDONR207, then is carried the segment from introduction by LR reaction It is transferred in body pDONR207 in over-express vector pEarleyGate 205, constructs p35S: as shown in Figure 3A:NtRRS3- TAP Overexpression vector.

4. tobacco CC-NBS-LRR class disease-resistant gene described in claim 1NtRRS3RNAi interference carrier, RNAi interference The construction method of carrier is: using digestion connection method, passes through the primer pair of forward direction insertionNtRRS3- RNAi-Xho I-F:5 '- ATTACTCGAGTCTAATTGATATATGGAGGTGTG-3’、NtRRS3- RNAi-NcoI-R:5 '-ATTACCATGGACAGAG GATTGTTAATGTTAGAG-3 ' and the primer pair being reversely inserted intoNtRRS3- RNAi-PacI-F:5 '-ACCTTAATTAATCTAA TTGATATATGGAGGTGTG-3 ' andNtRRS3- RNAi-BamHI-R:5 '-ATTAGGATCCACAGAGGATTGTTAATGTTA GAG-3 ' is expanded from 97 leaf cDNA template of rock cigarette obtain respectivelyNtRRS3Forward and reverse Insert Fragment of gene, is inserted into In RNAi expression vector pGSA2285 after to digestion, construct as shown in Figure 3BNtRRS3-RNAi expression vector.

5. tobacco CC-NBS-LRR class disease-resistant gene described in claim 1NtRRS3Or albumen as claimed in claim 2 is in cigarette Application in careless resistance to bacterial wilt.

6. application according to claim 5, which is characterized in that the method for the application includes: (1) building containing having the right to want Tobacco CC-NBS-LRR class disease-resistant gene described in asking 1NtRRS3Plant expression vector；(2) constructed plant is expressed and is carried Body is gone in tobacco；(3) the transgene tobacco new varieties of screening resistance to bacterial wilt are cultivated.