CN108948161B - Application of four plant NAC transcription factor NAC089 genes in soybean and N. benthamiana and their expression vectors - Google Patents

Abstract

The invention belongs to the field of genetic engineering, and discloses the application of four plant NAC transcription factor NAC089 genes in soybean and N. benthamiana and their expression vectors in plant resistance to Phytophthora infection. Take Ben's cigarettes as an example. Transient expression of the NAC089 gene in soybean and N. benthamiana can significantly improve the resistance of N. benthamiana to Phytophthora. In the current situation where there is no particularly good method for controlling Phytophthora, the cloning of NAC089 gene is of great significance for cultivating Phytophthora-resistant crop varieties by means of genetic engineering.

Description

Application of four plant NAC transcription factor NAC089 genes in soybean and N. benthamiana and their expression vectors

technical field

The invention belongs to the field of genetic engineering, and discloses the application of four plant NAC transcription factor NAC089 genes in soybean and N. benthamiana and their expression vectors in plant resistance to Phytophthora infection.

Background technique

Phytophthora capsicum is a broad-spectrum pathogen that can cause serious damage to various economic crops such as pepper and tobacco. Phytophthora capsicum belongs to Oomycetes. Although this type of pathogen is similar to fungi in morphology, it is closely related to diatoms and cyanobacteria in evolution. Other oomycetes are ineffective. The application of plant disease-resistant varieties has become an important means of achieving disease resistance in agricultural production due to overcoming the problems of phytotoxicity and environmental pollution.

In the long-term co-evolution of plants and pathogens, plants have evolved a large number of disease resistance-related genes in order to resist pathogens, including many NAC transcription factors that play a role in disease resistance. NAC089 is one of the more special transcription factors, which contains not only NAC domain, but also transmembrane domain, which plays an important role in the immune system mediated by the endoplasmic reticulum. The introduction of NAC089 into crops can improve the resistance of crops to Phytophthora capsici. Taking N. benthamiana as an example, transient expression of the NAC089 gene in soybean and N. benthamiana can significantly improve the resistance of N. benthamiana to Phytophthora capsici. Under the current situation that there is no particularly good method for controlling Phytophthora capsici, the cloning of NAC089 is of great significance for cultivating Phytophthora capsici-resistant crop varieties by means of genetic engineering.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of four plant NAC transcription factor NAC089 genes in soybean and N. benthamiana and their expression vectors in plant resistance to Phytophthora infection in order to make up for the deficiency of current crop blight control.

The object of the present invention can be realized through the following technical solutions:

Four NAC transcription factor NAC089 genes from two plant materials of Nicotiana benthamiana and Glycine max Williams 82, NbNAC089-1, NbNAC089-2, GmNAC089-1, GmNAC089-2, the NbNAC089-1 The nucleotide sequences of , NbNAC089-2, GmNAC089-1 and GmNAC089-2 genes are shown in SEQ ID NO.1-4 in turn.

The proteins encoded by the above-mentioned four NAC transcription factor NAC089 genes are NbNAC089-1, NbNAC089-2, GmNAC089-1 and GmNAC089-2, and the amino acid sequences thereof are SEQ ID NO.5-8.

The expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant bacteria containing the above-mentioned NAC transcription factor NAC089 gene.

The recombinant expression vectors containing the above-mentioned NAC transcription factor NAC089 gene are pBinGFP ₂ :NbNAC089-1, pBinGFP ₂ :NbNAC089-2, pBinGFP ₂ :GmNAC089-1 and pBinGFP ₂ :GmNAC089-2. Construct the recombinant expression vector pBinGFP ₂ :NbNAC089-1, pBinGFP ₂ :NbNAC089-2, pBinGFP ₂ :GmNAC089-1, pBinGFP ₂ of the NAC transcription factor genes NbNAC089-1, NbNAC089-2, GmNAC089-1, GmNAC089-2: GmNAC089-2. Using the plant expression vector pBinGFP ₂ as the starting vector, the NbNAC089-1, NbNAC089-2, GmNAC089-1 and GmNAC089-2 genes were inserted into the Sam1 restriction site of pBinGFP ₂ respectively.

The NAC transcription factor NAC089 gene and its expression vector can be used to construct crop varieties resistant to Phytophthora capsicum and Phytophthora sojae.

A method for constructing a Phytophthora-resistant crop variety comprises the steps of introducing the above-mentioned gene or a recombinant expression vector into a crop plant, obtaining a positive transformed plant through resistance screening, and obtaining a Phytophthora-resistant crop variety.

The advantages and positive effects of the present invention are shown in:

The present invention clones four plant NAC transcription factor NAC089 genes from soybean and N. benthamiana, and inserts them into the expression vector pBinGFP ₂ . Introducing the vector into crops can improve the resistance of crops to Phytophthora. The NAC transcription factor can be used in genetic engineering to construct disease-resistant crops, and at the same time, it can provide powerful guidance for us to formulate disease-resistant strategies and control diseases in agricultural production.

Description of drawings

Figure 1. Transient expression of NAC089 gene in N. benthamiana improves the resistance of N. benthamiana to Phytophthora capsici.

Fig. 2 Statistics of lesion diameter of N. benthamiana leaves after expression of GFP and NAC089 protein respectively after inoculation with Phytophthora capsicum for 36 hours.

Detailed ways

The present invention is further described below in conjunction with the examples, but does not limit the protection scope of the present invention.

Example 1 Transient expression of NbNAC089-1, NbNAC089-2, GmNAC089-1 and GmNAC089-2 in N. benthamiana to improve the resistance of N. benthamiana to Phytophthora capsici

1. Test strains

The test strain Phytophthora capsicum LT263 was preserved by the Plant and Phytophthora Interaction Laboratory of the Department of Plant Diseases, Nanjing Agricultural University (a conventional strain widely used by those skilled in the art). The strains were stored in 10% V8 solid medium at 10°C.

2. Preparation of N. benthamiana seedlings for testing

Nicotiana benthamiana was sown in plastic flowerpots (d=10cm) filled with vermiculite (2-4mm), and grown in a 14h light (strong light)/10h dark greenhouse. Let it grow for 7 days, after growing two true leaves, it is transplanted to the culture soil with the volume ratio of vermiculite black soil of 5:1 and grown for 30 days, and the leaves at the same 3, 4, and 5 leaf positions are used for expression GFP and NAC089 proteins were then inoculated with Phytophthora capsicum.

3. Preparation of Soybean Seedlings for Test

Identifying the host soybean was sown in plastic flowerpots (d=10cm) with vermiculite (2-4mm), 15 seeds per pot, and grown in a greenhouse under 14h light (strong light)/10h dark, 7 10 seedlings were left for inoculation after the two true leaves grew. The variety used was Williams.

4. Extract soybean and N. benthamiana RNA, and obtain the cDNA of NAC089 gene

The leaves of soybean and Nicotiana benthamiana were collected, quick-frozen in liquid nitrogen after picking, and then placed in a mortar for cooling and grinding. The total RNA extraction and purification of soybean and N. benthamiana were carried out according to the method recommended by the Tiangen Total RNA Extraction and Purification Kit. Referring to the method provided by the RNA reverse transcription kit of Novozymes, cDNA was synthesized by reverse transcription with Oligo(dT) as the primer. The above-mentioned cDNA is used as a template, and the corresponding upstream and downstream primers are used for PCR amplification. The sequences of the primers are shown in SEQ ID NO. 9-16.

The PCR reaction system was: 2.5 μL 10×PCR reaction buffer; 1.5 μL 1.5 mM MgCl ₂ ; 0.5 μL 2.5 mM dNTPs; 0.25 μL Taq DNA polymerase (5.0 U/μL); 0.5 μL primers; 0.5 μL template; sterile water Make up to 25 μL.

The reaction program was as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 15 s, annealing at 58 °C for 15 s, extension at 72 °C for 1:30 min, 35 cycles; extension at 72 °C for 10 min; storage at 4 °C.

Electrophoresis: The PCR product was detected by 1% agarose gel electrophoresis, and the target fragment (about 1023 bp in size) was cut out on a UV gel imager. PCR products were recovered by TaKaRa gel recovery kit and sent to GenScript for sequencing. Amplification to obtain four NAC transcription factor NAC089 genes, NbNAC089-1, NbNAC089-2, GmNAC089-1, GmNAC089-2, sequencing results show that the sequence of the NbNAC089-1 gene is shown in SEQ ID NO.1, the NbNAC089 The sequence of the -2 gene is shown in SEQ ID NO.2, the sequence of the GmNAC089-1 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the GmNAC089-2 gene is shown in SEQ ID NO.4 . The amino acid sequences of the proteins encoded by the four NAC transcription factor NAC089 genes NbNAC089-1, NbNAC089-2, GmNAC089-1 and GmNAC089-2 are shown in SEQ ID NO.5-8 in turn.

5. Construction of pBinGFP ₂ : NbNAC089-1, pBinGFP ₂ : NbNAC089-2, pBinGFP ₂ : GmNAC089-1, pBinGFP ₂ : GmNAC089-2 expression vector

The pBinGFP ₂ empty vector plasmid SamI was digested. The cDNA and vector were ligated using homologous recombinase from Novezan.

The reaction system is as follows: 2 μL of 10×CEII reaction buffer; 2 μL of the above PCR product; 2 μL of the above-cut empty vector; 1 μL of homologous recombinase; sterile water to make up to 10 μL.

The reaction procedure was as follows: 30 min at 37°C. The ligated product was transformed into Escherichia coli DH5α, and the transformants were screened on Kan-resistant medium, and positive clones were picked to extract plasmids for colony PCR identification. The identified positive clones were sequenced, and the correct pBinGFP ₂ :NbNAC089-1, pBinGFP ₂ :NbNAC089-2, pBinGFP ₂ :GmNAC089-1, pBinGFP ₂ :GmNAC089-2 expression vectors were obtained.

6. Obtain Agrobacterium competent cells

Streak the Agrobacterium GV3101 strain on the LB solid medium plate, and invert at 28°C for 18-20h; place a single colony in LB liquid medium (containing 100mg/L Rif), and shake it at 28°C for 18-20 hours. 20h; 100 times the volume of antibiotic-free LB liquid medium (containing 100mg/L Rif) was added to the bacterial solution, and the OD600 was shaken to 0.3-0.5 at 28°C; after cooling on ice, 4000r/min at 4°C Centrifuge for 5 min, discard the supernatant; add 20 mmol/L CaCl ₂ solution to suspend the precipitate, centrifuge at 4000 r/min for 5 min at 4°C, discard the supernatant; add 20 mmol/L CaCl ₂ solution to suspend the cell pellet again for use.

7. pBinGFP ₂ : NbNAC089-1, pBinGFP ₂ : NbNAC089-2, pBinGFP ₂ : GmNAC089-1, pBinGFP ₂ : GmNAC089-2 expression vector and pBinGFP ₂ empty vector were transformed into Agrobacterium

The pBinGFP ₂ :NbNAC089-1, pBinGFP ₂ :NbNAC089-2, pBinGFP ₂ :GmNAC089-1, pBinGFP ₂ :GmNAC089-2 expression vectors and pBinGFP ₂ empty vector were transformed into Agrobacterium cells by electroporation; the electroporation cup was washed with 70% alcohol three times, then air dry the alcohol completely. 100ng of the expression vector was added to Agrobacterium-competent, and placed on ice for 30min. The Agrobacterium-competent cells were transferred into electroporation cups and electroporated at a voltage of 2.5 kV. After the electric shock was completed, Agrobacterium competent cells were added to 400 μL of LB liquid medium, and cultured with shaking at 28° C. for 2 h. Pipette 20 μL of bacterial solution, spread it evenly on a solid LB medium plate containing 50 mm kanamycin and 25 mm rifampicin, and invert at 28 °C for 48 h. Pick a single colony for colony PCR to obtain positive clones.

8. Transient expression of GFP and NAC089 proteins in N. benthamiana

The above-mentioned positive clones were placed in LB liquid medium and cultured with shaking at 28°C for 30h. The bacterial liquid was collected, centrifuged at 8000 rpm for 2 min, and washed three times with 10 mM MgCl ₂ . Finally, dilute the bacterial solution with ₁₀ mM MgCl to an OD ₆₀₀ value of 0.3. The bacterial solution was mixed with the bacterial solution containing the silencing inhibitor P19 in a volume ratio of 1:1. The left and right sides of the same N. benthamiana leaf were injected with a 1 mL syringe, respectively.

9. Detection of N. benthamiana disease resistance by transiently expressing GFP and NAC089 proteins

The leaves confirmed to express the protein were cut out and placed in a tray moistened with filter paper. Use a 7mm hole puncher to punch holes on the growing edge of the plate of Phytophthora capsicum LT263, and place the punched bacterium disc on the left and right sides of the tobacco leaves symmetrically. Place the tray in a 25°C incubator, dark, for 36h. The leaves were removed and then irradiated with a hand-held UV fluorometer. The darkened area is the infection area of Phytophthora capsicum LT263. The diameter of the infected area was measured with a ruler, and the leaf surface expressing GFP protein was used as the control to compare the effects of the four proteins on the resistance of N. benthamiana. Four NAC089 proteins from soybean and N. benthamiana were found to significantly increase the resistance of N. benthamiana to Phytophthora capsici LT263.

sequence listing

<110> Nanjing Agricultural University

<120> Four plant NAC transcription factor NAC089 genes and their expression vectors in soybean and N. benthamiana

<130> 2018

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1411

<212> DNA

<213> Nicotiana benthamiana

<400> 1

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ggaaggaagt atccaaacgg gtcgcaaagt aaaagggcaa ctgaatctgg ttactggaag 120

gccacaggaa aagaacgtaa tgtgaaatct ggttcgaatc tcattggcac aaagagaact 180

ctggtgttcc acactggtcg agcacctaaa ggacagagga cacattggat aatgcatgaa 240

tattgcatga gcggaaacac taattatcag aagccttcac caggacttga caggttccga 300

aattcaatta tgttaccgct tccgcactct ggagattttt attatgttac cactttccgc 360

actctggaga ttttttattgc acatattgat tctatggttg tctgccgcct ccggaagaat 420

agtgagtttc atttgaatgg caccccaaga aatcaaagga atcaactgtc tgctaccgct 480

actgcaaccg ctcagtctgg agcaggacaa ttgggcagct tggaattggt cactgtaggg 540

ggttgctgtt gttcaaagga agggagtagc agtttttatt cccattcggt tgagcagatg 600

gactctggat cggaatctga taaaccaact aaagagttct ctcagcacga ttcttctggc 660

cacttcaagg actgtgatgg tgaagaggac tggtttgctg atataatgaa agatgatatc 720

attaagctag acgattcttc gttgaatgac caacctattt ccatggttcc cagtaggcct 780

gaatcctcga tatcaactca tgaagctaga gctgcgatgt ccggtgtggc tcctttccag 840

ggcactgcta atcgaagact cagactagta agggaaaaag tagtggtgtg tccagtaaag 900

ggatccagaa gatatgaagc taccaaaaag aataaaatgg gagtgagcac aagtagttca 960

ggaagatggc tgagaaacat gttttcagta aaaacaatga agcaatacgt gatgcctata 1020

tttctggttg cctttatatt actggtcatg cttcttagta tgttgggagt ctcacagcaa 1080

gtgaaatggc ttaggtgtgt tttcgttttg acgagcagct gaagttaaat acctatctgg 1140

tatgttgttt tagtaatatt tagtgtggga gttgaaaaag gaatgagttg tttgtggtat 1200

ttgagcttaa cacttaatta tttcaattca gaagtacagg gctgtaaata gaatgtgtaa 1260

tagaggtaga actttagatg gtggctataa acaaccattt gtttgtactt tgatttgcct 1320

gcatgaatta ttacaacagc tgaaatgtta tgcccttgaa aaatctgaat ttttacaagg 1380

aaaatccctt gtgttacaac atataattgt g 1411

<210> 2

<211> 1257

<212> DNA

<213> Nicotiana benthamiana

<400> 2

atggaagatg tatcaggttc tacagatgag aaaaaaggca gagaaatagt agaaagaagt 60

gaaaataatg gtaatgacga caagaatgtg gaaatttcag tagctacagc ttctactatg 120

tttccggggt ttcggttttg tcccacggat gaagagctga tatcgtatta cttaaagaag 180

aaagttgaag ggtctgatag atgtgttgaa gttatttctg aagttgagat ttggaaacat 240

gaaccttggg atctcccaga taaatccatt gttcaatcag ataatgaatg gttcttcttc 300

tctcctcgtg gaaggaaata cccgaatggc tcgcaaagta aaagggcgac tgagtctggt 360

tactggaagg ccacaggaaa aaaacgtaat gtgaagtctg gttcaaatat gattggcaca 420

aaaagaaccc tggtgttcca cacgggtagg gcaccgaaag gacagagaac acaatggata 480

atgcatgaat attgcatgac tggaaactct tattatcagg attcagtggt tgtttgccgc 540

ctccggaaga ataatgagtt tcatttgaat gacacccacg aaaacagaag gaatcatctg 600

ggaaatgact gcgccactgc tttgtctgga gcaggaaagt tgaacagttt ggagttgata 660

aatggagggg attgctgttc aaaagagggt agcagcagtt ttagttctca ttcggttgag 720

aagattgaat ctgggtcaga ttccgataag ctgactaaag agttgccgca gcaacagtcc 780

tctagtcaat ggaaggattg tgatgaagag gactgttttg ctgatataat gaaagatgat 840

atcgttaagc tagatgattc ttcatataag gcaagataca atgtgttgcc aatagccaat 900

aggaagcttg aatcatctac aaattcatca acccaggaat tccaagatgt aatgtctcat 960

aggctgcctt tccagggtac tgctaatcga agactaagac tacaaacgga aatagtgccc 1020

cgtcgagtag ggagatatga agctaataag aagaatggcc atgctgtgga tgctagtaga 1080

catttaagga ataacattat tccagtaaga agaataaagc aatggtcgat acctctgctt 1140

ctggtttcct tggcgttact ggtcatattt ctttgtatgt ttggaattcc ccacctaaaa 1200

tggcttaggc atcttcttgt tattactcca gctgaggttg ataccagacc tgcatag 1257

<210> 3

<211> 1683

<212> DNA

<213> Soybean (Glycine max)

<400> 3

atgggtgagg cttcaggagc tggttctgct gattgtttca gccagatgat gtcgtcgatg 60

ccgggttttc gcttccatcc cacggatgag gagttggtga tgtactatct gaagcggaag 120

atttgtggga agaggctgaa gctcgacgtg attcgtgaaa ccgatgtgta taagtgggat 180

cctgaggatt tgcctgggca atctatattg aaaactggag ataggcaatg gttctttttc 240

tgtcacagag ataggaagta tcctaatggt ggaaggtcta accgagcaac cagacgtggg 300

tattggaaag caacgggaaa ggatcgtaac gtgatatgca attctaggtc agttggagtg 360

aaaaagaccc tggttttcta tgctggcaga gctcctagtg gtgagcggac tgattgggtt 420

atgcatgaat ataccttgga tgaagaggag cttaagagat gcctgggtgt taaggactat 480

tatgcacttt acaaggttta caagaaaagt ggacctggtc caaaaaatgg tgaacagtat 540

ggcgcaccat ttaatgaaga acagtgggca gatgatgaca tagtagattt taatatcaac 600

tcagctgatc aggaggctcc aaatgatgtt gataatggta atgtactgca gcctttgctt 660

gatgatgaaa tcgacaacat cattagggga attttggatg atgagcttgt ccttgatcag 720

cagcatgtga atggttatcc tgactttcct caggttgtta gtgaagaaac acaaagtact 780

gttgtggatc agttctctga ggctgtgatg ttccctgagc cagtcagaat cacacagcct 840

agcaaccaat tttttgatgc acagcctagc tttgacttca atcagccagt tacttctcat 900

ttgcatgttt ctgaagcatc tgaggtcact tctgcttcca gcattaaagc aaaggagctt 960

gattttaatg aggatggctt cttggaaatt aatgatctca ttgatactga acctatattg 1020

gcaaatatgg aaaatcctgt ggagtaccag cagtttgaag atgggttaag tgaacttgat 1080

ctgttccaag atgcacagat gtttcttcgt gacctggggc caattattca cgaaacagat 1140

tcacatgcat atacaaatgc atttgtcagc agcaatattg aaagtcaaag ttaccagttg 1200

ctgccaaatc cagaggatgc caatcaaact gtcggtgaat tctggatgca cggtgaaaga 1260

aacaccccaa gtgcatcaga aggctttgtt gattctttct cgctatcatc cccaggtgtt 1320

gtatatgaat ctgttggctt tcctacggaa ggcaataaca atcaaagtag cactgtggaa 1380

gatgttgcta caagtagctt ctcctctgct ctctgggcct ttgttgagtc aatacctaca 1440

actcctgcat cagctgctga aagtgctttg gtgaaccgag cattgaatcg aatgtctagc 1500

ttcagcaggt tgaagatcaa gcacacaaac attgctgcag caggtaaaga cactgcaact 1560

atgaagagag caggcagaaa gggacttcca ttcctcttct tccctattat tattgcttta 1620

tgtgcttttt gttgggtttt tgttggaaac ttaagactat tggggagatg tgtctctcct 1680

tga 1683

<210> 4

<211> 1812

<212> DNA

<213> Soybean (Glycine max)

<400> 4

atgggttcgg tggactgtta tccgtctcgt gtggacgatg cggccgtggt gtctctagat 60

tcgctgccgt tagggttccg attccgacct accgacgagg aactcgtcaa ttactacctg 120

cgacagaaga tcaacggtaa tggtcgcgag gtttgggtta tccgagaaat tgatgtctgc 180

aaatgggagc cttgggacat gcccggtttg tcggtggtac agactaagga tccggagtgg 240

ttcttcttct gtccacagga ccgaaagtat ccaaatgggc atcgattgaa cagagcgacg 300

aataatgggt attggaaggc gacggggaag gatcgtaaaa tcaagtctgg gacgattttg 360

attggaatga agaagacttt ggtgttctac actggtcgag cacccaaggg gaataggacc 420

aattgggtta tgcatgagta tcgccccact ttgaaggagc ttgatggtac caatcctgga 480

cagaatccat atgtcctttg tcgcttgttt aagaaacatg atgagagcct tgaagtttca 540

cactgtgatg aagcagagcc gactgcttcc actcctgtgg ctgcctatta ctccacagag 600

gaaatacaat ctgatctggc tgttgttgca ggatcgccgt cacaagttac agaagatgat 660

aagcatcagt caatgatccc tgcacactct gaggaagcaa tatccaatgt tgtaaccccg 720

gttgatcgcc gtactgatgg atatgatgct tgtgatgcac aaaatcaaat tgagttacca 780

actgcggagg agtttcaacc attgaacttt gatatatatt atgacccaag aagcgagcta 840

ctggatggca aattattctc ccctgtactt gcacatattc aaccagaatt tcattatcaa 900

gcaaacatcg aatcagatgg tcgatatggg cttcaatatg gtacaaatga gacaaacatg 960

tcagactttt tgaattcagt tgttaactgg gatcaagtac cctttgagga tcccaattgt 1020

caacagcaga gctacccctt gtttaatgtt aaggataaca tattaaacag cgacttagat 1080

tctgaacttg ccaatatgac atgtatgcaa gctggttatg cagagggggc aattgacgga 1140

aggattcctt tgttgataac ttcagaattt tgcagcacca ctggcatccc cgttgactgt 1200

gttggtgacg agcagaaaag caatgttggg ttatttcaaa acaattccca gagagctttt 1260

actgatgtta atatgggtca agtatacaat gtagttgatg attatgagca acaaagaatc 1320

tgtagtgcag ttgctagtgg caatactgga atcataagga ggcatcgtga ggtacgaaat 1380

gaacagctaa gcataaactc aacacaaggt actgcacaga ggagaattcg tttgtcaatg 1440

gctacacatg gctcaaataa gacggtgaaa agtgaaagtt gtgcacaaga agagctttat 1500

gcaaaaccag tcattgctgt gggggagaaa ggttcagaaa atcatgcttc tgatgaaagt 1560

gctactctta ctaatgatgg gaatgaactg cagaagacac ccgagtcaac cggcaaaaga 1620

aagatttctc aacaagttac aaaagcaggc tccaccttgg ggttgaaaga ccttttcttg 1680

ctcagaaggg tgcctcacat ttcaaaggct tcctcaaatc gcaccaagtg ctcttctgtt 1740

tttgtacttt ctgcctttgt aatggtctca ttagtggtct ttactgacat ttggggatat 1800

cttaaatttt aa 1812

<210> 5

<211> 373

<212> PRT

<213> Nicotiana benthamiana

<400> 5

Met Leu Pro Ala Lys Ser Val Ile Gln Ser Asp Asn Glu Trp Phe Phe

1 5 10 15

Phe Ser Pro Arg Gly Arg Lys Tyr Pro Asn Gly Ser Gln Ser Lys Arg

20 25 30

Ala Thr Glu Ser Gly Tyr Trp Lys Ala Thr Gly Lys Glu Arg Asn Val

35 40 45

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

50 55 60

Thr Gly Arg Ala Pro Lys Gly Gln Arg Thr His Trp Ile Met His Glu

65 70 75 80

Tyr Cys Met Ser Gly Asn Thr Asn Tyr Gln Lys Pro Ser Pro Gly Leu

85 90 95

Asp Arg Phe Arg Asn Ser Ile Met Leu Pro Leu Pro His Ser Gly Asp

100 105 110

Phe Tyr Tyr Val Thr Thr Phe Arg Thr Leu Glu Ile Phe Ile Ala His

115 120 125

Ile Asp Ser Met Val Val Cys Arg Leu Arg Lys Asn Ser Glu Phe His

130 135 140

Leu Asn Gly Thr Pro Arg Asn Gln Arg Asn Gln Leu Ser Ala Thr Ala

145 150 155 160

Thr Ala Thr Ala Gln Ser Gly Ala Gly Gln Leu Gly Ser Leu Glu Leu

165 170 175

Val Thr Val Gly Gly Cys Cys Cys Ser Lys Glu Gly Ser Ser Ser Phe

180 185 190

Tyr Ser His Ser Val Glu Gln Met Asp Ser Gly Ser Glu Ser Asp Lys

195 200 205

Pro Thr Lys Glu Phe Ser Gln His Asp Ser Ser Gly His Phe Lys Asp

210 215 220

Cys Asp Gly Glu Glu Asp Trp Phe Ala Asp Ile Met Lys Asp Asp Ile

225 230 235 240

Ile Lys Leu Asp Asp Ser Ser Leu Asn Asp Gln Pro Ile Ser Met Val

245 250 255

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

260 265 270

Met Ser Gly Val Ala Pro Phe Gln Gly Thr Ala Asn Arg Arg Leu Arg

275 280 285

Leu Val Arg Glu Lys Val Val Val Cys Pro Val Lys Gly Ser Arg Arg

290 295 300

Tyr Glu Ala Thr Lys Lys Asn Lys Met Gly Val Ser Thr Ser Ser Ser

305 310 315 320

Gly Arg Trp Leu Arg Asn Met Phe Ser Val Lys Thr Met Lys Gln Tyr

325 330 335

Val Met Pro Ile Phe Leu Val Ala Phe Ile Leu Leu Val Met Leu Leu

340 345 350

Ser Met Leu Gly Val Ser Gln Gln Val Lys Trp Leu Arg Cys Val Phe

355 360 365

Val Leu Thr Ser Ser

370

<210> 6

<211> 418

<212> PRT

<213> Nicotiana benthamiana

<400> 6

Met Glu Asp Val Ser Gly Ser Thr Asp Glu Lys Lys Gly Arg Glu Ile

1 5 10 15

Val Glu Arg Ser Glu Asn Asn Gly Asn Asp Asp Lys Asn Val Glu Ile

20 25 30

Ser Val Ala Thr Ala Ser Thr Met Phe Pro Gly Phe Arg Phe Cys Pro

35 40 45

Thr Asp Glu Glu Leu Ile Ser Tyr Tyr Leu Lys Lys Lys Val Glu Gly

50 55 60

Ser Asp Arg Cys Val Glu Val Ile Ser Glu Val Glu Ile Trp Lys His

65 70 75 80

Glu Pro Trp Asp Leu Pro Asp Lys Ser Ile Val Gln Ser Asp Asn Glu

85 90 95

Trp Phe Phe Phe Ser Pro Arg Gly Arg Lys Tyr Pro Asn Gly Ser Gln

100 105 110

Ser Lys Arg Ala Thr Glu Ser Gly Tyr Trp Lys Ala Thr Gly Lys Lys

115 120 125

Arg Asn Val Lys Ser Gly Ser Asn Met Ile Gly Thr Lys Arg Thr Leu

130 135 140

Val Phe His Thr Gly Arg Ala Pro Lys Gly Gln Arg Thr Gln Trp Ile

145 150 155 160

Met His Glu Tyr Cys Met Thr Gly Asn Ser Tyr Tyr Gln Asp Ser Val

165 170 175

Val Val Cys Arg Leu Arg Lys Asn Asn Glu Phe His Leu Asn Asp Thr

180 185 190

His Glu Asn Arg Arg Asn His Leu Gly Asn Asp Cys Ala Thr Ala Leu

195 200 205

Ser Gly Ala Gly Lys Leu Asn Ser Leu Glu Leu Ile Asn Gly Gly Asp

210 215 220

Cys Cys Ser Lys Glu Gly Ser Ser Ser Phe Ser Ser His Ser Val Glu

225 230 235 240

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

245 250 255

Gln Gln Gln Ser Ser Ser Gln Trp Lys Asp Cys Asp Glu Glu Asp Cys

260 265 270

Phe Ala Asp Ile Met Lys Asp Asp Ile Val Lys Leu Asp Asp Ser Ser

275 280 285

Tyr Lys Ala Arg Tyr Asn Val Leu Pro Ile Ala Asn Arg Lys Leu Glu

290 295 300

Ser Ser Thr Asn Ser Ser Thr Gln Glu Phe Gln Asp Val Met Ser His

305 310 315 320

Arg Leu Pro Phe Gln Gly Thr Ala Asn Arg Arg Leu Arg Leu Gln Thr

325 330 335

Glu Ile Val Pro Arg Arg Val Gly Arg Tyr Glu Ala Asn Lys Lys Asn

340 345 350

Gly His Ala Val Asp Ala Ser Arg His Leu Arg Asn Asn Ile Ile Pro

355 360 365

Val Arg Arg Ile Lys Gln Trp Ser Ile Pro Leu Leu Leu Val Ser Leu

370 375 380

Ala Leu Leu Val Ile Phe Leu Cys Met Phe Gly Ile Pro His Leu Lys

385 390 395 400

Trp Leu Arg His Leu Leu Val Ile Thr Pro Ala Glu Val Asp Thr Arg

405 410 415

Pro Ala

<210> 7

<211> 560

<212> PRT

<213> Soybean (Glycine max)

<400> 7

Met Gly Glu Ala Ser Gly Ala Gly Ser Ala Asp Cys Phe Ser Gln Met

1 5 10 15

Met Ser Ser Met Pro Gly Phe Arg Phe His Pro Thr Asp Glu Glu Leu

20 25 30

Val Met Tyr Tyr Leu Lys Arg Lys Ile Cys Gly Lys Arg Leu Lys Leu

35 40 45

Asp Val Ile Arg Glu Thr Asp Val Tyr Lys Trp Asp Pro Glu Asp Leu

50 55 60

Pro Gly Gln Ser Ile Leu Lys Thr Gly Asp Arg Gln Trp Phe Phe Phe

65 70 75 80

Cys His Arg Asp Arg Lys Tyr Pro Asn Gly Gly Arg Ser Asn Arg Ala

85 90 95

Thr Arg Arg Gly Tyr Trp Lys Ala Thr Gly Lys Asp Arg Asn Val Ile

100 105 110

Cys Asn Ser Arg Ser Val Gly Val Lys Lys Thr Leu Val Phe Tyr Ala

115 120 125

Gly Arg Ala Pro Ser Gly Glu Arg Thr Asp Trp Val Met His Glu Tyr

130 135 140

Thr Leu Asp Glu Glu Glu Leu Lys Arg Cys Leu Gly Val Lys Asp Tyr

145 150 155 160

Tyr Ala Leu Tyr Lys Val Tyr Lys Lys Ser Gly Pro Gly Pro Lys Asn

165 170 175

Gly Glu Gln Tyr Gly Ala Pro Phe Asn Glu Glu Gln Trp Ala Asp Asp

180 185 190

Asp Ile Val Asp Phe Asn Ile Asn Ser Ala Asp Gln Glu Ala Pro Asn

195 200 205

Asp Val Asp Asn Gly Asn Val Leu Gln Pro Leu Leu Asp Asp Glu Ile

210 215 220

Asp Asn Ile Ile Arg Gly Ile Leu Asp Asp Glu Leu Val Leu Asp Gln

225 230 235 240

Gln His Val Asn Gly Tyr Pro Asp Phe Pro Gln Val Val Ser Glu Glu

245 250 255

Thr Gln Ser Thr Val Val Asp Gln Phe Ser Glu Ala Val Met Phe Pro

260 265 270

Glu Pro Val Arg Ile Thr Gln Pro Ser Asn Gln Phe Phe Asp Ala Gln

275 280 285

Pro Ser Phe Asp Phe Asn Gln Pro Val Thr Ser His Leu His Val Ser

290 295 300

Glu Ala Ser Glu Val Thr Ser Ala Ser Ser Ser Ile Lys Ala Lys Glu Leu

305 310 315 320

Asp Phe Asn Glu Asp Gly Phe Leu Glu Ile Asn Asp Leu Ile Asp Thr

325 330 335

Glu Pro Ile Leu Ala Asn Met Glu Asn Pro Val Glu Tyr Gln Gln Phe

340 345 350

Glu Asp Gly Leu Ser Glu Leu Asp Leu Phe Gln Asp Ala Gln Met Phe

355 360 365

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

370 375 380

Thr Asn Ala Phe Val Ser Ser Asn Ile Glu Ser Gln Ser Tyr Gln Leu

385 390 395 400

Leu Pro Asn Pro Glu Asp Ala Asn Gln Thr Val Gly Glu Phe Trp Met

405 410 415

His Gly Glu Arg Asn Thr Pro Ser Ala Ser Glu Gly Phe Val Asp Ser

420 425 430

Phe Ser Leu Ser Ser Pro Gly Val Val Tyr Glu Ser Val Gly Phe Pro

435 440 445

Thr Glu Gly Asn Asn Asn Gln Ser Ser Thr Val Glu Asp Val Ala Thr

450 455 460

Ser Ser Phe Ser Ser Ala Leu Trp Ala Phe Val Glu Ser Ile Pro Thr

465 470 475 480

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

485 490 495

Arg Met Ser Ser Phe Ser Arg Leu Lys Ile Lys His Thr Asn Ile Ala

500 505 510

Ala Ala Gly Lys Asp Thr Ala Thr Met Lys Arg Ala Gly Arg Lys Gly

515 520 525

Leu Pro Phe Leu Phe Phe Pro Ile Ile Ile Ala Leu Cys Ala Phe Cys

530 535 540

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

545 550 555 560

<210> 8

<211> 604

<212> PRT

<213> Soybean (Glycine max)

<400> 8

Met Gly Ser Val Asp Cys Tyr Pro Ser Arg Val Asp Asp Ala Ala Val

1 5 10 15

Val Ser Leu Asp Ser Leu Pro Leu Gly Phe Arg Phe Arg Pro Thr Asp

20 25 30

Glu Glu Leu Val Asn Tyr Tyr Leu Arg Gln Lys Ile Asn Gly Asn Gly

35 40 45

Arg Glu Val Trp Val Ile Arg Glu Ile Asp Val Cys Lys Trp Glu Pro

50 55 60

Trp Asp Met Pro Gly Leu Ser Val Val Gln Thr Lys Asp Pro Glu Trp

65 70 75 80

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

85 90 95

Asn Arg Ala Thr Asn Asn Gly Tyr Trp Lys Ala Thr Gly Lys Asp Arg

100 105 110

Lys Ile Lys Ser Gly Thr Ile Leu Ile Gly Met Lys Lys Thr Leu Val

115 120 125

Phe Tyr Thr Gly Arg Ala Pro Lys Gly Asn Arg Thr Asn Trp Val Met

130 135 140

His Glu Tyr Arg Pro Thr Leu Lys Glu Leu Asp Gly Thr Asn Pro Gly

145 150 155 160

Gln Asn Pro Tyr Val Leu Cys Arg Leu Phe Lys Lys His Asp Glu Ser

165 170 175

Leu Glu Val Ser His Cys Asp Glu Ala Glu Pro Thr Ala Ser Thr Pro

180 185 190

Val Ala Ala Tyr Tyr Ser Thr Glu Glu Ile Gln Ser Asp Leu Ala Val

195 200 205

Val Ala Gly Ser Pro Ser Gln Val Thr Glu Asp Asp Lys His Gln Ser

210 215 220

Met Ile Pro Ala His Ser Glu Glu Ala Ile Ser Asn Val Val Thr Pro

225 230 235 240

Val Asp Arg Cys Thr Asp Gly Tyr Asp Ala Cys Asp Ala Gln Asn Gln

245 250 255

Ile Glu Leu Pro Thr Ala Glu Glu Phe Gln Pro Leu Asn Phe Asp Ile

260 265 270

Tyr Tyr Asp Asp Pro Arg Ser Glu Leu Leu Asp Gly Lys Leu Phe Ser

275 280 285

Pro Val Leu Ala His Ile Gln Pro Glu Phe His Tyr Gln Ala Asn Ile

290 295 300

Glu Ser Asp Gly Arg Tyr Gly Leu Gln Tyr Gly Thr Asn Glu Thr Asn

305 310 315 320

Met Ser Asp Phe Leu Asn Ser Val Val Asn Trp Asp Gln Val Pro Phe

325 330 335

Glu Asp Pro Asn Cys Gln Gln Gln Ser Tyr Pro Leu Phe Asn Val Lys

340 345 350

Asp Asn Ile Leu Asn Ser Asp Leu Asp Ser Glu Leu Ala Asn Met Thr

355 360 365

Cys Met Gln Ala Gly Tyr Ala Glu Gly Ala Ile Asp Gly Arg Ile Pro

370 375 380

Leu Leu Ile Thr Ser Glu Phe Cys Ser Thr Thr Gly Ile Pro Val Asp

385 390 395 400

Cys Val Gly Asp Glu Gln Lys Ser Asn Val Gly Leu Phe Gln Asn Asn

405 410 415

Ser Gln Arg Ala Phe Thr Asp Val Asn Met Gly Gln Val Tyr Asn Val

420 425 430

Val Asp Asp Tyr Glu Gln Gln Arg Ile Cys Ser Ala Val Ala Ser Gly

435 440 445

Asn Thr Gly Ile Ile Arg Arg His Arg Glu Val Arg Asn Glu Gln Leu

450 455 460

Ser Ile Asn Ser Thr Gln Gly Thr Ala Gln Arg Arg Ile Arg Leu Ser

465 470 475 480

Met Ala Thr His Gly Ser Asn Lys Thr Val Lys Ser Glu Ser Cys Ala

485 490 495

Gln Glu Glu Leu Tyr Ala Lys Pro Val Ile Ala Val Gly Glu Lys Gly

500 505 510

Ser Glu Asn His Ala Ser Asp Glu Ser Ala Thr Leu Thr Asn Asp Gly

515 520 525

Asn Glu Leu Gln Lys Thr Pro Glu Ser Thr Gly Lys Arg Lys Ile Ser

530 535 540

Gln Gln Val Thr Lys Ala Gly Ser Thr Leu Gly Leu Lys Asp Leu Phe

545 550 555 560

Leu Leu Arg Arg Val Pro His Ile Ser Lys Ala Ser Ser Asn Arg Thr

565 570 575

Lys Cys Ser Ser Val Phe Val Leu Ser Ala Phe Val Leu Val Ser Leu

580 585 590

Val Val Phe Thr Asp Ile Trp Gly Tyr Leu Lys Phe

595 600

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<400> 9

atgttaccag caaagtccgt 20

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<400> 10

cacaattata tgttgtaaca c 21

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<400> 11

atggaagatg tatcaggttc 20

<210> 12

<211> 19

<212> DNA

<213> Artificial sequence

<400> 12

ctatgcaggt ctggtatca 19

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<400> 13

atgggtgagg cttcaggagc 20

<210> 14

<211> 23

<212> DNA

<213> Artificial sequence

<400> 14

tcaaggagag acacatctcc cca 23

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<400> 15

atgggttcgg tggactgtta 20

<210> 16

<211> 22

<212> DNA

<213> Artificial sequence

<400> 16

ttaaaattta agatatcccc aa 22

Claims (9)

1. NAC transcription factorNAC089A gene which isNbNAC089-2SaidNbNAC089-2The nucleotide sequence of (A) is shown in SEQ ID NO. 2.

2. The protein encoded by the gene of claim 1, which has the amino acid sequence shown in SEQ ID No. 6.

3. Comprising the NAC transcription factor of claim 1NAC089An expression cassette for a gene.

4. Comprising the NAC transcription factor of claim 1NAC089Recombinant expression vectors for genes.

5. Comprising the NAC transcription factor of claim 1NAC089Transgenic recombinant bacteria of the gene.

6. The recombinant expression vector according to claim 4, wherein the recombinant expression vector is the recombinant expression vector of claim 1NbNAC089-2Insert pBinGFP₂SmaI cleavage sites of the vector.

7. The NAC transcription factor of claim 1NAC089The gene is applied to the construction of a phytophthora capsici resistant Nicotiana benthamiana variety.

8. Use of the recombinant expression vector of claim 4 or 6 for the construction of P.capsorum resistant Nicotiana benthamiana.

9. A method for constructing a tobacco of Nicotiana benthamiana resistant to Phytophthora capsici, characterized in that the gene of claim 1 or the recombinant expression vector of claim 4 is introduced into a tobacco plant of Nicotiana benthamiana, and a positive transformed plant is obtained by resistance selection, thereby obtaining the tobacco of Nicotiana benthamiana resistant to Phytophthora capsici.

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