CN114058626B - Application of Nup50A gene in improving resistance of plants to botrytis cinerea infection - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering, and specifically relates to the application of a Nup50A gene in improving plant resistance to Botrytis cinerea infection. By isolating and cloning the Nup50A gene from tomato, the tomato Nup50A gene knockout recombinant vector pTX-Nup50A, Gene knockout recombinant strain LBA4404-pTX-Nup50A and its tomato Nup50A gene knockout homozygous strain, the present invention has confirmed that knockout of Nup50A gene can effectively improve the ability of tomato to resist Botrytis cinerea infection, tomato Nup50A gene knockout strain and Compared with the wild-type strain, the lesion diameter on the leaves and the amount of gray mold in the leaves were significantly reduced. The invention proves that the Nup50A gene has application value in improving tomato gray mold resistance through experiments, and lays a good theoretical and application foundation for using the Nup50A gene to cultivate disease-resistant plants.

Description

Application of a Nup50A Gene in Improving Plant Resistance to Botrytis Botrytis Infection

Technical field:

The invention belongs to the field of plant genetic engineering, and specifically relates to an application of Nup50A gene in improving plant resistance to Botrytis cinerea infection.

Background technique:

The eukaryotic nucleoporin complex mediates the nucleoplasmic shuttling of macromolecules such as proteins and RNAs, and plays an extremely important role in the maintenance and regulation of cell life activities. The nuclear pore complex (NPC) is the largest multiprotein complex in cells, and studies have shown that it is composed of at least 30 nucleoporins (Nup) in multiple copies. Its basic composition is highly conserved in vertebrates, yeast and plants. The relevant research on plant nucleoporins is still rare, and the functions of only a few plant nucleoporins have been initially identified. Existing research results show that they are widely involved in various physiological and biochemical processes.

1. Hormone signal transduction

Arabidopsis SAR1 and SAR3, mutations that affect the nuclear export of mRNA and the subcellular localization of the auxin-responsive transcriptional repressor IAA17 protein, can partially restore the auxin-resistant phenotype of axr-1, suggesting their involvement in auxin Signal transduction pathway. In addition, Arabidopsis Trp/Mlp1p/Mlp2p mutants also exhibited similar phenotypes to sar1 and sar3. In addition to participating in auxin signal transduction, the Nup160 mutation also enhanced the response of Arabidopsis to ethylene, suggesting that it may be involved in the interaction between auxin and ethylene signaling.

2. Response to chilling stress

Arabidopsis Nup160 mutation can inhibit the expression of CBF3 under chilling stress and reduce the resistance of plants to chilling stress. As a negative regulator of low temperature signal transduction in plants, HOS1 can regulate the expression of genes related to chilling injury and freezing stress. Overexpression of HOS1 in Arabidopsis can inhibit the expression of CBF family genes and enhance the sensitivity of Arabidopsis to freezing injury, while hos1 mutation will lead to high-level expression of low temperature response genes and improve plant resistance to chilling stress. At the same time, HOS1 protein has a RING-finger domain, which can perform the function of E3 ubiquitin ligase, and can specifically mediate the degradation of ICE1 protein under cold-induced conditions, thereby weakening the response of Arabidopsis to low temperature.

3. Flowering and growth of plants

Arabidopsis Nup160, Nup96 and NUA/TPR are not only involved in the above physiological processes, but also affect other plant development processes, such as flowering time and fertility.

4. Plant responses and symbiosis to pathogens

Mutation of MOS3 in Arabidopsis (homologous gene of animal Nup96) will enhance the sensitivity of Arabidopsis to pathogens. Mutations in MOS7 (an ortholog of animal Nup88) lead to defects in Arabidopsis R protein-mediated immunity as well as basal and systemic acquired resistance. Mutations in Seh1 and Nup160 also disrupt basal plant resistance to pathogens. Nup75 in Nicotiana benthamiana is involved in ethylene-mediated phytodefensin production after Phytophthora infestans infection. Nucleoporin mutations also affect plant-microbe symbiosis. For example, mutations of Nup85, Nup133, and NENA (the homologous gene of SEC13) in japonicus japonicus will affect nodulation.

It can be seen that nucleoporins play a pivotal role in various aspects of plant growth and development and responses to environmental stress. However, our studies on the function and molecular mechanism of plant nucleoporins are very few and mainly focus on Arabidopsis. In mouse cells, Nup50A is a hydrophilic protein containing 5 FG repeats, its localization can move between NPC and nucleoplasm, it is necessary for cell differentiation and survival of primordial germ cells and participates in DNA damage repair. The biological function of Nup50A in plants has not been reported.

Invention content:

The purpose of the present invention is to overcome the deficiencies and defects in the prior art, and provide an application of Nup50A gene in improving plant resistance to Botrytis cinerea infection. The invention isolates and clones the Nup50A gene from tomato, constructs the Nup50A gene knockout recombinant vector pTX-Nup50A and its homozygous strain, and confirms that the Nup50A gene can effectively improve the ability of tomato to resist Botrytis cinerea infection.

To achieve the purpose of the above invention, the present invention provides an application of Nup50A gene in improving plant resistance to Botrytis cinerea infection, the nucleotide sequence of the Nup50A gene is shown in SEQ ID NO.1.

Further, the application includes the following steps:

(1) Construction of the Nup50A gene knockout recombinant vector: design primers according to the CDS sequence of the Nup50A gene, and use the plasmid pTX-043 as a template to carry out PCR amplification; after the amplification product is purified, use the restriction endonuclease Bsa I to performing enzyme digestion, recovering the enzyme digestion product and connecting it with the pTX-041 plasmid to obtain the NUP50A gene knockout recombinant vector;

(2) Constructing a Nup50A gene knockout recombinant strain: transforming the Nup50A gene knockout recombinant vector into Agrobacterium to obtain a Nup50A gene knockout recombinant strain;

(3) Construction of Nup50A gene knockout strain: transform the Nup50A gene knockout recombinant strain into plant cotyledons, use the antibiotic Kan to screen the transformed callus, and cultivate until regenerated plants are obtained, that is, the Nup50A gene knockout strain is obtained. Remove strains.

Further, the sequence of the primer in the step (1) is

Nup50A-F0:

5'-ATATATGGTCTCGTTTGCCCCTTTGCAGCAATCCGCTGTTTTGAGCTAGAAATAGC-3';

Nup50A-R0:

5’-ATTATTGGTCTCGAAACGGCTGAAACCAAGGAAGGCACAAACTACA

CTGTTAGATTC-3'.

Further, the plasmid is pTX-Nup50A.

Further, the gene knockout recombinant vector is pTX-Nup50A.

Further, the Agrobacterium is LBA4404.

Further, the culture conditions in the step (3) are 16h light (26°C)/8h dark (18°C).

Further, compared with the wild-type strain of the Nup50A gene knockout strain, the lesion diameter on the leaves is significantly reduced.

Furthermore, compared with the wild-type line, the amount of Botrytis cinerea on the leaves of the tomato Nup50A gene knockout line was significantly reduced.

Further, the plant is tomato.

Compared with the prior art, the advantages and technical effects of the present invention: the present invention isolates and clones the Nup50A gene from tomato, and further connects the Nup50A gene target point information to the gene knockout vector pTX-041 to obtain the Nup50A gene knockout In addition to the recombinant vector pTX-Nup50A, the gene knockout recombinant strain LBA4404-pTX-Nup50A was obtained by using Agrobacterium, and the cotyledon of tomato was infected and transformed to obtain a homozygous Nup50A gene knockout strain. By studying the gene knockout transgenic strain phenotypes and compared them to lesions on leaves of wild-type tomatoes grown under the same conditions. The present invention proves for the first time that the Nup50A gene has the function of regulating the resistance of tomato to Botrytis cinerea through experimental analysis, and knocking out the Nup50A gene can effectively improve the ability of tomato to resist Botrytis cinerea; Lesions were significantly reduced, and the amount of gray mold was also significantly lower than that of wild-type tomato lines. The technical scheme of the invention has application value for Nup50A gene in improving the disease resistance of tomato to Botrytis cinerea, and also lays a good theoretical and application foundation for cultivating disease-resistant and high-yielding tomato varieties using Nup50A gene.

Description of drawings:

Figure 1 shows the PCR amplification of the Nup50A target sequence, where M is a DNA marker, and 1-3 are the PCR products of the pTX-Nup50A target sequence.

Figure 2 is the result of the knockout vector construction, where M is the DNA marker, and 1-4 are the screening of recombinant plasmids.

Fig. 3 is the sequence comparison result of the mutation site of the homozygous knockout mutant of T2 generation of Nup50A gene.

Fig. 4 is the result of the disease resistance change of the Nup50A gene knockout homozygous line;

Fig. 5 is the result of the lesion diameter on the leaves of the Nup50A gene knockout homozygous line, (**p<0.01).

Fig. 6 shows the results of Botrytis cinerea in the leaves of the Nup50A gene knockout homozygous line (**p<0.01).

Detailed ways:

The present invention will be further described below through embodiments and in conjunction with the accompanying drawings.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments, etc. used in the following examples are commercially available unless otherwise specified. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

The PQB vector, pTX-041 vector and pTX-043 vector described in the following examples, the public can obtain this biological material from the applicant, this biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes .

Example 1:

Obtaining of Tomato Nup50A Gene Knockout Mutants and Homozygous Lines

1. Obtainment of tomato Nup50A gene knockout mutant

(1) Vector construction

Obtain the CDS sequence of tomato NUP50A gene in GeneBank database, its length is 1347bp, the protein that code contains 449 amino acids, its nucleotide sequence is as shown in SEQ ID NO.1 (underline is target sequence), the amino acid of its code The sequence is shown in SEQ ID NO.2. Enter the full sequence of the Nup50A gene on the CRISPR direct website, and screen suitable targets according to the results. The selected targets are: CCCCTTTGCAGCAATCCGCT and GGCTGAAACCAAGGAAGGCA, and design primers accordingly. The primer sequence is as follows: Nup50A-F0:

5'-ATATATGGTCTCGTTTGCCCCTTTGCAGCAATCCGCTGTTTTGAGCTAGAAATAGC-3 (SEQ ID NO. 3);

Nup50A-R0:

5’-ATTATTGGTCTCGAAACGGCTGAAACCAAGGAAGGCACAAACTACA

CTGTTAGATTC-3' (SEQ ID NO.4).

The plasmid pTX-043 was used as a template, NUP50A-F0 and Nup50A-R0 were used as primers, and the target sequence was amplified by PCR with high-fidelity enzymes. The electrophoresis results were shown in Figure 1, and the amplified products were recovered and purified; Restriction endonuclease Bsa I digested the purified PCR product and the pTX-041 plasmid, recovered them separately, and ligated overnight under the catalysis of T4 ligase; after the ligation was completed, the ligated product was transformed into Escherichia coli JM109 and selected containing The positive clone of the recombinant plasmid was sent to the company for sequencing, and finally the recombinant plasmid with correct sequencing was named pTX-Nup50A. Wherein, the sequences of the primers used during plasmid screening are as follows:

pTX-Fw:

5'-AGCGGATAACAATTTCACACAGGA-3' (SEQ ID NO.5);

pTX-Rv:

5'-GCAGGCATGCAAGCTTATTGG-3' (SEQ ID NO. 6).

(2) Preparation of transgenic plants

The recombinant vector pTX-Nup50A was transformed into Agrobacterium LBA4404 to obtain the recombinant strain LBA4404-pTX-NUP50A.

According to the Agrobacterium-mediated transgenic method, the recombinant strain LBA4404-pTX-Nup50A was used to transform the tomato cotyledon; then the antibiotic Kan was used to screen the transformed callus, and an empty vector was set as a control; the culture was replaced every 2-3 weeks The culture conditions were 16h light (26°C)/8h dark (18°C) until regenerated shoots were obtained.

The genomic DNA of the regenerated seedlings was extracted respectively, and the partial sequence of the Nup50A gene including the target site was subjected to PCR amplification. The sequences of the primers used were:

Nup50A-F1261:

5'-GACAATCCTG GTCTCGATGA TGAT-3' (SEQ ID NO.7);

Nup50A-R1920:

5'-CCCAGTCCCT GAAAATCCTG TG-3' (SEQ ID NO. 8).

The obtained PCR product was detected by electrophoresis and then cut into a gel and sent to the biological company for sequencing; if there was a set of peaks near the sequence target or a partial deletion of the sequence between the two target points, it indicated that the plant was a Nup50A knockout positive plant. The sequencing results showed that the present invention obtained three Nup50A knockout positive plants, which were labeled as Nup50A-4-12, Nup50A-7-6 and Nup50A-11-1 respectively.

2. Obtainment of tomato NUP50A gene knockout homozygous lines

Harvest the seeds of T0 generation knockout positive plants Nup50A-4, Nup50A-7 and Nup50A-11 as the T1 generation; sow the obtained T1 generation seeds respectively, and take 5-10 plants for each knockout line after 2-3 weeks Genomic DNA was extracted from the plants, and PCR amplification was performed with Nup50A-F1261 and Nup50A-R1920 primer pairs. After the PCR products were recovered, they were connected to the pMD-18T vector, and positive clones were selected and sent to the company for sequencing. Four samples of plants of each knockout line were sent for cloning. The sequencing results showed that the 4 clone samples of each knockout line had no set peaks, and the position of the target site was mutated or there was a partial deletion between the two target sites, so it was determined that the samples were all Nup50A gene knockout homozygous strains Tie.

The three positive Nup50A gene knockout homozygous lines identified were named knockout lines Nup50A-4-12, Nup50A-7-6 and Nup50A-11-1, respectively. Compared with the wild-type plants, the knockout line Nup50A-4-12 gene has an extra T behind the 528th position, and a frameshift mutation occurs; the knockout line Nup50A-7-6 gene lacks the 528th position of the gene, and a frameshift occurs Mutation; in the knockout line Nup50A-11-1 gene, positions 528-531 of the gene are deleted, and a frameshift mutation occurs. See Figure 3 for the specific alignment sequence information. The obtained Nup50A gene knockout homozygous lines were transplanted into large pots, and the seeds were harvested for subsequent experiments.

Example 2:

Phenotype Identification of Positive Tomato Nup50A Gene Knockout Plants

1. The seeds of Nup50A gene knockout lines Nup50A-4-12, Nup50A-7-6 and Nup50A-11-1 and wild-type tomato (WT) were sown in nutrient soil respectively, and cultivated in a greenhouse, wherein the culture conditions It is 16h light (26°C)/8h dark (18°C). Then, select 3 groups of NUP50A knockout homozygous lines and wild type of about 4 weeks old with the same size and growth potential, each group contains 3 tomato plants, inoculate tomato leaves with spore liquid with a spore concentration of 106, and place the leaves in In a plastic box, put a layer of plastic wrap on it to maintain 100% humidity, and keep it at 22°C for 24-48 hours to observe the incidence of tomato leaves and make statistics. The results are shown in Figure 4 and Figure 5, compared with the wild type, the lesion diameter on the leaves of the Nup50A gene knockout homozygous line was significantly reduced.

2. Total RNA was extracted from the tomato leaves infected by Botrytis cinerea, and the amount of Botrytis in the leaves was detected by qRT-PCR, and tomato Actin was used as an internal reference. The primer sequences used were as follows:

β-Tubulin-F:

5'-ACCGTTCCAGAGTTGACTCAA-3' (SEQ ID NO.9);

β-Tubulin-R:

5'-GCAAGAAAAGCCTTTCTTCTGA-3' (SEQ ID NO. 10).

The results are shown in Figure 6, the amount of Botrytis cinerea in the NUP50A gene knockout homozygous line was significantly lower than that of wild-type tomato.

The above evidence shows that Nup50A gene plays an important role in tomato resistance to Botrytis cinerea infection, and knocking out Nup50A gene can effectively improve the ability of tomato to resist Botrytis cinerea infection, so tomato Nup50A gene has the function of regulating tomato resistance to Botrytis cinerea infection.

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still understand the foregoing embodiments. Modifications are made to the technical solutions described, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed in the present invention.

Sequence listing:

SEQ ID NO.1

ATGGGAGATGCCGAGAACTTCTCTTCAACCATCAAAAGAAAAGGGCAGCTAT

AAAGGAATTATCACGAGACAATCCTGGTCTCGATGATGATGAAAATGAAGC

TGAGCTAGAGACTGGGTCTTTTCAAGAAAGCAAGTGAGGAGGTGATGGCAA

GTAGAAGAATTGTCAAAGTTCGTCGCAGTCAAACTACTTCATCTACAGCTA

CACCTTCAGCTAACCCCTTTGCAGCAATCCGCTTGGTTCCACCCACTGAGT

CCAGTATCCCGTCTACTGTGATAACAAGTGAAGTTGAGAGTGGGATA

ACAAGTTCAGGTAAACCAGAAGATAAAAATGACATTGGTGAGGCAACTAG

AAAGGAGACAAATGATGTGTGTAAGGAAGAGTTGAAGGAATCTGATCAAA

TTTCTAAGCCATCAGAAGGTAATGTTGATGAATCCAATGTTGTCAAGGAGA

AAGTTGAAACTCCTAATGAGGCTGACAAACCTGAATCTGCTGAAGAGAAG

GTAGCAGATGATGAGAAAGTCCAGGCTGAAACCAAGGAAGGCACAGTAGT

TGAGAAGAGCGAAAATGACAGTAAGAAGGATGTGGAAGTCGAGAAAACA

AAGAATGAAGAACAAAATGATGCTGGTGGTGAGAAAAGTGAGAAGGGTG

CAGAAACTGCTTCTTTTAGCTCATTCCAACAACTCTCAAGTGGCCAAAATG

CTTTCACAGGATTTTCAGGGACTGGGTTTCCAGCACTACTTTTCTCCTTTGG

AGGTATTTCAAAAAGAGGGATCTTTCTCTAGGTTTTGGTTCTGAATCAGGTGCT

GGTTCCTCTCTTTGGAGCAAAAAGTGACCAGTCACCATTTGGACTTAATCTT

CCTACTAATGGAAGTACTTCTCTGTTTGGAAACTCGGGGTCATCC

CTTGTGAATAAGAGGTGAGGGTACTGGATTTCCTTCCAAAGAAGAGGTCACT

GTTGAAACAGGGGAGGAAAATGAAAAACCCGTATTCGCAGCTGATTCCGT

GCTGTTTGAATATCTTAATGGAGGGTGGAAAGAGCGGGGGAAGGGAGAAC

TAAAGGTCAATGTTTCTACAACAGGGGAAGGAAAAGGTAGACTTGTTATGA

GGACCAAAGGAAATTACAGATTGATCTTGAATGCCAGCCTTTTTCCAGAAA

TGAAGCTTGCTAATATGGACAAAAGAGGGGTCACTTTCGCTTGCTTGAATA

GTGCTGCTGACGGAAAAGGACTTTCTACTATTGCTCTGAAGTTCAAGGATG

CCTCCATCGTGGAAGATTTTCGTGCTGCTGTAGTGGAACATAAAGGTACTA

CAACTGGTTCTTTGAAGACACCAGAAAACTCTCCTAAAAGCTTAA

SEQ ID NO.2

MGDAENSLQPSKKRAAIKELSRDNPGLDDDENEAELETGSFKKASEEVMASR

RIVKVRRSQTTSSTATPSANPFAAIRLVPPTESSIPSTVITSEVESGITSSGKPEDK

NDIGEATRKETNDVCKEELKESDQISKPSEGNVDESNVVKEKVETPNEADKPE

SAEEKVADDEKVQAETKEGTVVEKSENDSKKDVEVEKTKNEEQNDAGGEKS

EKGAETASFSSFQQLSSGQNAFTGFSGTGFSSTTFSFGGISKEGSSLGFGSESGA

GSLFGAKSDQSPFGLNLPTNGSTSLFGNSGSSLVNKSEGTGFPSKEEVTVETGE

ENEKPVFAADSVLFEYLNGGWKERGKGELKVNVSTTGEGKGRLVMRTKGNY

RLILNASLFPEMKLANMDKRGVTFACLNSAADGKGLSTIALKFKDASIVEDFR

AAVVEHKGTTTGSLKTPENSPKA

SEQ ID NO.3

ATATATGGTCTCGTTTGCCCCTTTGCAGCAATCCGCTGTTTTTAGAGCTAGAAATAGC

SEQ ID NO.4

ATTATTGGTCTCGAAACGGCTGAAACCAAGGAAGGCACAAACTACACTGTTAGATTCSEQ ID NO.5

AGCGGATAACAATTTCACACAGGA

SEQ ID NO.6

GCAGGCATGCAAGCTTATTGG

SEQ ID NO.7

GACAATCCTGGTCTCGATGATGAT

SEQ ID NO.8

CCCAGTCCCT GAAAATCCTG TG

SEQ ID NO.9

ACCGTTCCAGAGTTGACTCAA

SEQ ID NO.10

GCAAGAAAGCCTTTCTTCTGA

sequence listing

<110> Qingdao Agricultural University

<120> Application of a Nup50A gene in improving plant resistance to Botrytis cinerea infection

<130> 2020

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1350

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgggagatg ccgagaactc tcttcaacca tcaaagaaaa gggcagctat aaaggaatta 60

tcacgagaca atcctggtct cgatgatgat gaaaatgaag ctgagctaga gactgggtct 120

ttcaagaaag caagtgagga ggtgatggca agtagaagaa ttgtcaaagt tcgtcgcagt 180

caaactactt catctacagc tacaccttca gctaacccct ttgcagcaat ccgcttggtt 240

ccaccactg agtccagtat cccgtctact gtgataacaa gtgaagttga gagtgggata 300

acaagttcag gtaaaccaga agataaaaat gacattggtg aggcaactag aaaggagaca 360

aatgatgtgt gtaaggaaga gttgaaggaa tctgatcaaa tttctaagcc atcagaaggt 420

aatgttgatg aatccaatgt tgtcaaggag aaagttgaaa ctcctaatga ggctgacaaa 480

cctgaatctg ctgaagagaa ggtagcagat gatgagaaag tccaggctga aaccaaggaa 540

ggcacagtag ttgagaagag cgaaaatgac agtaagaagg atgtggaagt cgagaaaaca 600

aagaatgaag aacaaaatga tgctggtggt gagaaaagtg agaagggtgc agaaactgct 660

tcttttagct cattccaaca actctcaagt ggccaaaatg ctttcacagg attttcaggg 720

actgggttct ccagcactac tttctccttt ggaggtattt caaaagaggg atcttctcta 780

ggttttggtt ctgaatcagg tgctggttct ctctttggag caaaaagtga ccagtcacca 840

tttggactta atcttcctac taatggaagt acttctctgt ttggaaactc ggggtcatcc 900

cttgtgaata agagtgaggg tactggattt ccttccaaag aagaggtcac tgttgaaaca 960

ggggaggaaa atgaaaaacc cgtattcgca gctgattccg tgctgtttga atatcttaat 1020

ggagggtgga aagagcgggg gaagggagaa ctaaaggtca atgtttctac aacaggggaa 1080

ggaaaaggta gacttgttat gaggaccaaa ggaaattaca gattgatctt gaatgccagc 1140

ctttttccag aaatgaagct tgctaatatg gacaaaagag gggtcacttt cgcttgcttg 1200

aatagtgctg ctgacggaaa aggactttct actattgctc tgaagttcaa ggatgcctcc 1260

atcgtggaag attttcgtgc tgctgtagtg gaacataaag gtactacaac tggttctttg 1320

aagacaccag aaaactctcc taaagcttaa 1350

<210> 2

<211> 449

<212> PRT

<213> Tomato (Solanum lycopersicum)

<400> 2

Met Gly Asp Ala Glu Asn Ser Leu Gln Pro Ser Lys Lys Arg Ala Ala

1 5 10 15

Ile Lys Glu Leu Ser Arg Asp Asn Pro Gly Leu Asp Asp Asp Glu Asn

20 25 30

Glu Ala Glu Leu Glu Thr Gly Ser Phe Lys Lys Ala Ser Glu Glu Val

35 40 45

Met Ala Ser Arg Arg Ile Val Lys Val Arg Arg Ser Gln Thr Thr Ser

50 55 60

Ser Thr Ala Thr Pro Ser Ala Asn Pro Phe Ala Ala Ile Arg Leu Val

65 70 75 80

Pro Pro Thr Glu Ser Ser Ile Pro Ser Thr Val Ile Thr Ser Glu Val

85 90 95

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

100 105 110

Gly Glu Ala Thr Arg Lys Glu Thr Asn Asp Val Cys Lys Glu Glu Leu

115 120 125

Lys Glu Ser Asp Gln Ile Ser Lys Pro Ser Glu Gly Asn Val Asp Glu

130 135 140

Ser Asn Val Val Lys Glu Lys Val Glu Thr Pro Asn Glu Ala Asp Lys

145 150 155 160

Pro Glu Ser Ala Glu Glu Lys Val Ala Asp Asp Glu Lys Val Gln Ala

165 170 175

Glu Thr Lys Glu Gly Thr Val Val Glu Lys Ser Glu Asn Asp Ser Lys

180 185 190

Lys Asp Val Glu Val Glu Lys Thr Lys Asn Glu Glu Gln Asn Asp Ala

195 200 205

Gly Gly Glu Lys Ser Glu Lys Gly Ala Glu Thr Ala Ser Phe Ser Ser

210 215 220

Phe Gln Gln Leu Ser Ser Gly Gln Asn Ala Phe Thr Gly Phe Ser Gly

225 230 235 240

Thr Gly Phe Ser Ser Thr Thr Phe Ser Phe Gly Gly Ile Ser Lys Glu

245 250 255

Gly Ser Ser Leu Gly Phe Gly Ser Glu Ser Gly Ala Gly Ser Leu Phe

260 265 270

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

275 280 285

Gly Ser Thr Ser Leu Phe Gly Asn Ser Gly Ser Ser Leu Val Asn Lys

290 295 300

Ser Glu Gly Thr Gly Phe Pro Ser Lys Glu Glu Val Thr Val Glu Thr

305 310 315 320

Gly Glu Glu Asn Glu Lys Pro Val Phe Ala Ala Asp Ser Val Leu Phe

325 330 335

Glu Tyr Leu Asn Gly Gly Trp Lys Glu Arg Gly Lys Gly Glu Leu Lys

340 345 350

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

355 360 365

Thr Lys Gly Asn Tyr Arg Leu Ile Leu Asn Ala Ser Leu Phe Pro Glu

370 375 380

Met Lys Leu Ala Asn Met Asp Lys Arg Gly Val Thr Phe Ala Cys Leu

385 390 395 400

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

405 410 415

Lys Asp Ala Ser Ile Val Glu Asp Phe Arg Ala Ala Val Val Glu His

420 425 430

Lys Gly Thr Thr Thr Gly Ser Leu Lys Thr Pro Glu Asn Ser Pro Lys

435 440 445

Ala

<210> 3

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atatatggtc tcgtttgccc ctttgcagca atccgctgtt ttagagctag aaatagc 57

<210> 4

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

attattggtc tcgaaacggc tgaaaccaag gaaggcacaa actacactgt tagattc 57

<210> 5

<211> 24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

agcggataac aatttcacac agga 24

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gcaggcatgc aagcttattg g 21

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gacaatcctg gtctcgatga tgat 24

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cccagtccct gaaaatcctg tg 22

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

accgttccag agttgactca a 21

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gcaagaaagc ctttcttctg a 21

Claims (7)

1. the application of knocking out Nup50A gene in improving tomato resistance Botrytis cinerea infection, it is characterized in that, the nucleotide sequence of described Nup50A gene is as shown in SEQ ID NO.1. 2. the application of knockout Nup50A gene according to claim 1 in improving tomato resistance Botrytis cinerea infection, is characterized in that, described application comprises the following steps: (1) Construction of Nup50A gene knockout recombinant vector: design primers according to the CDS sequence of Nup50A gene and carry out PCR amplification; after the amplification product is purified, it is digested with restriction endonuclease, and after the digestion product is recovered, it is combined with the plasmid Connecting to obtain the Nup50A gene knockout recombinant vector; (2) Constructing a Nup50A gene knockout recombinant strain: transforming the Nup50A gene knockout recombinant vector into Agrobacterium to obtain a Nup50A gene knockout recombinant strain; (3) Construction of a Nup50A gene knockout strain: the Nup50A gene knockout recombinant strain was transformed into tomato cotyledons, screened with antibiotics and cultured until regenerated tomatoes were obtained, that is, a Nup50A gene knockout strain was obtained. 3. the application of knockout Nup50A gene according to claim 2 in improving tomato resistance Botrytis cinerea infection, is characterized in that, the sequence of primer in the described step (1) is: Nup50A-F0: 5’-ATATATGGTCTCGTTTGCCCCTTTGCAGCAATCCGCTGTTTTAGAGCTAG AAATAGC-3'; Nup50A-R0: 5’-ATTATTGGTCTCGAAACGGCTGAAACCAAGGAAGGCACAAACTACA CTGTTAGATTC-3'. 4. The application of knocking out Nup50A gene according to claim 2 in improving tomato resistance to Botrytis cinerea infection, characterized in that, the plasmid is pTX-Nup50A. 5. The application of knocking out Nup50A gene according to claim 2 in improving tomato resistance to Botrytis cinerea infection, characterized in that, the gene knockout recombinant vector is pTX-Nup50A. 6. The application of knocking out the Nup50A gene according to claim 2 in improving tomato resistance to Botrytis cinerea infection, characterized in that the Agrobacterium is LBA4404. 7. The application of knockout Nup50A gene according to claim 2 in improving tomato resistance to Botrytis cinerea infection, is characterized in that, the condition of cultivating in described step (3) is: every day 26 ℃ of 16 hours of light and 8 hours in the dark at 18°C.

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