CN114058626B

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

Info

Publication number: CN114058626B
Application number: CN202010741639.0A
Authority: CN
Inventors: 宋丽敏; 李晓霞; 徐阳; 梁文星
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2020-07-29
Filing date: 2020-07-29
Publication date: 2023-05-02
Anticipated expiration: 2040-07-29
Also published as: CN114058626A

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to application of a Nup50A gene in improving resistance of plants to botrytis cinerea infection, a tomato Nup50A gene knockout recombinant vector pTX-Nup50A, a gene knockout recombinant strain LBA4404-pTX-Nup50A and a tomato Nup50A gene knockout homozygous strain thereof are constructed by separating and cloning the Nup50A gene from tomatoes, and the invention proves that the Nup50A gene knockout capability of the tomato against the botrytis cinerea infection can be effectively improved, and compared with a wild type strain, the spot diameter on leaves and the botrytis cinerea amount in the leaves of the tomato Nup50A gene knockout strain are obviously reduced. Experiments prove that the Nup50A gene has application value in improving the gray mold resistance of tomatoes, and lays a good theory and application foundation for cultivating disease-resistant plants by using the Nup50A gene.

Description

Application of Nup50A gene in improving resistance of plants to botrytis cinerea infection

Technical field:

the invention belongs to the field of plant genetic engineering, and particularly relates to application of a Nup50A gene in improving plant resistance to botrytis cinerea infection.

The background technology is as follows:

eukaryotic nucleoporin complexes mediate the shuttling between the nucleophiles of macromolecular substances such as proteins and RNAs, playing an extremely important role in the maintenance and regulation of cellular vital activities. The nucleopore complex (nuclear pore complex, NPC) is the largest polyprotein complex in cells, and studies have shown that it consists of at least 30 more nucleoporins (Nup) in multiple copies. The basic composition is highly conserved among vertebrates, yeasts and plants. The related research of plant nucleoporins is not so much seen, and only a few functions of plant nucleoporins are primarily identified, and the existing research results show that the plant nucleoporins are widely involved in various physiological and biochemical processes.

1. Hormone signaling

Arabidopsis SAR1 and SAR3, after mutation, affect nuclear output of mRNA and subcellular localization of auxin response transcription repressor IAA17 protein, so that the phenotype of axr-1 resistance to auxin can be partially restored, indicating that they are involved in auxin signal transduction pathways. In addition, the Arabidopsis Trp/Mlp1p/Mlp2p mutation also exhibited a phenotype similar to that of sar1 and sar 3. In addition to being involved in auxin signaling, nup160 mutations also enhance the response of arabidopsis to ethylene, suggesting that it may be involved in the interaction between auxin and ethylene signaling.

2. Response to Cold injury stress

The Arabidopsis Nup160 mutation can inhibit the expression of CBF3 under the cold damage stress, and reduce the resistance of plants to the cold damage stress. HOS1 is used as a negative regulator of low-temperature signal transduction of plants, and can regulate the expression of cold injury and cold injury stress related genes. Overexpression of HOS1 in Arabidopsis can inhibit the expression of CBF family genes, enhance the sensitivity of Arabidopsis to cold injury, and mutation of HOS1 can cause high-level expression of low-temperature response genes, thereby improving the resistance of plants to cold injury stress. Meanwhile, the HOS1 protein has a RING-finger structural domain, can perform E3 ubiquitin ligase function, and can specifically mediate the degradation of ICE1 protein under cold induction conditions, so that the response of arabidopsis to low temperature is weakened.

3. Flowering and growth of plants

The Arabidopsis Nup160, nup96 and NUA/TPR are involved in the physiological processes and influence other development processes of plants, such as flowering time and fertility.

4. Response and symbiosis of plants to pathogens

MOS3 (homologous gene of animal Nup 96) in Arabidopsis thaliana, and the sensitivity of Arabidopsis thaliana to pathogen is enhanced after mutation. MOS7 (homologous gene of animal Nup 88) mutation results in Arabidopsis thaliana R protein mediated immunity and underlying and systemic acquired resistance deficiency. Seh1 and Nup160 mutations also disrupt the underlying resistance of plants to pathogens. Nup75 in nicotiana benthamiana is involved in ethylene-mediated production of phytosanitary elements after phytophthora infestations. Nucleoporin mutations also affect plant and microbial symbiosis. Mutations such as Baimaigen Nup85, nup133, and NENA (i.e., the homologous gene of SEC 13) affect nodulation.

It can be seen that the nucleoporin plays a role in various aspects of plant growth and development, response to environmental stress, and the like. However, at present, little research on the function and molecular mechanism of plant nucleoporins is focused mainly on Arabidopsis thaliana. In mouse cells Nup50A is a hydrophilic protein containing 5 FG repeats, whose localization can move between NPC and the nucleus, necessary for cell differentiation and primordial germ cell survival and involved in DNA damage repair. The biological function of Nup50A in plants was not reported.

The invention comprises the following steps:

the invention aims to overcome the defects and the shortcomings existing in the prior art and provides an application of a Nup50A gene in improving the resistance of plants to gray mold infection. The Nup50A gene is separated and cloned from tomatoes, a Nup50A gene knockout recombinant vector pTX-Nup50A and a homozygous line thereof are constructed, and the Nup50A gene is proved to be capable of effectively improving the capability of tomatoes against gray mold infection.

In order to achieve the aim of the invention, the invention provides application of a Nup50A gene in improving the resistance of plants to botrytis cinerea infection, and the nucleotide sequence of the Nup50A gene is shown as SEQ ID NO. 1.

Further, the application comprises the steps of:

(1) Constructing a Nup50A gene knockout recombinant vector: designing a primer according to the CDS sequence of the Nup50A gene, and carrying out PCR amplification by taking plasmid pTX-043 as a template; after the amplified product is purified, restriction enzyme Bsa I is used for enzyme digestion, and the enzyme digestion product is recovered and then connected with pTX-041 plasmid to obtain a NUP50A gene knockout recombinant vector;

(2) Constructing a Nup50A gene knockout recombinant strain: the Nup50A gene knockout recombinant vector is transformed into agrobacterium to obtain a Nup50A gene knockout recombinant strain;

(3) Constructing a Nup50A gene knockout line: and (3) transforming the Nup50A gene knockout recombinant strain into plant cotyledons, screening the transformed callus by using an antibiotic Kan, and culturing until a regenerated plant is obtained, thus obtaining the Nup50A gene knockout strain.

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

Nup50A-F0：

5’-ATATATGGTCTCGTTTGCCCCTTTGCAGCAATCCGCTGTTTTAGAGCTAGAAATAGC-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 conditions of the culture in the step (3) are 16h light (26 ℃ C.)/8 h dark (18 ℃ C.).

Further, compared with a wild type strain, the Nup50A gene knockout strain has obviously reduced lesion diameter on leaves.

Furthermore, compared with a wild type strain, the tomato Nup50A gene knockout strain has obviously reduced gray mold quantity on leaves.

Further, the plant is tomato.

Compared with the prior art, the invention has the advantages and technical effects that: the invention separates and clones Nup50A gene from tomato, and further connects Nup50A gene target spot information to gene knockout carrier pTX-041 to obtain Nup50A gene knockout recombinant carrier pTX-Nup50A, then uses agrobacterium to obtain gene knockout recombinant strain LBA4404-pTX-Nup50A, and infects transformed tomato cotyledon to obtain homozygous Nup50A gene knockout strain, and compares it with disease spots on leaves of wild tomato grown under the same condition by researching phenotype of gene knockout transgenic strain. According to the invention, experimental analysis shows that the Nup50A gene has the function of regulating and controlling the disease resistance of tomatoes to gray mold for the first time, and the capability of the tomatoes against gray mold infection can be effectively improved by knocking out the Nup50A gene; the leaf spot of the Nup50A gene knockout homozygous strain is obviously reduced, and the gray mold amount is obviously lower than that of a wild tomato strain. The technical scheme of the invention has application value on the Nup50A gene in improving the disease resistance of tomatoes to gray mold, and lays a good theoretical and application foundation for cultivating disease-resistant and high-yield tomato varieties by using the Nup50A gene.

Description of the drawings:

FIG. 1 shows PCR amplification of Nup50A target sequence, wherein M is DNA marker, and 1-3 are PCR products of pTX-Nup50A target sequence.

FIG. 2 shows the construction result of knockout vector, wherein M is DNA marker, and 1-4 are the recombinant plasmid screens.

FIG. 3 shows the sequence comparison result of mutation sites of the T2 generation homozygous knockout mutant of the Nup50A gene.

FIG. 4 shows the disease resistance variation results of Nup50A knockout homozygous lines;

fig. 5 shows the results of leaf spot diameter on leaf in Nup50A knockout homozygous lines, (< 0.01).

Fig. 6 is the results of the amount of botrytis in leaves of Nup50A knockout homozygous lines, (< 0.01).

The specific embodiment is as follows:

the invention is further described below by way of examples and with reference to the accompanying drawings.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments and the like used in the examples described below are commercially available products unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

The PQB vector, pTX-041 vector and pTX-043 vector described in the examples below are publicly available from the applicant and are used only for the experiments related to the repeated invention and are not used for other purposes.

Example 1:

tomato Nup50A gene knockout mutant and acquisition of homozygous lines

1. Acquisition of tomato Nup50A Gene knockout mutant

(1) Vector construction

CDS sequences of tomato NUP50A genes are obtained in a GeneBank database, have the length of 1347bp, code 449 amino acid-containing protein, have the nucleotide sequence shown in SEQ ID NO.1 (underlined is a target sequence), and have the encoded amino acid sequence shown in SEQ ID NO. 2. Inputting Nup50A gene complete sequence in a CRISPR direct website, and screening proper targets according to the result, wherein the selected targets are respectively: CCCCTTTGCAGCAATCCGCT and GGCTGAAACCAAGGAAGGCA, and designing primers based thereon, the primer sequences are as follows: nup50A-F0:

5’-ATATATGGTCTCGTTTGCCCCTTTGCAGCAATCCGCTGTTTTAGAGCTAGAAATAGC-3(SEQ ID NO.3)；

Nup50A-R0：

5’-ATTATTGGTCTCGAAACGGCTGAAACCAAGGAAGGCACAAACTACA

CTGTTAGATTC-3’(SEQ ID NO.4)。

taking plasmid pTX-043 as a template, NUP50A-F0 and Nup50A-R0 as primers, carrying out PCR amplification on a target sequence by high-fidelity enzyme, and recovering and purifying an amplified product as shown in a figure 1; the purified PCR product and pTX-041 plasmid are digested with restriction enzyme Bsa I, recovered separately and ligated overnight under the catalysis of T4 ligase; after ligation, the ligation product was transformed into E.coli JM109, and positive clones containing the recombinant plasmid were selected and sent to the company for sequencing, and finally the recombinant plasmid with no error in sequencing was named pTX-Nup50A. Wherein, the sequence of the primer used in plasmid screening is 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 give recombinant strain LBA4404-pTX-NUP50A.

Transforming tomato cotyledons by utilizing a recombinant strain LBA4404-pTX-Nup50A according to an agrobacterium-mediated transgenic method; screening the transformed callus by using an antibiotic Kan, and setting a trans-empty vector as a control; the medium was changed every 2-3 weeks until regenerated seedlings were obtained, wherein the culture conditions were 16h light (26 ℃ C.)/8 h dark (18 ℃ C.).

Extracting genome DNA of the regenerated seedlings respectively, and carrying out PCR amplification on partial sequences of the Nup50A gene containing targets, wherein the sequences of the used primers are as follows:

Nup50A-F1261：

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

Nup50A-R1920：

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

the PCR product is cut into gel and sent to biological company for sequencing after electrophoresis detection; if a sleeve peak starts to exist near the sequence target point or the sequence is partially deleted between the two target points, the plant is a Nup50A knockout positive plant. Sequencing results show that 3 Nup50A knockout positive plants are obtained, and are respectively marked as Nup50A-4-12, nup50A-7-6 and Nup50A-11-1.

2. Acquisition of tomato NUP50A Gene knockout homozygous lines

Harvesting seeds of T0-generation knockout positive plants Nup50A-4, nup50A-7 and Nup50A-11 as T1 generation; sowing the obtained T1 generation seeds, taking 5-10 plants from each knockout line after 2-3 weeks, extracting genome DNA of the plants, carrying out PCR amplification by using Nup50A-F1261 and Nup50A-R1920 primer pairs, connecting the PCR products with pMD-18T vectors after recovering the PCR products, and selecting positive clones to send to a company for sequencing. 4 samples were taken for each knockdown line plant for cloning. Sequencing results show that 4 clone samples of each knockout line plant have no set peak, and the positions of target points are mutated or partial deletions are generated between the two target points, so that the samples are determined to be Nup50A gene knockout homozygous lines.

The identified three positive Nup50A gene knockout homozygous lines are named as knockout lines Nup50A-4-12, nup50A-7-6 and Nup50A-11-1 respectively. Compared with a wild plant, the knocked-out line Nup50A-4-12 gene 528 th bit is subjected to one more T frame shift mutation; knocking out 528 th site of the gene deleted in Nup50A-7-6 gene to generate frame shift mutation; the 528 th to 531 th positions of the gene deleted from the Nup50A-11-1 gene undergo frame shift mutation, and specific comparison sequence information is shown in figure 3. Transplanting the obtained Nup50A gene knockout homozygous strain into a large pot, and collecting seeds for subsequent experiments.

Example 2:

identification of phenotype of positive tomato Nup50A Gene knockout plant

1. Seeds of Nup50A knockout lines Nup50A-4-12, nup50A-7-6 and Nup50A-11-1 and wild type tomato (WT) were sown in nutrient soil, respectively, and cultured in a greenhouse under the conditions of 16h light (26 ℃) and 8h dark (18 ℃). Then, selecting 3 groups of NUP50A gene knockout homozygous lines and wild type strains which are about 4 weeks and have the same growth vigor, inoculating tomato leaves with spore liquid with the spore concentration of 106, placing the tomato leaves in a plastic box, applying a preservative film on the leaves to maintain the percentage humidity, and observing the disease condition of the tomato leaves and carrying out data statistics after the tomato leaves are maintained at 22 ℃ for 24-48 hours. The results are shown in FIGS. 4 and 5, where the diameter of lesions on leaves of the Nup50A knockout homozygous strain is significantly reduced compared to the wild type.

2. Extracting total RNA from tomato leaves infected by botrytis cinerea, detecting the amount of botrytis cinerea in the leaves by qRT-PCR, and using tomato action as an internal reference, wherein the sequence of the used primer is as follows:

β-Tubulin-F：

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

β-Tubulin-R：

5’-GCAAGAAAGCCTTTCTTCTGA-3’(SEQ ID NO.10)。

as a result, as shown in FIG. 6, the amount of Botrytis cinerea in the NUP50A knockout homozygous line was significantly lower than that of the wild type tomato.

The evidence shows that the Nup50A gene plays an important role in resisting the infection of the botrytis cinerea, and the capability of the tomatoes in resisting the infection of the botrytis cinerea can be effectively improved by knocking out the Nup50A gene, so that the Nup50A gene of the tomatoes has the function of regulating and controlling the tomatoes in resisting the infection of the botrytis cinerea.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.

And (3) a sequence table:

SEQ ID NO.1

ATGGGAGATGCCGAGAACTCTCTTCAACCATCAAAGAAAAGGGCAGCTAT

AAAGGAATTATCACGAGACAATCCTGGTCTCGATGATGATGAAAATGAAGC

TGAGCTAGAGACTGGGTCTTTCAAGAAAGCAAGTGAGGAGGTGATGGCAA

GTAGAAGAATTGTCAAAGTTCGTCGCAGTCAAACTACTTCATCTACAGCTA

CACCTTCAGCTAACCCCTTTGCAGCAATCCGCTTGGTTCCACCCACTGAGT

CCAGTATCCCGTCTACTGTGATAACAAGTGAAGTTGAGAGTGGGATA

ACAAGTTCAGGTAAACCAGAAGATAAAAATGACATTGGTGAGGCAACTAG

AAAGGAGACAAATGATGTGTGTAAGGAAGAGTTGAAGGAATCTGATCAAA

TTTCTAAGCCATCAGAAGGTAATGTTGATGAATCCAATGTTGTCAAGGAGA

AAGTTGAAACTCCTAATGAGGCTGACAAACCTGAATCTGCTGAAGAGAAG

GTAGCAGATGATGAGAAAGTCCAGGCTGAAACCAAGGAAGGCACAGTAGT

TGAGAAGAGCGAAAATGACAGTAAGAAGGATGTGGAAGTCGAGAAAACA

AAGAATGAAGAACAAAATGATGCTGGTGGTGAGAAAAGTGAGAAGGGTG

CAGAAACTGCTTCTTTTAGCTCATTCCAACAACTCTCAAGTGGCCAAAATG

CTTTCACAGGATTTTCAGGGACTGGGTTCTCCAGCACTACTTTCTCCTTTGG

AGGTATTTCAAAAGAGGGATCTTCTCTAGGTTTTGGTTCTGAATCAGGTGCT

GGTTCTCTCTTTGGAGCAAAAAGTGACCAGTCACCATTTGGACTTAATCTT

CCTACTAATGGAAGTACTTCTCTGTTTGGAAACTCGGGGTCATCC

CTTGTGAATAAGAGTGAGGGTACTGGATTTCCTTCCAAAGAAGAGGTCACT

GTTGAAACAGGGGAGGAAAATGAAAAACCCGTATTCGCAGCTGATTCCGT

GCTGTTTGAATATCTTAATGGAGGGTGGAAAGAGCGGGGGAAGGGAGAAC

TAAAGGTCAATGTTTCTACAACAGGGGAAGGAAAAGGTAGACTTGTTATGA

GGACCAAAGGAAATTACAGATTGATCTTGAATGCCAGCCTTTTTCCAGAAA

TGAAGCTTGCTAATATGGACAAAAGAGGGGTCACTTTCGCTTGCTTGAATA

GTGCTGCTGACGGAAAAGGACTTTCTACTATTGCTCTGAAGTTCAAGGATG

CCTCCATCGTGGAAGATTTTCGTGCTGCTGTAGTGGAACATAAAGGTACTA

CAACTGGTTCTTTGAAGACACCAGAAAACTCTCCTAAAGCTTAA

SEQ ID NO.2

MGDAENSLQPSKKRAAIKELSRDNPGLDDDENEAELETGSFKKASEEVMASR

RIVKVRRSQTTSSTATPSANPFAAIRLVPPTESSIPSTVITSEVESGITSSGKPEDK

NDIGEATRKETNDVCKEELKESDQISKPSEGNVDESNVVKEKVETPNEADKPE

SAEEKVADDEKVQAETKEGTVVEKSENDSKKDVEVEKTKNEEQNDAGGEKS

EKGAETASFSSFQQLSSGQNAFTGFSGTGFSSTTFSFGGISKEGSSLGFGSESGA

GSLFGAKSDQSPFGLNLPTNGSTSLFGNSGSSLVNKSEGTGFPSKEEVTVETGE

ENEKPVFAADSVLFEYLNGGWKERGKGELKVNVSTTGEGKGRLVMRTKGNY

RLILNASLFPEMKLANMDKRGVTFACLNSAADGKGLSTIALKFKDASIVEDFR

AAVVEHKGTTTGSLKTPENSPKA

SEQ ID NO.3

ATATATGGTCTCGTTTGCCCCTTTGCAGCAATCCGCTGTTTTAGAGCTAGAAATAGC

SEQ ID NO.4

ATTATTGGTCTCGAAACGGCTGAAACCAAGGAAGGCACAAACTACACTGTTAGATTCSEQ ID NO.5

AGCGGATAACAATTTCACACAGGA

SEQ ID NO.6

GCAGGCATGCAAGCTTATTGG

SEQ ID NO.7

GACAATCCTG GTCTCGATGATGAT

SEQ ID NO.8

CCCAGTCCCT GAAAATCCTG TG

SEQ ID NO.9

ACCGTTCCAGAGTTGACTCAA

SEQ ID NO.10

GCAAGAAAGCCTTTCTTCTGA

sequence listing

<110> Qingdao university of agriculture

<120> application of Nup50A gene in improving resistance of plants 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

ccacccactg 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

1. The application of the Nup50A gene in improving the resistance of tomatoes to the infection of botrytis cinerea is characterized in that the nucleotide sequence of the Nup50A gene is shown as SEQ ID NO. 1.

2. Use of the knockout Nup50A gene according to claim 1 for increasing resistance of tomatoes to botrytis cinerea infection, said use comprising the steps of:

(1) Constructing a Nup50A gene knockout recombinant vector: designing a primer according to the CDS sequence of the Nup50A gene and carrying out PCR amplification; after the amplified product is purified, restriction enzyme is used for carrying out enzyme digestion on the amplified product, and the digested product is recovered and then connected with a plasmid to obtain a Nup50A gene knockout recombinant vector;

(3) Constructing a Nup50A gene knockout line: and (3) transforming the Nup50A gene knockout recombinant strain into tomato cotyledons, screening by using antibiotics, and culturing until regenerated tomatoes are obtained, thus obtaining the Nup50A gene knockout strain.

3. The use of the Nup50A gene knockout according to claim 2 for increasing resistance of tomato to botrytis cinerea infection, wherein the primer sequence in step (1) is:

Nup50A-F0：

5’-ATATATGGTCTCGTTTGCCCCTTTGCAGCAATCCGCTGTTTTAGAGCTAG

AAATAGC-3’；

Nup50A-R0：

5’-ATTATTGGTCTCGAAACGGCTGAAACCAAGGAAGGCACAAACTACA

CTGTTAGATTC-3’。

4. use of the knockout Nup50A gene according to claim 2 for increasing resistance of tomato to botrytis cinerea infection, wherein the plasmid is pTX-Nup50A.

5. The use of the knocked-out Nup50A gene according to claim 2 for increasing resistance of tomato to botrytis cinerea infection, wherein the gene knocked-out recombinant vector is pTX-Nup50A.

6. Use of the knockout Nup50A gene according to claim 2 for increasing resistance of tomato to botrytis cinerea infection, wherein said agrobacterium is LBA4404.

7. Use of the knockout Nup50A gene according to claim 2 for increasing resistance of tomato to botrytis cinerea infection, wherein the conditions of the culturing in step (3) are: light at 26℃for 16 hours and darkness at 18℃for 8 hours per day.