CN111500579A - Cotton miR164a and NAC 100L and application thereof in regulation and control of verticillium wilt resistance of plants - Google Patents

Abstract

The invention discloses cotton miR164a and NAC100L and their application in regulating plant verticillium wilt resistance. The present invention provides GhNAC100L protein (SEQ ID No. 2) and ghr-miR164a (SEQ ID No. 1). Experiments have proved that the ghr-miR164a and GhNAC100L provided by the present invention, as a disease resistance module, can positively regulate disease resistance and can be used as candidate genes for cotton verticillium wilt resistance breeding.

Description

Cotton miR164a and NAC100L and their application in regulating plant verticillium wilt resistance

technical field

The invention relates to the field of biotechnology, in particular to cotton miR164a and NAC100L and their application in regulating plant verticillium wilt resistance.

Background technique

Cotton belongs to the Malvaceae cotton genus, and its cultivars currently have 4 species, including upland cotton, sea island cotton, grass cotton and Asian cotton, of which upland cotton is the main species. Cotton is an important economic crop, cotton fiber is an important raw material for the textile industry, cottonseed oil is one of the main sources of food and chemical oil, and cottonseed meal is a protein additive component of ruminant animal feed such as cattle and sheep. The yield and quality of cotton are easily affected by various biotic and abiotic stresses. Among them, cotton Verticillium wilt severely restricts cotton production. Cotton Verticillium wilt is a soil-borne vascular disease, and its pathogen is mainly Verticillium dahliae, which belongs to the genus Verticillium dahliae. Pathogens invade from the roots of cotton, causing plant disease and seriously affecting the fiber yield and quality of cotton. Due to the characteristics of cotton verticillium wilt, such as wide distribution, serious harm, many transmission routes and long survival time, in order to solve this thorny problem, cultivating disease-resistant varieties is the most economical and effective method. However, the difficulty in cultivating disease-resistant cotton varieties lies in the acquisition of disease-resistant germplasm resources. At present, due to the lack of germplasm resources for disease resistance and immunity, it is difficult to effectively breed disease-resistant varieties by conventional breeding methods. Therefore, the use of genetic engineering technology to mine disease resistance-related genes and create new disease-resistant varieties has become an important research direction to solve the harm of cotton verticillium wilt. In a word, improving the disease resistance of cotton is of great significance to the safe production of cotton and the better life of the people.

MicroRNAs (miRNAs) are a family of small RNAs of approximately 19-24 nucleotides in length that are non-coding proteins and regulate target gene expression post-transcriptionally. miRNAs play important roles in plant fiber development, signal transduction, stress response, hormone synthesis, and morphogenesis.

The mining of plant disease resistance genes mainly involves some regulatory factors that regulate gene expression, including regulatory proteins and transcription factors. Among them, transcription factors, also known as trans-acting factors, specifically bind to cis-acting elements in the promoter region of eukaryotic genes, and activate or inhibit the transcription of downstream genes. These transcription factors mainly include NAC, MYB, bZIP, WRKY, etc., and they play an important role in plant growth, development and defense responses. The NAC transcription factor involved in the present invention is an important type of transcription factor family in the plant transcription factor family, and participates in processes such as plant growth and development, defense response and the like.

In recent years, some literatures have reported that miRNA and NAC transcription factors are involved in plant disease resistance. However, there are few studies on miRNA-NAC transcription factors involved in cotton resistance to verticillium wilt, especially the in-depth analysis of disease resistance of these genes.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide cotton miR164a and NAC100L and their application in regulating plant verticillium wilt resistance.

In a first aspect, the present invention claims a biological material.

The biological material claimed in the present invention consists of (A) and (B).

(A) A protein or its related biological material; the related biological material is a nucleic acid molecule capable of expressing the protein or an expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing the nucleic acid molecule.

(B) miRNA or its related biological material; the related biological material is a DNA molecule that can be transcribed into the miRNA or an expression cassette, a recombinant vector, a recombinant bacteria or a transgenic cell line containing the DNA molecule.

The protein is a protein shown in any of the following (A1)-(A4):

(A1) a protein whose amino acid sequence is shown in SEQ ID No.2;

(A2) The amino acid sequence defined in (A1) is subjected to the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function as a protein;

(A3) A protein that has 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology with the amino acid sequence defined in (A1) or (A2) and has the same function;

(A4) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein as defined in any one of (A1)-(A3).

In the above proteins, the tag refers to a polypeptide or protein that is fused and expressed with the target protein by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tags may be Flag tags, His tags, MBP tags, HA tags, myc tags, GST tags, and/or SUMO tags, and the like.

The miRNA is a mature miRNA or a precursor miRNA corresponding to the mature miRNA; the nucleotide sequence of the mature miRNA is SEQ ID No.1.

Further, in (A), the nucleic acid molecule can be any of the following:

(B1) the DNA molecule shown in SEQ ID No.3;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes the protein;

(B3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in (B1) or (B2) and encodes the protein.

In the above genes, the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl _sulfate (SDS), 0.5M Na3PO4 and 1mM EDTA, at 50°C, ₂ ×SSC, Rinse in 0.1% SDS; also: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M Na ₃ PO ₄ and 1mM EDTA, rinse in 1×SSC, 0.1% SDS at 50° C; : 50°C, hybridize in a mixed solution of 7% SDS, 0.5M Na ₃ PO ₄ and 1 mM EDTA, rinse at 50° C, 0.5×SSC, 0.1% SDS; also: 50° C, in 7% SDS, Hybridize in a mixed solution _of 0.5M Na3PO4 and 1mM EDTA, _wash in 0.1×SSC, 0.1% SDS at 50°C; also: 50°C, in ₇ % SDS, 0.5M _Na3PO4 and 1mM EDTA Hybridize in a mixed solution of 65°C, 0.1×SSC, 0.1% SDS; also: hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, then use 2×SSC, 0.1% The membrane was washed once with SDS and 1×SSC, 0.1% SDS.

Further, in (B), the nucleotide sequence of the DNA molecule can be as shown in SEQ ID No.5.

Wherein, the recombinant vector may be a recombinant expression vector or a recombinant cloning vector.

The recombinant expression vector can be constructed using existing plant expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, and the like. The plant expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal can guide the addition of poly(A) to the 3' end of the mRNA precursor, such as Agrobacterium crown gall inducing (Ti) plasmid genes (such as Nos synthase Nos gene), plant genes (such as soybean storage) The untranslated regions transcribed at the 3' end of protein genes) have similar functions.

When constructing a recombinant plant expression vector, any enhanced promoter or constitutive promoter (such as cauliflower mosaic virus (CAMV) 35S promoter, maize ubiquitin promoter can be added before its transcription initiation nucleotide. (Ubiquitin)), or tissue-specific expression promoters (eg, seed-specific expression promoters), which can be used alone or in combination with other plant promoters. In addition, enhancers, including translational or transcriptional enhancers, can also be used in the construction of plant expression vectors. The translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic. The translation initiation region can be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding a gene (GUS gene, luciferase gene, luciferase gene) that can be expressed in plants encoding an enzyme that can produce color change or a luminescent compound. Gene, etc.), antibiotic markers with resistance (gentamycin marker, kanamycin marker, etc.) or anti-chemical reagent marker gene (such as herbicide resistance gene) and so on.

In a second aspect, the present invention claims proteins or related biological materials.

The protein or its related biological material as claimed in the present invention is as shown in (A) in the first aspect above.

In the third aspect, the present invention claims to protect the biological material described in the first aspect above or the protein described in the second aspect above or its related biological material or the miRNA shown in (B) in the first aspect above or its related biological material in any of the following One application:

(C1) regulating the resistance of plants to Verticillium wilt;

(C2) regulating the resistance of plants to Verticillium dahliae;

(C3) Regulation of plant resistance to vascular parasitic pathogens.

In the application, the content and/or activity of the protein shown in (A) in the first aspect above is higher in the plant, and/or the miRNA shown in (B) in the first aspect above is in the plant. The lower the content in the plant, the weaker the resistance of the plant to Verticillium wilt; the lower the content and/or activity of the protein shown in (A) in the first aspect above in the plant, and/or the above The higher the content of the miRNA shown in (B) in the first aspect in the plant, the stronger the resistance of the plant to Verticillium wilt.

In the application, the content and/or activity of the protein shown in (A) in the first aspect above is higher in the plant, and/or the miRNA shown in (B) in the first aspect above is in the plant. The lower the content in a plant, the weaker the resistance of the plant to Verticillium dahliae; the lower the content and/or activity of the protein shown in (A) in the first aspect above in the plant, and/or Or the higher the content of the miRNA shown in (B) in the first aspect above in the plant, the stronger the resistance of the plant to Verticillium dahliae.

In the application, the content and/or activity of the protein shown in (A) in the first aspect above is higher in the plant, and/or the miRNA shown in (B) in the first aspect above is in the plant. The lower the content in a plant, the weaker the resistance of the plant to vascular parasitic pathogens; the lower the content and/or activity of the protein shown in (A) in the first aspect above in the plant, and/or The higher the content of the miRNA shown in (B) in the first aspect above in the plant, the stronger the resistance of the plant to vascular parasitic pathogens.

In a fourth aspect, the present invention claims a method of cultivating a plant variety.

The method for cultivating plant varieties claimed in the present invention can be any of the following:

Method A1: A method for cultivating plant varieties with improved resistance to Verticillium wilt, comprising the following steps (a1) and/or (a2):

(a1) reducing the expression level and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in the first aspect;

(a2) Elevating the expression level of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) in the first aspect above.

Method A2: A method for cultivating plant varieties with increased resistance to Verticillium dahliae, comprising the following steps (a1) and/or (a2):

(a1) reducing the expression level and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in the first aspect;

(a2) Elevating the expression level of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) in the first aspect above.

Method A3: A method for cultivating plant varieties with improved resistance to vascular parasitic pathogens, comprising the following steps (a1) and/or (a2):

(a1) reducing the expression level and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in the first aspect;

(a2) Elevating the expression level of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) in the first aspect above.

Method B1: a method for cultivating plant varieties with reduced resistance to Verticillium wilt, comprising the following steps (b1) and/or (b2):

(b1) increasing the expression level and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in the first aspect;

(b2) Reducing the expression level of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) in the first aspect above.

Method B2: A method for cultivating plant varieties with reduced resistance to Verticillium dahliae, comprising the following steps (b1) and/or (b2):

(b1) increasing the expression level and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in the first aspect;

(b2) Reducing the expression level of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) in the first aspect above.

Method B3: A method for cultivating plant varieties with reduced resistance to vascular parasitic pathogens, comprising the following steps (b1) and/or (b2):

(b1) increasing the expression level and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in the first aspect;

(b2) Reducing the expression level of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) in the first aspect above.

In a fifth aspect, the present invention claims a method of growing transgenic plants.

The method for cultivating transgenic plants claimed in the present invention can be any of the following:

Method C1: a method for cultivating transgenic plants with improved resistance to Verticillium wilt, comprising the steps of: inhibiting expression of a specific nucleic acid molecule in a recipient plant, and/or introducing a specific DNA molecule into the recipient plant, A transgenic plant is obtained; the transgenic plant has improved resistance to Verticillium wilt compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid molecule shown in (A) in the first aspect above; the specific DNA molecule is The DNA molecule shown in (B) in the first aspect above.

Method C2: A method for cultivating transgenic plants with increased resistance to Verticillium dahliae, comprising the steps of: inhibiting expression of a specific nucleic acid molecule in a recipient plant, and/or introducing a specific DNA into the recipient plant molecule to obtain a transgenic plant; the transgenic plant has improved resistance to Verticillium dahliae compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid molecule shown in (A) in the first aspect above; the The specific DNA molecule is the DNA molecule shown in (B) in the first aspect above.

Method C3: A method for cultivating transgenic plants with improved resistance to vascular parasitic pathogens, comprising the steps of: inhibiting expression of a specific nucleic acid molecule in a recipient plant, and/or introducing a specific DNA molecule into the recipient plant , to obtain a transgenic plant; the transgenic plant has improved resistance to vascular parasitic pathogens compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid molecule shown in (A) in the first aspect above; the specific DNA The molecule is the DNA molecule shown in (B) in the first aspect above.

Method D1: A method for cultivating transgenic plants with reduced resistance to Verticillium wilt, comprising the steps of: introducing specific nucleic acid molecules into recipient plants, and/or inhibiting expression of specific DNA molecules in said recipient plants , to obtain a transgenic plant; the transgenic plant has reduced resistance to Verticillium wilt compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid molecule shown in (A) in the first aspect above; the specific DNA molecule is the DNA molecule shown in (B) in the first aspect above.

Method D2: A method of cultivating transgenic plants with reduced resistance to Verticillium dahliae, comprising the steps of: introducing specific nucleic acid molecules into recipient plants, and/or subjecting the recipient plants to specific DNA molecules Suppressing expression to obtain a transgenic plant; the transgenic plant has reduced resistance to Verticillium dahliae compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid molecule shown in (A) in the first aspect above; The specific DNA molecule is the DNA molecule shown in (B) in the first aspect above.

Method D3: A method for cultivating transgenic plants with reduced resistance to vascular parasitic pathogens, comprising the steps of: introducing specific nucleic acid molecules into recipient plants, and/or inhibiting specific DNA molecules in said recipient plants Expression to obtain a transgenic plant; the transgenic plant has reduced resistance to vascular parasitic pathogens compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid molecule shown in (A) in the first aspect above; the specific nucleic acid molecule The DNA molecule is the DNA molecule shown in (B) in the first aspect above.

In the methods C1-C3, the "inhibiting expression of a specific nucleic acid molecule in a recipient plant" can be achieved by any technical means. In a specific embodiment of the present invention, it is realized by virus-induced gene silencing (VIGS) technology. The VIGS vector used is tobacco rattle virus (TRV), specifically the recombinant vector obtained by cloning the DNA fragment shown in SEQ ID No. 4 (ie, positions 451-781 of SEQ ID No. 3) into the pYL156 vector.

In the methods C1-C3, the "introducing a specific DNA molecule into the recipient plant" can be achieved by any technical means, such as introducing the specific DNA molecule into the recipient plant in the form of a recombinant expression vector middle. In a specific embodiment of the present invention, the recombinant vector is specifically a recombinant vector obtained by cloning the DNA fragment shown in SEQ ID No. 5 into the pYL156 vector.

In the methods D1-D3, the "introducing a specific nucleic acid molecule into the recipient plant" can be achieved by any technical means, such as introducing the specific nucleic acid molecule into the recipient plant in the form of a recombinant expression vector.

In the methods D1-D3, the "inhibiting expression of a specific DNA molecule in the recipient plant" can be achieved by any technical means. The VIGS vector used is tobacco rattle virus (TRV), specifically the recombinant vector obtained by cloning the DNA fragment shown in SEQ ID No. 6 into the pYL156 vector.

In the above method, each recombinant vector is introduced into the recipient plant, specifically: by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conductivity, Agrobacterium-mediated and other conventional biology Methods Plant cells or tissues are transformed and the transformed plant tissues are grown into plants.

A transformed cell, tissue or plant is understood to include not only the end product of the transformation process, but also its transgenic progeny.

In each of the above aspects, the plant may be a plant capable of being infected with Verticillium wilt or Verticillium dahliae or a vascular parasitic pathogen.

Further, the plant may be a Malvaceae plant.

Still further, the Malvaceae plant may be a cotton plant (eg, upland cotton).

In a specific embodiment of the present invention, the plant is specifically an upland cotton variety Zhongmian Institute 35.

In a specific embodiment of the present invention, the Verticillium dahliae is the Verticillium dahliae 'V991' strain.

In a specific embodiment of the present invention, the resistance to Verticillium wilt, the resistance to Verticillium dahliae, and the resistance to vascular parasitic pathogens are embodied in the rate of diseased plants and the / or disease index.

In the sixth aspect, the present invention claims the application of the miRNA shown in (B) in the first aspect above in regulating the expression level of the protein shown in (A) in the first aspect above.

The present invention designs specific primers according to the gene sequence published in the miRNA database (http://www.mirbase.org/), and uses PCR technology to clone and obtain ghr-miR164a (SEQ ID No. 1, its precursor coding) from cotton leaves The gene is shown in SEQ ID No. 5). Specific primers were designed from the gene sequences published in the cotton genome database (https://www.cottongen.org/), and the GhNAC100L gene (SEQ ID No. 3) was cloned from cotton leaves using PCR technology. Using qRT-PCR analysis, it was found that ghr-miR164a was expressed in both roots, stems and leaves of cotton, with dominant expression in roots and lower expression in leaves; on the contrary, GhNAC100L had the lowest expression in roots. At the same time, the expression of ghr-miR164a and GhNAC100L were induced by Verticillium dahliae, and the expression of ghr-miR164a was up-regulated under the induction of pathogenic bacteria, while the expression of GhNAC100L was inhibited. GhNAC100L gene-silenced plants were obtained by virus-induced gene silencing (VIGS) technology. The disease resistance analysis of gene-silenced plants inoculated with Verticillium dahliae showed that the diseased plant rate and disease index of the silenced plants were lower than those of the control plants, indicating that silencing the GhNAC100L gene could improve the disease resistance of cotton plants. At the same time, using VIGS technology to obtain ghr-miR164a overexpressing and silenced plants. The disease resistance analysis of these plants inoculated with Verticillium dahliae showed that ghr-miR164a-overexpressing plants could enhance the disease resistance of cotton plants, while ghr-miR164a-silenced cotton plants became susceptible to disease. The results of GUS staining showed that ghr-miR164a could directly cleave the mRNA of GhNAC100L after transcription. Therefore, ghr-miR164a and GhNAC100L, as a disease resistance module, can positively regulate disease resistance and can be used as candidate genes for cotton verticillium wilt resistance breeding.

Description of drawings

Figure 1 is a comparison diagram of the phylogenetic tree analysis of cotton GhNAC100L and other plants NAC100.

Figure 2 is a graph showing the specific expression analysis of ghr-miR164a and GhNAC100L in different tissues and organs of cotton.

Figure 3 is a schematic diagram of the expression profile analysis of GhNAC100L at different time points after Verticillium dahliae infects cotton.

Figure 4 is a schematic diagram of GUS staining analysis of ghr-miR164a and GhNAC100L in tobacco leaves.

Figure 5 is a schematic diagram of cotton GhPDS gene silencing analysis.

Figure 6 is a schematic diagram of expression analysis of cotton ghr-miR164a silenced and overexpressed plants.

Figure 7 is a schematic diagram of the disease situation of cotton ghr-miR164a silenced and overexpressed plants and control plants.

Figure 8 is a schematic diagram showing the statistics of disease index of cotton ghr-miR164a silenced and overexpressed plants and control plants.

Figure 9 is a schematic diagram of cotton GhNAC100L gene silencing analysis.

Figure 10 is a schematic diagram of the disease situation of cotton GhNAC100L gene-silenced plants and control plants.

Fig. 11 is a schematic diagram of disease index statistics of cotton GhNAC100L gene-silenced plants and control plants.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

For the use of the kit, please refer to the application instructions of the purchased kit; the restriction enzymes and other tool enzymes used are products of TaKaRa Company.

The cotton material is China Cotton Research Institute 35, from the Seedling Company of the Cotton Research Institute of Shanxi Academy of Agricultural Sciences. Tobacco material, Escherichia coli 'DH5α' competent cells and Agrobacterium tumefaciens strain 'GV3101' were preserved by the State Key Laboratory of Plant Genomics. Verticillium dahliae strain (V. dahliae) 'V991', gifted by researcher Jian Guiliang, Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Various restriction enzymes and Buffer are products of TaKaRa Company. All kinds of chemical reagents, culture medium, column-type plant total RNA isolation, extraction and purification kits and plant DNA extraction kits are all products of Shanghai Sangon Bioengineering Co., Ltd. The plasmid miniprep kit (upgraded spin column type) is a product of Shanghai Jierui Bioengineering Co., Ltd. TaqDNA polymerase, dNTPs, Taq Buffer, T4 DNA ligase, PCR purification and recovery kit; PCR gel cutting and recovery kit, cloning vector pEASY-T1 kit, EasyScript One-Step gDNA Removel and cDNA SynthesisSuperMix kit, TransStart Top Green The qPCR SuperMix kits are all products of Beijing Quanshijin Biotechnology Co., Ltd. The plant expression vector pBI121 is a product of Shanghai Jierui Bioengineering Co., Ltd.

Example 1. Cloning and expression analysis of cotton GhNAC100L gene

1. Extraction of total RNA and synthesis of the first strand of cDNA

The seeds of China Cotton Research Institute 35 were soaked in sterile water, soaked at 37°C overnight, then transferred to a culture box, and placed in a light incubator at 25°C for 48h, with a photoperiod of 12h, light, 8h in the dark, and other seeds. After germination, it was transferred to a water-filled incubator and placed in an incubator for cultivation. After the cotton seedlings grew true leaves, the roots, stems and leaf samples of the cotton seedlings were picked respectively, and the cotton RNA was extracted using a column-type plant total RNA isolation, extraction and purification kit. Synthesis of the first strand of cDNA was performed according to the instructions of the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit.

2. Cotton GhNAC100L gene cloning

According to the orthologous alignment of the Arabidopsis NAC100 sequence in the cotton genome database (https://www.cottongen.org/), a highly conserved orthologous gene with Arabidopsis NAC100 was found and named GhNAC100L. Primers are designed according to the GhNAC100L gene sequence, and the primers are:

GhNAC100L-F: 5'-ATGGCAAACATTGGTGAAGAAG-3';

GhNAC100L-R: 5'-TCAGTAATGCCAAAAACAAT C-3'.

All primers in this experiment were synthesized by Beijing Zixi Biotechnology Co., Ltd. The cDNA of cotton leaf was used as the template for PCR amplification, and the PCR product was connected to the T vector for sequencing. The sequencing was completed by Beijing Qingke Biotechnology Co., Ltd. The results show that the size of the obtained fragment is consistent with the expected size. The full length of the fragment is 999bp, including the complete ORF reading frame starting from ATG and ending with TGA, encoding 332 amino acid residues, with a molecular weight of 37.5KDa and an isoelectric point pI=5.11 . The GhNAC100L gene sequence is shown in SEQ ID No.3, which encodes the protein shown in SEQ ID No.2.

3. Construction of phylogenetic tree of GhNAC100L gene sequence

The amino acid sequences of NAC100 of different plants were selected in NCBI, they are: soybean GmNAC100 (XP_003524726), cassava MeNAC100 (XP_021614789), rubber tree HbNAC100 (XP_021642379), grape VvNAC100 (XP_002284825) and Arabidopsis 30 AtNAC100 (AT5G614). Using the MEGA5.2 program, they and cotton GhNAC100L were used to construct a phylogenetic tree. From Figure 1, we can see that GhNAC100L has a high genetic relationship with cassava MeNAC100 (XP_021614789) and rubber tree HbNAC100 (XP_021642379) proteins.

4. Expression analysis of GhNAC100L gene in different tissues and different treatment conditions

(1) Sample collection and processing settings

Inoculation of Verticillium dahliae: Plant the 35 seeds of the germinating plant in a hydroponic incubator, and place it in an incubator with 16 hours of light, 8 hours of darkness, and 28°C for cultivation. Cotton seedlings of leaves were inoculated with a spore suspension of Verticillium dahliae V991. The taproots of cotton plants were treated uniformly and then soaked in a spore solution containing 10 ⁶ /mL Verticillium dahliae V991 for 50 min, then transferred to a hydroponic box and placed in an incubator to continue culturing. Cotton plant root samples were taken at corresponding time points (1, 4, 7, 10 and 13 days) and stored in a -80°C refrigerator for later use.

(2) Quantitative Real-time RT-PCR (qRT-PCR) method

Using qRT-PCR method, the expression of ghr-miR164a and GhNAC100L in cotton roots, stems and leaves and their expression after induction by pathogenic bacteria and hormones were detected.

miRNA reverse transcription primers:

miR164-RT: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGCACG-3';

5.8S-R: 5'-TGTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTGTGA-3'.

The primers for amplifying ghr-miR164a are:

miR164-F: 5'-GCGGCGTGGAGAAGCAGGCA-3;

miR164-R: 5'-GTGCAGGGTCCGAGGT-3'.

Using cotton 5.8S as the internal reference gene, the amplification primers are:

5.8S-F: 5'-ATTAGGCCGAGGGCACGTCTG-3';

5.8S-R: 5'-GTGCAGGGTCCGAGGT-3'.

The reaction system used in qRT-PCR was 20 μL, and the cDNA of cotton samples after different treatments was used as the template. The specific reaction system was prepared according to the instructions of the kit. The amplification conditions were: pre-denaturation at 95°C for 2 min, 40 cycles: 95°C for 10s, 58°C for 15s, and 72°C for 15s. Taking 5.8S gene as the internal reference, the biological experiment was repeated three times.

The primers for amplifying GhNAC100L are:

qGhNAC100L-F: 5'-CCAATCCTATCGGTCCTCAAG-3';

qGhNAC100L-R: 5'-AAATCAGCAGCGTAGAACGG-3'.

Using cotton UBQ7 as the internal reference gene, the amplification primers are:

UBQ7-F: 5'-GAAGGCATTCCACCTGACCAAC-3';

UBQ7-R: 5'-CTTGACCTTCTTCTTCTTGTGCTTG-3'.

The reaction system used in qRT-PCR was 20 μL, and the cDNA of cotton samples after different treatments was used as the template. The specific reaction system was prepared according to the instructions of the kit. The amplification conditions were: 95°C, 5 min; 95°C, 10s; 60°C, 30s; 72°C, 30s; 40 cycles. Taking UBQ7 gene as the internal reference, the biological experiment was repeated three times.

The relative expression levels of genes were calculated by the 2 ^-ΔΔCT method.

(3) Tissue-specific expression analysis

The total RNA of cotton roots, stems and leaf organs was extracted and reverse transcribed into cDNA. The cDNA was used as a template for qRT-PCR analysis. The results showed that the ghr-miR164a and GhNAC100L genes were expressed in these tissues and organs (Figure 2). The expression of ghr-miR164a was higher in roots, followed by stems, and the lowest in leaves. The opposite was true for GhNAC100L, suggesting a negative correlation between the expressions of these two genes, and their modular functions may be tissue-specific.

(4) The expression analysis of cotton ghr-miR164a and GhNAC100L induced by Verticillium dahliae invasion

In order to analyze whether the ghr-miR164a and GhNAC100L genes are involved in the disease resistance of cotton, cotton seedlings were inoculated with the spore liquid of Verticillium dahliae, and the cotton root RNA was extracted and reverse transcribed into cDNA. qRT-PCR analysis of the responses of the ghr-miR164a and GhNAC100L genes to Verticillium dahliae showed a slight increase in ghr-miR164a expression until 7 days after inoculation, while at 10 and 13 days, compared with the control (parallel water treatment) Experiment) significantly up-regulated expression; compared with control (a parallel experiment of water treatment) treatment, GhNAC100L expression level was significantly decreased (Figure 3). It indicated that ghr-miR164a and GhNAC100L genes were induced by Verticillium dahliae and may be involved in the resistance of cotton to Verticillium wilt.

Example 2. Analysis of expression of GhNAC100L regulated by ghr-miR164a after transcription

1. Construction of pBI121-MIR164 plasmid

Extract the 35 DNA from the plant DNA extraction kit, use the extracted DNA as a template, add forward and reverse primers (F: 5'-GC GGATCC CATGAAAACTTAGACCCCAGAGC-3', underlined BamH I restriction site, and R : 5'-CG GAGCTC CCGCTGATGATTGCAGGTG-3', Sac I restriction site is underlined) to amplify the precursor gene of ghr-miR164a (SEQ ID No. 5), and the amplification conditions are: 95℃, 5min; 95℃, 30s ; 60°C, 30s; 72°C, 30s; 35 cycles. The amplified product was inserted into the plant expression vector pBI121, replacing the GUS gene on the original pBI121 vector, to construct the pBI121-MIR164 plasmid, which was verified to be correct by sequencing.

2. Construction of pBI121-NAC100 and pBI121-NAC100mu plasmids

Using the cDNA of China Cotton Research Institute 35 as the template, the forward primer GhNAC100-F (5'-GC TCTAGA ATGGCAAACATTGGTGAAGAAG-3') was added, and the Xba I restriction site (underlined part) and the reverse primer GhNAC100 were introduced into the primer sequence. -R (5'-CG GGATCC TCAGTAATGCCAAAAACAATCAAG-3'), a BamH I restriction enzyme site (underlined part) was introduced into the primer sequence to amplify the gene GhNAC100. The amplification conditions were: 95°C, 5 min; 95°C, 30s; 60°C, 30s; 72°C, 60s; 35 cycles. The amplified product was inserted into the plant expression vector pBI121, fused with GUS gene expression, and the plasmid pBI121-NAC100 was constructed, which was verified to be correct by sequencing.

The mutant forward primer NAC100mu-F (5'-GTTTTTGCCGATTCAACGTATGTACCGTGTTTTAGTGATCCTATCGGTC-3'), and the reverse primer NAC100mu-R (5'-GACCGATAGGATCACTAAAACACGGTACATACGTTGAATCGGCAAAAAC-3') were designed. The plasmid pBI121-NAC100 was used as the template, and the primers GhNAC100-F and NAC100mu-R, NAC100mu-F and GhNAC100-R were used for amplification. The amplification conditions were: 95℃, 5min; 95℃, 30s; 60℃, 30s; 72℃ , 60s; 35 cycles, the amplification products α and β were obtained, respectively. Using the amplification products α and β as templates, primers GhNAC100-F and GhNAC100-R were added, and the amplification conditions were: 95°C, 5 min; 95°C, 30s; 60°C, 30s; 72°C, 60s; 35 cycles. The obtained amplification product (mutation of positions 639-659 of SEQ ID No. 3) was inserted into the plant expression vector pBI121, and the GUS gene was fused for expression to construct the pBI121-NAC100mu plasmid.

3. Cultivation of engineered Agrobacterium

The correctly constructed plasmids pBI121-MIR164, pBI121-NAC100 and pBI121-NAC100mu were transformed into Agrobacterium GV3101 by electroporation, and were treated with kanamycin (50mg/mL), gentamicin (50mg/mL) and rifampicin Screening at 25 mg/mL, positive clones were selected and verified by PCR and sequencing. The results showed that these plasmids had been transformed into Agrobacterium GV3101.

4. Agrobacterium-mediated transformation of tobacco leaves

Agrobacterium strains were inoculated into LB liquid medium containing kanamycin (50mg/mL), gentamicin (50mg/mL) and rifampicin (25mg/mL), and shaken at 28°C overnight; room temperature Centrifuge at 4000g for 10 min; discard the supernatant, resuspend the bacteria with MMA buffer, adjust the OD600 of the bacteria solution to 0.8, and let it stand for about 2 hours at room temperature; mix the resuspended bacteria according to the needs of the test before injection, and lightly tap the tobacco leaves with the needle of the syringe On the lower epidermis, the syringe absorbs the appropriate bacterial solution and injects it at the light point of the needle until the leaves are fully infiltrated with the resuspended bacterial solution; GUS reporter gene staining can be performed on the third day after injection.

5. GUS tissue staining

The whole piece of tobacco leaves was cut, soaked in 90% acetone, 4°C overnight; washed three times with 0.1M PBS buffer; the tobacco leaves were soaked in GUS staining solution (X-Gluc, Triton X-100, EDTA, PBS buffer) solution, potassium ferrocyanide, potassium hexacyanoferrate), overnight at 37 °C; repeatedly washed with 70% ethanol until the yellow leaves faded.

6. Transient expression of ghr-miR164a in tobacco leaves regulates its target gene GhNAC100L

The ghr-miR164a expression vector pBI121-MIR164, its target gene GhNAC100L expression vector pBI121-NAC100 and the mutant target gene mGhNAC100L expression vector pBI121-NAC100mu were transiently expressed in tobacco leaves mediated by Agrobacterium GV3101. Agrobacterium pBI121-NAC100 was transformed alone, and the reporter gene GUS could be expressed normally, with obvious blue color, indicating that the vector was constructed normally. Co-transformed Agrobacterium pBI121-MIR164 and pBI121-NAC100mu, the reporter gene GUS can also be expressed normally, with a clear blue color, indicating that the mutated target gene mGhNAC100L can interfere with the degradation of its target gene GhNAC100L by ghr-miR164a. However, the co-transformed Agrobacterium pBI121-MIR164 and pBI121-NAC100 reporter gene GUS expression was significantly inhibited, and the blue color was lighter, indicating that ghr-miR164a could degrade the expression of its target gene GhNAC100L in N. benthamiana leaves (Figure 4).

Example 3. Resistance analysis of ghr-miR164a and GhNAC100L gene silencing and overexpression plants to Verticillium dahliae

1. Cultivation of virus-induced gene silencing (VIGS) plants

(1) Construction of viral silencing vector pYL156-ST164a

Chemically synthesized by Shanghai Jierui Bioengineering Co., Ltd., STTM164a (5'-GG GGTACC TGCACGTGCCCAGTTGCTTCTCCAGTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATTGCACGTGCCCAGTTGCTTCTCCA CCCGGG GGGA-3'), and the 5' and 3' ends of the STTM164a synthetic fragment (SEQ ID No. Sites Kpn I and Xmal I. The synthetic fragment of STTM164a was digested and inserted into the corresponding restriction site of the viral vector pYL156 to construct a silent vector pYL156-ST164a, which was verified to be correct by sequencing.

(2) Construction of viral overexpression vector pYL156-OE164a

Taking the 35 DNA of the China Cotton Research Institute as the template, the forward primer MIR164a-F (5'-CC ACTAGT CATGAAAACTTAGACCCCAGAGC-3') was used, and the restriction enzyme cleavage site Spe I (underlined part) and the reverse primer MIR164a-R (5'-CCAGAGC-3') were introduced into the primer sequence. '-GGGGTACCCCGCTGATGATTGCAGGTG-3'), the restriction enzyme cleavage site KpnI (underlined) was introduced into the primer sequence, and the amplification conditions were: 95°C, 5min; 95°C, 30s; 60°C, 30s; 72°C, 30s; 35 cycles . The amplified product (SEQ ID No. 5) was inserted into the corresponding restriction site of the viral vector pYL156 to construct the viral vector pYL156-OE164a overexpressing ghr-miR164a, which was verified to be correct by sequencing.

(3) Construction of virus silencing vector pYL156-GhNAC100L

The virus silencing expression vector pYL156 was donated by Professor Liu Yule of Tsinghua University. Firstly, the target fragment of GhNAC100L gene was cloned by PCR technology, and the primers used for amplification were VGhNAC100L-F: 5'-CG GGATCC GCCAAGAATGAATGGGTG-3'; VGhNAC100L-R: 5'-GG GGTACC AATCAGCAGCGTAGAACGG-3' (underlined are BamH I and Kpn I restriction site). The amplification product was digested with restriction enzymes BamH I and Kpn I (the corresponding restriction site recognition sequences and protective bases were added at both ends of SEQ ID No. 4) and the pYL156 vector, and then a ligation reaction was carried out, and the The fragment was inserted between the BamH I and Kpn I sites of the pYL156 vector to construct the viral silencing vector pYL156-GhNAC100L. After the successfully constructed viral silencing vector pYL156-GhNAC100L was transformed into E. coli DH5α, positive clones were selected, digested with BamH I and Kpn I and sequenced for identification. The results showed that the obtained fragment was the target fragment of the GhNAC100L gene to be cloned.

(4) Cultivation of engineered Agrobacterium

The correctly constructed virus silencing vector pYL156-GhNAC100L, helper vector pYL192, positive control vector pYL156-PDS (preserved by the State Key Laboratory of Plant Genomics) and negative control vector pYL156 were transformed into Agrobacterium GV3101 by electroporation, and the The positive clones were selected and verified by PCR and sequencing. The results showed that the virus silencing vector pYL156-GhNAC100L, the helper vector pYL192 , The positive control vector pYL156-PDS and the negative control vector pYL156 have been transformed into Agrobacterium GV3101.

The pYL156-ST164a and pYL156-OE164a plasmids were also transformed into Agrobacterium GV3101 by the same method.

(5) Cultivation of GhNAC100L gene silenced plants, ghr-miR164a silenced and overexpressed plants

The above-validated Agrobacterium was added to the liquid LB medium containing kanamycin (50mg/mL), gentamicin (50mg/mL) and rifampicin (25mg/mL) at 28°C, 200rpm overnight Culture, the culture medium was centrifuged and the supernatant was poured off, and the cells were resuspended with MMA (10mM MES, 10mM MgCl2, 200mM AS) solution, adjusted to a final concentration of OD600 of 1.2, placed in the dark for 2-3 hours, and then The Agrobacterium suspensions containing pYL156-GhNAC100L, pYL156-ST164a, pYL156-OE164a, pYL156-PDS and pYL156 plasmids were mixed with the Agrobacterium suspensions containing pYL192 plasmids at 1:1 for use. After the two cotyledons of the Chinese Cotton Plant 35 are fully expanded, they can be used for Agrobacterium injection infection; first, use a needle to gently scratch the wound on the back of the cotyledon, and then use a 1 mL sterile syringe with the needle removed to inject the bacterial solution from the wound on the back of the cotyledon. Go in, try to make the whole cotyledon infected as much as possible, put the injected plants in a dark place for 12 hours, and then put them in a light incubator for cultivation.

2. Resistance analysis of plants overexpressing ghr-miR164a to Verticillium dahliae

Two weeks after Agrobacterium-transformed cotton plants, an albino phenotype was observed in the newly grown true leaves of the positive control TRV:PDS plants, suggesting that other genes in the same batch may also be silenced (Figure 5). TRV: 00 plants in the negative control group and young leaves of TRV: OE164a plants in the experimental group were taken respectively, total RNA was extracted, reverse transcribed into cDNA, and the diluted cDNA after reverse transcription was used as a template to detect ghr- Expression of miR164a in overexpressed cotton plants (see above for specific methods). The relative expression level of ghr-miR164a in TRV:OE164a plants increased by nearly 250% compared with the control group TRV:00 plants (Figure 6). The overexpressed plants were inoculated with Verticillium dahliae V991 spore fluid, the details were the same as 4(1) in Example 1. After the 21st day, the disease resistance phenotype of cotton plants was observed and the disease index was calculated.

The disease index (DI) of cotton is calculated by the leaf classification method, and the severity of the infection by pathogenic bacteria is divided into 5 grades:

Grade 0: The total leaves of the plant grow normally, without wilting and chlorosis;

Grade 1: The total leaf area of the plant is yellow and wilted <25%;

Grade 2: The yellowing and wilting area of the total leaves of the plant is greater than or equal to 25% and less than 50%;

Grade 3: The yellowing and wilting area of the total leaves of the plant is ≥50% and <75%;

Grade 4: The total leaves of the plant are yellow, the area with wilting is ≥ 75%, or fall off and die.

The results showed that compared with the control group TRV:00 plants, the TRV:OE164a plants were more resistant to the invasion of V991, and the TRV:00 plants in the control group had more serious leaf yellowing, wilting and necrosis (Figure 7). At the same time, the statistical results of disease index also showed that TRV:OE164a plants had stronger resistance (Fig. 8).

3. Resistance analysis of ghr-miR164a silenced plants to Verticillium dahliae

After two weeks of VIGS treatment, the young leaves of the negative control group TRV:00 and the experimental group TRV:ST164a plants were taken to extract total RNA, reverse transcribed into cDNA, and use cDNA as a template to detect whether ghr-miR164a was present in TRV:ST164a plants. Silencing occurred (qRT-PCR method, see above for details). The relative expression of ghr-miR164a in TRV:ST164a plants decreased by about 60% compared with the control group silenced plants (Fig. 6). After confirming that ghr-miR164a was silenced, the silenced plants and the control plants were respectively inoculated with the spore fluid of Verticillium dahliae V991, the details were the same as 4(1) in Example 1. The disease resistance phenotype of cotton plants was observed on day 21 and the disease index was calculated (ibid.).

The results showed that compared with TRV:00 plants, TRV:ST164a had poorer resistance to the infection of V991, and the TRV:ST164a plants had more serious leaf dehydration and necrosis, and even withered cotton plants (Fig. 7). At the same time, the statistical results of disease index also showed that compared with the control group TRV:00 plants, TRV:ST164a silenced plants were more likely to be infected by pathogenic bacteria (Fig. 8).

4. Resistance analysis of GhNAC100L silenced plants to Verticillium dahliae

Using qRT-PCR technology to detect the expression of GhNAC100L gene in silenced plants and control plants (from young leaves, see above for the specific method of qRT-PCR), the results showed that the expression level of this gene in silenced plants decreased by about 60% ( Figure 9). Plants with better silencing effect were selected and inoculated with Verticillium dahliae V991 spore liquid, the details were the same as 4(1) in Example 1. After 21 days of inoculation, the control plants showed obvious disease symptoms such as leaf yellowing, wilting and shedding, while the GhNAC100L silenced plants showed stronger disease resistance (Figure 10). The disease index of GhNAC100L-silenced plants was consistent with the inoculated phenotype (Figure 11), indicating that GhNAC100L-silenced plants were more resistant to Verticillium dahliae. It indicated that GhNAC100L negatively regulates the resistance of cotton to Verticillium dahliae.

<110> Jiushenghe Seed Industry Co., Ltd.; Institute of Microbiology, Chinese Academy of Sciences

<120> Cotton miR164a and NAC100L and their application in regulating plant verticillium wilt resistance

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Claims (10)

1. A biological material, consisting of (A) and (B); (A) protein or its related biological material; said related biological material is a nucleic acid molecule capable of expressing said protein or an expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing said nucleic acid molecule; (B) miRNA or its related biological material; said related biological material is a DNA molecule that can be transcribed into said miRNA or an expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing said DNA molecule; The protein is a protein shown in any of the following (A1)-(A4): (A1) a protein whose amino acid sequence is shown in SEQ ID No.2; (A2) The amino acid sequence defined in (A1) is subjected to the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function as a protein; (A3) A protein that has 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology with the amino acid sequence defined in (A1) or (A2) and has the same function; (A4) a fusion protein obtained by attaching a tag to the N-terminal and/or C-terminal of the protein as defined in any one of (A1)-(A3); The miRNA is a mature miRNA or a precursor miRNA corresponding to the mature miRNA; the nucleotide sequence of the mature miRNA is SEQ ID No.1. 2. The biological material according to claim 1, wherein in (A), the nucleic acid molecule is any of the following: (B1) the DNA molecule shown in SEQ ID No.3; (B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes the protein; (B3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in (B1) or (B2) and encodes the protein. 3 . The biological material according to claim 1 , wherein in (B), the nucleotide sequence of the DNA molecule is shown in SEQ ID No.5. 4 . 4. A protein or a related biological material thereof, as shown in (A) of any one of claims 1-3. 5. The biological material according to any one of claims 1-3 or the protein according to claim 4 or its related biological material or the miRNA or its related biological material shown in any one of claims 1-3 (B) in the following Applications in either: (C1) regulating the resistance of plants to Verticillium wilt; (C2) regulating the resistance of plants to Verticillium dahliae; (C3) Regulation of plant resistance to vascular parasitic pathogens. 6. The application according to claim 5 is characterized in that: the protein shown in (A) in any one of claims 1-3 has higher content and/or activity in the plant, and/or claim 1 The lower the content of the miRNA shown in (B) in any one of -3 in the plant, the weaker the resistance of the plant to Verticillium wilt; the protein shown in (A) in any one of claims 1-3 The lower the content and/or activity in the plant, and/or the higher the content of the miRNA shown in (B) in any one of claims 1-3 in the plant, the more resistant the plant is to Verticillium wilt. The stronger the resistance; The content and/or activity of the protein shown in any one of (A) in claims 1-3 is higher in the plant, and/or the miRNA shown in any one of claims 1-3 (B) is in the plant. The lower the content in the plant, the weaker the resistance of the plant to Verticillium dahliae; the lower the content and/or activity of the protein shown in (A) in any one of claims 1-3 in the plant , and/or the higher the content of the miRNA shown in (B) in any one of claims 1-3 in the plant, the stronger the resistance of the plant to Verticillium dahliae; The content and/or activity of the protein shown in any one of (A) in claims 1-3 is higher in the plant, and/or the miRNA shown in any one of claims 1-3 (B) is in the plant. The lower the content in the plant, the weaker the resistance of the plant to macrovascular parasitic pathogens; the lower the content and/or activity of the protein shown in (A) in any one of claims 1-3 in the plant , and/or the higher the content of the miRNA shown in (B) in any one of claims 1-3 in the plant, the stronger the resistance of the plant to vascular parasitic pathogens. 7. A method for cultivating a plant variety, which is any of the following: Method A1: A method for cultivating plant varieties with improved resistance to Verticillium wilt, comprising the following steps (a1) and/or (a2): (a1) reducing the expression and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in any one of claims 1-3; (a2) increasing the expression of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) in any one of claims 1-3; Method A2: A method for cultivating plant varieties with increased resistance to Verticillium dahliae, comprising the following steps (a1) and/or (a2): (a1) reducing the expression and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in any one of claims 1-3; (a2) increasing the expression of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) in any one of claims 1-3; Method A3: A method for cultivating plant varieties with improved resistance to vascular parasitic pathogens, comprising the following steps (a1) and/or (a2): (a1) reducing the expression and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in any one of claims 1-3; (a2) increasing the expression of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) in any one of claims 1-3; Method B1: a method for cultivating plant varieties with reduced resistance to Verticillium wilt, comprising the following steps (b1) and/or (b2): (b1) increasing the expression and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in any one of claims 1-3; (b2) reducing the expression of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) in any one of claims 1-3; Method B2: A method for cultivating plant varieties with reduced resistance to Verticillium dahliae, comprising the following steps (b1) and/or (b2): (b1) increasing the expression and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in any one of claims 1-3; (b2) reducing the expression of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) in any one of claims 1-3; Method B3: A method for cultivating plant varieties with reduced resistance to vascular parasitic pathogens, comprising the following steps (b1) and/or (b2): (b1) increasing the expression and/or activity of a specific protein in the recipient plant; the specific protein is the protein shown in (A) in any one of claims 1-3; (b2) reducing the expression level of a specific miRNA in the recipient plant; the specific miRNA is the miRNA shown in (B) of any one of claims 1-3. 8. A method for cultivating transgenic plants, which is any of the following: Method C1: a method for cultivating transgenic plants with improved resistance to Verticillium wilt, comprising the steps of: inhibiting expression of a specific nucleic acid molecule in a recipient plant, and/or introducing a specific DNA molecule into the recipient plant, A transgenic plant is obtained; the transgenic plant has improved resistance to Verticillium wilt compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid molecule shown in any one of (A) of claims 1-3; the specific nucleic acid molecule is The DNA molecule is the DNA molecule shown in (B) in any one of claims 1-3; Method C2: A method for cultivating transgenic plants with increased resistance to Verticillium dahliae, comprising the steps of: inhibiting expression of a specific nucleic acid molecule in a recipient plant, and/or introducing a specific DNA into the recipient plant molecule to obtain a transgenic plant; the transgenic plant has improved resistance to Verticillium dahliae compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid molecule shown in (A) in any one of claims 1-3 ; Described specific DNA molecule is the DNA molecule shown in any one of claim 1-3 (B); Method C3: A method for cultivating transgenic plants with improved resistance to vascular parasitic pathogens, comprising the steps of: inhibiting expression of a specific nucleic acid molecule in a recipient plant, and/or introducing a specific DNA molecule into the recipient plant , to obtain a transgenic plant; the transgenic plant has improved resistance to vascular parasitic pathogens compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid molecule shown in (A) in any one of claims 1-3; Described specific DNA molecule is the DNA molecule shown in any one of claim 1-3 (B); Method D1: A method for cultivating transgenic plants with reduced resistance to Verticillium wilt, comprising the steps of: introducing specific nucleic acid molecules into recipient plants, and/or inhibiting expression of specific DNA molecules in said recipient plants , to obtain a transgenic plant; the transgenic plant has reduced resistance to Verticillium wilt compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid molecule shown in (A) in any one of claims 1-3; the The specific DNA molecule is the DNA molecule shown in any one of claims 1-3 (B); Method D2: A method of cultivating transgenic plants with reduced resistance to Verticillium dahliae, comprising the steps of: introducing specific nucleic acid molecules into recipient plants, and/or subjecting the recipient plants to specific DNA molecules Inhibiting expression to obtain a transgenic plant; the transgenic plant has reduced resistance to Verticillium dahliae compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid shown in (A) in any one of claims 1-3 molecule; the specific DNA molecule is the DNA molecule shown in any one of claims 1-3 (B); Method D3: A method for cultivating transgenic plants with reduced resistance to vascular parasitic pathogens, comprising the steps of: introducing specific nucleic acid molecules into recipient plants, and/or inhibiting specific DNA molecules in said recipient plants Expression to obtain a transgenic plant; the transgenic plant has reduced resistance to vascular parasitic pathogens compared with the recipient plant; the specific nucleic acid molecule is the nucleic acid molecule shown in (A) in any one of claims 1-3; The specific DNA molecule is the DNA molecule shown in (B) of any one of claims 1-3. 9. The application or method according to any one of claims 5-9, wherein the plant is a plant capable of infecting Verticillium wilt or Verticillium dahliae or a vascular parasitic pathogen; Further, the plant is a Malvaceae plant; Further, the Malvaceae plant is a cotton plant. 10. Use of the miRNA shown in any one of claims 1-3 (B) in regulating the expression level of the protein shown in any one of claims 1-3 (A).

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