VdNOB1 gene and its target gene fragments against pathogens, interference vectors, and applications in disease resistance.

Abstract

This invention discloses VdNOB1 Genes and their target gene fragments against pathogens, interference vectors, and applications in disease resistance. This invention discovers that... VdNOB1 Gene knockout mutations can significantly reduce the pathogenicity of *Verticillium dahliae* to host plants. This invention also provides *Verticillium dahliae*. VdNOB1 The gene fragment targeting pathogenic bacteria, whose nucleotide sequence is shown in SEQ ID No. 4, was used to construct an RNA interference vector that, when transformed into plants, significantly reduced the pathogenicity of *Verticillium dahliae* to the host plant. This invention further provides a gene containing... VdNOB1 RNA interference vector for a gene or a fragment of a gene targeting pathogenic bacteria. The *Verticillium dahliae* provided in this invention... VdNOB1 Genes or their target gene fragments against pathogens and their RNA interference vectors have promising applications in improving crop resistance to diseases caused by Verticillium dahliae and in breeding new plant varieties resistant to Verticillium dahliae.

Description

Technical Field

[0001] This invention relates to pathogenicity-related genes in *Verticillium dahliae*, and more particularly to *Verticillium dahliae*. VdNOB1 The application of genes and their target gene fragments against pathogens in the prevention and control of plant diseases caused by Verticillium dahliae belongs to the field of pathogenicity-related genes and their target gene fragments against pathogens in Verticillium dahliae and their application of disease resistance. Background Technology

[0002] Cotton Verticillium Wilt, often called "cotton cancer," causes cotton yield reductions of 10-35% in many countries, severely damaging cotton production and causing huge economic losses. The causative agent is *Verticillium dahliae*, a highly pathogenic fungus that can infect cotton throughout its entire growth cycle, leading to wilting and yellowing of leaves. In severe cases, the entire cotton leaf withers and breaks, ultimately causing death. Furthermore, it has a wide host range, infecting over 600 plant species, including annuals, perennials, and woody plants. These include many economically valuable crops, seedlings, and flowers from the Brassicaceae, Solanaceae, Asteraceae, and Rosaceae families, and the range of host plants it can infect is constantly expanding. Because *Verticillium dahliae* is a soil-borne plant pathogen, control is difficult, and currently there are no effective pesticides for its application in production.

[0003] Screening genes related to the pathogenicity of Verticillium dahliae and fragments of resistance target genes from the Verticillium dahliae gene and constructing corresponding interference vectors will have important application prospects for improving plant resistance to diseases caused by Verticillium dahliae. Summary of the Invention

[0004] One of the objectives of this invention is to [dispose of] Verticillium dahliae. VdNOB1 Genes and their target gene fragments against pathogens are used to control plant diseases caused by Verticillium dahliae;

[0005] A second objective of this invention is to provide a product containing the aforementioned *Verticillium dahliae*. VdNOB1 RNA interference vectors for genes or their anti-pathogen target gene fragments;

[0006] The third objective of this invention is to [dispose of] the aforementioned *Verticillium dahliae*. VdNOB1 Genes or their target gene fragments against pathogens, as well as RNA interference vectors containing such target gene fragments, can be used to improve plant resistance to diseases caused by Verticillium dahliae or to construct new plant varieties that are resistant to disease.

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] One aspect of the present invention is to use Verticillium dahliae VdNOB1 Genes are applied to control plant diseases caused by *Verticillium dahliae*; a preferred embodiment of the present invention includes: constructing *Verticillium dahliae*. VdNOB1 RNA interference vectors or gene editing vectors for genes; the constructed Verticillium dahliae VdNOB1 Gene interference vectors or gene editing vectors are transformed into plants.

[0009] Among them, the *Verticillium dahliae* VdNOB1 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0010] The present invention further uses Verticillium dahliae VdNOB1 RNA interference vectors were constructed using the target gene fragments shown in SEQ ID No. 2, SEQ ID No. 3, or SEQ ID No. 4 of the gene. These RNA interference vectors were then transformed into cotton and inoculated with *Verticillium dahliae*. The results showed that the RNA interference vector constructed using the target gene fragment shown in SEQ ID No. 4 significantly reduced the pathogenicity of *Verticillium dahliae*. In contrast, the RNA interference vector constructed using the target gene fragments shown in SEQ ID No. 2 or SEQ ID No. 3 did not significantly reduce the pathogenicity of *Verticillium dahliae* after transformation into cotton.

[0011] Therefore, another aspect of the present invention provides Verticillium dahliae. VdNOB1 The gene contains a target gene fragment for resistance to pathogens, the nucleotide sequence of which is shown in SEQ ID No. 4.

[0012] Another aspect of the present invention is to provide a product containing the aforementioned *Verticillium dahliae*. VdNOB1 RNA interference vectors of genes or their anti-pathogenic target gene fragments and host cells containing said RNA interference vectors.

[0013] In a preferred embodiment of the present invention, the *Verticillium dahliae* VdNOB1 The nucleotide sequence of the gene targeting the pathogen is shown in SEQ ID No. 4.

[0014] Those skilled in the art can use various conventional techniques to construct a compound containing *Verticillium dahliae*. VdNOB1 Various RNA interference vectors for genes or their anti-pathogenic target gene fragments, the construction methods of these RNA interference vectors are all conventional techniques in the field and are well known to those skilled in the art.

[0015] For reference, the present invention provides a method for constructing Verticillium dahliae. VdNOB1Methods for using RNA interference vectors of genes or their anti-pathogenic target gene fragments, including: using *Verticillium dahliae*... VdNOB1 Genes or their anti-pathogen target gene fragments are used Eco RI and Bam HI enzyme is ligated to the pTRV2 vector to obtain the HIGS vector; or, the *Verticillium dahliae* is ligated to the pTRV2 vector via a BP reaction. VdNOB1 The gene or its anti-pathogen target gene fragment is linked to pDONR207, and then constructed into pK7GWIWG2(I),0 through LR reaction to obtain the Gateway interference vector.

[0016] Furthermore, dsRNA transcribed from the target gene fragments shown in SEQ ID No. 2, SEQ ID No. 3, or SEQ ID No. 4 is also included within the scope of protection of this invention.

[0017] Another aspect of the present invention is to use the aforementioned Verticillium dahliae VdNOB1 Genes or their anti-pathogen target gene fragments are used to improve plant resistance to various diseases caused by Verticillium dahliae, including: (1) constructing a structure containing the aforementioned Verticillium dahliae. VdNOB1 (1) RNA interference vector of a gene or its anti-pathogenic target gene fragment; (2) Transform the constructed RNA interference vector into a plant or plant cell.

[0018] Another aspect of the present invention provides a method for cultivating a new plant variety resistant to Verticillium dahliae, comprising the following steps: (1) constructing a plant variety containing the aforementioned Verticillium dahliae. VdNOB1 (1) RNA interference vector of gene or its anti-pathogenic target gene fragment; (2) Transform the constructed RNA interference vector into plants or plant cells; (3) Screen to obtain new plant varieties with improved resistance to Verticillium dahliae.

[0019] The disease caused by Verticillium dahliae described in this invention is preferably Verticillium wilt of cotton.

[0020] The transformation schemes and the schemes for introducing the nucleotides into plants described in this invention can vary depending on the type of plant or plant cell being transformed; specifically, suitable methods for introducing the nucleotides into plant cells include: microinjection, electroporation, Agrobacterium-mediated transformation, and direct gene transfer, etc.

[0021] The plant described in this invention is a host plant of Verticillium dahliae, and is more preferably an agricultural crop or vegetable, including any one of tobacco, cotton, tomato, potato, melon, watermelon, cucumber or peanut, with cotton being the most preferred.

[0022] This invention first identifies VdNOB1Gene expression patterns during Verticillium dahliae infection of cotton, and calculations. VdNOB1 Gene expression levels, the results showed VdNOB1 Gene expression levels varied significantly at different times, showing an overall trend of activation. This was particularly evident 2-8 hours after pathogen infection. VdNOB1 Gene expression levels increased significantly; this invention uses, respectively VdNOB1 Three target gene fragments in the coding region (their nucleotide sequences are shown in SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively) were used to construct HIGS vectors, which were then transformed into cotton using the HIGS transformation method before being inoculated with *Verticillium dahliae*. The fungal biomass and transcription level of the target genes were detected by disease index and molecular biology methods to screen for the interference fragment with the best effect. The results showed that the target gene shown in SEQ ID No. 4 could significantly reduce the pathogenicity of *Verticillium dahliae* infection in cotton.

[0023] This invention further constructs VdNOB1 Gene knockout mutants and complement strains were observed. dNOB1 Gene knockout mutants and VdNOB1 Phenotypic and pathogenicity of functional complement strains; results showed that... VdNOB1 The gene knockout mutant showed significantly lower colony diameter and sporulation rate on multi-carbon source media compared to the V991 wild-type strain. VdNOB1 The colony diameter and sporulation of the complement strains reverted to wild-type levels, indicating that... VdNOB1 Gene knockout mutations can significantly reduce the ability of Verticillium dahliae to utilize multiple carbon sources.

[0024] VdNOB1 The gene knockout mutant showed significantly lower colony diameter and sporulation rate on culture media under multiple stress compared to the V991 wild-type strain. VdNOB1 The colony diameter and sporulation of the complement strains reverted to wild-type levels, indicating that... VdNOB1 Gene knockout mutations can significantly reduce the ability of Verticillium dahliae to resist stress from NaCl, SDS, Sorbitol, CFW, CR, and H2O2.

[0025] VdNOB1 The disease index of cotton infected by the gene knockout mutant strain was about 80% lower than that of cotton infected by the V991 wild-type strain, and the biomass of Verticillium dahliae in the cotton root tissue was about 40% lower than that of the V991 wild-type strain, indicating that its pathogenicity to cotton was significantly reduced compared to the V991 wild-type strain; while VdNOB1The disease index of cotton infected with the complement strain and the biomass of *Verticillium dahliae* in the root tissue both recovered to the level of the wild-type strain V991, indicating that its pathogenicity to cotton is comparable to that of the wild-type strain; in summary, this invention further confirms VdNOB1 Gene knockout mutations can significantly reduce the ability of Verticillium dahliae to infect and cause disease in cotton.

[0026] This invention has significant application prospects in improving plant resistance to diseases caused by Verticillium dahliae and in cultivating new plant varieties resistant to Verticillium dahliae.

[0027] Terminology definitions involved in this invention

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] The terms "polynucleotide" or "nucleotide" mean deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (including (but not limited to) degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.

[0030] The terms "recombinant host cell" or "host cell" refer to a cell containing the nucleotides of the present invention, regardless of the method used for insertion to produce the recombinant host cell. The host cell may be a prokaryotic cell or a eukaryotic cell.

[0031] The term "RNA interference (RNAi)" refers to the phenomenon of silencing the expression of homologous genes induced by exogenous or endogenous double-stranded RNA within cells. Attached Figure Description

[0032] Figure 1 is VdNOB1 Expression pattern identification heatmap.

[0033] Figure 2 For target fragment amplification and pTRV2:: VdNOB1Recombinant plasmid construction; Note: M: DL2000 DNA marker; lines 1: VdNOB1-1 Fragment; lines 1: VdNOB1-2 Fragment; lines 1: VdNOB1-3 Excerpt.

[0034] Figure 3 pTRV2:: VdNOB1 Disease phenotypes in cotton after silencing; A: Cotton whitening phenotype; B: pTRV2 14 days after V991 infection. 00 and pTRV2:: VdNOB1-1-3 Plant phenotypic identification, bar=2 cm; C: Real-time quantitative PCR detection VdNOB1-1-3 Silencing efficiency D: 14 ​​days after vaccination, pTRV2:: 00 and pTRV2:: VdNOB1-1, 2, 3 Cotton plant disease index statistics; E: 14 days after inoculation, pTRV2:: 00 and pTRV2:: VdNOB1-1, 2, 3 Relative fungal biomass of cotton plants was determined; MOCK represented uninoculated cotton grown naturally in the laboratory, while WT represented cotton grown naturally in the laboratory until it developed two true leaves before being directly inoculated. pTRV2:: 00 To allow the organism to grow naturally in the laboratory until both cotyledons have unfolded, pTRV2 was injected. 00 Cotton was inoculated with Agrobacterium tumefaciens solution; ** indicates extremely significant difference at the P<0.01 level; * indicates significant difference at the P<0.05 level.

[0035] Figure 4 for VdNOB1 Mutant validation results; Note: 1: ΔVdNOB1-1 ,2: ΔVdNOB1-2 N: negative control, WT: wild type, U: upstream gene segment, D: downstream gene segment.

[0036] Figure 5 for VdNOB1 Complement verification results; Note: 1: ΔVdNOB1-C-1 ,2: ΔVdNOB1-C-2 N: negative control, WT: wild type.

[0037] Figure 6 WT on different carbon source media ΔVdNOB1 and ΔVdNOB1-C Growth diameter and strain morphology; Note: A: WT, ΔVdNOB1 , ΔVdNOB1-C Colony morphology with different carbon sources; B: WT, ΔVdNOB1 , ΔVdNOB1-C Colony diameters for different carbon sources; C: WT, ΔVdNOB1 ,ΔVdNOB1-C Number of spores with different carbon sources.

[0038] Figure 7 WT on different stress media ΔVdNOB1 and ΔVdNOB1-C Growth diameter and strain morphology; Note: A: WT, ΔVdNOB1 , ΔVdNOB1-C Different stress colony morphologies; B: WT, ΔVdNOB1 , ΔVdNOB1-C Different stress colony diameters; C: WT, ΔVdNOB1 , ΔVdNOB1-C Number of spores under different stresses.

[0039] Figure 8 For WT, ΔVdNOB1 and ΔVdNOB1-C Disease phenotype in cotton after inoculation; Note: A: WT, ΔVdNOB1 , Δ VdNOB1-C The phenotype of cotton 14 days after inoculation with the strain; B: WT, ΔVdNOB1 , ΔVdNOB1-C Disease index of the strain 14 days after inoculation; C: WT, ​ , ​ Relative fungal biomass 14 days after inoculation. Detailed Implementation

[0040] The present invention will be further described below with reference to specific experimental examples, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the experimental examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0041] 1. Biomaterials and reagents

[0042] 1.1 Experimental materials, vectors and strains

[0043] Upland cotton variety 'Zhongmian 49'; cotton VIGS carriers pTRV1, pTRV2, pTRV2:: ​The highly pathogenic Verticillium dahliae strain V991; Escherichia coli DH5α strain, Agrobacterium GV3101, and EHA105 competent cells were all preserved in the Key Laboratory of Ecological Adaptation and Evolution of Extreme Environment Biology, College of Life Sciences, Xinjiang Agricultural University. The growing conditions for upland cotton were as follows: plump seeds were selected, washed with sterile water, and soaked in a greenhouse at 37℃ for 24-36 h. Seeds showing signs of sprouting were planted in pots containing a mixture of autoclaved vermiculite and black soil (1:2). The growing conditions were indoor culture at 28℃ with a photoperiod of 16 h light and 8 h darkness.

[0044] 1.2 Test Reagents

[0045] The polysaccharide and polyphenol plant total RNA extraction kit was purchased from Hangzhou Bori Technology Co., Ltd.; the plasmid mini-preparation kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; the DNA Marker and reverse transcription kit MonScript™ RTIII all-in-one Mix (with dsDNase) were purchased from Monad Biotechnology Co., Ltd.; the seamless cloning kit pEASY®-UniSeamless Cloning and Assembly Kit was purchased from Beijing TransGen Biotech Co., Ltd.; the 2×Taq PCR Mix was purchased from Beijing Adley Biotechnology Co., Ltd.; and restriction endonucleases were also purchased. ​ RI ​ HI、 ​ I, ​ d III was purchased from Thermo Fisher Scientific; the 2x ChamQ Universal SYBR qPCR Master Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.; the synthesis of primers and DNA sequencing for PCR were performed by Xinjiang Youkang Biotechnology Co., Ltd. CAR, Cef, and 5-fluorouracil were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Hyg and G418 were purchased from Beijing Coolplay Technology Co., Ltd.; IM medium was purchased from Shanghai Ruichu Biotechnology Co., Ltd.; and PDA medium was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.

[0046] Experimental Example 1 ​ Gene expression pattern identification test

[0047] 1. Experimental Methods

[0048] In order to identify ​ Gene( ​ The expression pattern of the gene (as shown in SEQ ID No. 1) during the infection of cotton by *Verticillium dahliae* was investigated by inoculating cotton at the two-leaf-one-heart stage with 10 spores. 7Verticillium dahliae strain V991 was used, and cotton root tissue was collected at 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 120 h, and 144 h after inoculation and stored at -80 ℃ for total RNA extraction and reverse transcription into cDNA. The actin (VDAG_00941) of Verticillium dahliae was used as an internal reference gene, and the expression level at 0 h after inoculation was set as 1.

[0049] The specific experimental method is as follows:

[0050] 'Zhongmian 49' cotton was cultured in the soil of the growth chamber for 15 days. The roots of the cotton at the two-leaf-one-heart stage were then soaked in water with a spore count of 10. 7 In a CFU / mL *Verticillium dahliae* V991 bacterial culture, at 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 120 h, and 144 h, appropriate amounts of cotton root tissue were collected and stored at -80 °C. RNA was extracted using a total RNA extraction kit, and cDNA was prepared using a reverse transcription kit (MonScript™ RTIII all-in-one Mix). qRT-PCR primers were designed using NCBI. ​ -qRT-F / R (Table 1), using the actin (VDAG_00941) of *Verticillium dahliae* as an internal control gene, with three technical replicates for each sample. After the reaction, 2... -ΔΔCt The method calculates gene expression levels, where Ct is the cycle threshold, and the expression level at 0 h post-inoculation is set to 1.

[0051] 2. Experimental Results

[0052] calculate ​ See gene expression level results. ​ ;according to ​ The results show that ​ Gene expression levels varied significantly at different times, showing an overall trend of activation. This was particularly evident 2-8 hours after pathogen infection. ​ Gene expression levels increased significantly.

[0053] Experimental Example 2: Silence ​ Test to reduce the ability of Verticillium dahliae to infect cotton

[0054] 1. Experimental Methods

[0055] 1.1 Construction of HIGS vector

[0056] Design specific primers using Primer5 ​ -HIGS-F / R (Table 1), amplified using KOD DNA polymerase (Toyobo, Shanghai, China) ​The three target gene segments in the coding region are: ​ ​ The nucleotide sequences are shown in SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively. The PCR amplification products were then detected by 1% agarose gel electrophoresis, and fragments were recovered. ​ RI and ​ HI enzyme was ligated into the pTRV2 vector to obtain pTRV2:: ​ pTRV2:: ​ pTRV2:: ​ The carrier, pTRV2:: was used in a freeze-thaw process. ​ pTRV2:: ​ pTRV2:: ​ The plasmid was transformed into Agrobacterium GV3101.

[0057] Table 1 Primer names and sequences

[0058]

[0059] Note: The bases of lowercase letters are homologous arms.

[0060] 1.2 HIGS Conversion Method

[0061] A single Agrobacterium colony containing a positive plasmid was placed in LB liquid medium (25 µg / mL Rif and 50 µg / mL Kan) and incubated overnight at 28°C with shaking. The next day, the bacterial culture (2% concentration) was added to LB liquid medium and incubated again with shaking until OD reached. 600 When the OD value is 0.5-0.6, collect the bacterial cells by low-temperature centrifugation. Discard the waste liquid and resuspend the bacterial cells in the injection matrix (10 mM MMES, 10 mM MgCl2, 100 µM acetylsylgenone), adjusting the OD value accordingly. 600 To 0.8-1.0. Agrobacterium bacterial suspension containing pTRV1 vector was mixed separately with suspension containing pTRV2:: ​ plasmid 、 pTRV2:: ​ plasmid 、 pTRV2:: ​ Plasmid containing pTRV2:: ​ Plasmid (causing cotton leaves to turn white), pTRV2:: 00 The Agrobacterium tumefaciens in the empty carrier was mixed at a 1:1 ratio and allowed to stand at room temperature for 3-5 hours. Finally, the Agrobacterium tumefaciens mixture was injected into cotton seedlings with flat cotyledons using a syringe.

[0062] 1.3 HIGS Silent Plant Pathogenicity Determination and Fungal Biomass Determination

[0063] The newly emerged true leaves of cotton seedlings exhibited a complete whitening phenotype. Using the method described in Example 1, the seedlings were inoculated with *Verticillium dahliae* V991 bacterial suspension. Fourteen days after inoculation, the disease incidence in the cotton was observed, and the cotton disease index was recorded (Table 2) and calculated according to Formula 1. The silencing effect was detected by qPCR on the root tissue samples. Genomic DNA was extracted from the root tissues and analyzed using *Verticillium dahliae*. ​ Primers ​ -qPCR-F / R (Table 1) and internal reference genes ​ Primers ​ -qPCR-F / R (Table 1) was used to quantitatively detect the biomass of Verticillium dahliae in cotton roots using qPCR.

[0064] Table 2. Statistics of Disease Indices

[0065]

[0066] Formula 1: Disease index = [Ʃ (number × level) / (total plant × highestlevel)] ×100.

[0067] 2. Experimental Results

[0068] 2.1 Construction of HIGS Recombinant Vector

[0069] Amplification of cDNA containing *Verticillium dahliae* V991 was performed. ​ RI and ​ I restriction site ​ ​ ( ​ The amplified target fragment (approximately 300 bp) was sequenced and compared with the target gene CDS library. The gene sequence was found to be consistent with the target sequence and was used for the construction of the HIGS vector. The target fragment was then compared with the target gene CDS library. ​ RI and ​ The HIGS vector pTRV2, digested with HI double enzyme, was ligated and transformed into E. coli. Subsequent colony PCR confirmed that the size of the HIGS recombinant plasmid matched the expected gene fragment, demonstrating successful construction. Further transformation into Agrobacterium competent cells GV3101 was then performed to conduct a cotton HIGS gene silencing experiment.

[0070] 2.2 HIGS Silence ​ Verify gene function

[0071] 14 days after injection of bacterial culture, pTRV2:: ​ The true leaves of the control plants showed leukoplakia. ​(A) Five silent and five control plants were selected, and RNA was extracted from their leaves for Real-time quantitative PCR detection. ​ ​ Silencing efficiency of fragment genes ( ​ (C), the results show ​ Genes are silenced, with a silencing efficiency of over 50%, which can [result in] [the following]. ​ The seedling stage function of the gene was studied. Control group (negative control pTRV2:: 00 ) and silent group plants were inoculated with V991 14 days later, ​ The silent group showed yellowing of individual leaves with downward curling leaf margins, while the control group (negative control pTRV2:: 00 This results in more severe symptoms of Verticillium wilt, such as plant wilting and leaf drop. (Take...) ​ Cotton plants in the silent group and the control group (negative control pTRV2:: 00 The cotyledon junction of cotton plants was dissected and observed under a stereomicroscope. In the control group, the vascular bundles showed browning, with more pronounced brown streaks. ​ (B). Meanwhile, the control group (negative control pTRV2:: 00 The disease index and relative fungal biomass of the plants were significantly higher than those of the silent group. ​ (DE). Therefore, ​ The gene plays a positive regulatory role in the infection of cotton by *Verticillium dahliae*. Among the three target gene fragments for resistance to the pathogen... ​ The fragment significantly reduced the disease index and fungal biomass, showing significant differences compared to the negative control group and wild type. ​ Fragments and ​ The fragment did not significantly reduce the disease index and fungal biomass, and there was no significant difference compared with the negative control group and wild type.

[0072] Experimental Example 3 ​ Construction, phenotypic and pathogenicity observation experiments of gene knockout mutants and complement strains

[0073] 1. Experimental Methods

[0074] 1.1 ​ Construction of gene knockout mutants and complement strains

[0075] 1.1.1 ​ Construction of gene knockout mutant strains

[0076] Using the wild-type genomic DNA of *Verticillium dahliae* strain as a template (genome DNA extracted by the CTAB method), corresponding primers were designed for amplification. ​ Fragments approximately 1000 bp each upstream and downstream of the gene were used in-fusion technology to...​ Approximately 1000 bp fragments upstream and downstream of the gene are inserted into the vector pGKO- ​ ( ​ At the corresponding position (i.e., linearized restriction enzyme digestion), the corresponding pGKO- is finally obtained. ​ - ​ Knock out the plasmid. Take pGKO- ​ - ​ Plasmids were used to transform Agrobacterium EHA105 competent cells using heat shock, and the cells were plated on LB agar plates containing Kan and Rif resistance and cultured for 48 h. Clones were randomly selected for PCR identification. Positive clones were preserved and co-incubated with Verticillium dahliae on IM solid medium containing acetylsyleugenol for 48 h. Afterward, the cells were transferred to PDA medium containing Carb, Cef, Hyg, and 5-fluorouracil. Once single colonies appeared, they were transferred to fresh PDA medium containing Carb, Cef, Hyg, and 5-fluorouracil for another round of selection, followed by PCR amplification and identification. Two strains of each were ultimately obtained. ​ Knockout mutant strains.

[0077] 1.1.2 ​ Construction of gene complement strain

[0078] Using wild-type Verticillium dahliae cDNA as a template, specific primers were designed (see Table 1) for amplification. VdNOB1 Gene CDS sequence. In-Fusion technology was used to sequence the gene in the pMC vector (…). Bam H Ⅰ / Sal Insertion before the GFP CDS sequence (linearized by enzyme digestion) VdNOB1 The gene CDS sequence (excluding the stop codon) was finally used to obtain the complement plasmid pMC- VdNOB1 The obtained replenishment plasmid pMC- VdNOB1 The two previously obtained strains were transformed using the Agrobacterium co-culture transformation method. VdNOB1 After multiple rounds of G418 resistance screening and PCR amplification identification, two corresponding mutant strains were finally obtained from the knockout mutant strain. VdNOB1 complement strain.

[0079] 1.2 VdNOB1 Phenotypic and pathogenicity observation of gene knockout mutants and complement strains

[0080] 1.2.1 Knockout VdNOB1 Effects on colony diameter and sporulation on various carbon source media

[0081] Preparation of Verticillium dahliae V991 strain, VdNOB1 Knockout mutant strains and VdNOB1 Functional complement strain spore suspension, adjusted spore concentration to ×10 6CFU / mL. 10 µL of spore suspension was added dropwise to the center of Czapek's agar plates containing Sucrose, Pectin, Xylose, Starch, and Galactoose as carbon sources, respectively, and incubated in the dark at 25°C for 12 days. After 12 days, the colony diameter was observed, counted, and photographed. 2 mL of sterile water was added to the plate, and colonies were scraped from the plate using a sterile spreader. The plates were filtered through a 40 μm cell filter, and the number of spores in the filtrate was counted using a hemocytometer to obtain the sporulation yield.

[0082] 1.2.2 Knockout VdNOB1 Effects on colony growth inhibition rate and sporulation yield on culture media under various abiotic stresses

[0083] Verticillium dahliae V991 strain was prepared separately. VdNOB1 Knockout mutant strains and VdNOB1 Functional complement strain spore suspension, adjusted spore concentration to 10. 6 CFU / mL. 10 µL of spore suspension was added dropwise to the center of PDA medium and stress medium containing NaCl, SDS, Sorbitol, CFW, CR, and H2O2, and incubated in the dark at 25°C for 12 days. After 12 days, the colony diameter was observed and photographed. 2 mL of sterile water was added to the plate, and colonies were scraped from the plate using a sterile spreader. The plate was filtered through a 40 μm cell filter, and the number of spores in the filtrate was counted using a hemocytometer to obtain the sporulation yield.

[0084] NaCl and Sorbitol exert their stress primarily by creating a high osmotic pressure environment, while SDS, CFW, and CR exert their stress primarily by interfering with fungal cell wall synthesis, and H2O2 exerts its stress primarily by causing oxidative damage.

[0085] 1.2.3 Knockout VdNOB1 Impact on the pathogenicity of cotton

[0086] Preparation of Verticillium dahliae V991 strain, VdNOB1 Knockout mutant strains and VdNOB1 The spore suspension of the functional complement strain was prepared by adding 2 mL of spore suspension to 200 mL of CM liquid medium and incubating at 25 ℃ on a shaker at 200 rpm. After 5 days, the spores were filtered through a 40 μm cell filter to adjust the spore concentration to 10. 7CFU / mL. When cotton seedlings have developed two true leaves, they are inoculated using the root-dipping method: the seedlings are gently removed from the soil, minimizing root damage; the roots are then immersed in a spore suspension of each strain for 5 minutes, and the inoculated seedlings are replanted in new culture pots. Thirty days after inoculation, the disease incidence of each strain is observed. The disease index is calculated as [∑(number of diseased plants at each level × corresponding disease level) / (total number of plants × highest disease level)] × 100%, and photographed. After completion, a 2 cm long stem segment from the cotyledon area is cut longitudinally, and the vascular bundle color is observed and photographed under a stereomicroscope.

[0087] 2. Experimental Results

[0088] 2.1 VdNOB1 Construction of gene knockout mutant strains

[0089] Knockout of *Verticillium dahliae* using homologous recombination VdNOB1 The gene underwent multiple Hyg resistance screenings and PCR identification. Figure 4 It could not be amplified in the mutant strain. VdNOB1 The gene was successfully amplified. HPT and VdNOB1 By examining upstream and downstream gene fragments, it can be concluded that two strains have been successfully obtained. VdNOB1 Gene knockout strain.

[0090] 2.2 VdNOB1 Construction of gene complement strain

[0091] The obtained replenishment plasmid pMC- VdNOB1 The two previously obtained strains were transformed using the Agrobacterium co-culture transformation method. VdNOB1 Gene knockout mutant strains were identified through multiple rounds of G418 resistance screening and PCR amplification. Figure 5 ), can amplify GFP , VdNOB1 Genes cannot be amplified. HPT Two plants were obtained. VdNOB1 complement strain.

[0092] 2.3 VdNOB1 Phenotypic and pathogenicity observation of gene knockout mutants and complement strains

[0093] 2.3.1 Knockout VdNOB1 Effects on colony diameter and sporulation on various carbon source media

[0094] Depend on Figure 6It was found that the colony diameter and sporulation of both mutant strains on multi-carbon source media were significantly reduced compared to the wild-type V991 strain, while the colony diameter and sporulation of both complement strains returned to wild-type levels. These results indicate that the knockout mutation of this gene can significantly reduce the ability of Verticillium dahliae to utilize multiple carbon sources.

[0095] 2.3.2 Knockout VdNOB1 Effects on colony growth inhibition rate and sporulation yield on culture media under various abiotic stresses

[0096] Depend on Figure 7 It was found that the colony diameter and sporulation of both mutant strains on multi-stress media were significantly lower than those of the V991 wild-type strain, while the colony diameter and sporulation of both complement strains returned to wild-type levels. These results indicate that the knockout mutation of this gene can significantly reduce the resistance of Verticillium dahliae to stresses from NaCl, SDS, Sorbitol, CFW, CR, and H2O2.

[0097] 2.3.3 Knockout VdNOB1 Impact on the pathogenicity of cotton

[0098] Two plants VdNOB1 The disease index of cotton infected with the knockout mutant strain decreased by approximately 80% compared to cotton infected with the V991 wild-type strain, and the biomass of *Verticillium dahliae* in the cotton root tissue decreased by approximately 40% compared to the V991 wild-type strain, indicating that its pathogenicity to cotton was significantly reduced compared to the V991 wild-type strain; while the two strains VdNOB1 The disease index of cotton infected with the complement strain and the biomass of Verticillium dahliae in the root tissue both recovered to the level of the V991 wild-type strain, indicating that its infection and pathogenicity in cotton are comparable to those of the wild-type strain. Figure 8 Therefore, the VdNOB1 Gene knockout mutations can significantly affect the ability of Verticillium dahliae to infect and cause disease in cotton.

Claims

1. Verticillium dahliae VdNOB1 Gene anti-pathogen target gene fragment, characterized in that, Its nucleotide sequence is shown in SEQ ID No.

4.

2. Containing the *Verticillium dahliae* as described in claim 1 VdNOB1 RNA interference vector for gene anti-pathogen target gene fragments, characterized in that, The RNA interference vector is a HIGS vector, and its construction method includes: using Verticillium dahliae... VdNOB1 Utilization of gene anti-pathogen target gene fragments Eco RI and Bam The HI enzyme was ligated into the pTRV2 vector to obtain the HIGS vector.

3. The Verticillium dahliae according to claim 1 VdNOB1 Application of gene anti-pathogen target gene fragments in improving cotton resistance to diseases caused by Verticillium dahliae.

4. The application according to claim 3, characterized in that, include: (1) Constructing a structure containing the aforementioned Verticillium dahliae VdNOB1 (1) RNA interference vector for gene anti-pathogenic bacteria target gene fragment; (2) Transform the constructed RNA interference vector into cotton or cotton cells.

5. The application according to claim 3, characterized in that, The disease caused by Verticillium dahliae is Verticillium wilt of cotton.

6. The application of the RNA interference vector according to claim 2 in improving the resistance of cotton to diseases caused by Verticillium dahliae.

7. The application according to claim 6, characterized in that, include: The RNA interference vector was transformed into cotton or cotton cells.

8. The application according to claim 6, characterized in that, The disease caused by Verticillium dahliae is Verticillium wilt of cotton.

9. A method for cultivating transgenic plant varieties resistant to Verticillium dahliae, characterized in that, include: (1) Constructing a structure containing Verticillium dahliae VdNOB1 An RNA interference vector for a gene targeting a pathogenic bacteria; the method for constructing the RNA interference vector includes: using *Verticillium dahliae*... VdNOB1 Utilization of gene anti-pathogen target gene fragments Eco RI and Bam (1) HI enzyme is ligated to pTRV2 vector to obtain HIGS vector; (2) the constructed RNA interference vector is transformed into plants or plant cells; (3) transgenic plant varieties with improved resistance to Verticillium dahliae are screened; the Verticillium dahliae VdNOB1 The nucleotide sequence of the gene targeting the pathogen is shown in SEQ ID No. 4; the plant is cotton.

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