Cucumber fusarium oxysporum FocPel1 gene and application thereof in creating cucumber disease-resistant germplasm

Abstract

The invention discloses a cucumber fusarium oxysporum FocPel1 gene and application thereof to creation of cucumber disease-resistant germplasm, the sequence of the cucumber fusarium oxysporum FocPel1 gene is as shown in SEQ ID NO 1, and the sequence of an encoding protein of the cucumber fusarium oxysporum FocPel1 gene is as shown in SEQ ID NO 2. The cucumber fusarium oxysporum FocPel1 gene knockout and complementary mutants are created, and it is clear that FocPel1 is a key pathogenic gene of cucumber fusarium oxysporum. The FocPel1-RNAi transgenic cucumber material is constructed, and the material can silently invade the FocPel1 gene in fusarium oxysporum of cucumbers and shows relatively strong fusarium wilt resistance. The pathogenic function of the fusarium oxysporum FocPel1 is defined for the first time, a cucumber germplasm material with high fusarium wilt resistance is constructed, and a new thought and theoretical basis are provided for fusarium wilt resistance molecular breeding of cucumbers and even other cucurbitaceae crops.

Description

Technical Field

[0001] The invention belongs to the fields of genetic engineering and molecular biology, and in particular relates to a FocPel1 gene of cucumber wilt pathogen and its application in creating disease-resistant cucumber germplasm. Background Art

[0002] Cucumbers, originating from the southern foothills of the Himalayas, are annual climbing herbs belonging to the Cucurbitaceae family and the genus Cucumis. They are widely cultivated vegetables worldwide. However, the expansion of continuous cucumber cultivation in greenhouses has led to the frequent occurrence of cucumber wilt diseases, which has seriously hampered the high-quality, safe, and efficient cultivation of cucumbers.

[0003] Cucumber wilt is a devastating soil-borne fungal disease caused by Fusarium oxysporum f. sp. cucumerinum (Foc). This pathogen can infect cucumbers at all stages of growth and development, especially in the middle and late stages of growth. As plant vigor declines, the incidence increases, leading to total crop failure in severe cases. Furthermore, the pathogen has an extremely long incubation period. Due to the continuous cropping model of greenhouse cultivation, the number of Fusarium wilt bacteria in the soil accumulates year by year, leading to an increasingly severe incidence of cucumber wilt. However, the current lack of highly resistant cucumber germplasm resources has limited the development of new wilt-resistant cucumber varieties through traditional breeding methods. Therefore, using modern molecular biology techniques to identify pathogenicity-related genes of Fusarium oxysporum f. sp. cucumerinum and then using these genes to develop wilt-resistant cucumber germplasm and subsequently breed new wilt-resistant cucumber varieties is a green, safe, and effective approach to combating cucumber wilt. Summary of the Invention

[0004] Purpose of the invention: In order to solve the above technical problems, the present invention aims to provide a cucumber Fusarium wilt pathogen FocPel1 gene, which can effectively regulate the resistance of cucumber to Fusarium wilt and solve the problem of limited creation of new cucumber varieties resistant to Fusarium wilt.

[0005] The present invention also provides the application of the FocPel1 gene of cucumber wilt pathogen in creating disease-resistant cucumber germplasm.

[0006] Technical solution: In order to achieve the above purpose, the present invention provides a FocPel1 gene of cucumber wilt pathogen, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The protein sequence encoded by the FocPel1 gene of the cucumber wilt pathogen of the present invention is shown in SEQ ID NO.2.

[0008] The invention discloses an interference expression vector WMV076-FocPel1 for the FocPel1 gene of the cucumber wilt pathogen.

[0009] Construction method of interfering expression vector WMV076-FocPel1 of Fusarium oxysporum f. sp. cucumerinum FocPel1 gene of the present invention includes the following steps:

[0010] (1) Extract total RNA from Fusarium oxysporum f. sp. cucumerinum, and then reverse transcribe it into cDNA;

[0011] (2) Using the cDNA product obtained in step (1) as a template, amplify the FocPel1 target fragment through primers FocPel1-F / R;

[0012] (3) Design primers WMV076-FocPel1-F / R to amplify the FocPel1 target fragment;

[0013] (4) The amplified product is homologously recombined with the linearized vector WMV076, and after correct sequencing, the expression vector WMV076-FocPel1 is obtained, and the nucleotide sequence of its vector WMV076 is shown in SEQ ID NO.3.

[0014] Furthermore, the primers FocPel1-F / R in step (2) are:

[0015] FocPel1-F: 5’-CCAACAGCTCGTCTCACGACG-3’,

[0016] FocPel1-R: 5’-CTTTGAAGTGCCGTTGCGATA-3’.

[0017] Furthermore, the primers WMV076-FocPel1-F / R in step (3) are:

[0018] WMV076-FocPel1-F: 5’-ACGGGGGACGTCGACCCAACAGCTCGTCTCACGACG-3’

[0019] WMV076-FocPel1-R: 5’-GCGGTATATACGTACCTTTGAAGTGCCGTTGCGATA-3’.

[0020] Application of Fusarium oxysporum f. sp. cucumerinum gene FocPel1 or the encoded protein or the interfering expression vector WMV076-FocPel1 of the present invention in regulating cucumber Fusarium wilt resistance.

[0021] Furthermore, application of improving cucumber Fusarium wilt resistance by silencing Fusarium oxysporum f. sp. cucumerinum gene FocPel1 or transferring the interfering expression vector WMV076-FocPel1 into cucumbers.

[0022] Furthermore, the application process is as follows: The interference expression vector WMV076-FocPel1 of the constructed FocPel1 gene is transformed into Agrobacterium tumefaciens, and then it infects cucumbers. The obtained FocPel1-RNAi transgenic lines can silence the FocPel1 gene in the invading pathogens, significantly improving the resistance of cucumbers to Fusarium wilt.

[0023] Use of the Fusarium wilt pathogen gene FocPel1 or the encoded protein or the interference expression vector WMV076-FocPel1 in creating cucumber germplasm resistant to Fusarium wilt.

[0024] In this invention, gene knockout and complementary vectors of FocPel1 of cucumber Fusarium wilt pathogen are respectively constructed. Using the Fusarium oxysporum cucumerinum (Foc) strain as the receptor, the FocPel1 gene knockout vector is transformed by PEG-mediated genetic transformation to create the FocPel1 gene deletion mutant (△FocPel1); then, the △FocPel1 strain is used as the receptor to transform the FocPel1 complementary vector to obtain the FocPel1 complementary mutant (FocPel1.com). By analyzing the pathogenicity of the Foc, △FocPel1 and FocPel1.com strains, it is found that FocPel1 is a key pathogenic gene regulating the pathogenicity of cucumber Fusarium wilt pathogen.

[0025] Construct an RNA interference (RNAi) expression vector of FocPel1 using the specific fragment of the FocPel1 gene, and genetically transform cucumbers. Through antibiotic screening, PCR detection, self-pollination, etc., obtain the cucumber FocPel1-RNAi transgenic T2 lines. By inoculating cucumber Fusarium wilt pathogen and observing the phenotypic differences, investigating the disease incidence and disease index, the resistance of different cucumber seedlings to Fusarium wilt is reflected. It is found that the FocPel1-RNAi transgenic lines can silence the FocPel1 gene in the invading pathogens, significantly improving the resistance of cucumbers to Fusarium wilt.

[0026] Through this invention, the complete sequences of the FocPel1 gene of cucumber Fusarium wilt pathogen and the protein encoded by it are provided. By creating FocPel1 gene deletion and complementary mutants, it is proved that FocPel1 can regulate the pathogenicity of cucumber Fusarium wilt pathogen. At the same time, through the application of creating cucumber germplasm resistant to Fusarium wilt using the FocPel1 gene, construct the FocPel1-RNAi cucumber transgenic lines. This material can silence the FocPel1 gene in the invading Fusarium wilt pathogen, improving the resistance of cucumbers to Fusarium wilt. This invention first clarifies the function of FocPel1 of cucumber Fusarium wilt pathogen, and also first creates and applies cucumber germplasm resistant to Fusarium wilt by constructing FocPel1-RNAi cucumber transgenic materials.

[0027] Beneficial effects: Compared with the prior art, this invention has the following remarkable advantages:

[0028] The present invention utilizes a gene knockout and complementary mutant creation system for Fusarium oxysporum f. sp. cucumerinum, obtains Fusarium oxysporum f. sp. cucumerinum FocPel1 deletion and complementary mutants, and through pathogenicity analysis, for the first time clarifies that FocPel1 is a key pathogenic gene of Fusarium oxysporum f. sp. cucumerinum. By using cucumber transgenic technology, FocPel1-RNAi transgenic lines are constructed. Through inoculation with Fusarium oxysporum f. sp. cucumerinum for analysis, it is clarified that the FocPel1-RNAi transgenic lines can silence the FocPel1 gene in the invading Fusarium oxysporum f. sp. cucumerinum and exhibit strong fusarium wilt resistance, providing new ideas and theoretical basis for molecular breeding of cucumber and other cucurbit crops against fusarium wilt, and having good application prospects. Brief Description of the Drawings

[0029] Figure 1 It is a schematic diagram of the gene knockout and complementary vectors for Fusarium oxysporum f. sp. cucumerinum and mutant verification. Among them, a is the schematic diagram of the gene knockout and complementary vectors for Fusarium oxysporum f. sp. cucumerinum; b is the PCR verification of the DNA of the FocPel1 deletion / complementary mutant strains. Foc represents the Fusarium oxysporum f. sp. cucumerinum strain, △FocPel1 represents the FocPel1 gene knockout mutant, and FocPel1.com represents the FocPel1 complementary mutant; Tublin marked on the figure represents the internal reference primer, and FocPel1 and the hygromycin gene represent the detection primers;

[0030] Figure 2 It is the functional analysis of the FocPel1 deletion / complementary mutants of Fusarium oxysporum f. sp. cucumerinum. Among them, a is the colony morphology of the wild-type Fusarium oxysporum f. sp. cucumerinum Foc (left), the FocPel1 deletion mutant strain △FocPel1 (middle), and the FocPel1 complementary mutant strain FocPel1.com (right) after 5 days; b is the disease phenotypes of cucumber seedlings after inoculation with different strains of Foc, △FocPel1, and FocPel1.com for 20 and 30 days; c and d are the disease incidence rates and disease index of cucumber seedlings after inoculation with different strains of Foc, △FocPel1, and FocPel1.com for 20 and 30 days. The data are analyzed by T-test, and "**" represents a highly significant difference (p < 0.01).

[0031] Figure 3 It is a schematic diagram of the FocPel1-RNAi vector for Fusarium oxysporum f. sp. cucumerinum. Note: The forward and reverse of FocPel1 represent the target fragments of the FocPel1 gene of Fusarium oxysporum f. sp. cucumerinum.

[0032] Figure 4For the identification of FocPel1-RNAi transgenic cucumber plants, where a represents the detection of the antibiotic gene AADA fragment in FocPel1-RNAi transgenic plants; b represents the detection of the 35S promoter + target gene fragment + intron sequence fragment in FocPel1-RNAi transgenic plants; c represents the amplification and sequencing results of the target gene fragment in FocPel1-RNAi transgenic plants.

[0033] Figure 5 For the analysis of the Fusarium wilt resistance of FocPel1-RNAi transgenic cucumber plants, where a is the disease phenotypes of the receptor WT and FocPel1-RNAi transgenic cucumber lines at 10 and 20 days after inoculation with the Fusarium wilt pathogen Foc; b and c are the disease incidence rates and disease indices of the receptor WT and FocPel1-RNAi transgenic cucumbers at 10 and 20 days after inoculation with the Fusarium wilt pathogen Foc; d is the longitudinal section of the basal part of the stem of the receptor WT and FocPel1-RNAi transgenic cucumbers 15 days after inoculation, and the activation of the isolated Fusarium wilt pathogen; e is the analysis of the expression of the FocPel1 gene in the pathogens isolated from the basal parts of the stems of the receptor WT and FocPel1-RNAi transgenic cucumbers; f is the disease phenotype of the receptor WT and FocPel1-RNAi transgenic cucumbers at 35 days after inoculation with the Fusarium wilt pathogen during the adult plant stage; data were analyzed using the T-test, and "**" indicates a highly significant difference (p < 0.01). Detailed implementation manner

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0035] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The experimental methods without specific conditions noted in the examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers.

[0036] The cucumber inbred line Superina 'SUP11' and the cucumber inbred line Chinese Dragon '9930', as well as the cucumber Fusarium wilt pathogen strain Foc (Fusarium oxysporum f.sp. cucumerinum), were published in the article "Isolation and Identification of Cucumber Fusarium Wilt Pathogen and Analysis of Variety Resistance Differences" in the Journal of Molecular Plant Breeding in 2017 (Dong Jingping et al., 2017). The above materials and strains were provided by Yangzhou University.

[0037] Example 1

[0038] I. Cloning of the FocPel1 gene of the cucumber Fusarium wilt pathogen

[0039] Using the cDNA synthesized by reverse transcription of the total RNA extracted from the cucumber Fusarium wilt pathogen Foc as a template, and based on the genomic sequence of the cucumber Fusarium wilt pathogen, primers were designed with the cDNA of the cucumber Fusarium wilt pathogen Foc as a template:

[0040] FocPel1-F: 5'-CCAACAGCTCGTCTCACGACG-3',

[0041] FocPel1-R: 5'-CTTTGAAGTGCCGTTGCGATA-3'. The FocPel1 gene was cloned and verified by sequencing at Nanjing Tsingke Biotechnology Co., Ltd. The obtained FocPel1 nucleotide sequence is shown in SEQ ID NO.1, and the protein sequence is shown in SEQ ID NO.2.

[0042] II. Creation of FocPel1 gene deletion and complementation mutants of Fusarium oxysporum f. sp. cucumerinum

[0043] (1) Construction of the FocPel1 knockout vector of Fusarium oxysporum f. sp. cucumerinum

[0044] Based on the DNA sequence of FocPel1 of Fusarium oxysporum f. sp. cucumerinum, 1500 bp homologous arm sequences were selected before and after the gene coding region (5'UTR, 3'UTR), and overlapping extension PCR primers were designed with the hygromycin gene (HPH) sequence.

[0045] FocPel1-5’UTR-F: 5’-GCAACGCAGTCCTTTCCAC-3’;

[0046] FocPel1-5’UTR-R: 5’-AAAATGCTCCTTCAATATCAGGGTCAGGGATACGGAGGTT-3’;

[0047] FocPel1-3’UTR-F: 5’-TAGATGCCGACCGGGAACCAAGTGTTAGTTGGCGGCTGAT-3’;

[0048] FocPel1-3’UTR-R: 5’-GGTTTGACGCAGGTGCTTAT-3’;

[0049] HPH-F: 5’-AACCTCCGTATCCCTGACCCTGATATTGAAGGAGCATTTT-3’;

[0050] HPH-R: 5’-ATCAGCCGCCAACTAACACTTGGTTCCCGGTCGGCATCTA-3’;

[0051] Using the FocPel1-5’UTR-F / R and FocPel1-3’UTR-F / R primers respectively, the cDNA of Fusarium oxysporum f. sp. cucumerinum (Foc) was used as a template to clone the homologous arm DNA on both sides of FocPel1; using the HPH-F / R primers, the plasmid containing HPH was used as a template to clone the hygromycin resistance gene fragment HPH. The total volume of the PCR system was 50 μL: 25 μL of 2×Phanta Flash Master Mix, 2 μL of template, 2 μL of each upstream and downstream primer, and 19 μL of ddH2O. The PCR reaction program was: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 2 minutes, for a total of 35 cycles; finally, extension at 72°C for 1 minute. The obtained PCR reaction products were identified by 1% agarose gel electrophoresis, and then the target bands were purified using Takara's gel recovery kit. Using the mixture of the three recovered fragments as a template, PCR amplification was performed with FocPel1-5’UTR-F / FocPel1-3’UTR-R to obtain the FocPel1-5’UTR+HPH+FocPel1-3’UTR fragment as the gene knockout vector( Figure 1 a).

[0052] III. Creation of the FocPel1 knockout mutant of Fusarium oxysporum f. sp. cucumerinum

[0053] Inoculate Fusarium oxysporum f. sp. cucumerinum (Foc) on a potato dextrose agar (PDA) medium (200 g·L -1 potato, 20 g·L -1 dextrose, 15 g·L -1 agar) plate and culture it at 28°C for four to five days for activation; in a laminar flow hood, use a scalpel to cut a 2 mm×2 mm mycelium block of Fusarium oxysporum f. sp. cucumerinum and place it in a potato dextrose broth (PDB) (200 g·L -1 potato, 20 g·L -1)In the medium, shake-culture at 28°C in a constant-temperature shaker at 150 rpm / min for 12 hours until the growth of fine bacterial blocks into mycelia, presenting as extremely small woolly balls. Filter the bacterial liquid through a 1-layer filter cloth funnel and rinse it with 0.7 M / L NaCl until the filtrate is clear (to reduce the spore amount); after shaking well with 20 mL of lysis solution (0.15 g of Driselase fB, 0.11 g of Lysing Enzymes, 0.001 g of chitinase, 20 mL of 0.7 M / L NaCl), centrifuge at 700 rpm for 2 min; add the enzymatic hydrolysis solution to the mycelium in a new centrifuge tube and treat it at 30°C in a constant-temperature shaker at 65 - 70 rpm for 2 h to finally prepare the protoplasts of Fusarium oxysporum f. sp. cucumerinum (Foc). Add 20 μL of the FocPel1 gene knockout vector fragment to 180 μL of the protoplasts for transformation experiments, culture at 25°C for 3 - 10 d, pick the transformants onto PDA medium containing hygromycin; and extract the DNA of the transformants, design primers (FocPel1-T, HPH-T, Tublin) according to the FocPel1, HPH, and internal reference Tublin gene sequences for PCR detection to obtain the FocPel1 knockout mutant (△FocPel1)( Figure 1 b),

[0054] FocPel1-T-F: 5’-GAGGAAAGTTCGGGTGAGT-3’;

[0055] FocPel1-T-R: 5’-TGCCAGATAGTTTGGGACA-3’;

[0056] HPH-T-F: 5’-GCCCTTCCTCCCTTTATTTC-3’;

[0057] HPH-T-R: 5’-GATGTTGGCGACCTCGTATT-3’;

[0058] Tublin-F: 5’-GCCCAGGTTGTCAGCTCCA-3’;

[0059] Tublin-R: 5’-GAACTCGCCTTCCTCCATAC-3’.

[0060] IV. Creation of the FocPel1 Complementary Mutant of Fusarium oxysporum f. sp. cucumerinum

[0061] Primers (FocPel1-com-F / R) were designed according to the DNA sequence of Fusarium oxysporum f. sp. cucumerinum FocPel1. Using the cDNA of Fusarium oxysporum f. sp. cucumerinum Foc as a template, after amplifying the full-length sequence of the FocPel1 gene, it was constructed on the expression vector PYF11 (carrying the GFP tag) using a recombination kit (Nanjing Novoprotein Scientific Co., Ltd., product number: C112-01). The vector information can be found in "Study on the Regulation of Chitinase Activity and Virulence of Fusarium oxysporum by the Chitinase-encoding Gene FolCTS3" (Mao Huiying et al., 2020, Journal of Yangzhou University), forming a complementary vector of the FocPel1 gene ( Figure 1 a), which was used to create complementary mutants.

[0062] FocPel1-com-F: 5’-TTTCGTAGGAACCCAATCTTCAAAATGTGGTTACGATTCTTT TTA-3’;

[0063] FocPel1-com-R: 5’-CACCACCCCGGTGAACAGCTCCTCGCCCTTGCGCTAGCTACAAGATTCTTGT-3’.

[0064] △FocPel1 was inoculated on a potato dextrose agar medium PDA plate and cultured at 28 °C for four to five days for activation; and protoplasts were prepared by the above method for transformation experiments. The spores and hyphae of the complementary mutant transformants were observed under the GFP channel of a fluorescence microscope to preliminarily screen positive transformants, and the DNA of the transformants was extracted, and PCR detection was carried out using the detection primers (FocPel1-T, HPH-T). Finally, the FocPel1 complementary mutant (FocPel1.com) was obtained ( Figure 1 b).

[0065] Example 2

[0066] Pathogenicity analysis of Fusarium oxysporum f. sp. cucumerinum FocPel1 deletion and complementary mutants

[0067] The activated fungal blocks of Fusarium oxysporum f. sp. cucumerinum Foc and the △FocPel1 and FocPel1.com strains created in Example 1 were added to PDB and cultured in a constant temperature shaker at 28 °C at a rotation speed of 200 rpm / min for 3-4 days. After filtering the mycelium with non-woven fabric, the spore solution concentration was counted using a hemocytometer, and it was diluted with sterile water to a final concentration of 1×10 6A spore suspension of [[[number of spores]]] spores / mL. In the fusarium wilt inoculation experiment, 20 cucumber seedlings at the two-leaf and one-heart stage with consistent growth vigor and size were set in each group. They were inoculated with 5 mL of the spore suspension by the root irrigation method, and treated with an equal amount of sterile water as a control. The phenotypes of the cucumbers were continuously observed, and the disease incidence rate and disease index of the cucumbers were investigated. The results showed that 20 and 30 days after inoculation, the plants infected with the wild-type Foc first showed the fusarium wilt symptoms of leaf wilting and yellowing at the root base, and gradually withered and died after a period of time; the fusarium wilt symptoms of the plants infected with FocPel1.com were similar to those of the plants infected with Foc, and the seedlings wilted and finally died. However, no obvious fusarium wilt symptoms were found in the cucumber seedlings inoculated with △FocPel1( Figure 2 ), indicating that the deletion of FocPel1 in Fusarium oxysporum f. sp. cucumerinum causes a significant reduction in the pathogenic ability of Foc to cause fusarium wilt, and it is clear that FocPel1 is a key pathogenic gene of Fusarium oxysporum f. sp. cucumerinum.

[0068] Example 3

[0069] Construction of transgenic materials of Fusarium oxysporum f. sp. cucumerinum FocPel1-RNAi and disease resistance analysis

[0070] (1) Construction of the Fusarium oxysporum f. sp. cucumerinum FocPel1-RNAi expression vector

[0071] Using the cDNA of the wild-type Fusarium oxysporum f. sp. cucumerinum Foc as a template, the cloning primers FocPel1-F / R were designed, and PCR amplification was carried out with a high-fidelity DNA polymerase. The primer sequences are as follows:

[0072] FocPel1-F: 5’-CCAACAGCTCGTCTCACGACG-3’;

[0073] FocPel1-R: 5’-CTTTGAAGTGCCGTTGCGATA-3’;

[0074] The total volume of the PCR system was 50 μL: 25 μL of 2×PhantaFlash Master Mix, 2 μL of the template, 2 μL of each of the upstream and downstream primers, and 19 μL of ddH2O. The PCR reaction procedure was: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 0.5 minutes, for a total of 35 cycles; and finally extension at 72°C for 1 minute. The obtained PCR reaction products were identified by 1% agarose gel electrophoresis, and then the target bands were purified and reserved using Takara's gel recovery kit.

[0075] The RNA interference expression vector WMV076, whose sequence is shown in SEQ ID NO.3, was constructed and synthesized by Weimi Biotechnology (Jiangsu) Co., Ltd. Homologous recombination primers for vector construction were designed based on the nucleotide sequence (SEQ ID No.1) of the above-mentioned FocPel1 gene. The sequences are WMV076-FocPel1-F: 5’-ACGGGGGACGTCGACCCAACAGCTCGTCTCACGACG-3’; WMV076-FocPel1-R: 5’-GCGGTATATACGTACCTTTGAAGTGCCGTTGCGATA-3’. Using the cDNA of Fusarium oxysporum f. sp. cucumerinum as a template, the FocPel1 target fragment was amplified, subjected to agarose gel electrophoresis, and the gel was recovered for later use. Finally, the FocPel1 target forward + intron + FocPel1 target reverse fragment was assembled. The vector WMV076 was linearized using the restriction enzymes AatⅠⅠ and SpeⅠ, and the synthesized fragment and the linearized vector were used for vector construction through a homologous recombination kit (Nanjing Novoprotein Scientific Co., Ltd., product number: C112-01). After sequencing verification by Nanjing Tsingke Biotechnology Co., Ltd., the interference expression vector WMV076-FocPel1 was obtained ( Figure 3 ).

[0076] (2) Genetic transformation of cucumber with the FocPel1-RNAi expression vector of Fusarium oxysporum f. sp. cucumerinum

[0077] The successfully constructed WMV076-FocPel1 vector was transformed into the Agrobacterium tumefaciens strain LBA4404. After PCR identification, the cotyledons of the cucumber inbred line ‘9930’ were used as recipients for an Agrobacterium-mediated genetic transformation experiment. Transgenic seedlings were finally obtained through methods such as cucumber seed disinfection and germination, Agrobacterium infection and co-cultivation, and shoot and root induction culture. Transgenic lines of FocPel1-RNAi were obtained after PCR identification and sequencing analysis. Identification primers were designed using the spectinomycin antibiotic gene AADA of the WMV076 vector and the WMV076-35S promoter + FocPel1 target + intron sequence on the vector.

[0078] AADA-F: 5’-TCCGACATCGATCTCCTGGT-3’;

[0079] AADA-R: 5’-CAGGGTGAGGACCACATTCC-3’;

[0080] As Figure 4As shown in a-c, 35S promoter + FocPel1 target + intron - F: 5’-TGGTTAGAGAGGCTTACGCAG-3’; 35S promoter + FocPel1 target + intron - R: 5’-GGCGCGCCCTGCACACGTAC-3’; Finally, 6 transgenic positive plants were obtained by PCR detection, and T2 generation plants were obtained through self-crossing of single plants for subsequent analysis of Fusarium wilt resistance experiments on FocPel1-RNAi.

[0081] (3) Analysis of Fusarium wilt resistance of cucumber FocPel1-RNAi transgenic materials

[0082] In order to further analyze the Fusarium wilt resistance ability of FocPel1-RNAi transgenic plants, a Fusarium wilt pathogen inoculation experiment was carried out on the seedlings of FocPel1-RNAi transgenic and receptor WT cucumber plants at the two true leaf stage. Each seedling was inoculated with 5 ml of Fusarium wilt pathogen Foc spore suspension (10 6 spores / ml) by root irrigation. The results of phenotypic observation and disease index statistics showed that 10 days after inoculation with Fusarium wilt pathogen, the receptor cucumber WT began to show Fusarium wilt symptoms, the plants wilted and accompanied by slight discoloration at the base of the stem, while the FocPel1-RNAi transgenic seedlings hardly showed Fusarium wilt symptoms; 20 days after inoculation with Fusarium wilt pathogen, almost all of the receptor cucumber WT wilted and died, while the FocPel1-RNAi transgenic cucumber plants grew well, did not show obvious Fusarium wilt symptoms, and the disease index was much lower than that of the receptor cucumber WT ( Figure 5 a-c), indicating that FocPel1-RNAi transgenic cucumbers have strong Fusarium wilt resistance.

[0083] In addition, longitudinal sections were taken from the base of the stem of cucumber plants 15 days after inoculation with Fusarium wilt pathogen and observed and analyzed under a stereomicroscope. It was found that obvious reddish-brown lesions appeared on the longitudinal section of the base of the stem of the receptor cucumber WT after infection with Fusarium wilt pathogen, while no obvious lesions appeared on the base of the stem of FocPel1-RNAi transgenic plants. Next, the pathogens were isolated and activated from the stems of the receptor WT and FocPel1-RNAi transgenic cucumbers. It was found that the growth rate of the Fusarium wilt pathogen isolated from the receptor cucumber WT was relatively fast, and the whole surface of the PDA medium was covered after 48 hours of activation, while the amount of Fusarium wilt pathogen isolated from the stems of FocPel1-RNAi transgenic plants was small and the growth rate was slow, and less mycelial growth was observed after the same culture time ( Figure 5 d). RNA was extracted from the Fusarium wilt pathogens isolated from the above-mentioned receptor WT and FocPel1-RNAi transgenic cucumber plants. qRT-PCR primers were designed according to the FocPel1 gene sequence, and real-time fluorescence quantitative PCR (qRT-PCR) technology was used to verify the changes in the expression level of the FocPel1 gene.

[0084] FocPel1_qRT-PCR_F: 5’-GCTGTGGACGATGGTATA-3’;

[0085] FocPel1_qRT-PCR_R: 5’-GAGAGGAAGACTGATGTGATA-3’;

[0086] The results showed that the expression level of FocPel1 in the isolated and activated Fusarium oxysporum f. sp. cucumerinum in the stems of the receptor cucumber WT was relatively high, while the expression of the FocPel1 gene in the isolated and activated Fusarium oxysporum f. sp. cucumerinum in the FocPel1-RNAi transgenic cucumber was inhibited( Figure 5 e), indicating that the FocPel1-RNAi transgenic cucumber plants could silence the FocPel1 gene in the invading Fusarium oxysporum f. sp. cucumerinum, thereby enhancing the fusarium wilt resistance of cucumber plants.

[0087] In order to further verify the fusarium wilt resistance of the FocPel1-RNAi transgenic cucumber at the adult stage, the receptor WT and FocPel1-RNAi transgenic cucumber plants at the flowering and fruiting stage were inoculated with Fusarium oxysporum f. sp. cucumerinum Foc by root irrigation. As Figure 5 shown in f, the results showed that 35 days after inoculation, the receptor cucumber WT gradually showed fusarium wilt symptoms and almost all withered and died; while there were no obvious disease symptoms during the growth of the FocPel1-RNAi transgenic cucumber, and it could set seeds normally after pollination and obtain healthy and plump seeds, indicating that the FocPel1-RNAi transgenic cucumber had stronger fusarium wilt resistance than wild-type cucumber at the adult stage and could carry out seed propagation work, successfully creating a cucumber germplasm resistant to fusarium wilt.

Claims

1. A FocPel1 gene of cucumber wilt pathogen, characterized in that: The nucleotide sequence of the FocPel1 gene is shown in SEQ ID NO.

1.

2. A FocPel1 gene of the cucumber wilt pathogen according to claim 1, characterized in that: The protein sequence encoded by it is shown in SEQ ID NO.

2.

3. An interference expression vector WMV076-FocPel1 based on the FocPel1 gene of the cucumber wilt pathogen according to claim 1.

4. A method for constructing the interference expression vector WMV076-FocPel1 of the cucumber wilt pathogen FocPel1 gene according to claim 3, characterized in that: The steps include: (1) Total RNA was extracted from Fusarium wilt pathogen and then reverse transcribed into cDNA; (2) using the cDNA product obtained in step (1) as a template, amplifying the FocPel1 target fragment using primers FocPel1-F / R; (3) Design primers WMV076-FocPel1-F / R to amplify the FocPel1 target fragment; (4) The amplified product was homologously recombined with the linearized vector WMV076, and after correct sequencing, the expression vector WMV076-FocPel1 was obtained. The nucleotide sequence of the vector WMV076 is shown in SEQ ID NO.

3.

5. The method for constructing the interference expression vector WMV076-FocPel1 of the cucumber wilt pathogen FocPel1 gene according to claim 4, characterized in that: The primer FocPel1-F / R in step (2) is: FocPel1-F: 5'-CCAACAGCTCGTCTCACGACG-3', FocPel1-R: 5'-CTTTGAAGTGCCGTGCGATA-3'.

6. The method for constructing the expression vector WMV076-FocPel1 of the cucumber wilt pathogen FocPel1 gene according to claim 4, characterized in that: The primer WMV076-FocPel1-F / R in step (3) is: WMV076-FocPel1-F:5'-ACGGGGGACGTCGACCCAACAGCTCGTCTCACGACG-3'WMV076-FocPel1-R:5'-GCGGTATATACGTACCTTTGAAGTGCCGTTGCGATA-3'.

7. Use of the Fusarium wilt gene FocPel1 according to claim 1, the encoded protein according to claim 2, or the interference expression vector WMV076-FocPel1 according to claim 3 in regulating resistance to cucumber Fusarium wilt.

8. The application according to claim 7, characterized in that: The application of silencing the Fusarium wilt pathogen gene FocPel1 or introducing the interference expression vector WMV076-FocPel1 in improving the resistance of cucumber to Fusarium wilt.

9. The application according to claim 8, characterized in that: The constructed FocPel1 gene interference expression vector WMV076-FocPel1 was transformed into Agrobacterium and then infected into cucumber. The obtained FocPel1-RNAi transgenic strain was able to silence the FocPel1 gene in the invading pathogen and significantly improve the resistance of cucumber to Fusarium wilt.

10. Use of the Fusarium wilt gene FocPel1 according to claim 1, the encoded protein according to claim 2, or the interference expression vector WMV076-FocPel1 according to claim 3 in creating Fusarium wilt-resistant cucumber germplasm.

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