Cucumber Fusarium wilt resistance gene foc‑4 and its encoded protein and application
A cucumber fusarium wilt and disease-resistant gene technology, applied in the field of gene sequence and application, can solve the problem of successful isolation and cloning of the disease-resistant gene of physiological race 4 of cucumber fusarium wilt, and achieve the effect of improving breeding efficiency
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Embodiment 1
[0032] Example 1: Obtainment of Cucumber Fusarium Wilt Resistance Gene Foc-4
[0033] 1. Preliminary localization and fine localization of the resistance gene Foc-4 of cucumber Fusarium wilt race 4
[0034] The specific positioning method includes the following steps:
[0035] A. Cucumber Fusarium Wilt Resistance Gene Mapping Research Parents and Genetic Populations:
[0036] WI2757 (male parent) and Jinyan No. 2 (female parent) were selected as anti-infective parents to construct 90 RIL-F 8 strains, F of 130 strains 2:3 populations and more than 2000 strains of F 2 large groups. Among them, the male parent WI2757 and the female parent Jinyan 2 were purchased from the Beijing Crop Germplasm Resource Bank.
[0037] B. Cucumber Fusarium Wilt Disease Resistance Gene Location Research Disease Index Survey:
[0038] The seeds of the parent and each group were wrapped with gauze, soaked in warm soup, sown in 50-hole plug trays after germination at a constant temperature of 28°...
Embodiment 2
[0062] Example 2: Identification and Analysis of Cucumber Fusarium Wilt Resistance Gene Foc-4 cDNA and Protein Sequences
[0063] 1. Amplification of the full-length cDNA (mRNA) of the resistance gene Foc-4 of Fusarium wilt in cucumber:
[0064] Using the anti-infective parent materials WI2757 and Jinyan No. 2, the young leaf tissue was selected to extract total RNA, and reverse transcription was performed to synthesize the first strand of mRNA (cDNA). Amplify the upstream and downstream primers SEQ ID NO:7 and SEQ ID NO:8 for PCR amplification, and obtain the full-length cDNA (mRNA) sequence SEQ ID NO:3 and SEQ ID NO:4 in the anti / sense material, respectively. The DNAMAN software was used to compare and analyze the full length of the Foc-4 gene of the resistant / susceptible materials. For size differences, see Figure 1. Compared with the susceptible material, the cDNA sequence of the resistant material showed a deletion between 1915-2184 bp. It is speculated that the existenc...
Embodiment 3
[0071] Example 3: Functional verification of the disease-resistant gene Foc-4 and its application in the breeding of cucumber Fusarium wilt disease-resistant varieties
[0072] 1. Functional verification of cucumber Fusarium wilt resistance gene Foc-4
[0073] 1. Materials to be tested: Using WI2757 and GY 14 as parents, construct F 2 Genetic population, from which 100 individual plants were randomly selected for disease resistance identification and sequencing of Fusarium wilt. Among them, the male parent WI2757 and the female parent GY 14 were purchased from the Beijing Crop Germplasm Bank.
[0074] 2. The seeds of the above-mentioned materials to be tested were wrapped with gauze, soaked in warm water, sown in a 50-hole plug tray after germination at a constant temperature of 28°C, and the seedlings were raised in an air-conditioned greenhouse. The seedling substrate was sterilized perlite or nutrient soil. The genomic DNA of each material to be tested was extracted from ...
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