cspr3 gene and its application in cucumber wilt resistance
A fusarium wilt resistance and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of lowering the quality of cucumbers, unsatisfactory resistance to fusarium wilt, environmental pollution, etc.
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Embodiment 1
[0053] Example 1 Screening of Cucumber Fusarium Wilt Resistance Gene CsPR3
[0054] 1. Materials
[0055] The F 2 Among the isolated populations, 3461 were resistant to cucumber wilt and 3229 were susceptible to cucumber wilt. The Fusarium wilt pathogen used in the test inoculation was Race 4 (provided by the Plant Protection Department of China Agricultural University). The cucumber seeds used in the experiment were sterilized, placed in an incubator at 28°C for germination, and then seedlings were raised in 72 plug trays. Place them in the artificial climate chamber of China Agricultural University for normal cultivation.
[0056] 2. Preparation of spore suspension of cucumber wilt physiological race 4 and inoculation of cucumber wilt pathogenic bacteria
[0057] Use a sterilized inoculation needle to pick up the cucumber wilt pathogen stored on the slant of the PDA medium, transfer it to the central position of the new PDA medium, and place it in an incubator at 28°C fo...
Embodiment 2
[0074] Cloning of Example 2 Cucumber CsPR3 Gene
[0075] Using Cucumber 3461 and 3229 and F 2 The extreme phenotypes of the offspring were pooled and transcriptome sequencing was carried out. After BSA analysis, the Chr6:26077967-26079100 region was identified as a candidate region, and this region happened to be located on a gene, which was Csa6G507520. According to the annotation of the gene, the gene is tentatively named CsPR3, and the CDS sequence of the gene is found in the cucumber genome database (http: / / cucumber.genomics.org.cn / page / cucumber / index.jsp) according to the gene ID of CsPR3, according to Its CDS full-length sequence design primers:
[0076] Forward: 5'-ATGTTAATCACATCAAGGAAAAAT-3'
[0077] Reverse: 5'-CTAGAAAGACCTTTGATTGTAGC-3'
[0078] Using the cDNA of the third true cucumber leaf (from bottom to top) of 3461 and 3229 respectively as a template, primers were designed at both ends of the ORF region of the target gene for PCR amplification.
[0079] PCR ...
Embodiment 3
[0082] Example 3 Expression Analysis and Detection of CsPR3 in Cucumber Tissues
[0083] 1. Fluorescent quantitative PCR detection
[0084] Get female flower buds, male flower buds, leaves, stems, tendril bases, tendrils, fertilized ovaries, unfertilized ovaries and roots for all cucumber plants to extract RNA and synthesize the first strand of cDNA as in Example 1.
[0085] 2. The kit used for fluorescence quantitative analysis is SYBR Premix Ex Taq (TaKara, DRR041S), the instrument is ABI 7500, and the primers are as follows:
[0086] CsPR3F:5'-TATTGTAAAAATGGTTGTCAAAGC-3'
[0087] CsPR3R:5'-CCTCCTGTAGTCTCGTGAGAAGTT-3'
[0088] The reaction system for fluorescence quantification is:
[0089]
[0090] The amplification program was: 95°C, 30sec; (95°C, 5sec; 60°C, 40sec), 40 cycles. Taking the TUα gene as an internal reference, using 2 -△△Ct The algorithm calculates the changes in the expression of each gene.
[0091] The result is as Figure 7 As shown, CsPR3 was str...
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