Analysis And Screening Of SNP And Interacting Protein Of Fusarium Wilt Resistance Gene FOC1 In Brassica Oleracea

Based on the cloned fusarium wilt disease resistance gene FOC1, this study screened FOC1 related single nucleotide polymorphism(SNP) sites, and analysed the SNP gene sequences and their amino acid sequences, the fusarium wilt disease resistance gene FOC1 related specific SNP then were discoveryed,which provied theoretical basis to further develop the FOC1 specific molecular markers. At the same time, this study constructed the bait yeast strains Y2 HGold [pGBKT7-FOC1], and the autoactivation and toxicity of bait were tested by yeast two hybrid technology, Then prey proteins from cDNA library that interact with cabbage FOC1 protein were screened, research conclusions are as follows:1. The aim of this study was to uncover the specific SNPs of fusarium wilt resistance gene FOC1 of Brassica oleracea, which will lay the foundation for the development of specific molecular markers in the future. Based on the identification of fusarium wilt resistance in 11 cabbage self-bred lines, the variations of gene sequences and amino acid sequences of FOC1 and its alleles in 11 cabbage materials were analyzed. Results showed that there were 92 SNPs and 2 Indels between these sequences, including 18 SNPs related to fusarium wilt resistance, of which 86.11% were transition mutations and 13.89%were transversion mutations.The nucleotide substitutions of T/C(47.22%) and G/A(38.89%) showed higher mutation rates,while C/G and A/C accounting for about 13.89%. There were a total of 23 amino acid variations between the susceptible and resistant cabbage materials, among them, a common variant amino acid(H34D) was identified except for the P10, which resulted in little change of the variation of hydrophilic/hydrophobic and had no influence on the function of FOC1. In addition, the four specific SNPs identified between the susceptible and resistant cabbage materials led to the variations of three amino acids.2. To screen proteins that interact with FOC1, yeast two hybrid was performed. The bait plasmid pGBKT7-FOC1 was built, and then was successfully transformed into the yeast strain Y2 HGold named as bait yeast strains Y2 HGold [pGBKT7-FOC1], and the activation and toxic effects were detected.Results showed that the fusion protein itself does not have the ability to activate report genes, indicating pGBKT7-FOC1 didn’t have self-activating effect in yeast. The OD600 values of the bait carrier Y2HGold[pGBKT7-FOC1] microbial and the control Y2 HGold [pGBKT7] had no great difference and both were greater than 0.8, showing that the bait vector pGBKT7-FOC1 did not show toxicity to yeast cell. Thepurpose of the test showed that FOC1 didn’t have large influence on the growth of yeast Y2 HGold strains and can be used for screening of cDNA library.3. The yeast two hybrid results showed that after preliminary screening and confirmation of positive interactions, FOC1 A,FOC1 B,FOC1 C and FOC1 D were respectively interaction with FOC1.The sequences were submitted to NCBI, BRAD and TAIR database for homology comparison to get the candidate proteins. Gene FOC1 D annotated as phospho ribulokinase, may be associated with resistance to fusarium wilt in cabbage. This research provided clues for the future fusarium wilt disease resistance breeding and made full preparation for our next research work.