Cloning and evaluation of S locus F- box brother genes in genus Pyrus plants

Abstract:【Objective】In order to avoid self-pollination in the evolution process of Rosaceae, self-incompatibility is common. Studies have shown that the self-incompatibility of Rosaceae Maloideae Pyrus is achieved by the interaction of the pistil S-RNase gene and the pollen SFBB gene. In order to obtain the full-length sequence of the new pear SFBB gene, the situation of different pear SFBB genes involved in self-incompatibility was clarified, and the SFBB gene type that was more compatible with pear S-RNase gene was found.【Methods】DNA was extracted from the leaves of Yunnan Baozhu pear, Fu’an Jianba pear, and Pitai pear varieties, and specific primers were designed based on the conserved characteristics of the SFBB-gamma gene sequence of the Maloideae Pyrus, and the pear SFBB22-gamma genomic DNA Full-length sequence was amplified by PCR. The full-length sequences of all the isolated and identified SFBB- alpha, SFBB- beta, SFBB- gamma and SFBB- epsilon genes that can correspond one-to-one with the pear S-RNase gene were compared and analyzed. The two are respectively based on the physical and chemical properties of the gene, the proportion of variant sites, the proportion of different nucleotide compositions, the ratio of conversion and transversion, the pairwise genetic distance of the coding region sequence and the genetic diversity index, Tajima’s Neutrality Test D value, positive position The proportion of points, the proportion of neutral loci and the number of consecutive positive loci were compared and analyzed.【Results】The size of the coding region of pear SFBB22-gamma gene was 1191 bp, encoding 396 amino acids; the predicted molecular weight of ProtParam was 45.47 ku; the theoretical isoelectric point was 4.67 and thus it might be an acid-hydrophilic unstable protein. The N-terminus contained an F-box sequence consisting of 50 amino acids. The protein secondary structure had 5 α-helices and 24 β-sheets. The α-helix was mainly distributed in the F-box region, and the β-sheet was scattered throughout the protein. At present, a total of 52 full-length CDS sequences of SFBB gene corresponding to pear S-RNase gene have been registered in the NCBI database, including 15 SFBB-alpha, 10 SFBB-beta, 24 SFBB-gamma, and 3 SFBB-epsilon, corresponding to 30 full-length CDS sequences of pear S-RNase genes. Different SFBB gene in NJ phylogenetic trees were clustered into 4 subgroups, and there was no clustering with species as the boundary; the corresponding pear SRNase gene did not have obvious clustering with species as the unit either. The prediction of protein physicochemical properties showed that the proteins encoded by the pear SFBB- alpha, SFBB- beta, SFBB-gamma and SFBB-epsilon genes were all acidic proteins, and the basic proteins encoded by the pear S-RNase gene were theoretically attracted by positive and negative charges. At the same time, the proteins encoded by both were hydrophilic proteins with similar thermal stability. The molecular weight of the protein encoded by SFBB was about 1.75 times that of the protein encoded by the corresponding S-RNase gene. There was a theoretical possibility of interaction. The SFBB-alpha, SFBB-beta, SFBBgamma and SFBB- epsilon genes of pear plants were highly conserved. Further research and analysis showed that the pear SFBB- alpha, SFBB- gamma and SFBB- epsilon genes had the same correlation with the corresponding S-RNase. There was less evidence of gene co-evolution, and these SFBB genes exhibited lower sequence variation, less positive selection, and lower gene polymorphisms. The SFBBbeta gene showed a higher level of sequence diversity, although it was still lower than that shown by the corresponding S-RNase gene. If the four subtypes of pear SFBB gene are considered as a whole, the above indicators and corresponding S-RNase conditions are better than those of SFBB- alpha, SFBBgamma, SFBB-epsilon and SFBB-beta genes.【Conclusion】The design of specific primers can directly amplify the full-length sequence of pear SFBB gene. At present, this technology still has the defect of poor primer versatility and needs to be further improved. Finding a method comparable to amplifying the full-length sequence of the pear S-RNase gene is the key to the study of the pear SFBB gene. The existing SFBB gene full-length sequence family that can correspond one-to- one with the pear S-RNase gene has 4 members, and the sequences between each member are relatively conservative. All are smaller than S-RNase. Existing data show that pear SFBB-beta gene may participate in self-incompatibility alone, and pear SFBB-alpha and SFBB-gamma genes may not participate in self-incompatibility alone. From a theoretical point of view, whether all the four SFBB gene subtypes are involved in self-incompatibility or whether SFBB-beta alone is involved in self-incompatibility needs more research to verify.