Identification Of SFBBs In Yali And Heterologous Transformation Of Self Incompatibility Gene

Self-incompatibility（SI）is a genetic mechanism to encourage crosspollination and maintain species diversity in flowering plants.Pyrus belongs to Rosaceae fruit tree with typical SI.At present,it has been confirmed that the style S determinant is S-RNase,and SFBBs act as a candidate for pollen S determinant.It is speculated that the recognition mechanism of S-RNase and SFBBs belongs to the "non-self model".The verification of whether the S-RNase and SFBBs recognition mechanisms are non-self model faces three major challenges.First,Pyrus belongs to perennial woody plant that is difficult to genetically transform,making it difficult to verify the function of related genes in vivo;secondly,Pyrus are highly heterozygous and lacks homozygotes of S-loci,making it impossible to separate analyze the corresponding relationship between S-RNase and SFBBs;and finally,SFBBs have high homology with each other,and the distance between S-loci is long,making it difficult to identify SFBBs in S-loci.We used the traditional Chinese white pear variety ’Yali’ as the experimental material to solve the above problems and clarify whether the recognition mechanism of S-RNase and SFBBs belongs to the non-self model.We use de novo assembly of pollen transcriptome,PCR amplification and PCR enzyme-linked immunosorbent assay（PCR-ELISA）to identify SFBBs at S21 and S34 loci in ’Yali’,and used heterologous transformation technology to transfer the S-RNase and SFBB of’Yali’ into Arabidopsis thaliana,and single-S-RNase and single-SFBB transgenic plants were created to establish the groundwork for further research into S-RNase and SFBBs recognition mechanism.The relevant results are as follows:（1）27 SFBBs were identified in the two S-loci of ’Yali.’ In this experiment,F-box of ’Yali’ having more than 70%homology with the reported SFBBs in ’Yali’were selected in the trinity assemblies of ’Yali’ pollen tube transcriptome.According to the assembly results,universal primers and specific primers for PCR amplification were designed,and 27 SFBBs genes were obtained from ’Yali’pollen,of which 21 were not reported.The S genotype of SFBBs was identified by PCR-ELISA,and 12 SFBBs genes were identified to link with S21-RNase,and 15 SFBBs genes were linked to S34-RNase.（2）The genetic characteristics of SFBBs were clarified.The homology of SFBBs ranged from 74.3%-94.42%amino acid identity.At the same time,the domain and motif analysis showed that all genes included F-box domain and there was an FBA structure at the C end of these proteinand all contained the F-box domain.In addition,we found that all SFBBs genes were expressed in pollen,and the relative expression in ’Jinzhui’ was generally higher than that in ’Yali’.After evolutionary analysis,SFBBs genes of Pyrus were classified into 28 groups,significantly more than the 8 groups previously reported.（3）The SI heterologous system of Pyrus was initially constructed.S21RNase of ’Yali’ was cloned to construct pER8-S21-RNase vector;SFBB4 at the S21 locus of ’Yali’ was cloned to construct pLAT52-SFBB4-2301 vector（LAT52 is a pollen-specific promoter of tomato）.The two vectors were each inserted into the Arabidopsis thaliana genome,obtaining single-S-RNase and single-SFBB homozygotes,establishing the groundwork for further research into S-RNase and SFBBs recognition mechanism.