Identification Of S-RNase Gene In Camellia Vietnamensis And Its Effect On Cross-compatibility

Camellia is the general name of the tree species of Theaceae Camellia plants with high oil content and economic value of cultivation.It is a key woody oil crop in China.C.oleifera,C.meiocarpa and C.vieinamensis are the three main species of Camellia oleifera.Camellia oleifera has strict self-incompatibility characteristics,which leads to the phenomenon of "a thousand flowers and one fruit",resulting in the problem of low yield.C.oleifera belongs to post-gametophytic self-incompatibility.At present,the mechanism of self-incompatibility of Camellia oleifera is not clear.S-RNase gene is one of the pistil determinants of gametophyte self-incompatibility in plants.In this study,on the basis of clarifying the type of self-incompatibility of C.vieinamensis,the pistil determinant S-RNase of self-incompatibility of Camellia oleifera was cloned by homologous cloning.The function of Camellia oleifera S-RNase was verified by gene expression pattern analysis,subcellular localization and prokaryotic expression,and several C.vieinamensis S-RNase alleles were excavated to analyze the effect of S-RNase allele on C.vieinamensis hybridization incompatibility.The main results of this study are as follows:1.The self-incompatibility of C.vieinamensis was confirmed through the field pollination experiments of self-crossing and cross-crossing combined with the investigation of natural fruit-setting rate,and the pollen tube growth in the style was further observed to confirm that it was the self-incompatibility type in the late ovary stage.2.Ribonuclease activity in different tissues of C.vieinamensis was examined and found to be significantly higher in the style and ovary than in other tissue,and its activity was closely related to the state of pollen tube elongation.Thus its style may contain ribonuclease proteins(i.e.S-RNase)that regulate the self-incompatibility trait.3.Eight and six RNase T2 genes,namely TGYRNS1～TGYRNS8 and LGRNS1～LGRNS6,were identified through the identification and analysis of gene families of Camellia sinensis and Camellia sinensis var.sinensis genomes of Camellia.The physicochemical analysis and tissue-specific expression analysis of C.vieinamensis confirmed that TGYRNS8 was S-RNase of Camellia.4.The full-length c DNA sequence of S-RNase,named Cv S-RNase,was obtained through homologous cloning and RACE amplification using TGYRNS8 gene as the template in C.vieinamensis.The coding length of Cv S-RNase was 717 bp,and the protein length was 238 amino acids.The molecular weight of Cv S-RNase was 26.7k Da,and the isoelectric point was 5.28.5.Cluster analysis showed that Cv S-RNase had the closest genetic relationship with S-RNase of Solanaceae.Subcellular localization showed that Cv S-RNase gene was expressed on the cell membrane,and it was speculated that Cv S-RNase was a signal protein.QRT-PCR results showed that Cv S-RNase was specifically expressed in the style,and the expression was significantly up-regulated and reached the maximum during self-crossing 24h-48 h,and then down-regulated,which was significantly different from that in outcrossing and consistent with the growth of self-crossed pollen tubes in the style.The recombinant Cv S-RNase protein obtained by prokaryotic expression significantly inhibited the pollen germination and growth of Camellia oleifera.These results suggest that Cv S-RNase is the S-female determinant of self-incompatibility in C.vieinamensis.6.A total of 22 polymorphic amino acid locus mutant Cv S-RNase proteins were identified in different plants of C.vieinamensis,which accorded with the polymorphism characteristics of S-RNase as the determinant of S traits in plant self-incompatibility,and the fruit setting rate of pollination among plants with different Cv S-RNase variant protein was high,while that of plants with the same Cv S-RNase variant protein was low,indicating that S-RNase gene played an important role in the process of C.vieinamensis self-incompatibility.The phenomenon of self-incompatibility of C.vieinamensis was explored for the first time in this research,and the S-female determinant Cv S-RNase gene of self-incompatibility in Camellia oleifera was cloned and identified,and 22 Cv S-RNase mutant proteins were obtained.The research results have important theoretical and practical reference value for systematic analysis of Camellia oleifera self-incompatibility mechanism,hybrid breeding,flower and fruit preservation,etc.