SRK Gene Family Identification And Expression Analysis In Self-incompatibility Shatian Pummelo

Shatian pummelo have a characteristic of self- incompatibility, we do not know the molecular mechanism so far, and the key determining factor has not been cloned. SRK(S locus receptor kinase) gene has been confirmed it is the pistil decision factor in the cruciferous. Previous study indicates that Shatian pummelo(Citrus grandis Osbeck) contains SRK homologous gene, may plays a key role in the self- incompatibility. Because of the shatian pummelo genome sequence has not been public, this study based on sweet orange(Citrus sisensis Osbeck) genome information for reference, to carry out the whole genome wild members of SRK gene family, sequence structure and expression analysis. The main results are as follows:(1)Using the conservative structure domain for Blast, and screened we all obtain 113 genes which can located in 9 chromosomes of sweet orange, then the subcellular localization has been predicited, 61 genes can be located in plasma membrane, 36 genes in mitochondria, 9 genes in cytoplasm, 6 genes in the nucleus.(2)Homology analysis found that Cs SRK13 with At1g07650.1 and At5g39390.1 have higher homology, there were exprements proved the two genes specififtic expressed in the bud period of Arabidopsis thaliana.(3)The cis-elements of promoter divided into two categories, one is associat with growth and development, another one is associat with stress and hormone, and found 94 genes have elements relate to the growth and development, 57 genes have elements relate to the JA recation, 35 genes have e lements relate to SA, 31 genes have elements relate to ABA, 27 genes have elements relate to GA, 15 genes have elements relate to Eth, and 8 genes associat with the formation of endosperm and seed.(4) Shatian pummelo flower organ organizations as templates, semi-quantitative PCR was referenced with Actin, the results show that Cs SRK1 and Cs SRK85 specific expressed in Anther, Cs SRK96 and Cs SRK96 specific expressed in ovary, Cs SRK105 specific expressed in leaf, Cs SRK13 gene expressed in multiple templates, just have difference in the expression level. In the q RT-PCR analysis setting GAPDH and Actin as reference, there are 5 genes specific expressed in petal, 3 genes specific expressed in filament, 1 gene specific expressed in peduncle, 22 genes specific expres sed in anther, 2 genes expressed higher in ovary and style.(5) The pulp from 5 development periods of Jincheng as templates, the results of q RT-PCR show that the expression of Cs SRK3、Cs SRK20、Cs SRK40 to the peak in 210 DAF with fruit ripening, but the genes expression of Cs SRK53、Cs SRK54、Cs SRK58、Cs SRK73、Cs SRK94 more and more low and to the lowest of the expression in 240 DAF.(6)In the prokaryotic expression, recombinant Cs SRK with PGEX6P-1 a expression vector using double enzyme digestion method, induced 6h by 1.0m M/L IPTG can well express the purpose protein, this protein was connected with N-GST tag, molecular weight about 72 k Da.This protein may a transmembrane protein, have protein kinase domain structure, about 3 kinds of phosphorylation sites in the sequence, can encode 8 kinds of 13 kinases, mainly contain PKA and PKC.