Digital Gene Expression Profiling Analysis Of Gametophytic Self-incompatibility Styles In Citrus Grandis Var. Shatinyu

Citrus grandis var.shatinyu belongs to rutaceae,citrus plants,which is famous and high-quality fruits in Guangxi.It has won a great number of consumers with its beauty juice taste and high nutritional value.But Citrus grandis var.shatinyu need artificial cross-pollination to improve fruit--set rate because of its strict self-incompatibility and extremely low fruit rate.In this syudy,seven DGE libraries were constructed to screen differentially expressed genes using the one day,two days and three days styles of Citrus grandis var.shatinyu after self-pollination,cross-pollination and non-pollination,respectively.Differentially expressed genes between seven DGE libraries were used for genes expression patterns of clustering analysis,GO and KEGG pathway analysis.Then four selected differentially expressed genes related to self-incompatibility were verified by qPCR.And the gene Unigene2441_All was transducted to plasmid pET43.1a,and the recombinant plasmid pET43.1a-2441 was induced expression by IPTG in DL21.Then separated and purfied proteins were uesd for pollen germination and the growth of pollen tube in vitro.The results were as follows:(1)We analyze the seven DGE libraries of one day,two days and three days styles of Citrus grandis var.shatinyu after self-pollination,cross-pollination and non-pollination,respectively.We got more than 11754903 clean reads in each DGE library and 90%of clean reads were mapped to reference genes.After screening differentially expressed genes,we knowed about that:there were 244 up-regulated expressed genes and 27 down-regulated expressed genes in comparing 1 day styles after self-pollination with 1 day styles after cross-pollination.265 up-regulated expressed genes and 244 down-regulated expressed genes wree in comparing 2 days styles after self-pollination with 2 days styles after cross-pollination.193 up-regulated expressed genes and 685 down-regulated expressed genes in comparing 3 days styles after self-pollination with 3 days styles after cross-pollination.(2)GO significant enrichment analysis indicted that differentially expressed genes were mainly involved in regulating pellen tubes elongation,development and modification of plant cell walls in SY1-VS-SZ1 and that differentially expressed genes in SY2-VS-SZ2 were mainly involved in responsing to all kinds of chemicals,plant hormones,the stimulation of endogenous and exogenous substances and signal transduction process.(3)KEEG significant enrichment analysis indicted that differentially expressed genes were significantly enriched in Plant hormone signal transduction and the major differentially expressed genes were down-regulated.(4)The protein encoded by Unigene2441_All gene presented significantly inhibited effect on pollen germination and pollen tube growth in vitro,which suggested that the gene could plays a critical role in self-incompatibility response of Citrus grandis var.shatinyu.(5)The qPCR results of 4 differentially expressed genes related to self-incompatibility were basic agreed with DGE statistics.In conclusion,the study was made of DGE to analyze the differentially expressed genes of 1 day,2 days,3 days styles after self-pollination,cross-pollination and non-pollination,respectively.Differentially expressed genes involved in different biological processes came into being an complex network.So the mechanism of gametophyte self-incompatibility is a complex process involved in most genes.