Transcriptome Sequencing And Cloning And Expression Analysis Ribonuclease Gene (cgSLA?cgSRA) In Citrus Grandis Var.Shatinyu

Citrus grandis var.Shatinyu is one of the famous fiuit in Guangxi,which is welcomed by people because of its good taste and high nutritional value.However,Citrus grandis var.Shatinyu needs to rely on artificial pollination to increase the yields as its strict self-incompatibility and low fruit setting rate.In this study,we performed transcriptome sequencing for the Self-pollinated 1-3days?Cross-pollinated 1-3days and non-pollinated styles of Citrus grandis var.Shatinyu using the high-throughput sequencing technique.Then,the sequencing results were analysed based on the GO function analysis,pathway analysis and calculation of expression.Two RNase gene Unigene34907_All(cgSLA)and Unigene2441_All(cgSRA),which might be relate to the self-incompatibility,were chosen for constructed the plant expression vector and transferred.Here are the results:1.Eventually,90214 of All-Unigene after the the extraction of the sample RNA and preperation of cDNA library were gotten.The total lenth is 142142835nt,and the average lenth is 1576nt,N50 lenth is 2413nt?2.There were 62679?59673?41897?39362?28317?50701 of the Unigene matching with the NR?NT?Swiss-Prot?KEGG?COG?GO database respectively.Comparing with the COG database,the obtained Unigene was devided into 25 functional annotations.And most of them involved in general function prediction only and transcription.3.The results of Go and Pathway enrichment analysis indicated that the Unigenes were classified to 55 functional categories and 128 metabolic pathways.62752CDs,of which 61707 was goten by comparing with the Blastx and the public database,while 1045was predicted by ESTScan software.4.29055 SSR were gotten through the SSR analysis,the most of them were mono-nucleotide repeats,which was 12746.The number of SNP of S,Y1,Y2,Y3,Z1,Z2,Z3 was 47145,53617,54143,52717,50606,54005,53816 respectively.5.38 RNase genes were obtained by differential expression analysis.Unigene34907_All(cgSLA)and Unigene2441_All(cgSRA)which might be related to the self-incompatibility were selected for constructed the plant expression vectors.Then,the genes were transferred into Arabidopsis thaliana by Agrobacterium tidbits impregnation method.The results of PCR and sequencing showed that the genes were successfully transferred into the genome of Arabidopsis thaliana.6.RT-PCR results indicated that the differential expression trend of the 8 Citrus grandis var.Shatinyu stylar agrees with the sequencing results.7.The differential expression analysis of transcriptome sequencing indicated that most of the genes were related to the plant hormone signal transduction pathway and stress response.