Screening And Identification Of Genes From Ovule Transcriptome During Free Nuclear Mitosis In Pinus Tabulaeformis Carr.

The female gametophyte(FG)is the core structure of the ovule,and the free nuclear mitosis(FNM)is also the indispensable process of ovule development.The failure of FNM impairs the generation of FG maturation and generation of the egg cell,leading to female sterility.Therefore,it is meaningful for research in molecular regulatory mechanism in ovule development.At present,the researches are mainly focused in angiosperm,while few researches are in gymnosperm.However,the ovule development of gymnosperm is different between angiosperm and gymnosperm,and more distinct in woody gymnosperm.Formerly,our research group found the ovule sterility in seed orchard of Xingcheng,and the reason is the arrest of free nuclear mitosis.It was considered that the phenomenon resulted from multi-number genes expression abnormality.Therefore,in this study,we took advantage of suppression subtractive hybridization(SSH)library construction and transcriptomic high-throughput sequencing technology,combined with bioinformatics analysis,to explore the differentially expressed genes between female fertile and sterile ovules at different stages and reveal the potential molecular regulatory mechanism.The main result is listed below.1.We constructed the forward and reverse cDNA SSH library,and analyzed the differentially expressed genes between fertile line(FL)and sterile line(SL)ovules in free nuclear mitosis.From the two libraries,a total of 756 clones were selected,and the average length of EST was 286 bp,ranging from 105 bp to 763 bp.Out of them,233 ESTs from FL and 197 ESTs from SL SSH libraries showed high similarity to BLASTX hits,and 336(47.9%)proteins were annotated to gene ontology(GO)terms.Through bioinfonnatics analysis,genes encoding CKX-like and MYB-like family protein were up-regulated,while genes encoding BEL1-like protein,CDK-like protein and kenesin-1-like protein were down-regulated.The total number of screened potential functional genes is 27.2.By using the quantitative real-time PCR(qRT-PCR),we analyzed expression level of 11 genes,involved in developmental process,signal transduction,transcriptional regulation,metabolism and cell component,confirming the result of' SSH libraries.The putative functions of differentially expressed transcripts were involved in most aspects of biological processes.3.In the present study,by using Illumina high-seq 2000 platform to analyze the female fertile and sterile ovules at different developmental stages in free nuclear mitosis,we acquired the transcriptomic data and gene expression profile of free nuclear mitosis of Pinus tabulaeformis ovules.Six cDNA libraries were constructed.A total of 71.0M high-quality reads were generated.After de novo assembly,63,449 unigenes were generated with average length of 740 bp.More than 48.5%unigenes had high similarity hits in GenBank non-redundant protein database.49.7%(3 1,321/63,449)had hits to proteins in public databases,including the NCBI non-redundant(NR)database,Swiss-Prot protein database,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Based on transcriptomic data,7174 genes were found differentially expressed at the three developmental stages.4.By using qRT-PCR analysis,we revealed gene expression pattern of 14 selected differentially expressed genes in phytohormone signal transduction,conforming the result from transcriptomic high-throughput sequencing analysis.After integration of the putative function of differentially expressed genes,it is suggested that the genes involved in cell cycle,phytohormone regulation and ubiquitination may play important roles in FNM process.A complicated regulatory network may be constructed by these candidate genes.The present study broadened our view of ovule development in woody gymnosperm,and provided potential reasons for sterility of Pinus tabulaeformis ovules.We reported the differentially expressed genes between female fertile and sterile ovules during free nuclear mitosis process,and these genes were responsible for most biological processes.The P.tabulaeformis transcriptome data is a valuable resource for future genomic studies and will be helpful for researchers working on other closely related species.The differentially expressed genes dataset will also provide useful candidate genes for functional analysis of ovule development in woody gymnosperms.