Analysis Of Differentially Expressed Genes At Pattern Formation And Maturation Stages Of Soybean Seed

Soybean(Glycine max(L.) Merr) is an important economic plant worldwide.The seed oil is an important source of fatty acids for human and animal nutrition and one of the major resources of the edible oil for the Food Processing Industry.It is accounts for 31%of the worldwide vegetable oil consumption quantity.The goals of genetic engineering of soybean fatty acid biosynthesis are to increase the fatty acid content and to improve the fatty acid composition with more nutritional and specific value to match the fast growing demanding of human being.For protein and fatty acid accumulation starting from pattern formation stage,stop at dehydrate desiccation stage of soybeen seed development,we can compare the differentially expressed genes between different seed development stages, find critical genes which can effect seed development,protein and fatty acid biosynthesis, furthermore,increase the seed oil content by genetic engineering.In order to identify the differentially expressed genes involved in seed development and protein and fatty acid biosynthesis metabolism,we compared the gene expression between pattern formation and maturation of a high oil soybean line,Zhongdou 32.Based on the measurement of fatty acid and protein contents at different development stages of soybean seed,the immature seeds which 15 and 36 days after flowering(DAF) were sampled and differentially expressed gene between these two different developmental stages were obtained by subtractive suppression hybridization(SSH).All SSH clones were arrayed and screened by dot blot hybridization,followed by PCR analysis.Then design the gene special primers with Primer Permier 5.The differential expressed genes were detected by RT-PCR and real-time quantitative PCR.We also detected the tissue-specific of these genes.All the results provide important information on the gene regulation of seed oil/protein synthesis and ovular development.The genes in this study may be suitable as novel targets for genetic improvement of seed oil content and may also provide molecular markers for studies for soybean breeding.The subtraction efficiency of the forward and reverse subtracted libraries all above 2~5.The subtracted libraries were verified by colony PCR,the lengths of the inserted fragments were between 100bp and 900bp,average 434bp.By the result of dot hybridization,476 clones in forward library and 471 clones in reverse library were sequenced,respectively.The sequences were analyzed on line by BLAST in the GeneBank dbase,the results showed that 13 genes have explicit results;there were related to signaling,fatty acid,sugar and protein metabolism,auxin-regulate.Then design the gene special primers with Primer permier 5.Finally the differential expressed genes were detected by RT-PCR and real-time quantitative PCR.Tissue-specific detection of these 13 genes showed that only sucrose-binding protein gene was found to be specially expressed in the seed.These results showed that,the differentially expressed genes were likely related to the soybean seed development and fatty acids biosynthesis and metabolism.It's necessary to the further studying.