The Analysis On Gene Expression In Seed Germination In Brassica Napus

Seed germination is an important event in ontogenetic development in higher plants, which is regulated by a series of genes and environment factors. Though a large number of genes related to germination were found by in-depth study on Arabidopsis thaliana mutants, it is still a mystery about the mechanism for seed germination. Several studies have been carried out on Brassica napus (B. napus) seed germination, although there are no systematic studies on spatial and temporal gene expression patterns in B. napus. In this study, we chose B. napus seed as material to characterize gene expression in seeds different organs, and analyse expression of ten AQP (aquaporin) genes during seed germination. Our research aims to provide a basis for understanding molecular mechanisms of seed germination.cDNA-amplified fragment length polymorphism (cDNA-AFLP) approach was used to screen differential gene expression in cotyledons, hypocotyl and radicle during B. napus seed germination. In total, one hundred and twenty-one primer combinations were obtained5,356transcript-derived fragments (TDFs), including1,324bands in cotyledons,1,601bands in radicle and2,431bands in hypocotyl, respectively. We chosed365TDFs for cloning and sequencing, including75differentially expressed TDFs in cotyledons;145in radicle and hypocotyl, respectively. Finally,175differential expression genes (37in cotyledons,66in radicle,72in hypocotyl) were homologous to genes with known function from NCBI database by BLAST analyses. The above175known genes were classified into14different functional categories, followed by metabolic processes, transcription, signal transduction, translation and transport. However, some gene expression were special, including genes related to regulation metabolism and protein function in cotyledons; genes related to cell rescue and defense in radicle; genes related to signal transduction in hypocotyl. Our results showed that, following with spatial and temporal change, seed germination was accompanied by complex molecular events:differential gene expression at various germination stages and characteristic gene expression in different seed organs. Besides, eleven identified genes were chosed to verifiy by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) in different organs. The RT-PCR results showed similarity with those of cDNA-AFLP.To investigate molecular mechanism of water regulation during seed germination, seven genes encoding plasma membrane intrinsic protein (PIP) were initially cloned and sequenced from germinating seed cDNA pool of B. napus. These genes belong to the PIP1and PIP2subfamilies. The transcription levels of seven cloned genes plus three previously identified AQP genes from B. napus were analyzed during germination by quantitative real-time PCR (qRT-PCR). These results showed that expression levels of ten AQP genes were lower or scarcely detected in dry seed, but were up-regulated during germination as well as early young seedlings. In addition, expression of these ten AQP genes in response to abiotic stress was investigated, and results showed differential responses to abiotic stress treatments, suggesting that these ten genes play different roles during plant development and response to abiotic stresses in B. napus.In our study, differential gene expression duirng seed germination was characterized by cDNA-AFLP. Further, we analysized the expression of AQP gene during seed germination and stresses treatment. These results will expand our view to the complicated biology events on seed germination, and provid a theoretical basis for future studies on mechanism for B. napus seed germination.