Cloning And Preliminary Functional Verification Of Rapeseed SPATULA Homologous Genes BnaA01g0073D And BnaC03g61610D

As we all know.plant oils have an important role in daily life,and it is one of the main objectives of breeders to improve crop yield and oil content.Nowadays,it is a general research method to explore and isolate the genes related to the pod and seed development of crops by genetic technology to improve crop genetics heredity.SPATULA(SPT),an important bHLH transcriptional regulator in Arahidopsi.s thaliana(L.),regulates various development processes,including carpel development,flower development,fruit development,and so on.Our previcious results showed that spt mutants not only generate abnormal siliques,but also result in changes of seed oil contents and fatty components,compared with wild types.In this study,we cloned Brassica napus(L.)SPT homologous genes,BnaA01g0073D(BnSPTl)and BnaC03g61610D(BnSPT4),from Brassica napus(L.).We used molecular biology methods to analyze their roles in controlling fruit development in Arabidopsis.These results will provide theoretical significance on further improving crop yield and oil contents by cloning technology.The main results are as follows:We cloned AtSPT homologous genes BnSPTl and BnSPT4 in Brassica napus.Furthermore,we investigated tissue expression patterns of BnSPT1 and BnSPT4 utilizing quantitative real-time PCR.The results showed that they were expressed in various organs,including roots,stems.leaves,buds,flowers and siliques.These results indicated that they might involved in flower and pod development regulation.We also analyzed BnSPTl and BnSPT4 promoter sequences using online tools and found that BnSPT1 and BnSPT4 promotors contain many domains involving in a variety of biological and abiotic stress responses.1)Overexpressions of BnSPT1 and BnSPT4 in Arabidopsis spt mutants completely restored their silique sterile phenotype.and partially restored the phenotypes of plant height and seed size.Otherwise.compared vs with wild types.overexpressions of BnSPT1 and BnSPT4 in mutants significantly increased siliques length and siliques number.which suggested that BnSPT1 and BnSPT4 are involved in fruits developement.2)Compared with the wild types,the seeds oil contents of spt mutants were significantly decreased,but they were significantly enhanced in BnSPT1-overexpressing and BnSPT4-overexpressing lines.Scanning transmission electron microscopy(STEM)results showed that,the volumes of the aleurone particles were significantly raised and the volumes of the oil bodies were significantly reducted in spt2 and spt12 seeds compared with the wild types.On contrary,overexpressing BnSPTl in spt2 and spt12 mutants reduced the volume of the aleurone grains and enlarge the volume of the oil bodies.3)To investigate the regulation mechanisms of BnSPTs,we implemented qRT-PCR to analyze relative expression patterns of several key transcription factors involved in protein,starch and fatty acid genes in seed development.The analysis revealed that BnSPTl irregularly affected expression patterns of these genes.We infered that BnSPTl might disorder the expressions of transcription factors and genes related to oil synthesis,seed storage proteins synthesis and proteins transport,and result in an enhancement of fatty acid contents and alternations of fatty components.In summary,these results have shown that BnSPT1 and BnSPT4 regulate silique and vegetative organs development in Arabidopsis.These results will provide the theoretical significance on improving crop yield and oil content by molecular technologies.