Study On The Function Of BnaM154 Gene In Brassica Napus L.

In order to further study the development process of Brassica napus seeds and elucidate the regulation mechanism of fatty acid metabolism during seed development,We screened a protein sequence encoding the Meprin and Traf-Chomology Domain from 35 d EST sequence of SSH library constructed during seed development in Brassica napus.Based on this sequence,a gene was found in Brassica napus seed development database.I’ll call it BnaM154.In this study,bioinformatics,genetic engineering and molecular biology methods were used to study the characteristics and functions of BnaM154 gene,and the following results were obtained:1.A series of bioinformatics analysis of BnaM154 gene and its coding protein showed that the full length of BnaM154 DNA was 1800 bp,encoding 345 amino acids.The relative molecular weight of the protein was 39395.61,and the theoretical isoelectric point was 8.53,indicating that it was a stable hydrophilic protein.Its structure consists of a stable transmembrane domain(22 amino acids)and two consecutive Math domains(106 amino acids and 107 amino acids,respectively),belonging to the first class of the Math family.2.The full-length DNA of Brassica napus BnaM154 gene was cloned,and the overexpression vector of BnaM154 gene was successfully constructed.The gene fragment of Brassica napus BnaM154 was cloned and inserted into RNAi interference vector containing seed specific expression of Napina promoter.3.Genetic transformation of Brassica napus XY15 was carried out by using the above two vectors respectively through Agrobacterium-mediated method.Among them,Brassica napus was transformed by overexpressing vector,and 57 resistant seedlings were obtained through resistance screening,and 8 transgenic plants with stable inheritance were obtained through molecular identification and detection,with a comprehensive conversion rate of 0.61%.52 resistant seedlings and 10 transgenic plants were obtained by resistance screening of RNAi interference vectors.4.The expression of BnaM154 gene in roots,stems,leaves,flowers and young fruit pod of XY15 was analyzed by quantitative PCR,and the results showed that BnaM154 gene was significantly expressed in leaves and flowers.At the same time,the expression of BnaM154 gene in BnaM154 overexpressed plants was detected,and the results showed that the expression of BnaM154 gene increased.5.The agronomic traits,fatty acid content and components of seeds were observed and analyzed.It was found that the 1000-seed weight of BnaM154 overexpressed seeds was lower than that of XY15,with an average of 1.709 g and 2.042 g,respectively.At the same time,there was no significant difference in morphology and size between BnaM154 overexpressed seeds and Xiangyou 15 seeds.The fatty acid composition of BnaM154 overexpressed seeds was the same as that of XY15,but the absolute fatty acid content of BnaM154 was lower than that of XY15,with90.72% and 92.36% respectively.These results suggested that BNA154 had a negative regulation effect on fatty acid content in Brassica napus seeds.