Study On The Unknown-function Gene Bna88 Of Brassica Napus L.

In order to understand the regulatory mechanism of fatty acid metabolism in Brassica campestris L.more clearly and to discover new fat metabolism regulation factor, we sifted a gene named Bna88 with unknown founction from the SSH library of genes expressed specifically at 35days after flowering in Xiangyou15 seeds. We cloned the CDS of Bna88 gene and finished its bioinformatic analysis and the tissue-specific expression profiling. The overexpression plasmid and the RNAi plasmid of Bna88 gene were constructed. Via agrobacterium-mediated plant genetical transformation, we obatined the RNAi knock-down plants of Bna88 gene,followed by invesitigation of the expression level of Bna88 gene in the transgenic plants to judge whether this RNAi plasmid worked. We analyzed the oil content and fatty acids composition in the transgenic plant seeds to find the influence by the alteration of the Bna88 gene expression, and explored the elementary mechanism.The detailed results are as following:1) The full length CDS of Bna88 was successfully cloned based on the previous study of our lab.a In silico analysis indicated that Bna88 belonged to the BraLCR family. The length of Bna88 CDS is 237bp, encoding a protein with 79 amino acid residues. Bna88 protein contains a transmembrane domain and a cysteine-rich region which is well characterized in AtLCR family. The result of RT-PCR and qPCR showed that Bna88 was expressed in silique specifically and during the seed development the expression level was the highest at 35 days after flowering.2) Construction of the overexpression plasmid named pFGC5941-Napin-Bna88CDS, and the RNAi plasmid named pFGC5941-Napin-Bna88RNAi. The overexpression plasmid was transferred into Brassica napus XY15 by Agrobacterium-mediatod transformation, and transgenic seedlings were regenerated from cotyledons petioles.3) Through qRT-PCR analysis, compared to the control, the expression level of Bna88 gene in the transgenic plants seeds obviously incrased at 35days after flowering.4) The content and composition of fatty acid from pFGC5941-Napin-Bna88CDS T2 generation transgenic seeds oil was analyzed by TLC and GC. The results showed that, compared with seeds oil of the non-transgenic plants, the content of TAG decreased, the content of linolenic acid and linoleic acid increased and the content of oleic acid decreased in the transgenic plants.5) Through qRT-PCR analysis,sucrose contents analysis and sugar induced experiment, Bna88 gene was confirmed can inhibit SPP,which was important in the sucrose synthesis pathway. And then the glycolytic pathway was inhibited, which lead to the repressing of fatty acid.In addition,Sucrose can induce PK and inhibit Bna88 gene in turn.