Construction Of Susiba2 Sense And Antisense Expression Vectors And Genetics Transformation To Rice

Starch is the main storage carbohydrate in higher plants, and also the most important ingredient in food crops. Several enzymes, such as ADP-glucose gyrophosphorylase (AGPase), Soluble Starch Synthase (SSS), Granule-bound Starch Synthase (GBSS), Starch Branching Enzyme (SBE), Debranching Enzyme (DBE), are directly relative to starch biosynthesis. Recent researches have demonstrated that a lot of starch biosynthesis genes were expressed under the inducement of sugar signaling. Prof. Christer, Sweden Agriculture University, gained SUSIBA2 (sugar signaling in barley) gene from Barley. The SUSIBA2 protein, as an activator bound the cis-element in the promoter of isoamylase and SBE, plays important role in the process of starch accumulation in Barley endosperm.In this paper, by means of constructing pTCK303- SUSIBA2(+) vector of SUSIBA2 gene sense sequence and pTCK303- SUSIBA2(-) vector of anti-sense sequence, SUSIBA2 gene was introduced into the genome of Nipponbare by Agrobacterium-mediated. It is hoped that SUSIBA2 gene could regulate activity of the enzymes about starch biosynthesis to change starch composition in rice. The main results are as follows:(1) SUSIBA2 gene, digested by BglII from vector pS1473, was constructed into pTCK303. We finally got the sense vector pTCK303- SUSIBA2(+) and antisense vector pTCK303- SUSIBA2(-) after identification by PCR.(2) The two vectors were introduced into LBA4404 by frozen-melt method respectively, and then we chose the Agrobacterium that were resistant to Rifamphicin and Kanamycin.(3) We got six transformed plants, including 4 plants transformed by pTCK303- SUSIBA2(+) and 2 plants transformed by pTCK303- SUSIBA2(-).(4) By PCR identification, 4 plants transformed by pTCK303- SUSIBA2(+) all were positive, while 2 plants transformed by pTCK303- SUSIBA2(-) both were false positive.