SBE2B Cloning, Expression Vector Construction And Rice Genetic Transformation System Established

Rice (oryza saliva L) is one of the most important crops; more than one half population in the world take it as their staple diet. In China it is planted 3 million ha per year, of which hybrid rice is about one half. Rice takes up as much as 90% of their totle planting area in some province, like Sichuan,Jiangxi,. However, the processing quality and food quality of hybrid rice is not good due to high amylose content and hardness , low gel viscosity and bad mouthfeel etc. These problems were improved through regular breeding methods but it was only slightly. The main aim of this study was to modify rice starch quality by molecular biology methods. Detailly, it was(l) to isolate and construct high efficiency expression vector of rice starch branching enzyme gene sbe2b and(2) to establish a high-effecient rice transformation system.Total RNA was extracted from maize endosperm 15DAP(Days After Pollination ).and cDNA of sbe2b was obtained through RT-PCR. Sequencing result showed that the full length cDNA was 2.4kbp, coding for 800 amino acids, with estimated MW 93KD. Maize sbe2b gene has very high homology with that of rice, barly and wheat, and high alignment with maize ae gene with sequence NO. L08065.1.Endosperm specific expression vector pEBE2 was constructed. This vector , including sbe2b gene, was 6.06kb long, containing endosperm specific promoter and a intron that enhanced the transcript efficiency. Both sense and antisense cDNA were transformed to upregulate and downregulate amylose content and relative branching characters. pEBE2 vector containing the target gene and 35SIH3 vector containing Bar gene were co-bombarded into several indica rice lines Shuhui527, Minghui86 and 612S etc. Resistant seedling were obtained finally. In order to establish rice mutation library, Agrobacteriwn vector pSHK22 containing Bar gene as screening maker was transformed iniojaponica Zhonghual 1. Large quantity of resistant calli were now in the process of regenerating.