Improving Cassava Protein Content By Specifically Targeting Of Storage Protein

Cassava (Manihot esculenta Crantz) is a perennial (tropical, subtropical) or annual (temperate) shrub of Euphorbiaceae. Cassava is widely planted for the rich starch of storage root, providing a vital source of carbohydrates for over800million people in developing countries. Despite the high starch content, cassava roots are deficient in nutritional value, lacking significant amounts of protein. It is hard to provide enough essential amino acids, resulting in people’s severe malnutrition in poor areas. Therefore it is very important to improve the protein content in cassava storage roots by conventional and molecular breeding methods. With the rapid development of biological technology, increased the protein content of cassava through transfer and expression of exogenous protein has already become an effective method.In this study, a plant vector drived by vascular-specific promoter p54/1.0was constructed, and sporamin introduced by spor SS and KDEL signal peptide would be located in cassava vacuole and/or endoplasmic reticulum. After PCR detection and Southern Blot hybridization, there were19single copy transgenic lines identified. Some transgenic lines were selected for extracting RNA of root. The results of semi-quantitative PCR and Real-Time PCR showed that the amount of Sporamin in transgenic lines RV8+9was the highest, and the expression of sporamin in single copy lines was higher than in multiple copy lines. Western blot detection was used for RV8+9leaf protein, but no hybridization signal was discovered. So, it’s speculated that the expression of sporamin in RV8is lower than1%of the expression of sweet potato. The leaf protein content of some transgenic lines was determined by A280absorption and Bradford.This study established4dual expression vectors contain psRbcS TP and sporamin. The starch-binding domains (SBD) of SBE Ⅰ, SBE Ⅱ gene were selected as the target sequence, two large fragments fused with EGFP gene have got by Overlap Extension PCR (SOE PCR), and inserted the4fragments into the vector RV10(containing sporamin) to get4new plant expression vectors:SBD48-1, SBD48-2, SBD48-1EGFP, SBD48-2EGFP. According to PCR amplification, restriction enzyme digestion and sequencing, the vectors were confirmed successfully constructed. Vector SBD48-1EGFP, SBD48-2EGFP were transferred into Arabidopsis thaliana mediated by Agrobacterium tumefaciens Gv3101, and Arabidopsis with hygromycin resistant had been screened. EGFP signal was found in Arabidopsis transformed by SBD48-2EGFP. It’s speculated that SBD has worked and the protein successfully bound to the starch granule. These four vectors transformed into cassava fragile embryo callus mediated by Agrobacterium tumefaciens LBA4404after resistance screening, rooting experiment, so far,8positive transgenic lines of SBD48-2have been obtained (from different embryo callus cluster), also2positive transgenic lines of SBD48-2EGFP have been obtained.The purpose of this study was to get cassava cultivars with high protein nutritive value, as to provide superior varieties and theoretical basis to solve the problem of protein malnutrition.