Cloning And Characterization Of Starch Synthesis Associated Genes, IbAATP And IbSSI, From Sweetpotato (Ipomoea Batatas (L.) Lam.)

Sweetpotato (Ipomoea batatas (L.) Lam.) is widely consumed both as important food, fodder, industrial raw material and a new kind of energy crop. The study and utilization of sweetpotato starch has significant value for improving the yield of sweetpotato and its industrial qualities which can make it more suitably used as industrial raw material and energy crop. In the present study, two genes associated with starch synthesis were cloned from sweetpotato high-starch line (Xu 781) by homology cloning. The functions of the two genes were characterized by their overexpression and RNA interference (RNAi) in sweetpotato low-starch cultivar (cv. Lizixiang). The roles they played in the starch synthesis of sweetpotato were analysed. The results of this study are as follows:1. The plastidic ATP/ADP transporter protein gene (IbAATP) was isolated from sweetpotato high-starch line (Xu 781) using rapid amplification of cDNA ends (RACE). This gene has five exons and four introns. IbAATP showed highest expression level in storage roots and then in leaves. Exogenous sucrose treatment could induce the expression of IbAATP. Meanwhile, this gene is under the control of circadian rhythm. Subcellular localization revealed that IbAATP protein was localized to chloroplasts (or plastids). Overexpression of IbAATP in sweetpotato significantly increased the starch content and amylose content in the storage roots and the average size of the starch granules became larger. Meanwhile, the degree of crystallinity of starch granules were reduced in the transgenic lines and all plant lines exhibit type-A starch granules. The physicochemical properties (DSC and RVA) of starch and chain length distribution (CLD) of amylopectin in the transgenic plants were altered. qRT-PCR analysis showed that the starch synthesis associated genes in transgenic plants were up-regulated or down-regulated with different extent, and the enzymes they encode showed significantly altered activity. These results demonstrated that overexpression of IbAATP could significantly enhance the capability of starch synthesis and affects the content, composition and properties of starch in transgenic sweetpotato through varied influences on starch synthesis associated genes.2. The expression patterns and functions of the formerly cloned soluble starch synthase I gene (IbSST) from sweetpotato high-starch line (Xu 781) were analyzed. The IbSSI gene was successfully expressed in E. coli and enzymatic assay proved that IbSSI encodes a functional starch synthase. Transcript of IbSSI was detected with highest abundance in leaves and then storage roots. The expression of IbSSI could be enhanced by treatment of exogenous sucrose and is not regulated by circadian rhythm. Subcellular localization of IbSSI showed that it’s a chloroplastic protein.5’-deletions of the IbSSI promoter into 7 fragments demonstrated that each fragment confers activity in initiating the transcription of the gusA gene. Transgenic sweetpotato plants overexpressing IbSSI showed increased level of starch content, decreased level of amylose content, enlarged starch granules and enhanced degree of crystallinity compared with wild-type plants (WT). In contrast, transgenic plants in which IbSSI was suppressed by RNA interference (RNAi) showed opposite alterations in the three traits. All transgenic lines and WT plants exhibited type-A starch granules. Meanwhile, the physicochemical properties (DSC and RVA) of starch and CLD of amylopectin in the transgenic plants were altered. Further analysis demonstrates that IbSSI gene is mainly responsible for the synthesis of glucan chains with a degree of polymerization (DP) 9-17. qRT-PCR analysis revealed that the expression of starch synthesis associated genes were differentially influenced in transgenic plants and the enzymes they encode showed significantly altered activity. It was the alterations in the expression levels of these starch synthesis associated genes and the corresponding enzymatic activity that caused the changes in the content, composition and properties of starch from storage roots of the transgenic plants.