Cloning Of Starch Synthase Ⅲ CDNA From Potato And Construction Of RNA Interference Vector

Starch is a macromolecule compound produced through plant photosynthesis. Potato tuber starch accounts for 9%-25% of fresh weight, which ranks second in the plant starch next to production amount and commodity availability of maize starch. At present, it was not ideal that gelatinization temperature, stickiness, and freeze-thawing stability of starch from popularized potato variety. In the study, the cDNA of potato soluble starch synthase III (SSIII) was cloned by RT-PCR technique. The SSIII gene will be inhibited by RNA interference method to change starch structure and increase the covalently linked phosphate of starch granule, and breed new potato variety which is more suitable for industry utilization. At the same time, the function of SSIII gene will be studied in the starch synthesis by RNA interference method. The main results as following:1. The cDNA of SSIII gene was cloned by RT-PCR from potato. The sequence analysis showed that the total length of the cDNA was 3967 bp with 3693 bp open reading frame encoded a 1230-amino acid polypeptide. Homology analysis of the nucleotide sequence and the deduced amino acid sequence showed that the cDNA shared 99% and 98% identity with the published sequence (X94400) and its protein (CAA64173.1) respectively.2. The analysis of bioinformatics software showed that the SSIII protein has several phosphorylation sites, alpha helix with 369 amino acids, beta sheet with 249 amino acids, beta turn with 102 amino acids, random coil with 510 amino acids in second structure, 3 repeated sequences of soluble starch synthase III gene family, 1 starch synthase catalytic conserved domain, no trans-membrane region, and a 75 amino acid transit peptide for transportation to chloroplast.3. RNA interference intermediate vector of SSIII gene was constructed. The analysis of RNAStructure software showed that its secondary structure was hairpin structure, enabling to induce RNA interference mechanism in organism successfully. The analysis of BLAST alignment showed that the interference fragment could specially inhibit SSIII gene expression. The RNA interference expression vectors pBI-SSIII-RNAi and pBIC-SSIII-RNAi for SSIII gene, driven by constitutive expression promoter CaMV 35S and tuber-specific expression promoter CIPP respectively, were constructed, and transformed into Agrobaterium tumefaciens LBA4404 by freeze-thawing method, which provided the foundation for further potato genetic transformation.