Functional Analysis On Promoter Of Soluble Starch Sythase Ⅳ And Screening Of Its Regulators In Manihot Esculenta

Starch synthase (SS) is one of the very important enzymes in starch biosynthesis. It is divided into soluble starch synthase (SSS) and granule-bound starch synthase (GBSS). It can catalyze adenosine diphosphate glucose (ADPG) forming a-1,4-glucoside bonds by glycosyl transfer to produce dextran. SSS mainly catalyzes the synthesis of amylopectin, and the accumulation of total starch and amylopectin showed a highly significant positive correlation with its expression. It meant SSS played an important role in the regulation on accumulation of total starch and amylopectin. Cassava starch is a very important food industry and bio-energy raw materials, however, The expressional regulation of the gene encoding soluble starch synthase from Manihot esculenta remains largely unknown.According to the published cassava MeSSS sequences information, the gene can at least divide into five subtypes:MeSSSⅠ, MeSSSⅡa, MeSSSⅡb, MeSSSⅢ and MeSSSⅣ analysis by NCBI Protein blast. Quantitative PCR analysis found that all of the five genes could express in cassava root tuber and functional leaves, but the expression of MeSSSⅠ, MeSSSⅡa, MeSSSⅣ was much more than the others. The results declared that MeSSS Ⅱa’expression was the highest in root tuber, and MeSSSIV was the highest in functional leaf.According to part of the known promoter sequences and cDNA sequences, primers were designed to obtain the complete promoter sequences of the MeSSSⅣ by PCR amplification. Bioinformatics analysis finds that MeSSSⅣ promoter region contains typical eukaryotic core promoter, in addition to containing lots of important sugar signal response element, such as CGACGOSAMY3, DOFCOREZM, SREATMSD, SURE and so on. That indicates MeSSSⅣ expression level may be affected by the sugar or sugar concentration.Having Built a plant expression vector containing MeSSSⅣ promoter sequence and transforming it to tobacco in order to identify activity of MeSSSⅣ promoter. The positive transformed plants were identified by PCR and GUS staining. The GUS staining of TO transgenic tobacco appeared blue in stems, leaves and roots, that meant MeSSSⅣ 5 ’regulatory sequences could start GUS gene expression, having promoter activity.The bait sequence was cloned based on the sequences of MeSSSN promoter from Manihot esculenta, the bait vector (pMeSSSⅣ-AbAi) for yeast one hybrid was constructed, and transformed it to Y1HGold yeast. Meanwhile, SMART III ds-cDNA libraries from cassva root tuber and functional leaves were constructed by SMART and Advantage technologies. And screening of the transcription factor by yeast one hybrid is performing.