Gene Cloning And Prokaryotic Expression Of Key Enzymes Of Sucrose Metabolism In Lilium Davidii Var.Unicolor

Starch is the key factor to determine the quality of bulbs in lily.Sucrose is the main form of soluble carbohydrate in bulbs,which is involved in starch synthesis under the regulation of sucrose synthase(SuSy)and soluble acid invertase(SAI).In order to explore the genes function of SuSy and SAI and their regulation mechanism in sucrose-starch metabolism pathway,the full-length cDNA sequences were cloned from Lilium davidii var.unicolor and analyzed by bioinformatics.And then by using the method of Agrobacterium-mediated transformation,their constructed overexpression and RNAi vectors were introduced into Lilium davidii var.unicolor.The prokaryotic expression vectors of SuSy and SAI gene were constructed,and the expression of fusion proteins were induced under optimized conditions.The major results of this study were listed as follows:1.Cloning and analysis of the full length cDNA of SuSyl and SuSy2By RT-PCR and RACE methods,SuSyl and SuSy2 were cloned successfully with the total RNA of Lilium davidii var.unicolor as the template,and the full lengths werer 2877 bp and 2865 bp,respectively.The results of analysis showed that the homology of the SuSyl and SuSy2 was 85.73%,and the homology with other plants of Liliaceae was from 65%to 88%.SuSyl encoded 807 amino acids with isoelectric point(pi)6.10.SuSy2 encoded 811 amino acids with isoelectric point 6.10.Based on the analysis of domain,the conserved domain containing 188 amino acids was encoded,which contained a domain of glycosyl transferase.SuSyl and SuSy2 have a high similarity with the glycosyl transferase family GT1.2.Study on SuSy genetic transformation of Lilium davidii var.unicolorUsing the method of Agrobacterium-mediated transformation,SuSyl and SuSy2 were introduced into Lilium davidii var.unicolor.The optimal conditions of genetic transformation were listed as follows:hygromycin as 20 mg L-1 was lethal concentration,the concentration of cefradine is 300 mg L-1,with the pre-culture for 2 d,OD600=0.6,infection time for 20min,co-cultured for 3 d,100?M AS conditions.And 3 transgenic plants were obtained.3.Construction of prokaryotic expression vectors and prokaryotic expressionThe SuSy1,SuSy2 and SAI genes from Lilium davidii var.unicolor were cloned into prokaryotic expression vector pET28a,and the expression vectors were constructed.They were all successfully expressed in prokaryotic cells rosetta(DE3)under IPTG inducement and preliminarily indentified by SDS-PAGE.