无机焦磷酸化酶融合基因克隆分析及表达载体的构建

Cloning of inorganic pyrophosphatase(PPA) gene and construction of its plant expression vetor

无机焦磷酸化酶(PPa)基因是植物糖代谢过程中的蔗糖合成调控基因,为将该基因运用于甘蔗转基因的研究,从面包酵母克隆了该基因,利用生物信息学方法对该基因进行组成成分和理化性质分析,预测分析其疏水性/亲水性,并将其核苷酸和编码氨基酸序列提交Genbank进行比对分析。利用从武运粳8号水稻中克隆得到Rub isco小亚基基因(rbcS)的5'上游调控区rbcS启动子,构建了由rbcS引导的无机焦磷酸化酶融合基因,并将其转入根癌农杆菌EHA105菌株中。

To use inorganic pyrophosphatase（PPa） regulating sucrose synthesization in transgenie sugarcane research, the PPa was cloned from Yeast. and its sequences were refered to Genbank and were confirmed by comparison with the known genome sequences of others. The sequence and bioinformatics analysis indicated that ,the ORF sequence were 864 bp in length and encode 287 amino acid. By comparison ,the homology of Nucleotide and amino acid were 99 % and 86 % respectively between the Saccharomyces cerevisiae, S. cerevisiae chromosome Ⅱ, Yeast PPA gene and other isolates from Candida glabrata chromosome H, Candida glabrata partial mRNA. The analysis of hydrophilicity showed that it was hydrophilic. The cloned rbcS promoter,which the 5 ＂-upstream regulation region of rice Rubisco small subunit gene （rbcS） was cloned from a Chinese cultivar Wuyunjing 8 ,was fused to the 5＂-upstream of PPa gene coding region in a binary vector,and the rbcS- PPa gene was transmited into Agrobacterium tumefaciens. The integration of the rbcS- PPa fusion gene in Agrobacterium tumefaciens was confirmed by PCR analysis.