马铃薯转化酶抑制子St-inh cDNA克隆及在大肠杆菌中的表达和功能测定

CLONING OF POTATO INVERTASE INHIBITOR St-inh cDNA AND ITS EXPRESSION IN E.COLI AND FUNCTIONAL ANALYSIS

用RT-PCR结合5’RACE方法从马铃薯(Solanum tuberosum L.)栽培种JH块茎中克隆了转化酶抑制子St-inh 全长cDNA。序列分析表明,St-inh 基因编码区全长663bp,编码221个氨基酸。将含St-inh 基因cDNA 的DNA 片段克隆到pET28a(+)上,转化大肠杆菌BL21(DE3)后成功实现了表达。基因表达产物与马铃薯栽培品种(系)E1、JH 试管块茎以及番茄果实的转化酶提取物共孵育结果显示,转化酶活性分别下降了34.3%、21%和33.8%,说明St-inh 的翻译产物具有转化酶抑制子功能。BLAST 基因序列分析表明,St-inh 与Kunitz-type C 类基因序列同源性达95%以上,T-COF-FEE 氨基酸序列对比分析显示,该基因编码的蛋白质具有典型的Kunitz-type 结构域[L,I,V,M]-X-D-X-[E,D,N,T,Y]-[D,G]-[R,K,H,D,E,N,Q]-X-[L,I,V,M]-X(5)-Y-X-[L,I,V,M],因此,Sit-inh 基因可能为Kunitz-type 家族成员。

A full length cDNA clone encoding invertase inhibitor was isolated by RT-PCRcombined with 5'RACE from potato (S.tuberosum) tubers of cv.JH and designated as St-inh.The encoding region of St-inh is of 663bp encoding a protein of 221 amino acids.The DNA frag-menu including St-inh cDNA was cloned into the vector pET28a (+) and expressed successfully inE.coli.Co-incubation of the proteins produced by St-inh in E.coli and the invertase extracts frompotato tubers of cv.E1,JH and tomato fruits showed that the invertase activities of potato tubersand tomato fruits decreased by 34.3%,21% and 33.8% respectively.These results indicated thatproducts of St-INH protein had a function of invertase inhibitors.The analysises of the nucleotideand amino acid sequences using BLAST and T-COFFEE demonstrated that St-inh.cDNA was ofover 95% homologous to Kunitz-type C and there was a typical domain of Kunitz-type protein [L,I,V,M]-X-D-X-[E,D,N,T,Y]-[D,G]-[R,K,H,D,E,N,Q]-X-[L,I,V,M]-X(5)-Y-X-[L,I,V,M].There-fore,it was conjectured that St-inh could be a member of Kunitz-type gene family.