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

用RT-PCR结合5’RACE方法从马铃薯(Solarium 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-COFFEE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，St-inh基因可能为Kunitz-type家族成员。

Cloning of potato invertase inhibitor St-inh cDNA and its expression in E. coli and functional analysis]

A full length cDNA clone encoding invertase inhibitor was isolated by RT-PCR combined 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 fragment including St-inh cDNA was cloned into the vector pET28a (+) and expressed successfully in E. coli. Co-incubation of the proteins produced by St-inh in E. coli and the invertase extracts from potato tubers of cv. E1, JH and tomato fruits showed that the invertase activities of potato tubers and tomato fruits decreased by 34.3%, 21% and 33.8% respectively. These results indicated that products of St-INH protein had a function of invertase inhibitors. The analysises of the nucleotide and amino acid sequences using BLAST and T-COFFEE demonstrated that St-inh cDNA was of over 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. Therefore, it was conjectured that St-inh could be a member of Kunitz-type gene family.