天花粉蛋白基因的克隆及序列分析

Cloning and DNA Sequencing of the Gene Encoding Trichosanthin

本文应用ＤＮＡ多聚酶链式反应（ＰＣＲ）技术，从栝楼基因组ＤＮＡ中扩增并克隆了天花粉蛋白（ＴＣＳ）基因。核酸序列分析结果表明，克隆片段包括ＴＣＳ的前原蛋白的编码序列和５＇一侧翼区段。其编码序列与已发表的不同来源的３种ＴＣＳ基因的核苷酸序列的同源性分别为９９．２０％，９８．７４％和９８．６４％。推导出的氨基酸序列与已发表的４种ＴＣＳ的氨基酸序列的同源性分别为９８．６２％、９８．６２％、９７．４１％和９８．３８％。另外还发现，我们克隆的ＴＣＳ基因的５＇一侧翼区段比已发表的ＴＣＳ基因的相应区段多一个类似于ＴＡＴＡ盒的序列。我们进一步构建了该基因的一系列突变体，为深入研究ＴＣＳ基因结构、表达和调节机制以及ＴＣＳ的结构和功能关系奠定了重要的基础。

A gene encoding Trichosanth in (TCS)was amplified by means of DNA polymerase chain reaction(PCR) and cloned from the genomic DNA of Trichosanthes kirilowii.Its DNA sequence has been determined. Nucleotide sequence analysis indicates that the cloned TCS gene includes 5'-flanking region and the encoding sequence for TCS preproprotein. Comparison of its encoding sequence with the published nucleotide sequences of three TCS genes demonstrates that they have the sequence homologies of 99.20%, 98.74%and 98.64%respectively. Comparing its deduced amino acid sequence with four reported amino acid sequences of TCS demonstrates that they have the sequence homologies of 98.62%,97.62%, 97.4l%and 98.38%respectively.A new TA TA-like box sequence was found in the 5'-flanking region of cloned TCS gene.In order to further study the structure, expression, regulating mechanism of TCS gene and the structure-functional relationship of TCS, a series of mutants of TCS gene has been constructed. The expression of these mutants in bacteria and transgenic plant has being studied.