一个在麻疯树根和茎高表达的Kunitz型蛋白酶抑制剂基因JcKTI具有抗虫活性

通过RT-PCR和PCR技术,从麻疯树基因组中克隆得到一个Kunitz型蛋白酶抑制剂基因（JcKTI）的开放阅读框序列。对应开放读码框的基因组序列不含有内含子。该开放阅读框长度为540bp，编码一个含有179个氨基酸残基的成熟多肽，具有典型的Kunitz家族结构特征。组织特异性表达研究显示，JcKTI基因在根和茎中的表达丰度最高，在叶片和种子中表达较低。构建原核表达载体pET32-JcKTI在大肠杆菌BL21中表达，获得纯化的重组蛋白，该蛋白显示出一定的抑制牛胰蛋白酶的活性。将该基因在烟草中过表达，明胶酶法和BAEE法的结果均显示转基因植物的蛋白提取物对胰蛋白酶具有一定抑制作用，进一步的抗虫实验结果表明转基因烟草叶片可使进食后的棉铃虫幼虫生长发育受阻，并减少对叶片的吞食。上述结果暗示JcKTI基因可能在麻疯树根和茎的抗虫应答中扮演着一定的角色。

A Kunitz Type Protease Inhibitor Gene JcKTI High Expressed in the Roots and Stems of Jatropha curcas Showing Anti-pest Activity

The open reading frame (ORF) fragment of a Kunitz type protease inhibitor gene JcKTI was cloned by the RT-PCR and PCR technology, respectively, from the leaf cDNA and genomic DNA of Jatropha curcas. The genomic region corresponding to the open reading frame does not contain any introns. The open reading frame is of 540bp in length, encoding a mature peptide with 179 amino acid residues. The predictive protein contains the typical Kunitz structure exiting in the protein family members. Tissue specific expression study showed that the expression of JcKTI in root and stem was higher, and the expression was lower in leaf and seed. The prokaryotic expression vector pET32-JcKTI was constructed and expressed in E.coli BL21. The purified recombinant protein was obtained, which showed a certain degree of bovine trypsin inhibitory activity. The gene was over-expressed in the tobacco plantlets. The results of gelatin zymography and BAEE assays showed that the crude protein extracts from the transgenic plants exhibited stronger trypsin inhibitory activity than that of control. Further resistance experiment results showed that eating transgenic tobacco leaves led to the retardation larvae growth and reduced food intake. These results suggest that JcKTI gene may play a role in the roots and stems of Jatropha curcas response to pest attack.