河北毛白杨巯基蛋白酶抑制剂基因cDNA的克隆及序列分析

植物巯基蛋白酶抑制剂(cysteine proteinase inhibitor,CPI)在林木的抗虫基因工程中发挥着越来越重要的作用,分离和克隆植物CPI基因进而研究该基因的功能是CPI基因工程研究的热点.本文应用PCR技术从我国乡土抗天牛树种河北毛白杨形成层中分离和克隆到了一个710bp大小的cDNA片段,同时对序列进行测定和分析.测序和分析结果表明,该cDNA片段含有一个429 bp的完整开放阅读框,其编码143个氨基酸残基,具有LARFAVDEHN、QXVXG和YEAKVWVKPW三个典型的植物巯基蛋白酶抑制剂基因家族的高保守区段,同时在GenBank中进行了注册,核苷酸注册号为DQ020096,氨基酸注册号为AAY41807.氨基酸序列同源性分析表明,该基因与其他林木中的巯基蛋白酶抑制剂的同源性差异较大,同源性在48%～97%之间,其中与欧洲山杨的同源性最高,达到97%,与猕猴桃的同源性最低为48%.说明河北毛白杨形成层中存在CPI基因并能够有效表达,这为进一步研究该基因的抗虫功能奠定了良好的基础.

Cloning and Sequencing a cDNA of Cystatins from Poplar Landrace in Hebei

Cystatins, plant cysteine proteinase inhibitor (CPI) plays more and more important roles in the fi elds of tree genetic engineering for resistance to insect pest. It is one of hot points to isolate and validate the gene functions in the CPI gene engineering at present. In this research, a 710 bp cDNA fragment of cystatins was isolated from the cambium of poplar landrace in Hebei province (Populus tomentosa) using PCR methods . The cDNA also was sequenced that contained 429 bp length open reading frame (ORF) and encoded 143 amino acid residues (Accession number in GenBank: DQ020096 & AAY41807). Sequence analysis indicated that the cDNA contained three typical motif domains, LARFAVDEHN, QXVXG and YEAKVWVKPW, which usually exists in the members of the phytocystatin superfamily and the homology analysis of amino acid sequence of obtained CPI cDNA was very different with known CPI, which has highly 97% homologies with Populus tremula and 48% lowly homologies with Actinidia deliciosa. It would be suggested that CPI gene exist and express in the cambium of Populus tomentosa which could become the clue for cloning novel insect resistance gene.