植物Ⅴ类几丁酶苦瓜同源基因(McChi5)的克隆及特征分析  

Cloning and Characterization of a Homologous Gene of Plant Class Ⅴ Chitinase from Balsampear, Momordica charantia Linn.

作　　者：肖月华[1] 侯磊[1] 袁小红[1] 杨星勇[1] 裴颖 罗小英[1] 裴炎[1] 

机构地区：[1]西南农业大学生物技术中心,重庆400716

出　　处：《Acta Genetica Sinica》2002年第11期1028-1033,共6页

基　　金：科技部转基因植物专项 (ZJY A 0 1);重庆市教委科学技术研究项目资助~~

摘　　要：苦瓜是一种病害很少的蔬菜作物。用 3′RACE方法从苦瓜叶片RNA中扩增了一个与烟草Ⅴ类几丁酶序列相似的片段 ,进一步用Y RACE方法扩增了相应的 5′端序列。经序列拼接 ,获得了植物Ⅴ类几丁酶苦瓜同源基因(McChi5 )的全长cDNA基因。该基因全长 1348bp ,含有一个 10 4 4bp的ORF。推测的蛋白质由 347个氨基酸组成 ,计算所得的分子量为 38.3kD ,等电点为 5 .77。同源性分析表明 ,McChi5蛋白与烟草V类几丁酶、拟南芥的推测几丁酶和类几丁酶蛋白、以及哺乳动物和细菌的一些几丁酶有序列相似性 ,并具有第 18家族糖基水解酶的保守域。Southernblotting表明 ,在苦瓜基因组中存在 2个拷贝的McChi5基因 ,同时也存在少数同源基因。RNAdotblotting分析表明 ,McChi5基因有组成性表达的特性 ,伤害处理对其表达的影响不明显。对McChi5基因可能的生物功能和应用作了进一步的讨论。Balsampear ( M. charantia Linn.) is a vegetable crop, highly resistant to pathogens. Chitinases were proposed to play an important role in the defense response of this crop. Based on the N terminal sequence of a purified balsampear chitinase, a fragment (ChitB), similar to the tabacco class Ⅴ chitinase gene, was amplified from the leaf RNA using 3′RACE, and the corresponding 5′ sequence was further amplified by the Y RACE method. By joining the two amplified fragments, the full length cDNA of M. charatica homologous gene of plant class Ⅴ chitinase ( McChi5 ) was obtained. The 1348bp cDNA contained an ORF of 1044bp, which coded for a polypeptide of 347 amino acids. The deduced polypeptide had a predicted molecular weight of 38.3kD and a pI of 5.77. Homology analysis demonstrated that, McChi5 protein, which contained a conserved domain of family 18 glycosyl hydrolyse, had the sequence similar to tabacco class Ⅴ chitinases, several putative chitinases and chitinase like proteins of Arabidopsis thiliania , and some chitinases from mammals, insects and bacteria. Southern blotting suggested that two copies of McChi5 gene and several homologous genes existed in the M. charatica genome. By RNA dot blotting ananlysis, expression of the McChi5 gene wasdetected in cotyledons, roots, stems, and leaves, and it was not induced by wounding treatment. The biological functions and the potential applications of Mcchi5 gene were discussed.

关 键 词：植物 V类几丁酶 苦瓜 同源基因 克隆 特征分析 抗病性 

分 类 号：Q785[生物学—分子生物学]