香蕉穿孔线虫β-1，4-内切葡聚糖酶基因的克隆与特征分析  

Cloning and Characterization of a β-1,4-endoglucanase Gene from Burrowing Nematode Radopholus simili

作　　者：肖罗[1,2] 陈国华[2] 戴良英[1] 吕春花[2] 杨宇红[2] 谢丙炎[2] 

机构地区：[1]湖南农业大学生物安全科学技术学院,长沙410128 [2]中国农业科学院蔬菜花卉研究所,北京100081

出　　处：《园艺学报》2008年第10期1431-1440,共10页Acta Horticulturae Sinica

基　　金：国家‘973’计划项目(2002CB111400);‘十一五’‘863’计划项目(2006AA10Z432);‘十一五’国家科技支撑计划项目(2006BAD08A14);公益性行业(农业)科研专项经费项目(nyhyzx07-007)

摘　　要：植物寄生线虫的分泌物在线虫的侵染过程中具有重要的作用,其中纤维素水解酶是研究较多的主要分泌物之一。采用RT-PCR和RACE方法,从香蕉穿孔线虫(Radopholus similis)中获得编码β-1,4-内切葡聚糖酶基因的cDNA序列,命名为RS-eng-1(GenBank登录号为EU414839)。该cDNA序列全长1630bp,包含一个1404bp的开放阅读框(ORF),编码含467个氨基酸的蛋白,理论分子量与等电点分别为48.22kD和6.04。序列分析表明该酶含有糖基水解酶家族5(GHF5)的保守结构域,N端具有22个氨基酸残基组成的信号肽,C端含有细菌式样的纤维素结合域(CBDⅡ)。原位杂交结果显示此基因在香蕉穿孔线虫的食道腺部位表达,并且Southern结果显示其为多拷贝基因。cDNA与基因组DNA重叠分析表明,此基因包含6个内含子,切割点符合5′-GT...AG-3′的规律。进化分析表明该酶与细菌Bacillus subtilis和Erwina carotovora分泌的纤维素酶属于同一支,推测其来源于细菌的水平基因转移。Proteinaceous secretions from the oesophageal glands of plant-parasitic nematodes have crucial roles in nematode parasitism of plants, of which cellulases are among the best characterized. The full length cDNA sequence of a novel β-1, 4-endoglucanase gene from Radopholus similis was obtained by RT-PCR and RACE, named RS-eng-1 （GenBank： EU414839）. It consisted of 1 630 bp in length, with a 1 404 bp ORF encoding a 467 amino acid protein. The putative protein, with the oretical molecular weight 48.22 kD and pI 6.04, was classified as a member of glycosyl hydrolase family 5. Sequence analysis showed that it had a 22 amino acid signal sequence at N terminal and a bacterial type cellulose-binding domain Ⅱ at C terminal. Southern blot analysis showed that there were multiple copies of RS-eng-1 within Radopholus similis genome. In situ hybridization showed RS-eng-1 was expressed in Radopholus similis esophageal gland cells. Genomic analysis demon-strated that RS-eng-1 contained six introns demarcated by 5＇-GT... AG-3＇ in the slpice sites and phylogenic analysis suggested that RS-eng-1 had a strong homology to β-1, 4-endoglucanase genes from Bacillus subtilis and Erwina carotovora, which may be indicative of an ancient horizontal gene transfer.

关 键 词：香蕉穿孔线虫 香蕉 Β-1 4-内切葡聚糖酶 GHF5家族 原位杂交 

分 类 号：S436[农业科学—农业昆虫与害虫防治] Q78[农业科学—植物保护]