香蕉穿孔线虫组织蛋白酶B基因的克隆与分析

Cloning and analysis of cathepsin B gene of Radopholus similis

为了获得香蕉穿孔线虫的组织蛋白酶B基因,分析该基因的序列、结构及其功能,为进一步研究植物寄生线虫组织蛋白酶的功能及其在线虫防治中的应用提供科学依据。我们应用SMART技术构建了香蕉穿孔线虫cDNA文库;采用SL法,克隆得到香蕉穿孔线虫组织蛋白酶B基因全长cDNA,通过测序获得1 257bp全长序列,命名为Rs-cb-1(GenBank:GU360972),该基因cDNA全长序列包括1 071bp的完整ORF,编码356个氨基酸,蛋白质相对分子质量为41 400。对该基因的序列结构及其编码的蛋白2级结构和三维结构与功能进行分析和预测结果表明,Rs-cb-1序列与其他寄生虫的组织蛋白酶B基因序列相比,该基因与秀丽小杆线虫组织蛋白酶B基因的亲缘关系最近;其编码蛋白主要为细胞外分泌蛋白,定位于微体(过氧化物酶体)、内质网膜和内质网管腔上,约有25个氨基酸跨膜区段位于蛋白质的C端,其表面电荷呈明显的极性分布;另外,通过同源建模获得该蛋白的三维结构预测图,这些结构与已报道的组织蛋白酶B生物学功能相符。本研究分离克隆得到的Rs-cb-1,是首个分离克隆得到的香蕉穿孔线虫组织蛋白酶B基因,从而为该线虫组织蛋白酶的进一步研究奠定了基础。

Summary The banana burrowing nematode, Radopholus similis, is a migratory endoparasite plant nematode and severely harms many tropical and subtropical crops. It is the most important invasive species to banana worldwide, and is listed as a quarantine pest in many countries. Nematicide have been used as the main approach to control R. similis. However, due to the high toxicity, nernaticide has led to many environmental problems. Now, gene targeting has become a new sustainable strategy to control plant nematode. Cathepsin B is a lysosomal cysteine protease, and it occurs in a wide range of parasitic and free-living nematodes. It has been demonstrated that cathepsin B plays a very important role in egg hatching, development, reproduction, invading host, inducing hostpathogenesis and immune evasion in parasites. To our knowledge, there are few studies on plant parasitic nematode cathepsin. Until now only one cathepsin B gene from Bursaphelenchus xylophilus （GenBank： GU130153） has been cloned, but the detail gene function is still unclear. The R. similis cathepsin B gene was cloned and analyzed, to provide a scientific basis for the further research on the function of plant-parasitic nematodes cathepsin B, and also for developing new control strategies. A eDNA library of R. similis was constructed by switching mechanism at 5＇ end of the RNA transcript （SMART） technique. The full-length cDNA sequence of the cathepsin B gene was obtained by the method of splicing leader （SL）, and then sequenced. The homolog sequences of cathepsin B were analyzed to generate a phylogenetic tree. The three-dimensional structure and the function of the cathepsin B protein were also analyzed and forecasted by bioinformatics technology. A mixed stages of R. similis complementary eDNA library was constructed by SMART. Altogether 76 expressed sequence tags （ESTs） were analyzed by Blast in NCBI. Of the 76 ESTs, one 600 bp EST sequence showed high homology to the cathepsin B gene of Caenorhabditis elegans. Based on the 600 bp EST sequence, a 1 257 bp eDNA full-length sequence of the cathepsin B gene from R. sirnilis was cloned by SL, and named as Rs-cb-1 （GenBank accession No： GU360972）. A phylogenetic tree was constructed based on the cathepsin B gene sequences of various organisms. The complete Rs^cb-10RF is 1 071 bp, and encodes a polypeptide with 356 amino acids （the protein molecular weight is 41.4 ku）. The multiple sequence alignment between Rs-cb-1 and the cathepsin B gene of other parasites showed that Rs-cb-1 had the closest genetic relationship with the cathepsin B gene of C. elegans. The function analysis showed that the protein coded by Rs-cb-1 was mainly for cell external secretion protein. The protein was subcellular located in the microbody （peroxisome）, the endoplasmic reticulum membrane and the endoplasmic reticulum antrum. A 25 amino acids＇ transmembrane section was found at the C terminus of the protein. The protein surface charge was polarity distribution. Three-dimensional structure of the protein was drawn by SWISS-MODEL. The structure was consistent with the biological function of cathepsin B gene. Ranch-1 was the first record about cathepsin B gene from R. similis. Based on the analysis and forecast, it may have the important biological functions.