象耳豆根结线虫纤维素结合蛋白Me-cbp-1基因的克隆与功能分析

发布时间：2018-08-30 20:11

【摘要】：纤维素结合蛋白(cellulose binding protein,CBP)是植物寄生线虫在侵染过程中分泌的重要效应蛋白。为了获得象耳豆根结线虫(Meloidogyne enterolobii)纤维素结合蛋白序列信息、并分析其在线虫寄生致病过程中的功能地位,本研究利用已构建的象耳豆根结线虫二龄幼虫转录组数据库,结合cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术克隆了象耳豆根结线虫纤维素结合蛋白基因Me-cbp-1(Gen Bank登录号:KU350655)cDNA全长序列。生物信息学分析表明,Me-cbp-1 cDNA全长809bp,其5'末端和3'末端的非编码区长度分别为43和139 bp,以及含有1个长度为627 bp的完整开放阅读框(ORF),推导编码208个氨基酸。预测蛋白Me-cbp-1含有起分泌功能的信号肽结构和纤维素结合结构域。同源性搜索比对分析表明,象耳豆根结线虫Me-cbp-1与南方根结线虫(M.incognita)、爪哇根结线虫(M.javanica)及花生根结线虫(M.arenaria)纤维素结合蛋白具有88%~91%的相似性。原位杂交显示,Mecbp-1在象耳豆根结线虫二龄幼虫的亚腹食道腺细胞特异表达。利用RNAi技术对象耳豆根结线虫二龄幼虫的Me-cbp-1基因进行沉默后,导致二龄幼虫的侵染率显著降低。本实验结果初步证明了纤维素结合蛋白在象耳豆根结线虫二龄幼虫侵染寄主的过程中具有重要地位,推测其通过与细胞壁主要成分纤维素结合,促进植物细胞壁的软化和降解,从而有利于提高线虫的侵染和寄生能力。本研究为揭示象耳豆根结线虫分子寄生致病机制以及进一步研究防治根结线虫病害新策略提供了理论依据。
[Abstract]:Cellulose binding protein (cellulose binding protein,CBP) is an important effector protein secreted by plant parasitic nematode. In order to obtain the sequence information of (Meloidogyne enterolobii) cellulose-binding protein of root-knot nematodes and to analyze its functional position in the parasitic process of nematode, the transcriptome database of the second instar larvae of root-knot nematode nematodes was constructed in this study. CDNA terminal rapid amplification (rapid-amplification of cDNA ends,RACE) technique was used to clone the full-length sequence of cellulose binding protein gene Me-cbp-1 (Gen Bank accession number: KU350655 of root-knot nematode. Bioinformatics analysis showed that the full length of Me-cbp-1 cDNA was 809bp.The non-coding region length of 5'terminal and 3'terminal was 43 and 139 bp, respectively, and a complete open reading frame (ORF), containing 627 bp in length was deduced to encode 208 amino acids. Predictive protein Me-cbp-1 contains signal peptide structures and cellulose-binding domains that play a secretory role. Homology analysis showed that the cellulose-binding proteins of Me-cbp-1 were similar to those of M.incognita, M.javanica and M.arenaria. The results showed that the cellulose-binding proteins of RKN were 88% and 91% similar to those of RKN (M.incognita), M.javanica (RKN) and M.arenaria (Peanut Root-knot nematode). In situ hybridization showed that Mecbp-1 was specifically expressed in the subventral esophageal gland of the second instar larvae of root-knot nematodes. The RNAi technique was used to silence the Me-cbp-1 gene of the second instar larvae of root-knot nematode, and the infection rate of the second instar larvae was significantly decreased. The results showed that cellulose binding proteins play an important role in the infection of the second instar larvae of root-knot nematode nematodes, and the cellulosic binding proteins can promote the softening and degradation of plant cell walls by binding with cellulose, the main component of cell wall. Thus, the ability of infection and parasitism of nematodes was improved. This study provides a theoretical basis for revealing the mechanism of molecular parasitism of root-knot nematodes and for further study on new strategies for the control of root-knot nematode diseases.
【作者单位】：海南省农业科学院植物保护研究所/海南省植物病虫害防控重点实验室;中国热带农业科学院环境与植物保护研究所;
【基金】：国家重点基础研究发展计划(973)项目前期基础研究专项(No.2014CB160307) 国家自然科学基金(No.31401717和No.31360432)
【分类号】：S432.45

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