摘 要

松材线虫病又称松树萎蔫病（Pine Wilt Disease），是由松材线虫（Bursaphelenchus xylophilus）引起的松树上的毁灭性病害。由于该病涉及到寄主松树、传播媒介天牛、线虫、伴生细菌、真菌以及环境地理因素等多个方面，其致病机理尚未明确。从分子水平开展松材线虫的致病相关基因的研究可以为研究其致病机理提供理论依据。组织蛋白酶L（cathepsin L-like cysteine proteinase，CPL）是一种广泛存在于有机体组织细胞内（主要是溶酶体）的蛋白酶，与植物寄生线虫的发育和侵染毒力密切相关，但在松材线虫中研究较少。为了解松材线虫组织蛋白酶的生物学功能，克隆了一个松材线虫组织蛋白酶基因Bx-CPL-1并对其进行了生物信息学分析。发现该基因全长1377bp，含有1个长度为1176bp的开放阅读框。由Bx-CPL-1编码的蛋白质含有370个氨基酸，分子质量为41.27kDa。此蛋白存在Inhibitor_I29和Pept-C1两个功能域结构。对其进行的一级结构分析和二级结构分析中，表明了Bx-CPL-1蛋白是一个亲水性较强，疏水性较弱的蛋白，主要以α–螺旋，不规则盘绕和延伸链为蛋白结构元件，共有15个Ser、5个Thr和8个Tyr可能成为磷酸化位点。因此，推测这个基因翻译的蛋白是一种从膜内分泌的，可能有抗原提呈、组织器官发育、调节分泌蛋白、寄生虫营养摄取等功能的水溶性分泌蛋白。本研究为进一步验证该基因的功能打下了基础，有助于之后研究该基因与松材线虫致病性和生长发育等的关系。

关键词：松材线虫；Bx-CPL-1基因；组织蛋白酶；基因分析

Cloning and Bioinformatic analysis of cathepsin L-like cysteine proteinase in Bursaphelenchus xylophilus

ABSTRACT

Pine wilt disease is a kind of destructive disease to coniferous trees caused by the pine wood nematode (Bursaphelenchus xylophilus). For the disease’s complex relationship with hosts, vector insects, nematodes, associated bacteria, fungi and environmental geographical factors，the pathogenesis of the disease is unclear so far. Cathepsin L (cathepsin L-like cysteine proteinase，CPL) is a protease (mainly lysosome) widely existing in organism tissues. The protease is involved in plant parasitic nematode’s development and virulence. However, few researches are done on the role of CPL of the pine wood nematode. To find the biological function of cathepsin, a pine wood nematode cathepsin gene Bx-CPL-1 was cloned and the biological information analysis was performed. The result showed that the full-length cDNA of Bx-CPL-1 was 1377 bp long，which included an ORF of 1176 bp. The deduced amino sequence of protein Bx-CPL-1 contained 370 amino acids with molecular mass of 41.27 kDa. Protein Bx-CPL-1 contained an Inhibitor_I29 and a Pept-C1 conserved domains. The amino sequences of Bx-CPL-1 varied between different species. Concerning the primary structure prediction and the secondary structure prediction of the protein，we find protein Bx-CPL-1 has low hydrophilic property and mostly composed of α-helix, random coil and extended strand. Fifteen Ser、five Thr and eight Tyr were predicted to be phosphorylation targets of the protein. According to the result, we speculated the gene can be translated into a water-soluble secretory protein which secreted from intramembrane and may participate in the procedure of antigen present，organ development，secretory protein regulation and parasites’ nutrition. Our research provided support for studying on gene functions and facilate illustration of the relationship between CPL gene and pathogenesis and development of Bursaphelenchus xylophilus.

Key words：Bursaphelenchus xylophilus；Bx-CPL-1；cathepsin；Bioinformatic analysis

目 录

引言………………………………………………………………………………………1

1 材料与方法…………………………………………………………………………………3

1.1 材料………………………………………………………………………………3

1.1.1 供试线虫……………………………………………………………3

1. 1.2 主要试剂和引物…………………………………………………………3

1.1.3 所用培养基…………………………………………………………3

1.2 方法…………………………………………………………………………3

1.2.1 松材线虫的单异活体培养…………………………………………………3

1.2.2 Bx-CPL-1基因总RNA的提取………………………………………………3

1.2.3 第一链cDNA的合成…………………………………………………………………4

1.2.4 RACE法扩增Bx-CPL-1基因全长…………………………………………………4

1.2.5 克隆及序列测定………………………………………………………………………4

1.2.6 Bx-CPL-1基因的生物信息学分析 ……………………………………………4

2 结果与分析………………………………………………………………………………6

2.1松材线虫总RNA提取和RACE结果……………………………………………6

2.2松材线虫cDNA的RACE结果……………………………………………………………6

2.3松材线虫Bx-CPL-1基因全长cDNA序列分析…………………………………………7

2.4 松材线虫蛋白质序列的基础生物学分析………………………………………………7

2.5松材线虫组织蛋白酶Bx-CPL-1的信号肽预测…………………………………………8

2.6松材线虫组织蛋白酶Bx-CPL-1基因的功能域预测……………………………………9

2.7松材线虫组织蛋白酶Bx-CPL-1基因多序列比对分析…………………………10

2.8松材线虫组织蛋白酶L的系统进化树 ………………………………………………11

2.9 松材线虫组织蛋白酶L的疏水性分析……………………………………………12

2.10 磷酸化位点分析与跨膜结构分析………………………………………………………13

2.11 松材线虫Bx-CPL-1蛋白的二级结构预测…………………………………………14

3结论与讨论 ……………………………………………………………………………………15

致谢 …………………………………………………………………………………16

参考文献 ……………………………………………………………………………17

图1松材线虫总RNA提取………………………………………………………6

图2松材线虫cDNA的RACE结果………………………………………………………6

图3 松材线虫Bx-CPL-1基因全长cDNA序列及推测氨基酸序列……………………………7