大豆孢囊线虫Laccases基因功能验证及其调控雌虫表皮褐化机制

大豆孢囊线虫（SCN）是危害大豆的重要病原物之一，它繁殖速度快，孢囊阶段抗逆能力强，是该病害难以控制的关键因素。孢囊抗逆性强与孢囊皮的鞣化褐化密切相关，探讨该线虫由白色雌虫向褐色孢囊鞣化褐化分子机制，对明确抗逆机理具有重要理论意义。本研究在获得SCN白色雌虫和褐色孢囊蛋白质组中与Laccases较高同源蛋白的基础上，利用现代生物信息学、基因组学、RNAi和酵母真核表达等分子生物学技术，克隆Laccases基因全长，获得该酶基因功能信息；分析该酶基因遗传进化及在不同发育阶段虫态中的表达规律；明确该基因调控大豆孢囊线虫雌虫褐化机理及对卵孵化动力学影响，并进行酵母真核表达纯化，明确该酶的结构特点及功能。揭示大豆孢囊线虫从幼虫到成虫和孢囊过程中Laccases基因的表达规律和调控机制，为深入研究以该基因的启动因子和控制该基因耙点提供理论基础。以期为该病害防治提供新思路。

The soybean cyst nematode (SCN), Heterodera glycines, Ichinohe, is one of the most damaging pests of soybean. This nematode can reproduce quickly and abundantly，cyst stage has anti-adversity ability endurable with drought and cold, which is key factor of control disease. There is close relationship between anti-adversity ability of cyst nematode and its cuticle tanning (or sclerotization and pigmentation). However, little is known about the mechanism of browning from white female to brown cyst. Therefore, in the study, the project will study the molecular mechanism of browning of cyst base on our previous research results. The objectives of the current study are: to isolate, sequence and characterize Laccases gene from Heterodera glycines by RT-PCR and RACE techniques, and analyze nucleotide sequence, functional prediction and genetic evolution of gene and by using bioinformatics techniques; determine express differentia of different development stage nematode by the method of real time PCR; illustrate the function of the gene by using RNAi and effect of it on hatching. And the encode protein will be expressed in eukaryotic expression system, purify the protein and determine the function of the laccase protein by evaluating influence of laccase on the browning of the white female and extent of cyst browning. The results will be clarified natural law of the gene from juvenile to adult and cyst at molecular level, and provide important theoretical information on further study promoter factor of the gene and find the gene target of the gene, and develop new strategy for control of the nematode.