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

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.

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。

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，本研究探讨了大豆孢囊线虫白色雌虫表皮鞣化褐化分子机制。首次成功克隆了大豆孢囊线虫漆酶基因，利用RACE得到了大豆孢囊线虫漆酶基因的全长，大豆孢囊线虫漆酶基因的ORF区长度为2121bp，3’ 非翻译区为 94 bp，5’ 非翻译区为 279 bp编码了706个氨基酸，并进一步对大豆孢囊线虫漆酶蛋白进行生物信息学分析，发现它同时具有跨膜结构域和信号肽，预测蛋白分子量为80.2KDa;利用qRT-PCR检测了大豆孢囊线虫漆酶基因在不同龄期线虫中的表达量差异，其中白色胞囊中漆酶基因的表达量最高，二龄幼虫、三龄幼虫、四龄幼虫和褐色孢囊的漆酶表达量仅为白色孢囊时期的5.4%、0.8%、0.3%、和32.8%；通过体内RNAi技术降低大豆孢囊线虫二龄幼虫漆酶基因的表达量，显著降低了二龄幼虫的侵染率，二龄幼虫的侵染率仅为29.8%；构建了原核表达载体，并在大肠杆菌体内成功表达了漆酶基因，带有MBP标签蛋白(40 KDa)的漆酶蛋白分子量大约为120 KDa，与预测的漆酶蛋白分子量大小一致，并且去掉漆酶蛋白的信号肽后再进行表达增加了漆酶的表达量。大豆孢囊线虫漆酶基因的功能可通过In Planta RNAi 进一步验证。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。