利用VIGS技术研究棉花GhPPO1基因的抗虫作用  

Study on Insect Resistance of GhPPO1 Gene in Cotton(Gossypium arboretum) by VIGS Technique

作　　者：程慧[1,2] 张帅[2] 雒珺瑜[2] 荣伟[1] 崔金杰[1,2] 王登元[1] 

机构地区：[1]新疆农业大学农学院,乌鲁木齐830052 [2]中国农业科学院棉花研究所/棉花生物学国家重点试验室,安阳455000

出　　处：《农业生物技术学报》2017年第5期722-728,共7页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育科技重大专项(No.2016ZX08012-004)

摘　　要：多酚氧化酶(polyphenol oxidase,PPO)是一类含铜的氧化还原酶,能够抵御植食性昆虫或病原菌的危害。为了研究棉花(Gossypium arboretum)中PPO1基因的抗虫作用,本研究利用病毒介导的基因沉默(virus-induced gene silencing,VIGS),构建基因沉默重组载体pTRV2-GhPPO1,转化农杆菌(Agrobacterium tumefaciens)GV3101,并侵染子叶展平的棉花植株;通过qRT-PCR检测沉默效果,并用获得的棉花植株饲喂二龄初期棉铃虫(Helicoverpa armigera)幼虫,对沉默后棉花植株的抗虫性及GhPPO1基因表达量的变化进行检测。结果表明,利用烟草脆裂病毒(Tobacco rattle virus,TRV)体系成功抑制了棉花GhPPO1的表达,沉默后GhPPO1基因相对表达量仅为对照组(空载体pTRV0)表达量的20%(P<0.05)。与饲喂正常棉花叶片的棉铃虫相比,饲喂GhPPO1基因沉默后棉花叶片的棉铃虫72 h后,体重增加86%,表明GhPPO1是棉花抗虫物质中的一类防御机制基因。棉铃虫直接诱导18、72 h的叶片中GhPPO1的表达量高于间接诱导叶片,证明沉默该基因后,消弱了棉花植株直接防御的能力,致使棉铃虫增重效果显著。由此推测该基因可能参与棉花直接防御反应。本研究对于GhPPO1在棉花防御植食性昆虫中的作用具有重要意义,为棉铃虫与棉花诱导防御之间的关系提供基础资料。Polyphenol oxidase （PPO） is a class of copper-containing oxidoreductase, and it is resistant to phytophagous insects or pathogens. In order to study the insect resistance of GhPPO1 gene in cotton （Gossypium arboreum）, the gene silencing recombinant vector pTRV2-GhPPO1 was constructed, and was transformed into Agrobaoterium tumefaciens GV3101 by virus-mediated gene silencing （VIGS）. The cotton plants were fed to 2rd instar larvae of cotton bollworm （Helicoverpa armigera）, and the expression of GhPPO1 gene in cotton plants after GhPPO1 gene silencing was obtained by qRT-PCR. The results showed that the expression of GhPPO1 was inhibited by Tobacco rattle virus （TRV） system, and the expression of GhPPO1 gene was only 20% compared with the control group （empty vector pTRV0） （P〈0.05）, and the GhPPO1 gene in cotton had a significant silence effect. Compared with cotton bollworm which was fed with common cotton leaf, the weight of the cotton bollworm fed with cotton leaf which was treated by gene silencing increased by 86% after 72 h, which indicated that GhPPO1 was a kind of defense mechanism gene in cotton. The expression of GhPPO1 in the directly induced leaves with cotton bollworm was higher than that of the indirect induced leaves. The results showed that the ability of cotton plants to direct defenses was weakened, and weight of the cotton bollworm increased. It was speculated that GhPPO1 gene might be involved in cotton direct defense response. This study has important significance for the role of GhPPO1 in cotton herbivorous insects, and lays the foundation for the relationship between cotton bollworm and cotton induced defense.

关 键 词：棉花 病毒介导的基因沉默(VIGS) 多酚氧化酶基因(PPO) 抗虫性 基因表达 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]