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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 that is resistant to phytophagous insects or pathogenic bacteria. In order to study the anti-insect effect of PPO1 gene in cotton (Gossypium arboretum), we constructed a gene silencing recombinant vector pTRV2-GhPPO1 and transformed Agrobacterium tumefaciens by virus-induced gene silencing (VIGS) GV3101 and infecting cotton plants with cotyledons. The silencing effect was detected by qRT-PCR. The larvae of Helicoverpa armigera at the second instar larvae were fed with the obtained cotton plants, Changes in expression levels were detected. The results showed that the expression of GhPPO1 was successfully inhibited by the tobacco virus (TRV) system, and the relative expression level of GhPPO1 gene after silencing was only 20% of the control (empty vector pTRV0) (P <0.05) . Compared with the bollworm fed the normal cotton leaves, the bollworm fed the GhPPO1 gene silenced for 72 h, gaining a weight gain of 86%, indicating that GhPPO1 is a kind of defense mechanism gene in cotton insect-resistant material. The expression level of GhPPO1 in the leaves of bollworm directly induced 18 and 72 h was higher than that of the indirect induction leaves. The silencing of this gene showed that cotton bollworm attenuated the direct defense ability of cotton plants, resulting in significant effect of weight gain of cotton bollworm. It is speculated that the gene may be involved in direct cotton defense response. This study is of great significance for the role of GhPPO1 in cotton defense against phytophagous insects and provides the basic information for the relationship between cotton bollworm and the induction of defense.