利用VIGS技术对棉花防御相关基因的抗虫作用研究

棉花（Gossypium arboretum）是经济作物、纺织纤维、饲料以及油、生物燃料产品的一个显著来源，然而棉铃虫（Helicoverpa armigeraHübner）的危害日益加重，严重影响其产量的提高。从棉花中克隆得到一个可能与抗虫相关的多酚氧化酶GhPPO1基因及富亮氨酸蛋白激酶GhLRR1基因。为了探究该基因的抗虫作用，利用病毒介导的基因沉默（Virus-induced Gene Silencing, VIGS）手段，应用分子生物学和实验动物学的方法，构建基因沉默重组载体pTRV2-GhPPO1、pTRV2-GhLRR1，有效地解除了棉花防御机制，证明棉花中的防御相关基因具有抵制植食性昆虫的作用。本研究对于了解棉花防御过程中与棉铃虫的互作关系，明确棉花应对植食性昆虫时所产生的防御机制具有重要实际意义。具体研究内容如下：

（1）应用VIGS技术构建沉默载体，成功构建的pTRV2-GhPPO1、pTRV2-GhLRR1载体，有效地压制了棉花GhPPO1、GhLRR1表达量，降低了内源GhPPO1、GhLRR1含量。分别是空白对照组（CK）降低20%和45%，是空载体对照组（pTRV0）表达量的29%和54%，表达量达差异显著性。

（2）利用沉默GhPPO1、GhLRR1基因棉花饲喂2龄初期棉铃虫72 h，测定棉铃虫质量变化情况，结果表明：饲喂沉默GhPPO1棉花棉铃虫2龄初期的存活率、虫重增加量均显著高于对照组棉花饲喂的棉铃虫，分别为CK对照组的1.86倍（P<0.05），pTRV0对照组的2.91倍（P<0.05）。饲喂沉默GhLRR1棉花棉铃虫2龄初期的存活率、虫重增加量均显著低于对照组棉花饲喂的棉铃虫。分别为CK对照组的35%，差异达显著水平，pTRV0对照组的56%，差异达显著水平。

（3）明确了常规棉中棉所49受到2龄初期棉铃虫取食后，GhPPO1基因表达量在直接取食部位与间接取食部位变化情况。在处理18 h、72 h后，棉铃虫直接取食沉默GhPPO1植株叶片部位的表达量高于间接取食的叶片部位；该变化趋势与CK对照组和pTRV0对照组变化趋势结果一致，差异均达显著水平；表明棉花GhPPO1基因对机械损伤产生了应答反应。而GhLRR1在处理18 h、72 h后，间接取食表达量高于直接取食叶片部位中的基因表达量，差异均达显著水平；表明GhLRR1基因对棉铃虫的取食造成的损伤产生了间接部位的应答反应。

（4）应用荧光定量（qRT-PCR）方法测定沉默GhPPO1、GhLRR1基因在棉花不同部位叶片中的基因表达量情况，沉默GhPPO1棉花植株中，新增叶片中基因含量最低，沉默效果最好。沉默GhLRR1棉花植株中，老叶叶片中基因表达量最低，沉默效果最好。

关键词：棉花，棉铃虫，VIGS，GhPPO1基因，GhLRR1基因

Study on Insect Resistance of Cotton Defense - related Genes by Using VIGS Technique

Abstract

Cotton (Gossypium arboretum) is a significant source of cash crops, textile fibers, feedstuffs and oil and biofuel products. However, Helicoverpa armigera (Hübner) is becoming more and more serious, affecting its yield. Our group previously cloned insect may be associated with a polyphenol oxidase geneGhPPO1 and leucine-rich protein kinaseGhLRR1 gene from cotton. In order to study the insect resistance of this gene, the gene silencing recombinant vector pTRV2-GhPPO1, pTRV2-GhLRR1 was constructed by virus-induced gene silencing (VIGS), and the methods of molecular biology and experimental zoology were used this study is of great significance to understand the interaction between cotton and cotton bollworm and to clarify the molecular mechanism of cotton insect resistance. The specific research contents are as follows:

(1) Construction of silencing vector by VIGS technique, successfully constructed pTRV-GhPPO1, pTRV2-GhLRR1 vector, effectively silencedGhPPO1and pTRV2-GhLRR1, which reduced the contents of endogenousGhPPO1 andGhLRR1. Were 20% and 45% lower than the blank control group (CK), respectively, 29% and 54% of the empty vector control group (pTRV0).

(2) The quality of cotton bollworm was measured by silencingGhPPO1andGhLRR1cotton for 72 hours. The results showed that the survival rate ofHelicoverpa armigera (P<0.05), and 2.91 (P<0.05) times in blank control group, which was significantly higher than that of control group (P<0.05). The survival rate ofHelicoverpa armigerawas significantly lower than that of cotton bollworm (Helicoverpa armigera) 35% (P<0.05) in the blank control group and 56% in the empty vector control group (P<0.05).

(3) It was clear that the expression ofGhPPO1 gene in direct feeding site and indirect feeding site was obtained after feeding the 2st instar cotton bollworm. The expression ofGhPPO1 in the leaves ofGhPPO1 plants was higher than that of the indirect feeding leaves after 18 h and 72 h treatment. The trend of the changes in the control group (CK) and empty vector control group (pTRV0) And the difference was significant. The results showed that theGhPPO1 gene had a response to mechanical injury. The expression ofGhLRR1was significantly higher than that ofGhLRR1, and the expression ofGhLRR1was significantly higher than that ofGhLRR1. TheGhLRR1 gene had a significant response to the feeding injury of cotton bollworm

(4) The expression ofGhPPO1 andGhPPO1 cotton in different parts of cotton was investigated by qRT-PCR. The gene content of leaves inGhPPO1 cotton plants was the lowest, and the silencing effect was the best. A silentGhLRR1 cotton plant, the leaves of the old leaves the lowest gene content, the best silence effect.

Key words: cotton;Helicoverpa armigera; VIGS;GhPPO1 gene;GhLRR1 gene