丝氨酸蛋白酶SP8和SP105在亚洲玉米螟黑化反应中的功能和作用机制研究

Innate immune system in insects, including melanization reaction, is the important line to defend against the infection from foreign microbs. The deficiency in innate immune system would make insects more susceptible to the infection and die. Therefore, illumination on the molecular mechanisms involved in insect innate immune reaction could afford theoretical base for protecting beneficial insects and controlling pests by utilizing insect’s innate immune system. The Asian corn borer (Ostrina furnacalis (Guenée)) is the most serious corn pest in China. However, it is still largely unknown about the molecular mechanism involved in its melanization response. In this project we would focus on two serine protease genes named as SP8 and SP105. We would clone their full-lengthed cDNA, investigate their spatio-temporal and inducible expression profile, and study the role and molecular mechanism of SP8 and SP105 in melanization reaction of Asian corn borer, with multiple molecular biology and biochemical methods. The acquired knowledge will be helpful to deeply understand the innate immune system of Asian corn borer, and also provide potential target gene for biologically controlling this pest via disrupting its innate immune system.

包括黑化反应在内的天然免疫系统是昆虫抵御外源微生物侵染的重要防线，天然免疫系统不能正常工作则昆虫更易被感染致死，故阐明昆虫的天然免疫应答机制可为利用昆虫自身免疫系统来保护益虫和防治害虫提供理论依据。亚洲玉米螟是我国玉米生产上最具危害性的害虫，但是目前对其黑化反应的分子机制还知之甚少。为此，本项目将着眼于两个名为SP8和SP105的丝氨酸蛋白酶基因，采用多种分子生物学和生物化学手段，克隆基因的全长cDNA序列，分析该基因的时空和诱导表达模式，研究SP8和SP105在亚洲玉米螟黑化反应中的功能和作用机制。这为进一步深入研究亚洲玉米螟的天然免疫系统奠定基础，也为针对亚洲玉米螟自身免疫系统进行生物防治提供可能的靶标基因。

包括黑化反应在内的天然免疫系统是昆虫抵御外源微生物侵染的重要防线。黑化反应的发生依赖一系列级联激活的丝氨酸蛋白酶，尤其是一种叫做酚氧化酶原激活酶的丝氨酸蛋白酶。鉴定昆虫体内的酚氧化酶原激活酶，并研究其作用机制对深入了解昆虫黑化反应的发生机制有重要意义。本项目深入研究了丝氨酸蛋白酶SP8和SP105在亚洲玉米螟黑化反应中的功能和作用机制。得到如下结果：克隆了亚洲玉米螟丝氨酸蛋白酶SP8和SP105的全长序列并对其进行了详细的生物信息学分析；大肠杆菌、藤黄微球菌和球孢白僵菌均能诱导SP105的转录水，暗示它可能是一个免疫相关的基因；产生并纯化出了能在体外被商业化蛋白酶Factor Xa所切割激活的重组蛋白proSP105Xa；Factor Xa激活的SP105能直接切割激活亚洲玉米螟天然的酚氧化酶原PPO2以及体外的PPO2重组蛋白，说明SP105是一个酚氧化酶原激活酶；SP105本身的活性受到丝氨酸蛋白酶抑制剂Serpin-3的严格调控。所得研究成果为更好理解亚洲玉米螟自身的天然免疫系统奠定了基础。目前，已顺利完成了该项目的所有研究内容，并在国内外核心刊物上发表论文4篇。