东亚飞蝗FK506结合蛋白基因克隆及其免疫功能分析

为明确东亚飞蝗Locusta migratoria manilensis免疫相关FK506结合蛋白(FK506 binding protein,FKBP)的功能,了解FKBP对金龟子绿僵菌Metarhizium anisopliae侵染东亚飞蝗的影响,分离克隆获得FKBP46基因及其纯化蛋白,采用荧光定量PCR方法测定FKBP46基因在东亚飞蝗成虫不同组织以及不同虫态中肠内的表达量,通过饵剂饲喂方法测定FKBP46蛋白对金龟子绿僵菌致病力及对东亚飞蝗体内保护酶超氧化物歧化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)、酚氧化酶(phenoloxidase,PO)和解毒酶乙酰胆碱酯酶(acetyl cholinesterase,AchE)活性的影响。结果显示,东亚飞蝗FKBP46基因全长为1200 bp,编码蛋白分子量为55 kD。FKBP46基因在成虫不同组织中均有表达,其中在脂肪体中的相对表达量最高,为体壁中表达量的1.77倍;FKBP46基因在卵期和成虫期的相对表达量较高,分别为1龄蝗蝻期表达量的4.88倍和6.84倍。FKBP46蛋白与金龟子绿僵菌混合饲喂东亚飞蝗,第10天的累计死亡率为85.56%,显著高于金龟子绿僵菌单独处理,而东亚飞蝗体内的保护酶POD、SOD、PO和解毒酶AchE活性均较金龟子绿僵菌单独处理显著降低。表明FKBP46基因能够抑制东亚飞蝗的免疫功能,降低免疫相关保护酶和解毒酶的活性,促进金龟子绿僵菌的侵染。

Cloning and immune functional analysis of FK506 binding protein gene in Locusta migratoria manilensis

In order to clarify the function of FK506 binding protein (FKBP) in Locusta migratoria manilensis and illuminate the effect of FKBP in the resistance to Metarhizium anisopliae infection, the expression profile of the gene FKBP46 from different tissues of adults and in the midgut of nymphs with different instars was analyzed by using qRT-PCR technique. The FKBP46 gene was cloned, and its expressed protein FKBP46 was purified. The effect of FKBP46 protein on the infection of M. anisopliae and the activities of superoxide dismutase (SOD), peroxidase (POD), phenoloxidase (PO) and acetyl cholinesterase (AchE) were measured. It was found that the full length of FKBP46 gene was 1 200 bp, encoding a 55 kD protein. The FKBP46 gene was expressed in all tissues dynamically, and its relative expression was the highest in the fat body, which was 1.77 times higher than that in integument. The relative expressions of FKBP46 gene in the egg and adult stages were both higher than that during the firstinstar nymphal stage, which were 4.88 times and 6.84 times of the latter, respectively. The results of bioassay showed that, when FKBP46 protein was combined with M. anisopliae, the cumulative mortality of L. migratoria manilensis increased to 85.56% on the 10th day, which was significantly higher than that with M. anisopliae infection alone. Similarly, the activities of POD, SOD, PO and AchE in L. migratoria manilensis all decreased significantly. These results indicated that the gene FKBP46 could promote the infection of M. anisopliae by inhibiting the activity of protective enzymes and detoxifying enzymes in L. migratoria manilensis.