小菜蛾钙粘蛋白肽段对Cry1Ac的增效机制及二者协同对幼虫免疫的影响

Improvement of Bt insecticidal effect is essential for overcoming resistance of diamondback moth and other lepidopteran pests to Bt toxin, finding Bt synergistic substances is an important means to solve the problem of pest resistance . Our previous study indicated that expressed cadherin peptides in Plutella xylostella have different synergistic effects to Cry1Ac toxin. On the basis of purified synergistic peptides (PxCAD1)，Biochemical mechanism of synergistic effect of PxCAD1 to Cry1Ac would be cleared by assaying activity of midgut protease, esterase, esterase isozyme and alkaline phosphatase and ability of midgut protease activating Cry1Ac toxin. Biochemical mechanism of synergistic effect of PxCAD1 to Cry1Ac would be cleared by detecting binding ability of PxCAD1 to Cry1Ac toxin and BBMV, ability of Cry1Ac penetrating BBMV membrane, ability of BBMV membrane permeability, ability of Cry1Ac toxin oligomerization induced by PxCAD1 peptide. Influence of synergistic effect of PxCAD1 to Cry1Ac on immune status would be confirmed by assaying of hemolymph melanization and expression of peptidoglycan recognition protein (PGRP) gene of diamondback moth. These results will play an important theoretical and practical role in developing and persistantly applying Bt toxins to control insects and Bt resistance management in the field.

提高Bt的杀虫效果可有效改善小菜蛾等鳞翅目害虫对Bt的抗性，寻找Bt增效物质是解决害虫抗性问题的重要手段。申请者前期研究发现，表达的小菜蛾钙粘蛋白肽段对Cry1Ac毒素具有显著的增效作用。本项目通过制备大量钙粘蛋白肽段，检测小菜蛾幼虫在肽段与Cry1Ac协同作用后，中肠蛋白酶、碱性磷酸酶等抗性相关酶类活性、蛋白酶催化Cry1Ac的能力，检测肽段分别与Cry1Ac及小菜蛾中肠刷状缘膜囊的结合能力、Cry1Ac的膜穿孔能力、中肠刷状缘膜囊的膜渗透能力、肽段诱导Cry1Ac毒素寡聚化的能力等，明确钙粘蛋白肽段对Cry1Ac增效的生化与分子机理；在此基础上，测定小菜蛾幼虫血淋巴的黑化状态及免疫相关基因肽聚糖识别蛋白的表达量，阐明二者协同对小菜蛾免疫功能的影响。研究结果对新型Bt杀虫蛋白的开发与可持续应用及Bt抗药性的治理具有重要的理论和实践意义。

提高Bt的杀虫效果可有效改善小菜蛾等鳞翅目害虫对Bt的抗性，寻找Bt增效物质是解决害虫抗性问题的重要手段。.本研究表明，小菜蛾Bt 受体钙粘蛋白肽段PxCAD1（或碱性磷酸酶肽段PxALP）与Cry1Ac 毒素协同作用后，对Cry1Ac毒素起到了显著的增效作用。取食PxCAD1与Cry1Ac 毒素协同可导致小菜蛾幼虫的化蛹率下降，成虫羽化率与卵孵化率均显著降低，而PxALP与Cry1Ac 毒素协同可导致小菜蛾幼虫的化蛹率和羽化率下降；二者协同还可使小菜蛾幼虫体内的解毒、保护及消化酶活力显著升高或降低。..在增效蛋白PxALP的浓度为50 mg/mL的浓度下，可加快Cry1Ac原毒素的活化速率。PxALP对小菜蛾BBMV与Cry1Ac的结合有一定的促进作用，但随Cry1Ac浓度的增加呈减弱趋势；而PxCAD1对小菜蛾BBMV与Cry1Ac的结合存在一定的抑制或阻碍。.克隆得到三个PGRP基因（PGRP1，PGRP2及PGRP3），在中肠中，Cry1Ac抗性和武汉田间种群PGRP1的表达量分别较Cry1Ac敏感种群下降80 %和90 %；PGRP3分别下降90 %和70 %；而抗性种群中PGRP2的表达量是敏感种群中的3倍。在脂肪体与表皮混合物中，抗性种群PGRP1的表达量较敏感种群增加33 %，田间种群减少90 %；抗性和田间种群中PGRP3的表达量均较敏感种群下降超过90 %；田间种群PGRP2的表达量为敏感种群的17倍。抗性种群的酚氧化酶活力（50.58 ng/ml）显著高于敏感种群（39.80 ng/ml）。 PxALP（或PxCAD1）与Cry1Ac协同，对小菜蛾幼虫肠道可培养细菌种类无显著影响，但二者协同可使PGRPs基因的表达量均显著提高。.将原核表达所获得的小菜蛾幼虫体内其他免疫相关基因PxDef、PxMor及PxCec蛋白与Cry1Ac毒素混合后饲喂小菜蛾3龄幼虫，发现：在无Cry1Ac毒素时，PxDef、PxMor及PxCec的原核表达蛋白对小菜蛾有显著的保护作用（即死亡率显著低于空载对照，化蛹率较空载对照高，蛹重、成虫寿命等均显著高于空载对照）；在有Cry1Ac毒素时，PxDef、PxMor及PxCec对死亡率无显著影响，而PxPGRP2却能显著提高死亡率。.研究结果为继续筛选Bt 毒素增效剂的基因片段及新型Bt杀虫蛋白的开发与可持续应用提供依据。