中肠结合短肽改造Bt Cry 毒素以增强对褐飞虱的毒性

Transgenic Bt rice cultivars provide an ideal means to control major Lepidopteran and Coleopteran pests. However, the growth of transgenic Bt rice may potentially trigger outbreaks of hemipteran insect pests e.g. planthoppers and leafhoppers, which will compromise effective management of rice insect pests by use of transgenic Cry crops. Until now, very few Bt Cry toxins, which show toxicity to hemipteran insects, have been reported. It is believed that a low affinity to potential receptors in the midgut of hemipteran insects is most likely responsible for the poor toxicity of Bt Cry toxins. Using phage display technology, we previously identified P2S and other short peptides showing binding activity with the inner membrane of N. lugens midgut. In addition, a replacement of Cry1Ab Domain II loop2 with peptide P2S resulted in an ~11 fold increase of insecticidal activity against N. lugens nymphs. The first objective of this proposal is to determine the gut proteins that interacted with P2S, which may be putative receptors for the modified toxins. We will also screen for gut proteins that bind to other midgut binding peptides previously identified. The second objective is to further modify Cry1Ab and Cry11Aa, a Cry toxin showing low toxicity to hemipteran insect, with P2S or other gut binding peptides. Selection of peptides will be based on the determination of which gut proteins bind to the peptide. The peptides that interact with putative receptor proteins, e.g. cadherin, APN, ALP or other potential receptor proteins will be used for new toxin construction. We will either replace the functional domains of the Cry toxins with the peptides or insert the peptides as additional sequences of the functional domains to create new Cry molecules. We expect to select effective new Cry toxins that show toxicity against N. lugens. Knowledge gained from this work will contribute to better understanding of Cry toxin and N. lugens gut interaction. This work will also provide a novel approach for modifying toxins targeting specific insect pests and to expand the library of Cry toxins.

苏云金杆菌Cry蛋白是应用最广的杀虫毒素，转cry基因作物已成为防治鳞翅目及鞘翅目害虫的一种主要手段。然而Cry毒素对水稻重要害虫褐飞虱等半翅目刺吸性口器害虫不具毒性，究其原因是活化后的毒素蛋白无法与褐飞虱中肠内受体正确结合，因而导致难以应用Bt防治此类非靶标害虫。本研究在前期针对Cry1Ab毒素的改造工作基础上，对P2S及其它褐飞虱中肠结合短肽在褐飞虱中肠内的结合蛋白分子进行分析，进而了解改造Cry毒素对褐飞虱毒性作用的影响。此外，利用P2S及可能与褐飞虱中肠内Cry毒素潜在受体结合的短肽对Cry1Ab及Cry11Aa毒素决定受体识别的功能域进行改造，以期提高重构Cry毒素对褐飞虱的毒性。此工作将深入了解Cry毒素在非靶标半翅目昆虫体内的作用机制，为开发针对特定难防治的农业昆虫的新型蛋白毒素奠定理论基础。

转Bt杀虫基因作物已广泛应用于鳞翅目、鞘翅目害虫的防治中。然而，Bt Cry毒素对半翅目水稻褐飞虱的防治效果却十分有限。本研究计划从探究Cry毒素对褐飞虱等半翅目无效原因入手，分析了Cry1Ac，Cry3Aa及Cry4Ba蛋白在褐飞虱中肠酶作用下的稳定性及活化后的毒素蛋白与褐飞虱中肠上皮细胞膜结合的特异性，证明Cry毒素无法与中肠上皮细胞膜正确结合是造成褐飞虱对Cry毒素不敏感的重要因素。通过构建褐飞虱中肠转录组数据库，一方面利用已有褐飞虱中肠结合短肽替换Cry1A毒素受体识别域，构建改造毒素以求提升毒性作用。另一方面利用生物信息学技术筛选褐飞虱中肠内潜在Cry结合蛋白，并体外表达作为靶标蛋白筛选特异性结合短肽。利用已有短肽P3S改造Cry1A毒素后，部分重构毒素能够一定程度提升其对褐飞虱若虫的毒性作用，并对中肠上皮细胞膜造成损伤，生物测定结果显示毒性作用提升了5-10倍。为了进一步提升改造效率，增强重构毒素对褐飞虱若虫的毒性作用，计划在下一步实验中采用体外表达的褐飞虱中肠内潜在Cry毒素结合蛋白作为靶蛋白，筛选高亲合力短肽，并用这些短肽改造Cry1A毒素，以求进一步提升重构毒素对褐飞虱的毒性作用。本研究为扩展Cry毒素蛋白用于防治半翅目和同翅目害虫提供理论和应用基础，为进一步证明受体结合在Cry毒素作用机制中的重要作用提供依据，并为用分子工程手段发展新型杀虫毒素提供新的思路和方向。