Cry1Ia分泌相关序列的鉴定及其对蛋白稳定性和杀虫活性影响的研究

Bt杀虫晶体蛋白具有特异的杀虫活性，广泛用于农作物病虫害防治。其中，Cry1I类蛋白对两个目的昆虫具有杀虫活性，且表达形式也与其它Cry蛋白不同。目前，有关Cry1I的研究主要集中在杀虫谱和作用机制等方面，对其胞外分泌的序列调控机制尚无深入研究。我们前期研究表明N-端信号肽的缺失显著降低了Cry1Ia在Bt细胞内的稳定性，而非信号肽序列的改变也会影响胞外分泌。为揭示Cry1Ia分泌相关序列及其功能，本项目拟首先通过信号肽的梯度缺失，鉴定其中维持蛋白稳定的关键区域。然后，构建可调控分泌途径的突变体细胞，判定Cry1Ia的分泌途径；筛选该途径中与之互作的蛋白及结合位点。最后，研究这些位点的变化对蛋白分泌、稳定性和杀虫活性的影响。本项目的实施不仅能明确调控Cry1Ia蛋白胞外分泌的关键序列，阐明其具体功能，而且将从蛋白转运的角度揭示与其它Cry蛋白差异的本质，为改造和利用Cry蛋白提供新的思路。

Bt Cry toxins have been used widely in pest control due to their specific insecticidal activity to many agricultural insects. Among them, Cry1I toxins are secreted proteins and show a wide range of specificity for different insect orders which are distinct from other Cry toxins. Unlike the emphasized insecticidal activities and action mode, the sequence regulation of Cry1I during translocation is seldom investigated. Previously, we observed significant degradation of Cry1Ia after deleting the N-terminal secretion signal peptide when expressed in Bt strain. In addition, a large quantity of expression products of the domain exchanged Cry1Ia mutants are retained inside cells, respectively. For reveal the secretion related regions and their full function in Cry1Ia, the proposed project is to construct several amino acid deletion mutants in the N-terminal secretion signal peptide, and screening regions contributing in keeping Cry1Ia protein stability. We will further identify the type of secretion pathway for Cry1Ia and screen the molecular chaperones interacting directly with the toxic protein in this pathway. Thereafter the chaperone binding sites in Cry1Ia will be preyed by yeast two-hybrid system. Finally, we will substitute these critical sites with other Cry sequences and detect the secretion, stability and insecticidal activity of these substitution mutants. This present project expects not only to screen the secretion related regions of Cry1Ia protein, but also to identify their function on protein stability and insecticidal activity. These findings would generate a new insight into substantial differences between Cry1I and its counterparts, and in consequence may bring new ways to modify Cry toxins.