【作者】 周子珊；

【导师】 张杰；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2013， 博士

【摘要】 苏云金芽胞杆菌因其对多种害虫具有杀虫活性，不污染环境，对人畜无害，因而在害虫的生物防治中得到了广泛的应用。cry1Ah1基因是本实验室分离克隆的具有自主知识产权的杀虫基因，由于其编码的Cry1Ah蛋白对多种鳞翅目害虫具有较高的杀虫活性，目前cry1Ah1基因被应用于转基因玉米和水稻的研究中。为了评价cry1Ah1基因对非靶标昆虫的安全性，本文首先对Cry1Ah蛋白对鳞翅目中重要的经济昆虫家蚕（Bombyxmori）进行了活性测定，结果表明：Cry1Ah蛋白对家蚕的致死中浓度（LC50）大于500μg/mL。这不仅明确了对棉铃虫（Helicoverpaarmigera）等害虫高毒力的Cry1Ah蛋白（LC50=7.04μg/g）对非靶标昆虫（家蚕）是安全低毒（LC50>500μg/mL），更重要的是为cry1Ah1基因的推广及应用提供了理论依据。Cry1Ah的这种特性恰恰与本实验室分离克隆的另一个拥有自主知识产权杀虫基因cry1Ai相反，Cry1Ai蛋白对家蚕表现出很高的毒力（LC50=5.25μg/mL），但对棉铃虫则表现出较低的活性（LC50>500μg/g），而两者的氨基酸相似性为84%，并且拥有相似的三维结构。基于这种发现，本研究旨在揭示Cry1Ah蛋白对棉铃虫等农业害虫高毒力、对经济昆虫家蚕相对安全低毒的分子机制。本文以Cry1Ah蛋白为研究重点，以Cry1Ai蛋白为对照材料，一方面通过构建定点突变体、截短片段以及Loop突变体，明确Cry1Ah蛋白杀虫活性相关位点、Cry1Ai蛋白的杀虫活性区域，以及两者与杀虫特异性相关的Loop区域。另一方面，利用配体垂钓平台分别分析Cry1Ah蛋白和Cry1Ai蛋白在棉铃虫和家蚕刷状缘膜微囊泡（BrushBorderMembraneVesicles，简称BBMVs）上的特异性结合蛋白。主要研究结果如下：（1）利用定点突变的方法构建了Cry1Ah蛋白DomainⅠ和DomainⅡ上7个突变体，对比Cry1Ah蛋白对棉铃虫的生物活性发现，Cry1Ah蛋白DomainⅠ中α-6螺旋上第206位氨基酸突变为丙氨酸时，其对棉铃虫的杀虫活性略有降低，而在DomainⅡ上Loop3的第463位添加多个天冬酰胺时，能够降低其对棉铃虫的杀虫活性。（2）构建了26个Cry1Ai蛋白的截短片段，通过对小菜蛾的生物活性测定，将Cry1Ai蛋白对小菜蛾（Plutellaxylostella）的最小活性区域精准定位为第36I到605I氨基酸残基。（3）通过分析比较Cry1Ah/Cry1Ai毒素在棉铃虫和家蚕中肠BBMVs上的结合蛋白，探寻结合蛋白与杀虫特异性的关系。研究发现，Cry1Ah蛋白与棉铃虫BBMVs上的氨肽酶N（AminopeptidaseN，简称APN）能够特异的结合，而在家蚕的BBMVs上没有检测到这种高亲和力的结合，这可能是Cry1Ah蛋白对棉铃虫杀虫特异性的原因。而Cry1Ai蛋白在家蚕的BBMVs上也存在一些特异的结合蛋白，但这些结合蛋白仍需进一步验证。（4）为了进一步确定Cry1Ah蛋白与棉铃虫APN特异性结合的原因，本研究分析比较Cry1Ah蛋白和Cry1Ai蛋白的氨基酸序列和三维结构，研究发现，两者在受体结合区域的主要差别在DomainⅡ的Loop2和Loop3上，通过构建Loop互换突变体，以及比较突变体的生物活性，发现Cry1Ah和Cry1Ai蛋白Loop2的互换使得Cry1Ai蛋白获得了对棉铃虫的杀虫活性（Cry1Ai-hloop2：LC50=64.23μg/g），而Cry1Ah蛋白则失去了对棉铃虫的杀虫活性（Cry1Ah-iloop2：LC50>500μg/g）；但Loop2的互换并没有改变两种蛋白对家蚕原有活性（Cry1Ai-hloop2：LC50=11.60μg/mL；Cry1Ah-iloop2：LC50>500μg/mL），这说明DomainⅡ的Loop2与棉铃虫杀虫特异性密切相关。而两个蛋白Loop3互换时，不但没有使Cry1Ai蛋白没有获得对棉铃虫的杀虫活性（Cry1Ai-hloop3：LC50>500μg/g），反而导致Cry1Ah蛋白失去了对棉铃虫的杀虫活性（Cry1Ah-iloop3：LC50>500μg/g），这说明Loop3对Cry1Ah和Cry1Ai蛋白保持原有的对棉铃虫的杀虫活性比较重要。Cry1Ah蛋白对非靶标昆虫（家蚕）安全性的明确，将有利于cry1Ah1基因的推广应用，同时也为新型cry基因的发掘和应用提供筛选和评价标准；Cry1Ah蛋白杀虫特异性相关受体的确定，以及Cry1Ah蛋白上与杀虫特异性相关氨基酸区域的明确，初步揭示了Cry1Ah蛋白对棉铃虫杀虫特异性的分子机制，为cry1Ah1基因的推广应用奠定了坚实的理论基础，同时也为其它Cry蛋白的定向改造提供重要借鉴。更多还原

【Abstract】 Bacillus thuringiensis is widely used in insect control because it shows strong toxicity against many insects, but no toxicity to non-target organisms and safe for the environment. The novel gene crylAhl, intellectual property right owned, was cloned by our lab and was successfully transformed into corn and rice because of encoding a protein with highly toxic against many lepidopteran insect.To evaluate the safety of crylAhl gene against non-target insect, the bioassay of CrylAh protein against important economic lepidopteran insect-Bomfyx mori was tested. The results showed that the LC50of CrylAh against B. mori was more than500μg/mL, which confirmed that CrylAh highly toxicity against lepidopteran insect such as Helicoverpa armigera (LC50=7.04μg/g) was safe to economic insect B. mori (LC50>500μg/mL), moreover, it provided theory foundation for the promotion and application of crylAhl gene.These characteristic of CrylAh protein were just the opposite with that of another intellectual property right owned gene crylAi gene encoding a protein with low toxicity against H. armigera (LC50>500μg/g), but strong toxicity against B. mori (LC50=5.25μg/mL). The amino acid identity between CrylAh protein and CrylAi protein is84%. They share a very similar structure. In this study, we aimed to explore the molecular mechanism of CrylAh toxin with strong toxicity against H. armigera, and safe to economic insect B. mori.CrylAh protein is chose to be the key object, and CrylAi as control. On one hand, we plan to determine the amino acid sites related to insecticidal on CrylAh, the active fragment of CrylAi and the loop region of specific insecticidal activity of CrylAh and CrylAi protein by construction of site-directed mutants, truncated fragments and Loop mutants. On another hand, we plan to search the specific binding protein in BBMVs using an improved receptor-fishing method.The results are shown as follows:(1) Seven mutants on Domain I and Domain II of CrylAh were constructed by site-directed mutagenesis methods in this study. According to the bioassay results of mutants against H. armigera, the amino acid residue of206on helix a-6of Domain I was changed to Ala make the reduction of toxicity, and more than one additional Asn inserting in463on loop3of Domain I may reduce the toxicity against H. armigera.(2) Twenty six truncated fragments were constructed in this study. The active fragment of CrylAi toxin against P. xylostella was exactly located between amino acid residues361and6051, according to the bioassay results against P. xylostella.(3) We analyze the binding protein on BBMVs from H. armigera and B. mori to explore the relationship between binding proteins and insecticidal specificity. The results showed that there was a special binding band-APN of CrylAh toxin on BBMVs from H. armigera compared with B, mori. This difference may cause CrylAh toxin to show insecticidal specificity against H. armigera. However, there were also some different binding proteins of CrylAi toxin on BBMVs from B. mori compared with H. armigera, although this part of work need to be repeated further.(4) To determine the reasons of specific binding between CrylAh toxin and APN, we compared the structure and amino acid sequences with CrylAh and CrylAi toxin, the results indicated that the major differences on receptor binding region between two toxin was located on Loop2and Loop3of Domain Ⅱ. By constructing some Loops exchange mutants and comparing the bioassay results of Loops mutants against H. armigera and B. mori, we found that exchange of Loop2made CrylAi toxic against H. armigera (Cry1Ai-hloop2: LC50=64.23μg/g) and CrylAh lose toxicity against H. armigera (CrylAh-iloop2: LC50>500ug/g); while exchange of Loop2didn’t make two toxin exchange the toxiciry against B. mori (CrylAi-hloop2: LC50=11.60μg/mL; CrylAh-iloop2: LC50>500μg/mL), which indicated that Loop2is related to insecticidal specificity against H. armigera, however, exchange of Loop3not only made Cry1Ai non-increasing on toxiciry against H. armigera (Cry1Ai-hloop3: LC50>500μg/g), but also caused CrylAh lost the toxiciry against H, armigera (CrylAh-iloop3: LC50>500μg/g), which indicated Loop3was important for keeping original toxiciry of CrylAh and CrylAi against H. armigera.The determination of the safety of CrylAh to B. mori not only lays theory basis for the promotion and application of cry1Ah1gene, but also provides screen and evaluation standards for discovery and application of novel cry genes; The molecular mechanism of insecticidal specificity of CrylAh toxin was revealed by determination of receptor and amino acid region related to insecticidal specificity. All of these research progresses will lay foundation for the promotion and application of cry1Ah1gene, and provide the theoretical reference for modification of other Cry toxins.更多还原