转基因抗虫棉Bt Cry1Ac蛋白在土壤中的代谢及其产物的转移研究

N15 labeled Cry1Ac protein will be extracted and purified from the transgenic Bt cotton variety of SGK321/NUCOTON99B and added to the soil sample. After different interval of incubation, Bt parent protein and its degradation polypeptides will be extracted. Proteins and peptide extracts will be applied to SDS-PAGE and 2D-electrophoresis for sample cleanup and purification. We will primarily use three mass spectrometer (MS) systems to identify and characterize the intact Bt protein and its degradation peptides. One MS system is MALDI TOF-TOF MS. Another MS system is Agilent capillary LC interfaced to Bruker micrOTOF-Q MS system. The third MS system is Dionex UltimateTM 2D nano-LC system interfaced to Bruker esquire HCT ultra PTM Discovery nanoelectrospray ion trap MS system. Small Bt metabolized peptides in a mixture can be identified and characterized on the capillary LC-micrO TOF-Q MS system and 2D nano LC-HCT ultra system. Peptides can be identified via peptide mass fingerprint (PMF) to match the measured peptide masses with those of known Bt amino acid sequence. After the metabolic products of Bt protein have been identified, the main metabolized peptides will be synthesized and added to the cotton field. 2D nano LC-HCT ultra system will be used to identify the extracts of neighboring crop roots if the small Bt metabolized peptides could be absorbed by intercropping crop or afterculture crop. Further, we will study the influence of the Bt metabolized peptides on soil functional bacteria and growth and development of intercropping crop and afterculture crop. We will also confirm the metabolic pathway of Bt Cry1Ac in soil of Bt cotton field and study the difference of Bt Cry1Ac protein in different cotton area.

本研究通过向非抗虫棉田土壤中加入从抗虫棉SGK321/NUCOTON99B中提取的稳定同位素N15标记的Bt Cry1Ac蛋白，利用三种生物质谱技术详细研究Bt Cry1Ac蛋白在土壤中的代谢，确证Bt Cry1Ac代谢的路径及主要的代谢片段和多肽分子并研究其是否能够转移到套种作物和下茬作物，同时在质谱确证多肽分子结构的基础上，人工合成多肽片段并加入土壤，研究这些可转移片段对土壤中主要功能微生物及间作与后茬作物的影响。在土壤中Bt Cry1Ac蛋白代谢研究技术成熟的基础上，验证抗虫棉田土壤中Bt蛋白的主要代谢片断，明确不同棉区土壤中Bt Cry1Ac蛋白的代谢差异及差异导致对土壤生态系统的影响。

转Bt基因作物的安全性研究已有大量报道，但是对于Bt蛋白在土壤中的代谢及其代谢产物能否转移到非转基因的间作套种作物；代谢产物对土壤与肥力有关的微生物及间作套种作物的影响还未见报道。Bt蛋白进入土壤的主要途径是根系分泌和作物残渣降解。本研究中，为了研究Bt蛋白在土壤中的代谢，我们建立了Bt Cry1Ac蛋白的原核表达体系并纯化出Bt Cry1Ac蛋白。同时利用实验室已有的Bt Cry1Ac蛋白抗体，建立了亲和层析方法从抗虫棉的叶片与种子中纯化Bt Cry1Ac蛋白的方法。Bt Cry1Ac蛋白被土壤中普遍存在的链霉菌蛋白酶水解后， 降解成多个片段。通过氮端测序与质谱鉴定，定位了两个大片段多肽（1 ：N端30氨基酸起始, C端360-373；2：N端374氨基酸起始, C端602-648）。经过毛细管液相色谱-质谱（nanoLC-MS）鉴定了6种在降解产物中稳定存在的小片段多肽序列（A: 135-143 F.NDMNSALTT.A、B:269-275 Y.TNPVLEN.F、C: 342-349 M.GNAAPQQR.I、D: 552-564 F.SNTVPATATSLDN.L、E: 559-569 T.ATSLDNLQSSD.F、F: 561-569 T.SLDNLQSSD.F）。用其中的一种代谢片段多肽（NH2-YTNPVLENF-COOH）标记放射性125I，并且纯化得到了125I-peptide耦合物。将125I-peptide耦合物溶液添加到棉花的间作种套作物西瓜和小麦幼苗的土壤中。在1小时之内，两种作物幼苗组织中的放射性强度迅速增强，证明Bt Cry1Ac蛋白在土壤中代谢的多肽片段能够转移到间作套种作物组织中。为了研究转移的代谢多肽片段对植物的影响，将人工合成的3种Bt蛋白代谢多肽片段，喷施于小麦、西瓜、玉米幼苗。测定三种作物苗期的生理指标（可溶性蛋白、总糖、SOD、叶绿素），未发现有任何显著差异。我们也研究了三种代谢多肽片段对土壤肥力相关的土壤微生物的影响，发现其对解有机磷细菌、固氮菌和解钾细菌的生长速率无显著影响，即Bt蛋白代谢的多肽片段对间种套作作物和土壤肥力相关细菌无显著影响。