[目的]转基因育种是将对人类有益的外源基因通过生物技术整合进受体植物的基因组中,使其获得通过自然进化无法获得优异性状,这是转入外源基因的"预期效应".然而,外源基因插入受体植物还可能产生无法控制和预期的细胞、代谢或表型等方面的改变,即"非预期效应".非预期效应是转基因植物安全评价的核心内容之一,也是近年来研究的热点和难点.本研究以抗虫转基因玉米为材料,利用差异蛋白质组学技术比较研究转基因玉米与其非转基因玉米之间的非预期效应,为蛋白质组学技术解析转基因植物非预期效应提供理论参考.[方法]选取4份已经进入中国农业部安全评价阶段转苏云芽孢杆菌(Bacillus thuringeinsis,Bt)内毒素基因的抗虫玉米材料,即SK12-5zd、IE034z、Bt799z、Bt799zd,以及它们的对照玉米材料郑单958和郑58,分别种植于可控人工温室中.待玉米幼苗生长至5叶1心期时,每份材料取长势一致的6个单株最上部完全展开叶片,混合作为一个样本进行取样,并提取叶片蛋白质.双向电泳技术分别分离4种转基因玉米和它们各自对应的非转基因材料高丰度蛋白质组,利用Image Scanner扫描仪扫描脱色后的蛋白质凝胶,PD Quest 8.0软件(Biorad,USA)分析蛋白质凝胶图谱,以蛋白点相对体积(%Vol)来表述每一个匹配蛋白质的表达丰度,将电泳凝胶上特异性蛋白质和相对体积大于2倍的差异蛋白质点取样,依据相应蛋白质数据库进行质谱鉴定,并对鉴定出的特异性和差异性蛋白质进行细胞功能富集分析(GO)和代谢途径富集分析(KEGG),以评价转基因抗虫玉米的可能非预期效应.[结果]通过比较4种不同转基因抗虫玉米及其非转基因对照的高丰度蛋白质组,除目标抗虫基因外,共鉴定出61个蛋白质,其功能主要是富集于光合作用、碳固定、能量转运等基础细胞功能相关酶类,如核酮糖二磷酸羧化酶、ATP合成酶、丙酮酸磷酸双激酶等.仅有少数几个与光合作用、碳固定和ATP合成途径等基础代谢过程相关基因出现上调表达.KEGG分析表明,与对照ZD958相比,SK12-5zd的差异蛋白在光合生物碳固定途径中显著富集;Bt799zd的差异蛋白则分别在光合生物碳固定途径、光合作用和碳代谢途径中显著富集.与对照Z58相比,IE034z和Bt799z的差异蛋白均在光合作用代谢途径中显著富集.证明4种转基因材料与对照蛋白质组相似性较高,未见基因的异常表达.[结论]测试的4种转基因材料在蛋白质水平未发生影响其安全性的非预期效应;当前结果表明蛋白质组学技术可以用来验证转基因植物的非预期效应.%[Objective] Unintended effects of genetically modified (GM) crops are defined as the unexpected trait changes in GM breeding and research. Over the last decade, much attention has been taken to GM crops' intended effects that are attributed to the insertion of foreign gene, leaving the unintended effects of GM crops less investigated. In this study, a comparative proteomic study using two-dimensional electrophoresis and mass spectrometry was conducted to assess the high-abundant protein variation, so as to explore possible occurrence of their unintended effects.[Method] Four insect resistant GM maize varieties, i.e SK12-5zd, IE034z, Bt799z and Bt799zd, which were under approval of GM safety assessment by the Ministry of Agriculture, were planted in a condition-controlled greenhouse together with their corresponding CKs, i.e Zheng58 and Zhengdan958. When seedling growing into 5-leaf stage, the top-leaf blades of each maize variety were sampled and their leaf proteins were extracted. After profiling by two-dimensional electrophoresis, on-gel leaf protein spots of each GM maize were compared against their corresponding CKs using Imager Scanner and PD Quest 8.0 (Biored, USA). The resulted unique and differential protein (with a relative volume greater than 2-fold) spots were isolated from the gel and applied for protein identification by mass spectrometry against the relevant protein database. The identified proteins were analyzed in gene ontology (GO) and KEGG for their cellular functionalities and metabolism pathway enrichment.[Result] Through comparison of the unique and differential proteins between each GM maize and its corresponding no-GM CKs among 4 insect resistant GM maize varieties, a total of 61 differential proteins were identified in maize PDB (NCBI) alongside the 4 intended insect resistant gene products. The gene ontology analyses of the identified proteins revealed that they are popular cellular enzymes of fundamental metabolism, i.e Rubisco, ATP synthase, pyruvate orthophosphate dikinase, etc. Only a few genes relating to photosynthesis, ATP synthesis and carbon fixation were observed to be up-expressed, possibly due to the genetic background difference between the GM maize and its CK. KEGG analyses uncovered that, in comparison with the corresponding CKs, the differential proteins of SK12-5zd and Bt799zd were enriched similarly into photosynthesis, carbon fixation and metabolism pathways. In comparison with the CK Z58, the differential proteins of both IE034z and Bt799z were enriched into the photosynthetic metabolism pathway. Four GM maize varieties exhibited extensive similarity over their corresponding wild-type controls. It appeared to be no unintended effects observed.[Conclusion] The preliminary results revealed no obvious unintended effects in 4 insect resistant GM maize varieties in respect to high-abundant proteomic assessment.
展开▼
