蛋白质组学方法评估转基因抗虫玉米非预期效应

发布时间：2018-02-05 22:54

  本文关键词： 转基因玉米 蛋白质组学 非预期效应 双向电泳 差异表达蛋白　出处：《中国农业科学》2017年19期 　论文类型：期刊论文

【摘要】：【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。
[Abstract]:[objective] transgenic breeding is the integration of beneficial foreign genes into the genome of recipient plants through biotechnology, so that they can not obtain excellent traits through natural evolution. This is the "expected effect" of foreign gene transfer. However, exogenous gene insertion into recipient plants may also produce uncontrolled and anticipated changes in cell, metabolism, or phenotypes. That is, "unexpected effect". Unexpected effect is one of the core contents of transgenic plant safety evaluation, and it is also a hot and difficult research in recent years. In this study, insect-resistant transgenic maize was used as the material. The unanticipated effects of transgenic maize and its non-transgenic maize were studied by differential proteomics. To provide a theoretical reference for proteomics to analyze the unexpected effects of transgenic plants. [methods] four strains of Bacillus thuringiensis were selected and transferred to Bacillus thuringiensis in the safety evaluation stage of the Ministry of Agriculture of China. Bacillus thuringeinsis. BT) the insect-resistant maize material of endotoxin gene, SK12-5zdAZD IE034zT799zdBt799zd, as well as their control maize materials Zhengdan 958 and Zheng58. When maize seedlings grow to 5 leaves and 1 heart stage, the top leaves of 6 plants with the same growth rate are taken from each material, and mixed as a sample for sampling. The high abundance proteome of four transgenic maize and their corresponding non-transgenic materials were separated by two-dimensional electrophoresis. Image Scanner scanner was used to scan the protein gel profile after decolorization. PD Quest 8.0 software was used to analyze the protein gel map. The expression abundance of each matching protein was expressed by the relative volume of protein spots and the specific proteins on the gel electrophoresis and the differential protein spots whose relative volume was more than 2 times were sampled. The specific and differential proteins were identified by mass spectrometry according to the corresponding protein database. The specific and differential proteins were analyzed by cell function enrichment analysis and metabolic pathway enrichment analysis (KEGG). In order to evaluate the possible unexpected effect of transgenic insect-resistant maize. [results] by comparing the high abundance proteome of four different transgenic insect-resistant maize and its non-transgenic control, except the target insect-resistant gene. A total of 61 proteins were identified, whose functions are mainly concentrated in photosynthesis, carbon fixation, energy transport and other basic cell functional related enzymes, such as ribonose bisphosphate carboxylase and ATP synthase. Only a few genes related to basic metabolic processes such as photosynthesis, carbon fixation and ATP biosynthesis were up-regulated. Compared with the control ZD958, the differential protein of SK12-5zd was significantly enriched in the carbon fixation pathway of photosynthetic organisms. The differential proteins of Bt799zd were significantly enriched in photosynthetic biological carbon fixation pathway, photosynthesis and carbon metabolism pathway, respectively, compared with the control Z58. The differentially expressed proteins of IE034z and Bt799z were significantly enriched in the photosynthesis pathway, which indicated that the four transgenic materials were similar to the control in proteome. No abnormal gene expression was found. [conclusion] the four transgenic materials tested did not have unexpected effects on their safety at protein level. Current results suggest that proteomics can be used to verify the unintended effects of transgenic plants.
【作者单位】： 哈尔滨师范大学生命科学与技术学院;吉林省农业科学院;
【基金】：国家转基因生物新品种培育科技重大专项(2015ZX08013003)
【分类号】：S513
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本文编号：1492973