本文關(guān)鍵詞： 轉(zhuǎn)基因玉米 蛋白質(zhì)組學(xué) 非預(yù)期效應(yīng) 雙向電泳 差異表達(dá)蛋白　出處：《中國(guó)農(nóng)業(yè)科學(xué)》2017年19期 　論文類(lèi)型：期刊論文

【摘要】：【目的】轉(zhuǎn)基因育種是將對(duì)人類(lèi)有益的外源基因通過(guò)生物技術(shù)整合進(jìn)受體植物的基因組中,使其獲得通過(guò)自然進(jìn)化無(wú)法獲得優(yōu)異性狀,這是轉(zhuǎn)入外源基因的"預(yù)期效應(yīng)"。然而,外源基因插入受體植物還可能產(chǎn)生無(wú)法控制和預(yù)期的細(xì)胞、代謝或表型等方面的改變,即"非預(yù)期效應(yīng)"。非預(yù)期效應(yīng)是轉(zhuǎn)基因植物安全評(píng)價(jià)的核心內(nèi)容之一,也是近年來(lái)研究的熱點(diǎn)和難點(diǎn)。本研究以抗蟲(chóng)轉(zhuǎn)基因玉米為材料,利用差異蛋白質(zhì)組學(xué)技術(shù)比較研究轉(zhuǎn)基因玉米與其非轉(zhuǎn)基因玉米之間的非預(yù)期效應(yīng),為蛋白質(zhì)組學(xué)技術(shù)解析轉(zhuǎn)基因植物非預(yù)期效應(yīng)提供理論參考。【方法】選取4份已經(jīng)進(jìn)入中國(guó)農(nóng)業(yè)部安全評(píng)價(jià)階段轉(zhuǎn)蘇云芽孢桿菌(Bacillus thuringeinsis,Bt)內(nèi)毒素基因的抗蟲(chóng)玉米材料,即SK12-5zd、IE034z、Bt799z、Bt799zd,以及它們的對(duì)照玉米材料鄭單958和鄭58,分別種植于可控人工溫室中。待玉米幼苗生長(zhǎng)至5葉1心期時(shí),每份材料取長(zhǎng)勢(shì)一致的6個(gè)單株最上部完全展開(kāi)葉片,混合作為一個(gè)樣本進(jìn)行取樣,并提取葉片蛋白質(zhì)。雙向電泳技術(shù)分別分離4種轉(zhuǎn)基因玉米和它們各自對(duì)應(yīng)的非轉(zhuǎn)基因材料高豐度蛋白質(zhì)組,利用Image Scanner掃描儀掃描脫色后的蛋白質(zhì)凝膠,PD Quest 8.0軟件(Biorad,USA)分析蛋白質(zhì)凝膠圖譜,以蛋白點(diǎn)相對(duì)體積(%Vol)來(lái)表述每一個(gè)匹配蛋白質(zhì)的表達(dá)豐度,將電泳凝膠上特異性蛋白質(zhì)和相對(duì)體積大于2倍的差異蛋白質(zhì)點(diǎn)取樣,依據(jù)相應(yīng)蛋白質(zhì)數(shù)據(jù)庫(kù)進(jìn)行質(zhì)譜鑒定,并對(duì)鑒定出的特異性和差異性蛋白質(zhì)進(jìn)行細(xì)胞功能富集分析(GO)和代謝途徑富集分析(KEGG),以評(píng)價(jià)轉(zhuǎn)基因抗蟲(chóng)玉米的可能非預(yù)期效應(yīng)�！窘Y(jié)果】通過(guò)比較4種不同轉(zhuǎn)基因抗蟲(chóng)玉米及其非轉(zhuǎn)基因?qū)φ盏母哓S度蛋白質(zhì)組,除目標(biāo)抗蟲(chóng)基因外,共鑒定出61個(gè)蛋白質(zhì),其功能主要是富集于光合作用、碳固定、能量轉(zhuǎn)運(yùn)等基礎(chǔ)細(xì)胞功能相關(guān)酶類(lèi),如核酮糖二磷酸羧化酶、ATP合成酶、丙酮酸磷酸雙激酶等。僅有少數(shù)幾個(gè)與光合作用、碳固定和ATP合成途徑等基礎(chǔ)代謝過(guò)程相關(guān)基因出現(xiàn)上調(diào)表達(dá)。KEGG分析表明,與對(duì)照Z(yǔ)D958相比,SK12-5zd的差異蛋白在光合生物碳固定途徑中顯著富集;Bt799zd的差異蛋白則分別在光合生物碳固定途徑、光合作用和碳代謝途徑中顯著富集。與對(duì)照Z(yǔ)58相比,IE034z和Bt799z的差異蛋白均在光合作用代謝途徑中顯著富集。證明4種轉(zhuǎn)基因材料與對(duì)照蛋白質(zhì)組相似性較高,未見(jiàn)基因的異常表達(dá)�！窘Y(jié)論】測(cè)試的4種轉(zhuǎn)基因材料在蛋白質(zhì)水平未發(fā)生影響其安全性的非預(yù)期效應(yīng);當(dāng)前結(jié)果表明蛋白質(zhì)組學(xué)技術(shù)可以用來(lái)驗(yàn)證轉(zhuǎn)基因植物的非預(yù)期效應(yīng)。
[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.
【作者單位】： 哈爾濱師范大學(xué)生命科學(xué)與技術(shù)學(xué)院;吉林省農(nóng)業(yè)科學(xué)院;
【基金】：國(guó)家轉(zhuǎn)基因生物新品種培育科技重大專(zhuān)項(xiàng)(2015ZX08013003)
【分類(lèi)號(hào)】：S513
，

本文編號(hào)：1492973