利用PTN系统快速解析转基因水稻种子可溶性蛋白非预期变异的来源

【目的】建立转基因（genetically modified，GM)水稻安全性评价的亲本对照-转基因株系-非转基因对照（parent control-transgenic plant-nontransgenic control，PTN)系统。通过实质等同性比对分析，追溯GM水稻种子蛋白质非预期变异的技术根源，为GM水稻的安全性评价提供技术支持。【方法】根据GM水稻植株培育的技术原理，收集转基因株系（transgenic line，T）、亲本品种（parent variety，P）及其他遗传学背景相关的非转基因组培再生株系（non-transgenic regeneration line from tissue culture，NR）和非转基因遗传分离阴性后代株系（non-transgenic segregated negative offspring line，NS）等对照样本。以转2mG2-epsps抗草甘膦GM水稻株系T13和T23及各自的PTN系统对照样本（P、NR和NS）为试验材料，以种子蛋白质含量和组分的非预期变异为研究对象，根据PTN样本间的多重比较结果解析并追溯GM水稻非预期变异的技术根源。水稻种子可溶性蛋白的提取采用分级提取法，依次用蒸馏水、5% NaCl、70%乙醇和0.1 mol·L-1 NaOH提取稻米清蛋白、球蛋白、醇溶蛋白和谷蛋白。蛋白含量测定应用Bradford法，蛋白组分分析采用SDS-PAGE电泳法。【结果】GM水稻株系T13和T23种子总可溶性蛋白含量分别比亲本显著降低3.29%和6.84%，主要受谷蛋白变异影响，但GM水稻变异的最大幅度小于天然水稻亲本P1和P2品种间差异，说明GM水稻种子可溶性蛋白含量变异在安全范围之内。与各自亲本相比，GM水稻株系种子清蛋白和醇溶蛋白组分差异不显著，球蛋白和谷蛋白个别组分存在显著或极显著变异，主要表现为含量的增减。T13株系的56 kD和24 kD球蛋白含量比亲本P1显著增加，T23的65 kD球蛋白含量比亲本P2极显著降低。GM株系T13和T23的谷蛋白组分变异趋势相似，与相应亲本相比，主要表现为19-23 kD和33-38 kD谷蛋白含量显著增加，100 kD和9 kD谷蛋白含量显著减少。突出的变异是GM水稻新增加了41 kD和56 kD谷蛋白组分，但这些变异新组分也同样存在于NR和NS对照样本中。GM水稻株系T13和T23的可溶性蛋白非预期变异的特征与相应的NR对照样本基本一致。NS23对照株系的清蛋白含量和球蛋白组分存在不同于T23和NR23的独特非预期变异。【结论】GM水稻种子总可溶性蛋白质变异主要受谷蛋白含量变异影响，变异幅度小于天然水稻品种间差异。GM水稻种子的清蛋白、醇溶蛋白组分未发生显著变异，球蛋白和谷蛋白个别组分发生了显著变异。GM水稻种子蛋白质非预期变异主要来源于组织培养无性系变异，转基因插入突变的影响较小。

Rapid Identify the Sources of Unintended Variations of Seed Soluble Proteins in Genetically Modified Rice by Using PTN System

【Objective】The objective of this study is to establish a PTN（parent control-transgenic plant-nontransgenic control) system for the safety assessment of genetically modified (GM) rice, to provide technical supports for GM rice safety assessment by tracing the technical sources of unintended variations of soluble proteins in GM rice seed according to the comparative analysis of substantial equivalency.【Method】The transgenic rice lines (T), the parent variety controls (P) and other genetic background related non-transgenic controls including regeneration lines from tissue culture (NR) and segregated negative offspring lines (NS) were collected, according to the technological principle for GM rice development. Two glyphosate-resistant GM rice lines T13 and T23, which harboring 2mG2-epsps transgene, and their respective control samples (P, NR, NS) were used as experimental materials, while seed soluble protein contents and components as research subjects. The technical sources of unintended variations from GM rices were interpreted and traced on the basis of the multiple comparison analysis results of rice protein variations among PTN samples. The rice soluble proteins were prepared by sequential extraction method, the albumin, globulin, prolamin and glutelin were extracted successively with distilled water, 5% NaCl, 70% ethanol and 0.1 mol·L-1 NaOH. Bradford method was used for the seed protein content analysis. Analysis of protein components was done by SDS-PAGE method. 【Result】 The contents of seed total soluble protein of GM rice lines T13 and T23 were significantly decreased by 3.29% and 6.84%, respectively, and those variations are mainly affected by the glutelin content changes. While the top grade difference value from GM rice lines and their corresponding parents was less than that from the two natural parent rice varieties P1 and P2, indicating the variations of seed soluble protein content from GM rice are inside the scope of safety. Comparing to their respective parents, the components of albumin and prolamin proteins of GM rice lines T13 and T23 had no variation, but several components of globulin and glutelin varied significantly or extremely significantly, mainly shown as increase or decrease in content. The content of 56 kD and 24 kD globulin of T13 line increased significantly while the content of 65 kD globulin of T23 line decreased extremely significantly than their corresponding parent P1 and P2. The variation tendency of glutenin components from GM rice lines T13 and T23 was similar, mainly shown as the content of 19-23 kD and 33-38 kD glutelin components increased significantly as well as the content of 100 kD and 9 kD glutenin decreased significantly, when compared with their corresponding parent controls. The remarkable variation was that two new glutenin components of 41 kD and 56 kD presented in GM rice lines，while these new variation glutelin components also existed in the NR and NS control samples. The unintended variation characteristics of seed soluble proteins from GM rice lines T13 and T23 were basically consistent with that from their corresponding NR control samples. Additionally, there were unique unintended variations concerning seed albumin content and globulin components in NS23 line.【Conclusion】The content variations of seed total soluble protein from GM rice line are mainly affected by the glutelin content changes, with less variation range than the difference value of natural rice varieties. Components of albumin and prolamin proteins in GM rice seed did not vary notably, but several components of globulin and glutelin varied significantly. The unintended variations of soluble proteins in GM rice seed mainly derived from the somaclonal variations of tissue culture, with little compact caused by transgene insertion mutation.