转基因水稻中GUS蛋白质的检测及其表达特征

【目的】建立转基因水稻中GUS蛋白质的免疫学检测方法，并了解花椰菜花叶病毒（CaMV）35S启动子驱动的GUS蛋白质在转基因水稻中的表达特征。【方法】以细菌基因组DNA为模板，PCR扩增GUS基因后克隆到表达载体pET30a中，测序验证的重组子转入大肠杆菌表达菌BL21中，IPTG诱导获得重组表达的GUS蛋白质，用HIS-tag beads纯化后作为免疫原免疫小鼠制备GUS蛋白质特异的抗体，通过免疫印迹分析筛选高特异性的单克隆抗体，用Broadford法对重组的GUS蛋白质进行定量，对不同浓度的GUS蛋白质进行免疫印迹分析，绘制检测GUS蛋白质的标准曲线，通过与标准曲线的比较对水稻叶片中GUS蛋白质进行定量分析。提取不同时期、不同部位的水稻总蛋白质，包括苗期的地上部、地下部，分蘖期的茎、茎节、叶鞘、叶枕、叶片上部、叶片中部和叶片下部，孕穗期的茎、穗轴、叶鞘、叶枕、叶片、幼穗（长度分别为1、2、10和20 cm），开花期的茎、穗轴、叶鞘、叶片、穗子，成熟期的茎、叶片、授粉后不同时期的种子（分别为授粉后10、20、30和40 d）、乳熟期的胚、胚乳和颖壳、成熟种子的全种子、胚、胚乳和颖壳以及不同时期的叶片和根部材料等。SDS-PAGE分离后用抗体检测其GUS蛋白质的丰度。【结果】筛选获得了高特异性的抗GUS单克隆抗体（编号为#27），用该抗体检测转基因水稻中及重组的GUS蛋白质均呈现特异条带，没有可见的背景信号，用本研究建立的免疫印迹方法对重组GUS蛋白质的检测下限约为4 ng，可检出转基因水稻单粒大米2.5%样品中（约0.6 mg）的GUS蛋白质。在不同时期的转基因水稻叶片中GUS蛋白质的表达丰度基本稳定，而在水稻根部的GUS丰度随生长急剧减少，5叶期根中的表达量不到3叶期的三分之一，到6叶期检测不到GUS蛋白质。在水稻苗期叶片中，GUS蛋白质约占鲜重的0.02‰。另外，除分蘖期以后的根部之外，GUS蛋白质几乎在所有的水稻组织部位中呈组成型表达，只是不同组织中的表达量略有差异，如在孕穗期和开花期的茎及颖壳中的表达量较低。【结论】建立了具有应用价值的对转基因水稻中GUS蛋白质丰度检测的免疫印记方法。该方法特异性高、样品用量少、不依赖于GUS蛋白质的酶活性、测定结果易于在不同实验室间比较。证明了35S启动子驱动的GUS蛋白质在转基因水稻中基本呈组成型表达。

Detection of GUS Protein and Its Expression Pattern in Transgenic Rice Plants

【Objective】 The objective of the current research is to establish a method for detecting GUS protein in transgenic rice via immunoassay and reveal the expression pattern of GUS protein driven by cauliflower mosaic virus (CaMV) 35S promoter.【Method】Genomic DNA isolated from E. coli was used as template to amplify Gus gene, the PCR product was cloned into pET30a vector, sequencing verified recombinant was transferred into E. coli expression strain BL21, recombinant protein was induced by adding IPTG and His-tag beads purified recombinant protein was used as immunogen to generate monoclonal antibody. The GUS protein-specific antibody was selected using western blot analysis. Broadford assay was used to quantify recombinant GUS protein, a standard curve of GUS protein concentration versus the signal intensity of western blot analysis was generated and the quantification of GUS protein in rice leaves was carried out by comparing its intensity with the standard curve. Total proteins were isolated from different tissues at different developmental stages, including shoot and root at seedling stage, stem, node sheath, cushion, upper, middle and lower part of leaves at tillering stage, stem, stalk, sheath, cushion, leaves, spike (1 cm, 2 cm, 10 cm and 20 cm in length, respectively) at booting state, stem, stalk, sheath, leaves and spike at flowering stage, stem, leaves, seed at different days after pollination (10 d, 20 d, 30 d and 40 d, respectively) at mature stage, embryo, endosperm and lemma at milky mature stage, whole seed, embryo, endosperm and lemma of mature seed, leaves and roots at different developmental stages. SDS-PAGE separated total proteins were detected by GUS-specific antibody to detect the abundance of GUS protein.【Result】An anti-GUS monoclonal antibody with high specificity was obtained (clone number #27), specific band was detected by western blot analysis using the antibody for GUS protein in transgenic rice or recombinant GUS protein, while no visible background signal could be detected. The lower limit for recombinant GUS protein detection was about 4 ng, and GUS protein could be detected in about 2.5% single grain sample of transgenic rice. The abundance of GUS protein in transgenic rice leaves at different stages was pretty stable, while its abundance in roots was decreased dramatically, specifically, the abundance of GUS protein in 5-leaf stage was one third less than in that at 3-leaf stage, and it was undetectable at 6-leaf stage. GUS protein accounted for about 0.02 ‰ of the fresh weight in rice leave at seedling stage. Further more, GUS protein was constitutively expressed in almost all tested samples in transgenic rice except the roots after tillering stage. However, slight different in abundance was detected among tissues, e. g. the abundance in stems and lemmas at booting and flowering stages was lower than that in leaves.【Conclusion】A applicable immunoassay method was established for GUS protein detection in transgenic rice, which is specific to the abundance of GUS protein while independent of enzymatic activity, about 0.6 mg sample is needed for the detection and results obtained from different laboratories using same reference protein can be compared. The constitutive expression of GUS protein driven by CaMV 35S promoter in transgenic rice was demonstrated.