关键词: 水稻 , 转基因植物 , CaMV 35S启动子 , GUS蛋白质 , 免疫印迹

Abstract: 【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.

Key words: rice , transgenic plants , CaMV 35S promoter , GUS protein , western blotting