Western Blot Detection Of PMI Protein In Transgenic Rice

Background: With an increasing incidence of genetically modified products, the detection for genetically modified(gm) products become more important. PM I(phosphomannose isomerase) was widely used in transgenic plants as an emerging selection marker. At present, the method for marker gene detecting are confined to PCR, CPR etc. which limited the application range of gene. It is very important to establish immunological detection method with high sensitivity.Aim: Establishing high sensitivity detection method based on immunology for PMI(phosphomannose isomerase) protein, and exploring protein expression pattern in transgenic rice, enriching its application in the research.Material and methods: Genomic DNA isolated from E. coli was used as template to amplify Pmi gene, the PCR product was cloned into p ET30 a vector, turned to expressing strain BL21 to inducting expression and purification. Purified protein was used for preparation of PMI antibody. Screening for specific monoclonal antibody using Western Blot to analysis the content of PMI protein in rice leaves. To explore the bottom line of PMI protein in single grain of rice in seedling stage and understand expression features in different parts at tillering stage, booting stage, flowering period, mature period and full of seeds, seed embryo, endosperm, of glume shell etc. In addition, we also identified homozygous transgenic rice strain using T1 seeds, and analyzed PMI protein expressing abundance in 60 transgenic plants, and to the possible homozygous plant for further analysis by real-time fluorescent quantitative PCR.Results: PMI-specific monoclonal antibodies can be used in western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample account for 0.4 % of single rice grain(about 0.08 mg). PMI protein driven by Ca MV-35 S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. And the amount of PMI protein expression in rice leaves showed a trend of decline with the growth of rice, in the roots, expression quantity is higher at seedling stage than at tillering stage, booting stage, flowering period and mature period. In addition, screening a gm homozygous strain T1-10 through the detection of PMI protein in T1 generation of transgenic rice with the real-time fluorescent quantitative PCR detected it was a single copy. At the same time, batch testing T1 generation seedlings found T1-7-9 and T1-7-20 with high expression and peculated it may be a homozygous strain preliminary. We then analyzed copy number by real-time PCR using Sps as internal reference, the result indicate that there was one copy of Pmi gene, and are PMI positive plants, also the WB result in T2 generation seeds of T1-7-9 and T1-7-20 were positive.Conclusion: In this study, we established a high sensitivity immunological detection method for emerging PMI marker genes which has important significance on the basis research of PMI protein, and the identification for genetically modified(gm) homozygous plant, the development of genetically modified(gm) crops.