Studies on the expression of recombinant proteins in transgenic rice grains

• Main Authors: Chwan-Yang Hong, 洪傳揚
• Other Authors: Li-Fei Liu
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/89340948214043250221

博士 === 國立臺灣大學 === 農藝學研究所 === 91 === Abstract (I) Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals. Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and E. coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds. Phytase expression was controlled by two germinated inducible a-amylase gene (aAmy7 or aAmy8) promoters, and extracellular phytase secretion directed by aAmy7 or aAmy8 signal peptide sequences, respectively. A total of 8 plasmid constructs were transformed to rice via Agrobacterium — mediated transformation. Analysis of the T1 germinating rices showed that aAmy8 promoter leads two phytase genes to better phytase activities. The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally-controlled and tissue-specific manner. Phytase activity of SrPf6 and AppA peaked on day 3 and day5 following germination. Tissue specificity analysis revealed the expression levels of two phytases were highest in endosperms and next in embryos, very low phytase activity was detected in roots and shoots. Up to 0.6 and 1.4 U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 26 to 58 times of phytase activities as compared with the non-transformant. The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0 to 5.5 and 2.0 to 6.0, respectively. Phytase expression and inheritance of transgene of the highest transgenic line was stably expressed over four generations. Germinated transgenic rice seeds, producing a highly active recombinant phytase are also rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals. Abstract (II) Glutelin is the most abundant storage protein in rice, consisting 60-80％ of total endosperm protein in mature seeds. GluB-1 promoter is therefore a good candidate for expressing proteins in seeds and worth further improvement to increase protein yield. In this study, a highly efficient gene expression system was constructed based on GluB-1 promoter modified with enhancer and intron. Firstly, the TA-like sequence, TA-like flanking sequence, as well as the G-box flanking sequence of GluB-1 promoter were substituted by 2 copies of TA element, flanking sequences of TA element and G-box of rice aAmy3 promoter, respectively. The luciferase reporter gene was used for determining the promoter activities. Transgenic rice plants mediated by Agro-transformation possessed of 1-2 copy of transgene integrated into the rice genome detected by Southern blot analysis. Luciferase activity analysis revealed that the modifications conferred the sugar insensitive GluB-1 promoter to a sugar responsive promoter. Modifying promoters by substitution of TA-like sequence, flanking sequences of TA-like sequence and G-box resulted in highest sugar regulation to 32 fold and 650 fold in transformed calli treated with or without sucrose, respectively. Secondly, the maize ubiquitin1(Ubi1) first intron was inserted into 3’ end of modified GluB-1 promoters to enhance transgene expression. This remarkably boosted the luciferase activity to 500-8000 fold. Finally, expression of an E.coli phytase gene (appA) was evaluated by using modified GluB-1 promoter and Ubi1 intron. The results showed that 5 independently transformed calli contained modified GluB-1 promoter and Ubi1 intron expressed highly phytase activity as compared to the GluB-1 promoter .