Research Of Genetic Transferring Plant Sterols Key Enzyme OsSQS Gene Into Rice

Plant sterols are essential components of plant membranes, which possesses functions of anticancer, the capacity of lowering plasma cholesterol and relieving a cough etc. In plants, sterols which are triterpenoids are formed from various cyclation of squalene, which is formed via farnesyl diphosphate by the catalysis of squalene synthase (SQS). The branch point from isoprenoid central metabolism pathway to sterols synthetic metabolish is synthesis of squalene. SQS is the key enzyme for sterols biosynthesis. This study attempt to transform the OsSQS gene into rice with high yield but low plant sterols. The expression in the embryos of transgenic rice, can produce quality rice with high yield and plant sterols.The main results of this study are as follows:1.By PCR method from Oryza sativa subsp. Indica ("IR30") we cloned the open reading frame (1212bp) of plant sterols key enzyme OsSQS gene, encoding 403 amino acids.2.Double T-DNA plant expression vector pCDMAR-SQS-hpt was constructed.To make selectable genes in transgenic rice for safety vanishes, one T-DNA carring the gene hpt, another T-DNA carring the OsSQS gene driven by rice seed specific promoter pGt1.3. OsSQS gene was transferred into embryonic callus tissue of rice "T-9" with Agrobacterium-mediated method. By the resistant screening to hygromycin, we obtained 203 resistant plants, 25 of which were positive by PCR testing.4.Extract beta-sterol from seeds of transgenic plants with ultrasonic method. Get its results by ultraviolet spectrophotometer. The results showed that concentration of beta-sterol in16 samples above negative contrast.The highest content is 0.054%, the negative is 0.027%.5. Screened T1 generation of transgenic plants with OsSQS gene but not include the selection marker gene hpt by PCR testing.