| Starch, including amylose and amylopectin, is the main component of rice endosperm, and its composition and structure are the key determinants of grain cooking and eating quality, processing quality, industrial application, and even yielding. A serials of enzymes involved in the pathway of starch biosynthesis in plant, and the starch phosphorylase (EC2.4.1.1, Pho), which catalyzes the phosphorolysis of the terminal residue from the nonreducing ends of a-1,4-linked glucan chains, liberating Glc-1-phosphate (Glc-1-P), is one of the key enzymes. All plants studied so far have plastidial (L-form) and cytosolic isoforms (H-form) of starch phosphorylase, which are encoded by separate genes, Pho-L and Pho-H, respectively.Until today, the function of Pho and its encoded genes had been well elucidated in both dicot such as potato and monocot such as maize and wheat, but it was not in rice, especially their fuction for rice grain quality. In present study, the expression profile and allelic variation of two rice Pho genes, OsPho-L and OsPho-H, was carefully studied among different rice genotypes. Besides, transgenic analysis were performed to study the target gene's function through production of RNA interference transgenic rice. The main results were showed as followings.1. The sequence and structure of OsPho-L and OsPho-H genes were analyzed on the basis of public rice genomic sequences, and both contain fifteen exons and fourteen introns. The two deduced polypeptides, containing 951 and 841 amino acid residues,respectively, were predicted to have a conserved region of phosphorylase and high identity with those from other plants, such as wheat and Arabidopsis.2. Each Pho enzyme was confirmed to be encoded by a single copy gene in rice genome by Southern blotting analysis, and the allelic variation of both genes was carefully surveyed by using either RFLP or microsatellite molecular markers. The results showed that the polymorphism at both locus was obvious among different rice genotypes, especially for the OsPho-L gene between O.sativa and other wild rice.3. The transcript of each OsPho gene in different tissues or organs during different developmental stage was examined and subsequently compared through both Northern blotting and Real-Time quantitative RT-PCR. The data indicated that the OsPho-L gene seemed to tissue-specifically express in the developing seeds and the expression level was higher in the early and middle stages of grain filling than that of other stages.Whereas the expression level of OsPho-H gene was low in the developing seeds,there was little expression in root, leaf and germinating seed. The expression level of each gene was various among different rice genotypes, and the OsPho-L gene has a much higher expression than OsPho-H gene in all of the tested rice cultivars.4. The BAC clones containing the full length genomic sequence of OsPho-L gene were screened from a BAC library derived from the genome of a high quality waxy rice cv. Suyunuo, and the promoter, wich a size of 2.0kb upstream of the start codon, was subsequently cloned by PCR technique. Then, four GUS fusion genes, controlled by the cloned promoter with different size, were constructed and introduced into a japonica rice cv. Wuyunjing 9 by Agrobacterium-mediated transformation. The data from GUS activity assay in transgenic rice plants implied that the 2.0kb or 1.5kb promoter of OsPho-L gene could drive high expression of GUS in endosperm, leaf and stem, but low in embryo and germinated seed and no expression in anther and seed capsule. The GUS activity was highest in stem when the 2.0kb promoter was used, while it was in endosperm in the case of 1.5kb promoter. The 0.5kb promoter region could drive the GUS expression in transgenic rice plant, but the expression was not preferable in any tissue.5. The OsPho-L RNA interference vector was constructed and introduced into a japonica rice variety Nipponbare by Agrobacterium-mediated transformation, and lots of transgenic rice lines were regenerated. The results from both PCR and Southern blot analyses showed that the transgene(s) had integrated into the genome of rice plants. Northern blot analysis revealed that the expression level of endogenous OsPho-L gene in transgenic plants was descreased. The RNAi of target gene's expression had no effect on the grain shape and appearance as well as amylose content and gel consistency. But, the preliminary analysis of starch viscosity in primary transformants indicated that some of the starch RVA characteristics, especially the peak viscosity, hot viscosity and cool viscosity, were significantly descreased when compared with that of the negative control without the RNAi transgene.. |
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