Map-Based Cloning And Functional Analysis Of A Key Gene Ospdill-1Involved In The Starch Biosynthesis In Rice

Starch accretion determines final grain weight and thereby it contributes greatly to grain productivity. The enzymatic process model required for starch synthesis has been established by a series of mutants using maize and rice as model organisms. However studies on some new opaque endosperm mutants showed that these new genes can not be added into the previously established model, so we need to construct a bigger or a cross model using these novel mutants. To seek for more these kinds of mutant in rice, we performed a large-scale screen in T-DNA-inserted and60Co-irradiated mutant pool and isolated a series of mutant lines. T3612was one of those. One of the floury mutants T3612was selected for further research and and the detailed research progress which achieved in this study is as follows:T3612showed floury endosperm and the final mature seed weight was～33.7%lower than that of wild type. Scanning electron microscopic (SEM) examination revealed that the mutant endosperms contained round loosely packed starch granules which were probably responsible for the floury appearance. The amylose content in T3612was approximately80%that of Nipponbare. The total protein and lipid content was approximately15%higher than wild type.To genetically map the mutation, an F2population derived from a cross between T3612which is an indica variety, and japonica variety Nanjingll. Furthermore, we used1242floury segregants to located the mutation between marker T3612-31and T3612-54, and the mutation co-segregated with marker T3612-55, locating within a61kb region of BAC clone OSJNBa0058P12on chromosome11. RiceGAAS (http://ricegaas.dna.affrc. go.jp/) predicted eleven ORFs in this map region. The mutation gene was PDIL1-1. DNA-PCR amplification and sequencing revealed a4145bp deletion started from124bp in PDIL1-1CDS. Transgenic complementation test confirmed that the deletion of protein disulfide isomerase like1-1(PDIL1-1) was responsible for the floury phenotype. Expression analysis demonstrated that PDIL1-1was localized in the root, culm, sheath, leaf and greatly had the highest expression level at DAF12. T3612produced no OsPDIL1-1transcript or the OsPDIL1-1protein in those tissues. These results suggested that T3612was a deleted mutant of protein disulfide isomerase like1-1(PDIL1-1).The real-time RT-PCR was used to analyze the expression of31starch synthesis-related genes. Results suggested that genes expression for OsAGPL2、OsPHOL、 OsSuSy2and OsSuSy3, was double that of wild type, while expression of the genes for OsAGPL1、OsAGPL4、OsSSIIb, OsPHOH and OsDPE-1in T3612was decreased to half of those in wild type. Zymogram analyses were performed using T3612and the wild-type Nipponbare endosperm at9DAF and12DAF. We detected the activities of starch synthase (SS), branching enzyme (BE), Starch Phosphorylase (Pho) and debranching enzyme (DBE). The activity of pullulanase decreased largely and migration rate of PUL band decreased, whereas SSI increased significantly. The activity of Pho1was decreased significantly at9DAF. No significant differences in activities of BE isoforms (BEI, BEIIa, and BEIIb), SSIII, Pho1and ISA were found. AGPase and Suc synthase activity was higher than wild type.UGPase activity was also found not to be significantly different in the mutant than in the wild type Nipponbare. Then We determined the chain-length distributions of mutant endosperm amylopectin using DNA sequencer assisted fluorophore carbohydrate electrophoresis (DSA-FACE). The mutant endosperm had a higher proportion of short chains with a degree of polymerization (DP) of8to13and a decrease in chains with DP6to7.PDIL1-1encoded a protein disulfide isomerase. To determine whether PDIL1-1protein show disulfide reductase and molecular chaperone activity, we used insulin and citrate synthetase as substrate in our study. The results suggested that insulin was reduced more rapidly with the increase in the concentration of recombinant PDIL1-1. And recombinant PDIL1-1can effectively protect citrate synthetase from thermal denaturation in42℃in vitro.In the ER lumen, protein disulfide isomerase-like (PDIL) proteins assist in nascent immature secretary proteins folding. We detected the expression levels of genes encoding chaperones and oxidoreductases in mutant endosperm12DAF by employing Real-Time RT-PCR. In our study, ER stress-responsive genes were strongly up-regulated, such as PDIL, Bip, Hsp70, Hsp90, CRT, CNX, cochaperone (NEF, ERdj3, Stt3a and UDPG- glucose-transporteor), bZIP60and Derlins. Persistent or acute ER stress aims to initiate programmed cell death, and the PCD marker gene HSR203J was induced in T3612. So we speculated that programmed cell death which maybe happened in mutant endosperm influenced the starch synthesis.