Gene Map-Based Cloning And Mutation Mechanism Of Rice Floury Endosperm Mutant W24

Starch is the most abundant reserve polysaccharide in circle of vegetation. The chemical composition and content of starch are the decisive factors of cooking and eating quality of cereals. Understanding the starch metabolism of cereal crops is of great significance in improving the yield and quality of cereals. Rice mutant deficient in starch synthesis are well applied in studying the starch metabolic pathway. We obtained a floury endosperm mutant w24 from radiation mutants of the japonica variety cv. Koshihikari. Map-based cloning showed that w24 encodes a large subunit of adenosine diphosphate glucose pyrophosphorylase (AGPase). Phenotypic, physiological and biochemical analysis of w24 revealed the underlying mechanism of w24 mutation. These results throw new lights on the molecular mechanism of AGPase. The main results are as follows:1. The brown rice of w24 had an opaque endosperm appearance. Scanning electron microscopy of brown rice showed that the starch granules of wild-type were polyhedral, anomalistic and densely packed, while the abnormal endosperm was filled with spherical and loosely packed starch granules with large spaces. Compared with wild-type, w24 had the decreased 1000-grain weight, grain length, grain width and grain thickness as well as a slower filling rate at all the filling stages.2. Physiological and biochemical analyses indicated that compared with wild-type, amylose content and total starch content of w24 grains decreased, while the crude protein, sucrose, fructose and glucose contents increased. The differences of these indices between wild-type and w24 were all significant. The proportion of short chains (6^DP^ 10) was markedly elevated in the endosperm starch of w24, whereas proportions of intermediate-length chains (11≤DP≤35) were greatly depressed.3. The w24 locus was maped within an 88.6 Kb genomic region beween markers RM11383 and HY10 on the long arm of Chromosome 1 by crossing the mutant with an indica variety Nanjingll to generate a F2 mapping population. Gene sequencing showed a single nucleotide substitution in exon 5 of LOC_Os01g44220, resulting in the replacement of leucine by proline. Therefore, w24 encoded a large subunit 2 of AGPase, designated OsAGPL2.4. The results of real-time RT-PCR and Western blotting showed that the levels of OsAGPL2 mRNA and protein product in w24 were higher than those in the wild-type. However, the enzyme activity of AGPase was decreased to half of that of the wild-type. The lower AGPase activity might be responsible for the abnormal endosperm of w24.5. The mutation in the OsAGPL2 led to the changes in the expression of other genes encoding AGPase. Real-time RT-PCR showed the increased expression of seven types of mRNAs of the six genes encoding AGPase.6. Native-PAGE activity staining analysis was performed using extracts from endosperm. The activity bands for isoamylase (ISA), pullulanase (PUL), starch synthases(SSI, SSIIIa), starch phosphorylase(PHO1, PHO2), branching enzyme(BEI, BEIIa and BEIIb) had no significant differences between the wild-type and w24..7. The sucrose content of the mutant was significantly higher than that of the wild-type in the endosperm at 10-15 days after fertilization. However the starch content in the mutant leaves was dramatically lower than the wild-type in the same stage. Summarily, we speculated that there was a feedback inhibition in the endosperm of w24.