| Starch is the main storage component in rice endosperm, and accounts for about 85 percent of the dry weight of grain. Therefore, it is considered as the key determinant of rice quality. A serials of enzymes involved in the pathway of starch biosynthesis in rice endosperm, and the soluble starch synthases (SSSs), containing eight isoforms, are the key enzymes among them. But the functions of most of these enzymes and their encoded genes, in particular their effects on rice grain quality are insufficient. In present study, with the aim to understand the function of OsSSSâ… gene, which encoding the soluble starch synthaseâ… in rice, two approaches were carried out. Firstly, the allelic variation of the OsSSSâ… gene among rice cultivars and their effects on rice grain quality were studied by using the gene-tagged molecular markers and near isogenic lines (NILs). Secondly, transgenic analyses were performed to study the target gene's function through production of RNAi and over-expression transgenic rice lines in three rice varieties with different grain starch quality. The main results were showed as followings.1. The structure of OsSSSâ… gene was analyzed on the basis of public rice genomic sequences, and then the BAC clone containing the full length genomic sequence of OsSSSâ… gene was screened from a BAC library derived from the genome of a high quality waxy rice cv. Suyunuo. The full length cDNA of OsSSSâ… gene was cloned from the total RNAs of the developing rice seeds by RT-PCR, and sequencing analysis indicated the sequence of the cloned cDNA was the same as the reported sequence.2. The mRNA expression of OsSSSâ… gene in different tissues during different developing periods was examined and compared by Northern blot analysis, and the results showed that the OsSSSâ… gene seems 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.3. The allelic variation of OsSSSâ… gene was carefully analyzed by using both RFLP and gene-tagged molecular markers, and the wide polymorphism was detected in OsSSSâ… locus among different rice genotypes. Five OsSSSâ… -tagged markers were developed to investigate 165 rice varieties, and eight alleles, which could be classified into two groups (groupâ… andâ…¡), were detected. Among the eight alleles, two alleles, typesâ… -1 andâ…¡-1, were found to be the main genotypes. The results also showed that the polymorphism at OsSSSâ… locus was conservative among the japonica subspecies, while the allelic variation was abundant among the indica varieties.4. The genetic effect of two main allelic variation,â… -1 andâ…¡-1, on rice grain quality was analyzed by using NILs, in which the typeâ… -1 allele from rice variety 9311 was introgressed into two indica varieties Longtefu and Zhenshan97B with typeâ…¡-1 allele. The preliminary analysis of starch RVA profiles implied that several RVA characteristics, such as PKV, HPV and CPV, of mature seeds containing the typeâ… -1 allele were little higher than those of the typeâ…¡-1 allele with the background of Longtefu, however the difference was not significant with the background of Zhenshan97B. It seems, but not confirmed, that that the selected allelic variation might have some effects on the the quality of endosperm starch.5. Both the RNAi and over-expression vectors were constructed and introduced into three rice varieties with different grain quality, including two japonica rice varieties Nipponbare and Suyunuo and one indica rice variety Longtefu, by Agrobacterium-mediated transformation, and lots of transgenic rice lines were regenerated. The results from both PCR and Southern blot analysis showed that the transgene(s) had integrated into the genome of rice plants. Northern blot analysis implied that the expression of endogenous OsSSSâ… gene in the transgenic plants containing RNAi structure had been repressed.6. The grain appearance and quality were investigated in the transgenic rice plants with repressed or increased expression of OsSSSâ… gene. The results showed that there was no effect of the transgene on the shape and appearance, as well as the physicochemical properties of the mature grains. Besides, the preliminary data of the starch RVA profiles indicated that transgenic regulation of the target gene's expression had an effect on some RVA characteristics. In particular, after inhibition the OsSSSâ… gene's expression in transgenic rice plants, the PKV, HPV and CPV of starch RVA profiles were decreased, while the BDV and PaT increased, when compared with those of the un-transformed plants. However, the detail function of the target gene will be elucidated after more homozygous transgenic lines were selected and the quality and fine structure of endosperm starch were carefully analyzed. |
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