Studies On The Formation Mechanism Of Low Amylose Content In Four Yunnan Soft Rice Varieties

In this study we explored the formation mechanism of low amylose content(AC), and attempted to offer theoritical proof for molecular breeding of high qualitysoft rice.1. Inheritance and allelic analysis were analyzed with four Yunnan soft rice, and itshowed that the genes controlling low amylose content were recessive and were controlledby a major gene allelic to Wx. The Wx genome sequence of Haopi was sequencedcompletely with overlapping PCR and then compared with the counterpart ofNipponbare: in the promoter, the 5' acceptor motif in the first intron was AG/GT andthere were 10 (CT)_n repeats, both of which were characterized as the Wx^a allele; in theopen read frame (ORF) regions there was a A to G base change at nucleotide position497 from the coding start base, and it generated a miss-sense mutation (Asp to Gly) inthe fourth exon. Compared with the reported results, this is a novel Wx allele, and wasnamed Wx^hp.Then a dCAPs marker was designed, and it could select other Yunnan soft ricevarieties, the sequence analysis of these Yunnan soft rice Wx gene also proved theefficiency of this marker. Designing of dCAPs marker supplied basis for breeding ofexcellent soft rice varieties.2. GBSSI encoded by Wx gene catalyze the amylose synthesis, so quantity ofGBSSI accumulated and its activity could affect AC directly. In this study GBSSI ofHaopi, Nanjing11 and Nipponbare were extracted from the developing seeds at 14 daysafter flowering (DAF) and analyzed by SDS-PAGE. The result showed that GBSSIaccumulated in japonica variety Nipponbare was much less than indica varietyNanjing11, which was consistent with former research. The GBSSI accumulated and itsactivity in Haopi was less than Nipponbare dramatically, so this result showed that thelow AC in Haopi was resulted by the reduction of GBSSI quantity and its activity.3. RT-PCR was done with two primers to analyze the function of Wx^hp promoterby the control of Nipponbare and Nanjing11. The result showed the Haopi Wxtranscripts were the same to transcripts of Nipponbare and Nanjing11, and the firstintron can be spliced sufficiently.Transformed the Agrobacterium mentioned in method to the calli of Kaslath, calli were cultivated in culture medium which containing hygromicyn B andcephalosporin for 4 weeks, then the calli with shallow yellow color were used forGUS activity assay. The result showed the GUS activity of Wx^hp was much strongerthan that of Nipponbare, implying Wx^hp promoter was characterized as indica andhad ordinarily function to transcribe.4. The full length cDNA of Haopi and Nipponbare was cloned by RT-PCR, theenzyme activity of cDNA of Haopi and Nipponbare were assayed by the control ofempty PET vector, and there were no notable difference between Haopi andNipponbare. This result showed the single nucleotide mutation didn't affect theactivity of Haopi GBSSI.5. To make sure the single nucleotide mutated in the fourth exon of Wx^hp wasresponsible for the low amylose content in Yunnan soft rice, we construted the plantexpressing vectors pPZ::Wx^hp and pPZ::Wx^b, these vectors offered the basis for theconformation to rice.6. Starch granules of fourYunnan soft rice varieties were scanned by electronmicroscope. The super microstructure of Yunnan soft rice had the samecharacteristics: granules can bind each other tightly, the shape of granule is notstandard, and there were many small holes in the granule, while granules ofNipponbare were arrayed in order, and granule of Suyunuo were almost roundarrayed loosened.