Map-based Cloning And Functional Analysis Of A Chalkiness Gene OsBT1 In Rice (Oryza Sativa L.)

Starch content accounts for 60%-90%of the dry weight of seeds in cereal crops and is great importance for the formation of crop yields.Starch is composed of amylose and amylopectin,and its ratio affects the quality of rice as well as commodity value.So far,many starch synthase genes and other regulatory factors have been reported,but the molecular mechanism of starch synthesis is still not clear,therefore,identification and analysis of new genes involved in starch synthesis have important scientific significance.In this study,we screened a chalky mutant osbt1 from the mutants produced during the nipponbare tissue culture and conducted an in-depth study,the results were as follows:1.Rice appearance and physico-chemical quality has changed in the brittle1?osbt1?mutant.Seeds were observed a white-core phenotype;the starch grains in the inner endosperm cells of the mutant were loosely packed with spherical or oval and smooth surface;the total starch and amylose contents of the mutant were decreased 2.17%and 2.45%,respectively compare to wild-ype;the amylopectin with short and intermediate chains were elevated,whereas the proportion of intermediate and long chains were decreased;the viscosity was reduced to 68%of the wild-type;the gelatinization temperature was reduced 3°C;the swelling volume of the osbt1 mutant starch was the most obvious difference in the3-4M urea solution compared to the wild-type.These results indicated that the OsBT1 gene is important for the starch synthesis in the endosperm cells.2.The development of the compound starch granules was abnormal in the osbt1 mutant endosperm cells.A large number of scattered and smaller starch granules were filled with the mutant endosperm cells;the membrane structures of the amyloplasts were disintegrated at the early starge of the endosperm development,which did not form normal compound starch granules.The results revealed that OsBT1 gene had a very important effect on the normal formation of compound starch granules in rice endosperm cells.3.We crossed the osbt1 mutant with an indica varity NJ-11 to generate a F₂ mapping population and the osbt1 locus was mapped to a 39.5-kb genomic region between markers Ind3 and Ind10.Seven open reading frames?ORFs?were included in this region.Sequence analysis revealed that there was a18-bp repeat insertion in ORF2?Os02g0202400?in the mutant.Functional complementation demonstrated that Os02g0202400 was the gene that caused the mutant phenotype and named OsBT1.The OsBT1 gene encodes a transmembrane protein containing 425 amino acids which has three copy genes in rice.4.OsBT1 gene was specifically expressed in seeds and its relative expression level gradually increased after fertilization,with a maximum expression level at the mid-and late stages of grain filling.GUS staining results were approximately the same as that obtained using qRT-PCR.5.The OsBT1 encoded protein was subcellular localizated to plastid.6.The expression levels of most of the starch synthesis related genes were higher in osbt1 than that in the wild-type.The activities of SSI and Pho?Pho1 and Pho2?were increased in the osbt1 mutant while the activity of SSIIIa,ISA and PUL was found insignificant differences in the mutant compare to wild-type.These data indicated that the mutation of OsBT1 gene not only affects the expression of starch synthesis related genes,but also affects the activity of some starch synthesis related enzymes.