| Rice is one of the major food crops. About a third of the world's population take rice as staple food. Therefore, the research on the quality and quantity of rice are of great significance. Starch is the main component of rice grain, which is composed of amylose and amylopectin. The relative content of amylose is one of the key factors to determine the quality of rice. We found in the study of rice anion channel gene effects that over expression of OsAT1(Os02g0822100) resulted in a significant decrease of amylose content in rice grain. Therefore, it is suggested that this gene may be related to the synthesis of amylose. In this study.we studied the subcellular localization and tissue expression of OsAT1. The transgenic plants of OsAT1 gene promoter were constructed. We had analyzed the phenotype of OsAT1 over expression and interference transgenic plants, and the content of related elements and detected the differential gene expression by RNA-seq in transgenic rice. The study provided a foundation to further understand the biological function of OsAT1 and its regulating mechanism on amylose content. The main results are obtained as follows:(1) Subcellular localization and tissue expression of OsAT1According to the result of bioinformatics analysis, OsAT1 has a typical transmembrane domain, could encode a membrane protein which may be localized in the endoplasmic reticulum, Golgi or cytoplasmic membrane. The subcellular localization of OsAT1 was verified by the transformation of rice protoplasts. We found that OsAT1 was located in the endoplasmic reticulum and the Golgi apparatus and was not detected in the cytoplasm membrane. The expression of OsAT1 was high in stem and leaf and was lower in root by quantitative PCR. Further, We constructed the expression vector of OsAT1 gene promoter driven GUS and obtained transgenicplants. The expression of OsAT1 was higher in stem, leaf and sheath than in root by GUS tissue staining which was basically consistent with the quantitative PCR.(2) Over expression of OsAT1 resulted in the accumulation of boron in rice leavesAccording to the functional prediction of OsAT1 by bioinformatics, OsAT1 was a membrane protein which might have the transportation function of arsenic ion. In order to confirm whether OsAT1 had transportation function of related ions. We took the leaves of the lines of OsAT1 over expression and interference in heading stage as materials and measured the content of the 19 elements in rice. The results showed that the boron content in over expression plant of OsAT1 was 3.6 times as that in wild type plant while the boron content in the interference plant was only 64% as that in wild type plant. Copper content respectively had a little raise and fall in over expression and interference plant while the content of the other 17 elements had no difference.The results showed that the expression of OsAT1 affected the accumulation and transportation of boron in rice.(3) over expression of OsAT1 reducd amylose content in rice grainCompared to the wild type plant. We found that the height of OsAT1 over expressing plants was 7-15 cm Shorter, Grain obviously thined, The 1000 grain weight decreased 32%-38%, Starch content decreased 2%-5%, The amylose content decreased 22%-33% by testing the phenotype of over expression and interference of OsAT1 gene in transgenic plant, And the height of OsAT1 interference plant was higher 1~4 cm, The grain weight and starch content was slightly raised. But there was no obvious difference in amylose content. Therefore, the over expression of OsAT1 affected the rice phenotype significantly and reduced the amylose content in rice grain.(4) The relationship between the decrease of amylose content and the expression of related genes in rice grainIn order to confirm the over expression of OsAT1 decreased starch content of transgenic rice amylose. We analysised the differential expressions between OsAT1 transgenic plant and wild type after 5 days in the filling period by RNA sequencetechnology. The results showed that there was 804 genes expressed differentially in over expression of OsAT1 transgenic plant in total which 503 genes were up-regulated and 301 genes were down-regulated. According to clustering analysis, we found that these genes were involved in metabolism, genetic information processing, biological systems, environmental information processing and cell processing. Among these differential genes, there were some genes which took part in the synthesis of sugar and starch. GBSSI was not found to be differentially expressed, but we found GBSSII expression was up-regulated. In order to verify the results of RNA-sequence, we checked the expression of some genes related to sugar and starch metabolism by using quantitative real time PCR, the results was similar to RNA sequence analysis.The expression of GBSSII, BGLU6, BGLU22, 1-SSTwere up-regulated while the expression of BGLU1, SSSI, FKI were down-regulated. Therefore, we speculated that the reduced amylose content had nothing to do with the expression of GBSSI in OsAT1 over expression transgenic plant, which might be related to the differentially expression of GBSSII, BGLU, FKI, SSSI and other genes. |
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