Nitrogen use efficiency （NUE） was very low in China and a loss of as much as 7070 of the applied nitrogen fertilizers was reported in high-yielding rice fields. In order to investigate the molecular basis of high-af...Nitrogen use efficiency （NUE） was very low in China and a loss of as much as 7070 of the applied nitrogen fertilizers was reported in high-yielding rice fields. In order to investigate the molecular basis of high-affinity ammonium transport or uptake into rice （Oryza sativa L.）, we analyzed the expression profiles of nine ammonium transporters （AMT）, three each of OsAMT1, OsAMT2 and OsAMT3, at two different N requirement stages （young seedling stage and tillering stage） of rice growth as well as the changes in these expression patterns according to external N status using real-time reverse transcription polymerase chain reaction （RT-PCR）. The results suggested that the nine OsAMT genes were expressed in different organs of rice plants, including mature roots, new roots, stems, old leaves and new leaves and that the expression patterns were organ specific and independent of the positions of the corresponding proteins in the phylogenetic tree. OsAMT1;1, 3;2 and 3;3 were expressed in the roots and shoots, primarily old leaves, OsAMT1;2 and 1;3 mainly in the roots, and OsAMT2;1, 2;2, 2;3 and 3;1 mainly in the shoots, primarily in new leaves, and relatively more in the stems than other genes. The expression patterns at the two different N requirement stages were the same; however, at the tillering stage with greater N requirements, the OsAMTs transcript levels were greater than those at the young seedling stage with low N requirements. N starvation for 48 h up-regulated OsAMT1;1, 1;2, 3;1, 3;2, 3;3 and down-regulated OsAMT1;3 mRNA abundance. Following N starvation, NH4＋ and NH4NO3 re-supply down-regulated OsAMT1;2 and 3;3 and up-regulated OsAMT1;3, whereas NO3 re-supply down-regulated OsAMT1;1 and 1;2. These suggested that the organ-specific expression pattern of OsAMT could be regulated by N requirement and external N status.展开更多
A pair of primers containing BamHI and XhoI sites was designed to amplify n gene of 1149bp from the positive pMD18-TN plasmid of transmissible gastroenteritis virus n gene by PCR.PCR product was subcloned into multipl...A pair of primers containing BamHI and XhoI sites was designed to amplify n gene of 1149bp from the positive pMD18-TN plasmid of transmissible gastroenteritis virus n gene by PCR.PCR product was subcloned into multiple cloning sites of pET-30a containing 6 His·Tag.Recombinant plasmid pET-30a of n gene named pET30a-N was transfected into E.coli BL21 and the bacteria was induced with IPTG at 37℃.It was demonstrated by SDS-PAGE that recombinant N protein expressed in E.coli BL21 was soluble protein and its molecular weight was about 52kD.The result of Western blot test showed that immunoactivity of the recombinant N protein was the same to that of the natural N protein.展开更多
基金Supported by the National Natural Science Foundation of China (No. 30800702)the National Basic Research Program of China (No. 2007CB109303)the National Key Technology R&D Program of China (No. 2012BAD15B03)
文摘Nitrogen use efficiency （NUE） was very low in China and a loss of as much as 7070 of the applied nitrogen fertilizers was reported in high-yielding rice fields. In order to investigate the molecular basis of high-affinity ammonium transport or uptake into rice （Oryza sativa L.）, we analyzed the expression profiles of nine ammonium transporters （AMT）, three each of OsAMT1, OsAMT2 and OsAMT3, at two different N requirement stages （young seedling stage and tillering stage） of rice growth as well as the changes in these expression patterns according to external N status using real-time reverse transcription polymerase chain reaction （RT-PCR）. The results suggested that the nine OsAMT genes were expressed in different organs of rice plants, including mature roots, new roots, stems, old leaves and new leaves and that the expression patterns were organ specific and independent of the positions of the corresponding proteins in the phylogenetic tree. OsAMT1;1, 3;2 and 3;3 were expressed in the roots and shoots, primarily old leaves, OsAMT1;2 and 1;3 mainly in the roots, and OsAMT2;1, 2;2, 2;3 and 3;1 mainly in the shoots, primarily in new leaves, and relatively more in the stems than other genes. The expression patterns at the two different N requirement stages were the same; however, at the tillering stage with greater N requirements, the OsAMTs transcript levels were greater than those at the young seedling stage with low N requirements. N starvation for 48 h up-regulated OsAMT1;1, 1;2, 3;1, 3;2, 3;3 and down-regulated OsAMT1;3 mRNA abundance. Following N starvation, NH4＋ and NH4NO3 re-supply down-regulated OsAMT1;2 and 3;3 and up-regulated OsAMT1;3, whereas NO3 re-supply down-regulated OsAMT1;1 and 1;2. These suggested that the organ-specific expression pattern of OsAMT could be regulated by N requirement and external N status.
文摘A pair of primers containing BamHI and XhoI sites was designed to amplify n gene of 1149bp from the positive pMD18-TN plasmid of transmissible gastroenteritis virus n gene by PCR.PCR product was subcloned into multiple cloning sites of pET-30a containing 6 His·Tag.Recombinant plasmid pET-30a of n gene named pET30a-N was transfected into E.coli BL21 and the bacteria was induced with IPTG at 37℃.It was demonstrated by SDS-PAGE that recombinant N protein expressed in E.coli BL21 was soluble protein and its molecular weight was about 52kD.The result of Western blot test showed that immunoactivity of the recombinant N protein was the same to that of the natural N protein.
