Cloning and expression analysis of PpSUT2 encoding a sucrose transporter in pear

文献类型： 外文期刊

第一作者： Tang, J.

作者： Tang, J.;Lin, J.;Zhang, B. L.;Wang, Z. H.;Li, X. G.;Chang, Y. H.;Tang, J.

作者机构：

关键词： Sucrose transporter;PpSUT2;Subcellular localization;Real-time fluorescent quantitative polymerase chain reaction;Prokaryotic expression

期刊名称：GENETICS AND MOLECULAR RESEARCH （ 影响因子：0.764； 五年影响因子：0.912 ）

ISSN： 1676-5680

年卷期： 2014 年 13 卷 4 期

页码：

收录情况： SCI

摘要： A 1794-bp cDNA fragment was amplified from mRNA isolated from pear (Pyrus pyrifolia NaKai. Cuiguan) leaves by using primers based on the sequences generated during the analysis of the pear transcriptome. The 597-amino acid sequence encoded by the cDNA was compared with the sequences in GenBank, and it was found to be similar to that of members of the sucrose-proton cotransporter family. The hydrophobic protein, which was predicted to have 11 transmembrane domains, was designated as PpSUT2. Real-time fluorescent quantitative polymerase chain reaction analysis indicated the accumulation of PpSUT2 mRNA throughout the plant, with the highest levels in the buds. Analysis of the expression of PpSUT2 during fruit development showed that the abundance of its transcripts increased at the end of April and then decreased to the lowest level at the end of July. Subcellular localization studies with the pCXDG vector as a probe demonstrated that PpSUT2 localized to cell membranes. An expression vector was constructed by inserting the PpSUT2 cDNA into pET32(a), and the vector was expressed in Escherichia coli (strain BL21) after induction with 1 mM isopropyl beta-D-1-thiogalactopyranoside at 25 degrees C. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified the induction of a 71-kDa protein. Further analysis indicated that PpSUT2 might be not directly involved in sucrose transport, instead, functioning as a sucrose sensor on the cytoplasmic membrane.

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