title: Cloning and expression analysis of PpSUT2 encoding a sucrose transporter in pear.
abstract: 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 co-transporter 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 b-d-1-thiogalactopyranoside at 25°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.
authors.affiliation: Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
authors.forename: J
authors.initials: J
authors.lastname: Tang
authors.affiliation: Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
authors.forename: J
authors.initials: J
authors.lastname: Lin
authors.affiliation: Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
authors.forename: B L
authors.initials: BL
authors.lastname: Zhang
authors.affiliation: Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
authors.forename: Z H
authors.initials: ZH
authors.lastname: Wang
authors.affiliation: Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
authors.forename: X G
authors.initials: XG
authors.lastname: Li
authors.affiliation: Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Nanjing, China cyh@jaas.ac.cn.
authors.forename: Y H
authors.initials: YH
authors.lastname: Chang
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005638:Fruit / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D057927:Hydrophobic and Hydrophilic Interactions; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018519:Meristem / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D031989:Pyrus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012621:Seasons; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D026901:Membrane Transport Proteins; D010940:Plant Proteins; C078729:sucrose transport protein, plant; D013395:Sucrose