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甘蔗蔗糖磷酸合成酶基因(SofSPSA)的克隆及正反义植物表达载体的构建
黄东亮1,方锋学2,黎涛2,李双喜2,廖青2,李杨瑞3
1. 广西甘蔗研究所
2.
3. 广西农科院
Cloning of Sucrose Phosphate Synthase Gene (SofSPSA) from Sugarcane and Construction of Its Sense and Antisense Plant Expression Vector
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摘要 蔗糖磷酸合成酶(SPS)是植物体内控制蔗糖合成的关键酶。本研究通过搜索NCBI数据库获得迄今植物中所有的蔗糖磷酸合成酶基因序列共77条,其中全长序列43条;对全长序列进行进化分析表明,植物SPS基因分为A、B、C和D四个家族,同一植物体内有多个分别属于不同家族的SPS基因;比对A家族中进化关系很近的甘蔗(Saccharum officinarum)SofSPSII、水稻(Oryza sativa)OsSPS4、黑麦草(Lolium perenne)LpSPS和竹子(Bambusa oldhamii)BoSPS基因序列,并在保守区设计一对引物扩增获得甘蔗A家族SPS基因(SofSPSA)2 553 bp序列,结合5'-RACE和3'-RACE技术获得甘蔗SofSPSA基因全长cDNA序列3 906 bp;该序列包含一个3 183 bp的开放阅读框(ORF),编码1 060个氨基酸残基的蛋白,其理论分子量和等电点分别为118.4 kD和6.09。该序列的起始密码子(ATG)位于转录起始位点后359 bp处,终止密码子(TAA)后还有一段365 bp的非编码序列,并带有真核生物典型的polyA尾巴 (GenBank 登录号:HM854011);SofSPSA与SofSPSII、OsSPS4、LpSPS、BoSPS的氨基酸相似性分别为99.3%、91.8%、91.6%和91.9%,一致性分别为99.2%、87.9%、87.5%和87.8%;设计引物分别扩增正、反向SofSPSA基因ORF并分别连到pBI121上获得由组成型表达启动子35S驱动的正、反义植物表达载体pBI-SofSPSA和pBI-anti- SofSPSA,为进一步研究甘蔗SPS基因的生物学功能及利用甘蔗SPS基因改良作物品种提供科学依据。
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黄东亮
方锋学
黎涛
李双喜
廖青
李杨瑞
关键词 甘蔗蔗糖磷酸合成酶植物表达载体    
Abstract:Sucrose phosphate synthase (hereinafter as SPS) is the key enzyme that controls the sucrose biosynthesis in plants. By searching NCBI GenBank, 77 SPS genes identified to date from plants were obtained, in which 43 were in full length sequences. By phylogenetic analysis of the 43 full length sequences, the SPS in plants were classed into four families, named A, B, C and D family, respectively, and there were several SPS genes in the same plant belonging to different families. By alignment of the four genes, SofSPSII from sugarcane(Saccharum officinarum), OsSPS4 from rice(Oryza sativa), LpSPS from ryegrass (Lolium perenne) and BoSPS from bamboo(Bambusa oldhamii) had very close phylogenetic relation. A pair of primers were designed in conserved sequence region to amplify partial sequence of SofSPSA. With the primers, a 2 553 bp fragment was amplified and sequenced. According to this partial sequence of SofSPSA, a 3 906 bp full length cDNA sequence was obtained by 5'-RACE and 3'-RACE. The full length cDNA contained a 3 180 bp open reading frame (ORF) encoding a protein of 1 060 amino acids. The theoretical molecular weight and isoelectric point (PI) of the protein were 118.4 kD and 6.09, respectively. The start codon (ATG) lies 359 bp after the transcription start site, and a 364 bp non-coding sequence with a typical polyA tail lies after stop codon (TAA) on the full length cDNA sequence (GenBank accession No. HM854011). The amino acid sequense of SofSPSA showed 99.3%, 91.8%, 91.6% and 91.9% similarity, and 99.2%, 87.9%, 87.5% and 87.8% identify to that of SofSPSII, OsSPS4, LpSPS and BoSPS, respectively. Finally, sense and antisense SofSPSA gene ORFs were amplified and connected to pBI121 to construct the plant expression vectors, pBI-SofSPSA and pBI-anti-SofSPSA, respectively. This work will lay a foundation for further study on the biological function of SPS from sugarcane and variety impovement by transfer of sugarcane SPS gene.
Key wordsSugarcane    Sucrose Phosphate Synthase (SPS)    Plant Expression Vector
收稿日期: 2010-10-27     
通讯作者: 黄东亮   
引用本文:   
黄东亮1,方锋学2,黎涛2,李双喜2,廖青2,李杨瑞3. 甘蔗蔗糖磷酸合成酶基因(SofSPSA)的克隆及正反义植物表达载体的构建[J]. , 2011, 19(4): 624-633.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2011/V19/I4/624
 
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