甘蔗B家族蔗糖磷酸合成酶基因SofSPSB的克隆及原核表达

Cloning and prokaryotic expression of sucrose phosphate synthase gene (SofSPSB) in sugarcane

【目的】克隆甘蔗B家族SofSPSB基因并进行原核表达,为进一步研究甘蔗SPS酶学特性及SPS活性调控机制奠定基础。【方法】在进化分析的基础上,通过同源克隆获得甘蔗SofSPSB基因部分序列,再结合RACE技术获得全长cDNA序列。扩增SofSPSB基因ORF并连到原核表达载体pETBlue-2上,导入大肠杆菌BL21(DE3)中表达。【结果】通过比对B家族中进化关系很近的玉米(Zea mays)ZmSPS1和水稻(Oryza sativa)OsSPS1基因序列,并在保守区设计一对引物扩增获得甘蔗B家族SPS基因(SofSPSB)2330bp序列。结合5'-RACE和3'-RACE技术获得3481 bp SofSPSB基因全长cDNA序列,该序列包含一个3225bp的开放阅读框(ORF);起始密码子(ATG)位于转录起始位点后56bp处,终止密码子(TGA)后有一段201bp的非编码序列,并带有真核生物典型的polyA尾巴;编码1074个氨基酸,SofSPSB与Zm-SPS1、OsSPS1的核苷酸序列同源性分别为94.7%和81.3%,氨基酸序列同源性分别为96.0%和83.9%;其理论分子量Mw=118.96kDa,等电点pI=6.30。经原核表达后纯化获得带6×His标签的融合蛋白。【结论】克隆获得甘蔗B家族Sof-SPSB基因全长cDNA序列,成功构建了SofSPSB基因原核表达载体,使其在大肠杆菌BL21(DE3)中表达。

[Objective]Cloning and prokaryotic expression of SoPSPSB gene from sugarcane will lay a foundation for further study on the enzymatic characteristic and the regulation mechanism of SPS in sugarcane. [Method]On the basis of phylogenetic analysis, the partial sequence of SotSPSB was obtained by homologous cloning. Then, the full length cDNA sequence was ob- tained by RACE method. The ORF of SotSPSB was amplified and connected to pETBlue-2 to construct the prokaryotic expres- sion vectors, which was transfen＇ed into E.coli BL21 （DE3） for expression. [Result]The cDNA sequences of ZmSPS1 from maize （Zea mays） and OsSPS1 from rice （Oryza sativa） were aligned first, then a pair of primers was designed in conserved sequence region for amplification of partial sequence of SotSPSB. With this primer pair, a 2330 bp fragment was amplified and sequenced. According to this partial sequence of SotSPSB, a 3481 bp full length cDNA sequence was obtained by 5＇-RACE and 3＇-RACE. The full length cDNA contained a 3225 bp open reading frame （ORF） encoding a protein of 1074 amino acids. The start eodon （ATG） lied 56 bp after the transcription start site, and a 201 bp non-coding sequence with a typical polyA tail lied after stop codon （TAA） on the full length cDNA sequence （GenBank accession No. JN584485）. The theoretical molecular weight （MW） and isoelectric point （PI） of the protein were 118.96 kD and 6.30, respectively. The homology of nucleotide sequence between SotSPSB and ZmSPS1,0sSPS1 were 94.7% and 81.3%,respectively. The homology of amino acid sequence were 96.0% and 83.9%, respectively. After prokaryotic expression and purification, the fusion protein with the tag of 6~His was at- tained. [Conclusion]Full length cDNA sequence of SotSPSB was cloned from sugarcane B family. The prokaryotic expression vector of SotSPSB gene from sugarcane was constructed successfully and could be expressed in BL21 （DE3） of E.coli.