甘蔗B家族蔗糖磷酸合成酶基因SotSPSB的克隆及原核表达

【目的】克隆甘蔗B家族s0心PsB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（Zeamays）ZmSPS1和水稻（Oryzasativa）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（SolSPSB）2330bp序列。结合5’－RACE和3’－RACE技术获得3481 b...

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Bibliographic Details
Published in南方农业学报 Vol. 44; no. 4; pp. 545 - 551
Main Author 黄东亮 秦翠鲜 陈忠良 桂意云 李双喜 汪淼 廖青 李杨瑞
Format Journal Article
LanguageChinese
Published 2013
Subjects
SofSPSB
克隆
原核表达
甘蔗
蔗糖磷酸合成酶
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Summary:【目的】克隆甘蔗B家族s0心PsB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（Zeamays）ZmSPS1和水稻（Oryzasativa）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（SolSPSB）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（0I江）；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6xHis标签的融合蛋白。【结论】克隆获得甘蔗B家族Sol-SPSB基因全长cDNA序列，成功构建了SoPSPSB基因原核表达载体，使其在大肠杆菌BL21（DE3）中表达。
Bibliography:[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 read
ISSN:2095-1191