S-腺苷甲硫氨酸合成酶在大肠杆菌中的克隆与表达

目的:克隆与表达大肠杆菌S-腺苷甲硫氨酸合成酶的基因。方法:应用PCR技术从E.coliBL21(DE3)的总DNA中扩增出1.1kb的S-腺苷甲硫氨酸合成酶基因,将其连接到PGEMT载体上,测序证明正确后再将目的基因插入大肠杆菌高表达载体PET28a中,含有目的基因的重组质粒在大肠杆菌中进行表达与分析。结果:SAM合成酶在E.coliBL21(DE3)中的表达量达到23.6%,酶活达到4.17μmol/h.ml。结论:大肠杆菌表达出了具有生物活性的S-腺苷甲硫氨酸合成酶。     

烟草中一条新的S-腺苷甲硫氨酸合成酶基因的克隆及表达分析

利用差异显示技术从烟草中分离到一个受乙烯利诱导表达的cDNA片段,结合RACE扩增技术获得该基因的全长序列。该全长cDNA共含1350个碱基,编码区有1170 bp,通过GenBank同源性比较发现它与一条已知的烟草S-腺苷甲硫氨酸合成酶(SAMS1,EC2．5．1．6)的基因编码区存在90％的同源性,编码的氨基酸顺序96％同源,而在cDNA的5’及3’非编码区同源性很低,且有一些短片段的缺失。设计合适的引物分别对这两个SAMS基因进行RT-PCR分析表明,SAMS1基因受高温诱导表达增强,但不受甲基紫晶诱导的氧化胁迫及盐胁迫影响,而新分离到的这个基因(SAMS2)的表达同时受高温、甲基紫晶及高盐3种胁迫的诱导。     

红树植物杯萼海桑SAMS基因的克隆与生物信息学分析

红树植物杯萼海桑是最耐盐的红树植物之一。S-腺苷甲硫氨酸合成酶(S-adenosylmethionine synthetase,SAMS)是S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)生物合成途径的关键酶。SAMS作为一个逆境胁迫响应蛋白在植物的耐盐调控中发挥着极其重要的作用。本文结合杯萼海桑根的转录组注释,根据编码区序列设计引物,通过PCR克隆杯萼海桑SAMS基因的编码区cDNA,并对其进行生物信息分析,为研究杯萼海桑适应逆境的机制奠定理论基础。结果显示PCR扩增了一个长1 182 bp的基因片段,该片段编码由393个氨基酸组成的S-腺苷甲硫氨酸合成酶。同源性比对及进化树分析显示杯萼海桑的SAMS氨基酸序列进化上相对保守。本研究首次从红树林植物杯萼海桑中克隆S-腺苷甲硫氨酸合成酶基因,并获得其编码区序列,为进一步研究杯萼海桑应对逆境胁迫的分子生物学机制与胁迫相关基因调控网络奠定基础。     

多胺生物合成途径中两个关键酶基因研究进展

多胺是一类小分子生物活性物质,广泛存在于生物体内,与植物的生长发育、衰老及抗逆性都有着密切的联系。就多胺合成途径中的两个关键酶基因,即S-腺苷甲硫氨酸合成酶基因(SAMS)和S-腺苷甲硫氨酸脱羧酶基因(SAMDC)的克隆、表达,以及转S-腺苷甲硫氨酸合成酶基因(SAMS)和转S-腺苷甲硫氨酸脱羧酶基因(SAMDC)表达调控等方面的研究进行回顾总结,并对其应用前景进行展望。     

S-腺苷甲硫氨酸合成酶反应条件的优化

优化了重组毕赤酵母表达的S-腺苷甲硫氨酸合成酶催化L-甲硫氨酸(Met)和ATP合成 S-腺苷甲硫氨酸的条件,确定了该酶的最适酶活力检测条件为20mmol/L的L -Met,26mmol/ L的ATP,52mmol/L的MgCl2,300mmol/L的KCl,8mmol/L的还原型谷胱甘肽,100mmol/ L的Tris,反应液pH 8.5,35°C反应 1h,比活力达到23.84U/mg.该酶还可以催化以DL-Met代替L-Met为底物的S-腺苷甲硫氨酸合成反应,以降低生产成本.     

条斑紫菜SAMS基因克隆与生物信息学分析

干旱、高盐和低温是限制植物生长和农作物产量的主要逆境因子。S-腺苷甲硫氨酸合成酶（S-adenosylmethionine synthetase,SAMS）与植物抗逆境密切相关,而且超表达SAMS的转基因植物具有更高的抗干旱、高盐和低温能力。进行条斑紫菜（Porphyra yezoensis）S-腺苷甲硫氨酸合成酶基因（PySAMS）cDNA的克隆及序列特征分析。首先利用电子克隆技术得到基因相关的contig,预测全长并设计引物,最后利用RT-PCR技术成功克隆了PySAMS的cDNA序列（GenBank accession：FJ404748）。该cDNA序列含有长1155nt的完整ORF,编码蛋白（PySAMS）分子量41.8kDa,长384AA。PySAMS与盘基网柄菌（Dictyostelium discoideum）、莱茵衣藻（Chlamydomonas reinhardtii）、拟南芥（Arabidopsis thaliana）和水稻（Oryza sativa）等多种生物的SAMS具有较高的序列一致性,含有与SAMS酶活性密切相关的氨基酸残基和保守序列,与人（Homo sapiens）和大肠杆菌（Escherichia coli）的SAMS具有高度相似的三维结构。所以PySAMS与其它生物的SAMS一样,在条斑紫菜的抗逆过程中发挥重要作用。研究PySAMS有助于阐明条斑紫菜耐受非生物胁迫的作用机理,有助于获得高抗逆转基因植物。     

千里光S-腺苷甲硫氨酸合成酶(SAMS)的结构域与功能位点分析（英文）

基于前期研究工作中构建的千里光全长c DNA文库,我们分离到千里光S-腺苷甲硫氨酸合成酶(Sadenosylmethionine synthase,SAMS)基因,本研究分析了该基因开放阅读框,并对该多肽3个高度保守序列形成的结构域和功能位点之间的关系进行了探讨。结果表明,该基因(Gen Bank ID:KC149908.1)编码的蛋白质由394个氨基酸残基组成,理论等电点5.48,相对分子质量43.40 k D;结构域比对和3-D模型分析结果表明,α-helix/β-strand是该蛋白结构域的主要组分,且SPOUT结构与甲基转移酶反应密切相关,其功能可能涉及核酸、蛋白质和磷脂的合成,本研究揭示SAMS的氨基酸保守基序是形成高级结构并决定其生物学功能之关键所在。     

嗜热古菌S-腺苷甲硫氨酸合成酶的序列分析、基因克隆与异源表达

将Methanopyrus sp.SNP6进行功能基因组测序,获得S-腺苷甲硫氨酸合成酶基因(sam)序列,并进行序列分析。将sam基因扩增后连接至表达质粒p ET-28b(+),转化E.coli BL21(DE3),获得重组菌后进行诱导表达。生物信息学分析表明,sam基因编码蛋白的理论分子量为44 086.4Da,三级结构为同源四聚体,与其他相关古菌来源的S-腺苷甲硫氨酸合成酶的蛋白序列较保守。实验结果显示,构建的重组表达质粒p ET28b(+)-sam可在E.coli BL21(DE3)宿主菌中高水平表达。重组蛋白的分子量与预期值基本一致,部分为胞内可溶性表达,另一部分以包涵体形式存在。本研究首次实现了Methanopyrus sp.SNP6菌株S-腺苷甲硫氨酸合成酶的异源表达,为后期的蛋白纯化、酶学性质研究和酶促转化法生产S-腺苷甲硫氨酸奠定了理论基础。     

刚毛柽柳S-腺苷甲硫氨酸合成酶(ThSAMS)基因的克隆及表达分析

通过对刚毛柽柳转录组分析,克隆获得了一条与S-腺苷甲硫氨酸合成酶(SAMS)基因同源性高的基因,命名为ThSAMS。序列分析结果表明:ThSASM基因全长cDNA为1185bp,编码394个氨基酸,编码蛋白相对分子质量为97.85kDa,理论等电点为5.02。通过生物信息学分析表明,ThSASM基因编码的氨基酸与其他物种SAMS基因编码的氨基酸具有很高的同源性,其中与枣的同源性最高,达95%。实时荧光定量PCR(quantitativereal-timePCR,qRT-PCR)分析表明,ThSASM表达受NaCl、聚乙二醇(PEG)和ABA处理做出应答,暗示ThSASM可能参与了刚毛柽柳对盐和干旱的胁迫应答,为进一步研究SAMS基因在植物胁迫应答中的功能及作用机制提供了参考依据。     

硫腺苷甲硫氨酸合成酶基因的克隆及其在盐胁迫条件下的不同表达

硫腺苷甲硫氨酸作为甲基供体在转甲基反应中起到重要作用。为了解硫腺苷甲硫氨酸在盐地碱蓬(Suaeda salsa (L.) Pall)耐盐中的作用,我们对可能编码硫腺苷甲硫氨酸合成酶的基因(SsSAMS2)进行了分析.该基因在经400mmol/L NaCl处理的盐地碱蓬地上部分的λ-Zap cDNA文库中克隆到,其播入片段全长1531bp,包含一个395个氨基酸的开放阅读框架,该基因推断的分子量约为43kD.SsSAMS2与长春花(Catharanthus roseus)的SAMS2在氨基酸水平上的一致性为93%.Southern杂交显示,SsSAMS2在盐地碱蓬基因组中可能是两个拷贝.Northern分析显示硫腺苷甲硫氨酸合成酶基因受NaCl等胁迫的正调控.酶活性检测表明,NaCl胁迫条件下该酶活性增强.     

盐胁迫下过量表达腺苷甲硫氨酸合成酶基因的转基因烟草的生长

以转腺苷甲硫氨酸合成酶基因（SsSAMS2）烟草纯合子ST8-9为实验材料,研究盐胁迫下过量表达SsSAMS2对转基因烟草生长影响的结果表明,200mmol·L-1NaCl处理后的转基因烟草的光合速率和生物量都比野生型烟草高,积累的自由多聚胺比较多,同时精氨酸脱羧酶的转录本也更丰富。显示转基因烟草的耐盐性比野生型烟草高,多聚胺在烟株缓解盐胁中可能是起了重要作用。     

过量表达腺苷甲硫氨酸合成酶基因能提高转基因烟草中多聚胺的生物合成

以土壤农杆菌介导的转化方法将盐地碱蓬的腺苷甲硫氨酸合成酶cDNA（SsSAMS2）转化到烟草K326中。PCR分析的结果表明,SsSAMS2整合进K326的基因组内。共筛选到8个转基因纯合品系,分析其中3个品系（ST8．9、ST14—2、ST3．5）基因表达和多聚胺含量的结果表明,SsSAMS2可在转基因烟草中表达,转基因烟草中的多聚胺含量明显高于野生型烟草。这些结果表明,腺苷甲硫氨酸合成酶基因已在转基因烟草中过量表达并导致多聚胺含量提高。     

S-腺苷甲硫氨酸合成酶对埃博霉素生物合成的影响

【目的】研究S-腺苷甲硫氨酸合成酶(SAMs)对埃博霉素生物合成的影响。【方法】通过向发酵培养基中添加抑制剂和促进剂,比较分析纤维堆囊菌中SAMs的活性变化以及埃博霉素的产量变化。【结果】在埃博霉素的合成期,SAMs的活性较高。加入抑制剂吲哚乙酸(IAA)之后,SAMs的活性和埃博霉素的产量都不同程度的降低,而加入促进剂对甲苯磺酸钠(p-TSA-Na)之后,SAMs的活性和埃博霉素的产量在不同程度上都有提高。在纤维堆囊菌的次级代谢中,SAMs活性与埃博霉素的生物合成量呈正相关。【结论】S-腺苷甲硫氨酸合成酶在纤维堆囊菌的埃博霉素生物合成过程中发挥了重要的作用。     

Penicillium oxalicum S-adenosylmethionine synthetase is essential for the viability of fungal cells and the expression of genes encoding cellulolytic enzymes

As the universal methyl donor for methylation reactions, S-adenosylmethionine (AdoMet) plays an indispensable role in most cellular metabolic processes. AdoMet is synthesized by AdoMet synthetase. We identified the only one AdoMet synthetase (PoSasA) in filamentous fungus Penicillium oxalicum. PoSasA was widely distributed in mycelium at different growth stages. The absence of PoSasA was lethal for P. oxalicum. The misregulation of the PoSasA encoding gene affected the synthesis of extracellular cellulolytic enzymes. The expression levels of cellobiohydrolase encoding gene cbh1/cel7A, β-1-4 endoglucanase eg1/cel7B, and xylanase encoding gene xyn10A were remarkably downregulated as a result of decreased PosasA gene expression. The production of extracellular cellulases and hemicellulases was also reduced. By contrast, the overexpression of PosasA improved the production of extracellular cellulases and hemicellulases. A total of 133 putative interacting proteins with PoSasA were identified using tandem affinity purification and mass spectrometry. The results of functional enrichment on these proteins showed that they were mainly related to ATP binding, magnesium ion binding, and ATP synthetase activity. Several methyltransferases were also observed among these proteins. These results were consistent with the intrinsic feature of AdoMet synthetase. This work reveals the indispensable role of PoSasA in various biological processes.     

Evidence for Symbiont-induced Alteration of a Host's Gene Expression: Irreversible Loss of SAM Synthetase from Amoeba proteus

ABSTRACT. Symbiont-bearing xD amoebae no longer produce a 45-kDa cytoplasmic protein that functions as S-adenosylmethionine synthetase in symbiont-free D amoebae. The absence of the protein in xD amoebae is attributable to xD amoeba's failure to transcribe the corresponding gene as a result of harboring bacterial symbionts. However, xD amoebae have about half the level of enzyme activity found in D amoebae, indicating that they use an alternative source for the enzyme. xD amoebae originated from D amoebae by bacterial infection and now depend on their symbionts for survival. xD amoebae exhibit irreversible nucleolar abnormalities when their symbionts are removed, suggesting that X-bacteria supply the needed enzyme. A monoclonal antibody against the 45-kDa protein was produced and used as a probe in cloning its corresponding cDNA. The product of the cDNA was found to have S-adenosylmethionine synthetase activity. These results show how symbiotic X-bacteria may become essential cellular components of amoebae by supplementing a genetic defect for an amoeba's house-keeping gene that is brought about by an action of X-bacteria themselves. This is the first reported example in which symbionts alter the host's gene expression to block the production of an essential protein.     

Enhancing macrolide production in Streptomyces by coexpressing three heterologous genes

Antibiotic production in Streptomyces can often be increased by introducing heterologous genes into strains that contain an antibiotic biosynthesis gene cluster. A number of genes are known to be useful for this purpose. We chose three such genes and cloned them singly or in combination under the control of the strong constitutive ermE* promoter into a ?C31-derived integrating vector that can be transferred efficiently by conjugation from Escherichia coli to Streptomyces. The three genes are adpA, a global regulator from Streptomyces coelicolor, metK, encoding S-adenosylmethionine synthetase from S. coelicolor, and, VHbS, hemoglobin from Vitreoscilla. The substitutions with GC in VHbS was intended to convert codons from lower usage to higher, yet causing no change to the encoded amino acid. Plasmids containing either one of these genes or genes in various combinations were introduced into Streptomyces sp. FR-008, which produces the macrolide antibiotic FR-008-III (also known as candicidin D). The largest increase in FR-008-III production was achieved by the plasmid containing all three genes. This plasmid also increased avermectin production in Streptomyces avermitilis, and is likely to be generally useful for improving antibiotic production in Streptomyces.