Dunaliella tertiolecta中乙酰CoA合成酶的基因克

发布时间：2018-04-10 22:51

  本文选题：乙酰辅酶A合成酶 + 特氏杜氏藻　； 参考：《华南理工大学》2016年硕士论文

【摘要】：乙酰辅酶A合成酶(ACS)催化乙酰辅酶A的合成,它是油脂代谢和醋酸盐代谢的重要节点之一。据报道,过表达ACS会导致醋酸盐大量被转化为acetyl-CoA,从而支持后续的糖酵解途径、脂肪酸代谢、氨基酸代谢和糖异生作用,并加速细胞生长和物质积累。本研究以特氏杜氏藻(Dunaliella tertiolecta)为主要研究对象,首先利用反转录PCR(RT-PCR)技术得到了长度为1175 bp的DtACS EST序列,该片段经Blast序列比对推断为DtACS基因片段。然后通过cDNA末端快速扩增(RACE)技术获得了长为790 bp的DtACS 5’端序列和长为784 bp的DtACS 3’端序列,经序列拼接得到DtACS的全长cDNA序列为2464 bp,包含长为2184 bp的DtACS ORF序列,编码727个氨基酸。经氨基酸序列同源性分析,发现DtACS与绿藻ACS最为相似(与衣藻Chlamydomonas reinhardtii有68%一致;与团藻Volvox carteri f.nagariensis有70%一致)。生物信息学分析结果显示DtACS分子量为79.72 kDa,等电点为6.7,其不稳定指数预测为36.57,属于稳定蛋白质。其主要的结构域有ACS、AMP-binding_C、PRK00174、Ac_CoA_lig_AcsA以及AMP-binding。系统发生树显示Dt ACS与绿藻ACS和链形植物的ACS都较为亲近。选用带有硫氧还原蛋白标签(Trx-tag)的pET32a(+)作为原核表达载体,并将DtACS转入pET32a(+)中从而构建了pET-32a(+)-DtACS质粒。将其转入BL21(DE3)感受态细胞中,同时将pET-32a(+)空载体也转入BL21(DE3)感受态细胞中,分别得到重组菌pET-32a(+)-DtACS-BL21(DE3)和对照菌种pET-32a(+)-BL21(DE3)。将重组菌在18℃、终浓度为0.6 mmol/L的IPTG条件下诱导12 h,表达出带有Trx和His标签融合蛋白的DtACS,SDS-PAGE检测出其分子量约为100 kDa,与预测的分子量(79.72 kDa+20 kDa,标签蛋白约为20 kDa)一致。此外,将表达得到的重组蛋白经Ni2+柱纯化,纯化后蛋白比活力为52.8725 U/mg。乙酸钾为底物时,DtACS的Km值为3.597 mM,Vmax为61.73 U/mg。乙酸钠为底物时,DtACS的Km值为4.673 mM,Vmax为59.88 U/mg。DtACS的最适pH为8,最适温度为37℃。
[Abstract]:Acetyl coenzyme A synthase (ACSs) catalyzes the synthesis of acetyl coenzyme A, which is one of the important nodes of lipid metabolism and acetate metabolism.It is reported that overexpression of ACS can result in the conversion of acetate to acetyl-CoA, which supports the subsequent glycolysis pathway, fatty acid metabolism, amino acid metabolism and glycosylation, and accelerates cell growth and substance accumulation.In this study, Dunaliella tertiolecta (Dunaliella tertiolecta) was used as the main research object. Firstly, a 1175 BP DtACS EST sequence was obtained by reverse transcription PCR RT PCR technique, which was deduced to be a DtACS gene fragment by Blast sequence alignment.The 790bp DtACS 5'terminal sequence and the DtACS 3'terminal sequence (784bp) were obtained by rapid amplification of the cDNA terminal. The full-length cDNA sequence of DtACS was 2464 BP, including the DtACS ORF sequence of 2184 BP.Encoding 727 amino acids.The amino acid sequence homology analysis showed that DtACS was most similar to ACS (68% consistent with Chlamydomonas reinhardtii and 70% consistent with Volvox carteri f.nagariensis).Bioinformatics analysis showed that the molecular weight of DtACS was 79.72 kDa, the isoelectric point was 6.7, and its instability index was 36.57, which was a stable protein.The main domains are ACSA AMP-bindingsCPRK00174Acs / CoAlig-AcsA and AMP-binding.The main domains are ACSA AMP-bindingsCPRK00174Actit CoAlig-AcsA and AMP-binding.Phylogenetic tree showed that Dt ACS was close to ACS and ACS of chain plant.PET32a () with thioxy-reducing protein tag Trx-taga () was selected as prokaryotic expression vector, and DtACS was transferred into pET32a () to construct pET-32a (DtACS) plasmid.The pET-32a (pET-32a () empty vector was also transferred into BL21DE-3) and the recombinant strain pET-32a (DtACS-BL21DE3) and the control strain pET-32a (BL21DE3) were obtained respectively.When the recombinant strain was induced at 18 鈩，

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