發(fā)布時(shí)間：2018-04-10 22:51

  本文選題：乙酰輔酶A合成酶 + 特氏杜氏藻　； 參考：《華南理工大學(xué)》2016年碩士論文

【摘要】：乙酰輔酶A合成酶(ACS)催化乙酰輔酶A的合成,它是油脂代謝和醋酸鹽代謝的重要節(jié)點(diǎn)之一。據(jù)報(bào)道,過表達(dá)ACS會(huì)導(dǎo)致醋酸鹽大量被轉(zhuǎn)化為acetyl-CoA,從而支持后續(xù)的糖酵解途徑、脂肪酸代謝、氨基酸代謝和糖異生作用,并加速細(xì)胞生長(zhǎng)和物質(zhì)積累。本研究以特氏杜氏藻(Dunaliella tertiolecta)為主要研究對(duì)象,首先利用反轉(zhuǎn)錄PCR(RT-PCR)技術(shù)得到了長(zhǎng)度為1175 bp的DtACS EST序列,該片段經(jīng)Blast序列比對(duì)推斷為DtACS基因片段。然后通過cDNA末端快速擴(kuò)增(RACE)技術(shù)獲得了長(zhǎng)為790 bp的DtACS 5’端序列和長(zhǎng)為784 bp的DtACS 3’端序列,經(jīng)序列拼接得到DtACS的全長(zhǎng)cDNA序列為2464 bp,包含長(zhǎng)為2184 bp的DtACS ORF序列,編碼727個(gè)氨基酸。經(jīng)氨基酸序列同源性分析,發(fā)現(xiàn)DtACS與綠藻ACS最為相似(與衣藻Chlamydomonas reinhardtii有68%一致;與團(tuán)藻Volvox carteri f.nagariensis有70%一致)。生物信息學(xué)分析結(jié)果顯示DtACS分子量為79.72 kDa,等電點(diǎn)為6.7,其不穩(wěn)定指數(shù)預(yù)測(cè)為36.57,屬于穩(wěn)定蛋白質(zhì)。其主要的結(jié)構(gòu)域有ACS、AMP-binding_C、PRK00174、Ac_CoA_lig_AcsA以及AMP-binding。系統(tǒng)發(fā)生樹顯示Dt ACS與綠藻ACS和鏈形植物的ACS都較為親近。選用帶有硫氧還原蛋白標(biāo)簽(Trx-tag)的pET32a(+)作為原核表達(dá)載體,并將DtACS轉(zhuǎn)入pET32a(+)中從而構(gòu)建了pET-32a(+)-DtACS質(zhì)粒。將其轉(zhuǎn)入BL21(DE3)感受態(tài)細(xì)胞中,同時(shí)將pET-32a(+)空載體也轉(zhuǎn)入BL21(DE3)感受態(tài)細(xì)胞中,分別得到重組菌pET-32a(+)-DtACS-BL21(DE3)和對(duì)照菌種pET-32a(+)-BL21(DE3)。將重組菌在18℃、終濃度為0.6 mmol/L的IPTG條件下誘導(dǎo)12 h,表達(dá)出帶有Trx和His標(biāo)簽融合蛋白的DtACS,SDS-PAGE檢測(cè)出其分子量約為100 kDa,與預(yù)測(cè)的分子量(79.72 kDa+20 kDa,標(biāo)簽蛋白約為20 kDa)一致。此外,將表達(dá)得到的重組蛋白經(jīng)Ni2+柱純化,純化后蛋白比活力為52.8725 U/mg。乙酸鉀為底物時(shí),DtACS的Km值為3.597 mM,Vmax為61.73 U/mg。乙酸鈉為底物時(shí),DtACS的Km值為4.673 mM,Vmax為59.88 U/mg。DtACS的最適pH為8,最適溫度為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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