盐穗木HcUKPP原核表达及重组菌非生物胁迫耐受性研究

Procaryotic Expression of HcUKPP from Halostachys caspica and Analysis of Abiotic Stress Tolerance of the Recombinant Bacteria

该研究采用RT-PCR技术,从盐穗木cDNA文库中克隆获得未知功能多肽HcUKPP基因,构建了大肠杆菌Escherichia coli BL21∷pET30a-HcUKPP重组菌株,并检测了重组菌株在不同非生物胁迫下的耐受性。结果显示:HcUKPP基因开放阅读框为243bp,融合His的HcUKPP蛋白的分子量约为15kD。在37℃条件下,不同浓度的异丙基-β-D硫代半乳糖苷(IPTG)诱导4h后His-HcUKPP融合蛋白均可表达,且E.coli BL21∷pET30aHcUKPP重组菌在不同浓度NaCl(100~900mmol/L)、聚乙二醇(2.5%~20%,PEG 6000)和甲基紫精(25~200μmol/L)胁迫处理下,其生长均具有明显优势。尤其是在500mmol/L NaCl、10%PEG 6000和75μmol/L甲基紫精胁迫12h后,重组大肠杆菌BL21呈现出极显著的优势,分别达到了对照菌的1.81、1.47和3.48倍。研究表明,盐穗木HcUKPP可以显著提高重组大肠杆菌对不同非生物胁迫的耐受性,证明HcUKPP是一类新发现的能够响应非生物胁迫的多肽。

The recombinant strain Escherichia coli BL21∷pET30a-HcUKPP was obtained by the method of prokaryotic vector construction after cloning of unknown functional peptide HcUKPP gene,and then tested its tolerance under different abiotic stresses.The results revealed that open reading frame(ORF)of the gene was243bp,the molecular weight of the recombinant HcUKPP was approximately15kD.In addition,the expression of His-HcUKPP fusion protein could be induced with different concentrations of isopropylβ-D-1-thiogalactopyranoside(IPTG)for4h at37℃.Furthermore,the growth of recombinant strain E.coli BL21∷pET30a-HcUKPP have shown obvious advantages under treatment of different concentrations of NaCl(100-900mmol/L),polyethylene glycol(2.5%-20%,PEG6000)and methyl viologen(25-200μmol/L).Especially under the condition of500mmol/L NaCl,10%PEG6000and75μmol/L methyl viologen for12h,interestingly,recombinant E.coli BL21showed significant advantages,their growth reached1.81,1.47and3.48fold of control bacteria respectively.Overall,the HcUKPP gene of Halostachys caspica can significantly improve tolerance of recombinant E.coli BL21under different abiotic stresses,which proved that the HcUKPP is a kind of newly discovered polypeptide in response to abiotic stress.