发状念珠藻NfwspA3基因的克隆及其耐旱能力分析

Gene Cloning of NfwspA3 and Analysis of Its Drought Tolerance Capability in Nostoc flagelliforme

水分胁迫蛋白(WSP,Water stress protein,)在植物耐干旱过程中起着重要作用。发状念珠藻具有极强的耐旱、耐盐和抗辐射等能力。本研究以发状念珠藻基因组DNA为模板,利用PCR技术克隆到一个发状念珠藻水分胁迫蛋白基因的开放阅读框(ORF)序列,命名为NfwspA3,其序列全长1017 bp。生物信息学预测表明,该基因可编码338个氨基酸残基,预测等电点为4.85,分子量为37.48 kD,为亲水性稳定蛋白,分布在细胞质中。构建了NfwspA3基因的重组表达菌株pET32aNfwspA3,以聚乙二醇-6000(PEG-6000)对其模拟干旱胁迫,发现重组菌株最高可忍耐60%浓度的PEG-6000,表现出较强的耐旱性,且具有较高的半乳糖苷酶活性。研究结果为探知发状念珠藻WSP蛋白逆境响应机制及其遗传改良提供了理论依据和基因元件。

Water stress proteins play an important role in the process of drought tolerance in many plants.Nostoc flagelliforme showed strong tolerance to drought,salt and radiation stresses.In this study,using the genomic DNA of Nostoc flagelliforme as a template,the open reading frame(ORF)of a water stress protein gene was cloned by PCR amplification,and this gene was named NfwspA3 according to the online annotation via blastP in NCBI(https://www.ncbi.nlm.nih.gov/).Bioinformatic analysis showed that NfwspA3 gene contained 1017 base pair that encodes a putative protein consisted of 338 amino acid residues.The predicted isoelectric point was4.85 and the theoretical molecular weight was 37.48 kD.The encoded protein was predicted being a hydrophilic stable protein,and distributed in the cytoplasm.By analyzing the recombinant expression vector of pET32 aNfwspA3 under the condition of PEG-6000 simulating drought stress,it showed strong drought tolerance.Meanwhile,the recombinant expression strain showed higher activity of galactosidase.Thus,this work will provide a theoretical basis for detecting the molecular mechanism of water stress protein and a gene element that might be used for genetically modified breeding in Nostoc flagelliforme.