| 谢红艳，万鲁长，黄春燕，刘国霞，Jean-Francois Picimbon，宫志远,2017,烟粉虱MED隐种气味结合蛋白基因 BtabOBP2和BtabOBP4的cDNA克隆及原核表达[J].环境昆虫学报,(4):752-761 |
| 烟粉虱MED隐种气味结合蛋白基因 BtabOBP2和BtabOBP4的cDNA克隆及原核表达 |
| cDNA cloning and prokaryotic expression of odorant binding protein genes BtabOBP2 and BtabOBP4 from the Bemisia tabaci MED cryptic species |
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| DOI： |
| 中文关键词: 烟粉虱 气味结合蛋白 基因克隆 原核表达 纯化 |
| 英文关键词:Bemisia tabaci odorant binding protein (OBP) gene cloning prokaryotic expression purification |
| 基金项目:山东省农业科学院青年科研基金（2015YQN36）；山东省博士后创新项目专项资金（201203025）；山东省农业科学院农业科技创新工程（CXGC2017A01）山东省现代农业产业技术体系食用菌创新团队建设项目（SDAIT-07-01） |
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| 中文摘要: |
| 采用RT-PCR和RACE技术成功克隆烟粉虱Bemisia tabaci (Gennadius)气味结合蛋白（odorant binding protein, OBP）基因BtabOBP2和BtabOBP4的cDNA全长。BtabOBP2（GenBank 登录号：AIS71883）和BtabOBP4（GenBank 登录号：AIS71884）的cDNA序列全长分别为1107 bp和874 bp，完整开放阅读框（ORF）分别为744 bp和429 bp，分别编码247和142氨基酸，BtabOBP4有6个保守的半胱氨酸，属于典型OBP，而BtabOBP2除了具有典型OBP的6个半胱氨酸，增加了3个保守的半胱氨酸，属于Plus-C OBP。将得到的BtabOBP2和BtabOBP4重组到原核表达载体pET30a(+)，转化入BL21(DE3)大肠杆菌感受态细胞，用IPTG诱导融合蛋白表达。采用亲和层析和凝胶过滤层析纯化融合蛋白，并进行Western blot 分析。结果显示，BtabOBP2和BtabOBP4融合蛋白在大肠杆菌中均有可溶性表达，Western blot 结果证实所表达的融合蛋白确实为目的蛋白。BtabOBP2融合蛋白在SDS-PAGE中的表观分子量比预测的分子量大了6.53 kDa，用重组肠激酶切掉6×His标签后，目的蛋白的表观分子量与预测分子量相近，偏差降低，说明6×His标签是造成融合蛋白表观分子量偏差的原因。本研究明确了烟粉虱气味结合蛋白基因BtabOBP2和BtabOBP4的核苷酸、氨基酸序列特征，并成功进行了原核表达和纯化，为进一步研究这两个OBP基因的分子结构和功能奠定了基础。 |
| 英文摘要: |
| The full-length cDNAs of two odorant binding protein genes BtabOBP2 and BtabOBP4 from Bemisia tabaci (Gennadius)were cloned by using reverse transcription-polymerase chain reaction (RT-PCR)and rapid amplification of cDNA ends (RACE). The full-length cDNA sequences of BtabOBP2 (GenBank accession No. AIS71883)and BtabOBP4 (GenBank accession No. AIS71884)are 1107 bp and 874 bp, which contain 744 bp and 429 bp open reading frame encoding 247 and 142 amino-acid residues, respectively. The deduced amino acid sequence of BtabOBP4 contains six typical conservative cysteine residues, which are the hallmark of typical OBPs. Besides the six typical conservative cysteines, BtabOBP2 contains 3 more conservative ones and BtabOBP2 belongs to Plus-C OBP. BtabOBP2 and BtabOBP4 were then constructed into the pET30a(+)/BL21(DE3)prokaryotic expression vector and expressed with the induction of IPTG. The recombinant proteins were purified by affinity chromatography and gel filtration chromatography and analyzed by western blot. The results showed that recombinant proteins of BtabOBP2 and BtabOBP4 were expressed as soluble form in Escherichia coli.The results of western blot confirmed that the expressed recombinant proteins were the target proteins.The molecular weight of BtabOBP2 recombinant protein in SDS-polyacrylamide gel electrophoresis was 6.53 kDa higher than that of the value calculated from its amino acid sequence deduced from the encoding cDNA. After a 43 amino acid peptide including the 6×His tag in N-terminal was excised from the recombinant protein by digestion with the recombinant enterokinase, the deviation of molecular weight of this protein determined by SDS-PAGE was reduced, which indicated that the 6×His tag caused the deviation of the molecular weight. In this study, the sequence characteristics of nucleotides and amino acids of BtabOBP2 and BtabOBP4 were clarified. BtabOBP2 and BtabOBP4 recombinant proteins were successfully expressed and purified, which lay the foundations for studying the molecular structures and functions of BtabOBP2 and BtabOBP4. |
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