白木香AsMYC2蛋白的原核表达与纯化

Prokaryotic expression and purification of Aquilaria sinensis(Lour.) Gilg AsMYC2 protein

MYC2转录因子属于bHLH转录因子家族中重要的一员,它是植物茉莉酸(JA)信号途径中的核心调控元件之一,但在白木香中研究甚少。本实验以白木香叶片中分离的总RNA为模板,通过RT-PCR方法获得AsMYC2基因的编码区(CDS)全长,利用基因重组技术构建pGEX-4T-1-AsMYC2原核表达载体,并进行酶切和测序鉴定,将鉴定正确的克隆转化大肠杆菌BL21(DE3),在37℃下0.1 mmol·L^(-1)异丙基硫代β-D-半乳糖苷(IPTG)诱导4 h,AsMYC2蛋白的表达量最高,且主要为可溶性蛋白。利用谷胱甘肽亲和介质进行融合蛋白的纯化,最后应用SDS-PAGE电泳和Western blotting鉴定融合蛋白。结果表明,成功构建了pGEX-4T-1-AsMYC2原核表达载体,诱导了GST-AsMYC2(谷胱甘肽巯基转移酶-AsMYC2)融合蛋白的表达并进行了蛋白的纯化。高质量的GST-AsMYC2融合蛋白的获得,为多克隆抗体的制备提供材料基础,为筛选其互作蛋白因子和研究基因功能奠定基础。利用实时荧光定量PCR(qRT-PCR)技术对该基因进行组织表达特异性分析,结果显示AsMYC2基因在沉香形成的主要部位根和茎中的表达量最高,在叶中的表达量最低;该结果提示该基因可能与白木香中沉香的形成有关。

The MYC2 transcription factor is a member of the important plant b HLH transcription factor families, and it is also the core regulatory elements in jasmonate（JA） signaling pathway. However, there is a little information about AsMYC2 gene in Aquilaria sinensis. In this study, with the total RNA isolated from A. sinensis leave as template, the full-length coding sequence（CDS） of AsMYC2 gene was amplified using RT-PCR method and subcloned into pGEX-4T-1 vector by the gene recombination technique. The recombinant vector pGEX-4T-1-AsMYC2 was verified by restriction enzyme digestion and nucleotide sequencing, and was transformed into E. coli BL21（DE3） to express the protein. A maximum expression of soluble protein was observed with induction by 0.1 mmol·L^（-1） IPTG at 37 ℃ for 4 hours. The fusion protein was purified through a Sepharose-Glutathione column, and verified by SDS-PAGE and Western blotting using an anti-GST polyclonal antibody. We successfully constructed the GST-AsMYC2 plasmid, produced and purified the GST-AsMYC2 fusion protein, which would provide the basic material for polyclonal antibody preparation, interactive factors screening and gene function research. According to the tissue-specific expression pattern analysis by q RT-PCR method, the AsMYC2 gene in A. sinensis tissues is mainly expressed in roots and stems, the main agarwood formation parts, and lowest expressed in leaves. These results indicate that AsMYC2 gene likely play some roles in agarwood formation in A. sinensis.