桑树MaUBC-C基因的表达模式分析及原核表达

Expression Pattern Analysis and Prokaryotic Expression of MaUBC-C Gene in Mulberry

泛素结合酶E2(ubiquitin-conjugating enzyme, UBC)在植物中具有重要的作用,参与植物的生长发育、逆境胁迫、免疫响应等生理活动。本课题组前期通过分析桑脉带相关病毒(Mulberry vein banding-associated virus, MVBaV)侵染的桑树品种‘桂桑优62号’(Morus atropurpurea)转录组获得了上调表达的基因MaUBC-C。为探究泛素结合酶MaUBC-C基因的功能,本研究以‘桂桑优62号’桑树为材料,克隆MaUBC-C的CDS序列进行生物信息学和表达模式分析,并对其进行原核表达获得该蛋白。结果显示,MaUBC-C编码区全长为567 bp,编码蛋白含188个氨基酸,大小为21.03 kDa,属于亲水、酸性、不稳定的核定位蛋白,具有保守的UBC结构域。将其氨基酸序列与其他物种的UBC氨基酸序列进行比对和进化分析,发现MaUBC-C与川桑MnUBC-C的相似性最高为98.40%,且具有最近的进化亲缘关系。荧光定量PCR分析MaUBC-C基因表达模式,结果显示MaUBC-C在桑苗的根、茎、叶中均有表达,其中在根部表达量最高,分别是茎和叶的1.24倍和1.71倍;对外源激素(ABA、GA、MeJA、SA)和非生物胁迫(低温、高温、高盐、干旱)处理均产生响应,且在ABA和MeJA处理下表现出显著上调,在24 h时的表达量分别为正常水平的29.55、9.05倍,推测MaUBC-C在桑树的生长发育中具有广泛的调控作用,参与桑树的激素信号转导和对非生物胁迫的防御反应。利用无缝克隆技术构建MaUBC-C原核表达载体pET-30a-MaUBC-C并转化大肠杆菌BL21,0.5 mmol/L IPTG在37℃条件下进行诱导,获得约为25 kDa的融合蛋白,与预期大小相符,为今后进行MaUBC-C基因功能研究提供了基础材料。本研究为进一步探究桑树泛素蛋白酶体途径的作用机制奠定了基础。

Ubiquitin-conjugating enzyme(E2)plays an important role in plants and participates in physiological activi-ties such as plant growth and development,stress,immune response and so on.In previous research,we profiled the transcriptome of‘Guisangyou 62’which infested with mulberry vein binding-associated virus(MVBaV)to obtain the up-regulated expression gene MaUBC-C.In order to explore the function of MaUBC-C,the CDS sequence of MaUBC-C was cloned from mulberry for bioinformatics and expression pattern analysis,and MaUBC-C was obtained by prokary-otic expression.The results showed that the coding region of MaUBC-C was 567 bp,coding 188 amino acids,and it was predicted to target the nuclear.MaUBC-C was 21.03 KDa and belonged to hydrophilic,acidic and unstable protein with a conserved UBC domain.Multiple sequence alignment and phylogenetic analysis of UBC in Morus atropurpurea and other species showed that MaUBC-C had the highest similarity with the homologous MnUBC from M.notabilis,which was 98.40%.The real-time fluorescence quantitative PCR data showed that MaUBC-C was expressed in roots,stems and leaves, and the highest expression was in roots, which were 1.24 and 1.71 times higher than those in stems and leaves, respectively. And we found MaUBC-C responded to differently to different (ABA, GA, MeJA, SA) and abiotic stress (low temperature, high temperature, high salt, drought). Among them, MaUBC-C was significantly up-regulated after treatment with ABA and MeJA, as the expression at 24 h was 29.55 and 9.05 times of the normal level, respectively. It is speculated that MaUBC-C may extensively regulate the growth and development of mulberry, participating in the hormonal signal transduction and defense responses to abiotic stresses. The prokaryotic expression vector pET-30a-MaUBC-C was constructed and transformed into E. coli BL21 to induce the expression of MaUBC-C with 0.5 mmol/L IPTG at 37℃. SDS-PAGE gel showed that a band with a size of about 25 kDa appeared from 2 h, the size was basically consistent with the expectation. And Western-blot showed that it could react specifically with His-tag monoclonal antibody indicating that MaUBC-C was successfully expressed, which provided basic materials for func-tional research of MaUBC-C. The results of this study would provide a theoretical basis for further exploring the mechanism of action of the ubiquitin proteasome pathway in mulberry.