小麦蛋白激酶TaMAPK2互作蛋白的筛选与验证

Screening and Identification of Proteins Interacting with TaMAPK2 in Wheat

【目的】利用构建的干旱胁迫处理的小麦cDNA文库,通过酵母双杂系统筛选与小麦TaMAPK2互作的蛋白,并对其进行验证分析。【方法】以小麦cDNA为模板克隆得到TaMAPK2,构建pGBKT7-TaMAPK2诱饵载体,将诱饵载体pGBKT7-TaMAPK2质粒以及pGADT7和小麦cDNA文库共转化酵母AH109菌株感受态细胞,在SD/-Trp/-Leu/-His/-Ade营养缺陷型培养基上30℃培养3—5 d,挑选单克隆于YPDA培养基中培养,吸取1μL各候选克隆的菌液点至于SD/Raf/Gal/x-gal平板上培养,筛选蓝色单克隆。将筛出的单克隆经测序、序列比对分析,初步获得与TaMAPK2互作的候选蛋白。采用双酶切的方法构建pGADT7-HSP90以及pSPYNE-TaMAPK2和pSPYCE-HSP90双分子荧光互补表达载体,将重组载体质粒pGADT7-HSP90和pGBKT7-TaMAPK2共转化AH109酵母感受态细胞,利用酵母双杂交的方法验证TaMAPK2与候选蛋白HSP90的相互作用;采用PEG-Ca2+介导法将双分子荧光互补表达载体pSPYNE-TaMAPK2和pSPYCE-HSP90共转化小麦原生质体细胞,激光共聚焦显微镜下观察相互作用产生的YFP荧光信号。根据HSP90的保守序列设计特异引物,在SYBR GreenⅠ的检测模式基础上,构建HSP90的实时荧光定量PCR检测体系,检测HSP90在不同胁迫处理以及不同组织的小麦植株中的表达量。【结果】通过对候选蛋白的初步分析表明这些候选蛋白主要参与植物体的信号转导或免疫过程,表明TaMAPK2在植物的逆境信号转导、基因转录调控过程中发挥着重要作用。通过酵母双杂交显蓝系统和双分子荧光互补技术(BiFc)进一步确认HSP90与TaMAPK2的互作关系。HSP90定位在细胞核、细胞膜以及细胞质中,且在植物的根茎叶中均有表达,在根中的表达量最高。通过实时荧光定量PCR分析显示,HSP90基因受到高温、低温、高盐和ABA胁迫后表达量上调。【结论】HSP90作为分子伴侣,在降解这些受损或异常蛋白以及调控抗逆相关转录因子表达的过程中发挥着重要的作用。非生物胁迫会引起植物体内一些受损或异常蛋白聚合物的积累,HSP90能通过折叠已损坏的蛋白底物以及构象的维持来保持细胞的完整性。表明在植物逆境信号转导过程中,TaMAPK2功能的发挥可能需要HSP90蛋白的参与。

[Objective]Mitogen-activated protein kinases（MAPKs） play an important role in stress signal transduction process in plant. To provide data for exploring the functional mechanism of TaMAPK2, a wheat cDNA library was constructed and its interacting proteins were screened by yeast two-hybrid system.[Method]The wheat cDNA was used as the template for amplifying the TaMAPK2 gene, and the bait plasmid pGBKT7-TaMAPK2 was constructed. The mixture of recombinant plasmid pGBKT7-TaMAPK2, pGADT7 with wheat cDNA library was introduced into yeast cell AH109. Transformed cells were incubated on SD/-Trp/-Leu/-His/-Ade plate at 30℃ for 3-5 days before selection of clones and further incubated on SD/Raf/Gal/x-gal for screening blue clones. The candidate proteins that interacted with TaMAPK2 were obtained by sequencing and analyzing the blue clones. Enzyme digestion and ligation method was used to construct the expression vectors of pGADT7-HSP90, pSPYNE-TaMAPK2 and pSPYCE-HSP90. The recombinant plasmids pGADT7-HSP90 and pGBKT7-TaMAPK2 were transformed into yeast competent cells AH109, and the yeast two-hybrid system was used to analyze the interaction between TaMAPK2 and HSP90. The expression vectors pSPYNE-TaMAPK2 and pSPYCE-HSP90 were transformed into wheat protoplasts by using PEG-Ca2＋ method. At last the interactions of TaMAPK2 and HSP90 were observed using confocal laser scanning microscope. A real-time PCR testing system was constructed to quantify HSP90 in wheat by the specific primer, designed on the conservative sequences of HSP90 complete genome, and a testing mode based on SYBR Green I technology.The expression patterns of HSP90 gene in response to different abiotic stresses and in different organs were analyzed by quantitative real-time PCR.[Result] The proteins that interacted with TaMAPK2 were obtained, and most of the candidate proteins involved in signal transduction and immune process. This result suggests that TaMAPK2 possibly plays significant roles in stress signal transduction, transcription regulation of downstream genes and translation process in stress environments. Yeast two-hybrid and BiFc showed that HSP90 protein interacted with TaMAPK2. Subcellular localization analysis revealed that HSP90 is located on the plasma membrane, the cytoplasm and nucleus. The QRT-PCR showed that HSP90 expressed in roots, stems and leaves. The transcripts of HSP90 were more abundant in roots. Real-time PCR showed that the expression of HSP90 was up-regulated by the imposition of high-temperature, low-temperature, high-salt and ABA stresses.[Conclusion]The HSP90 is a widespread family of molecular chaperones. HSP90 can contribute to maintain cellular integrity by folding damaged proteins and maintaining protein conformation. Abiotic stress conditions cause the accumulation of aggregates containing damaged and abnormal proteins in plant. HSP90 is required for the degradation of damaged and misfolded proteins, and mediates expression of transcription factors. This result suggests that TaMAPK2 transmits signals by interacting with other proteins in plant abiotic stress responses.