甘蓝型油菜BnMAPK1的原核表达、亚细胞定位及酵母双杂交文库筛选

Prokaryotic expression,subcellular localization and yeast two-hybrid library screening of BnMAPK1 in B.napus

植物的丝裂原活化蛋白激酶(Mitogen-Activated Protein Kinases,MAPKs)级联是生长进程中多种信号跨膜传递的共同通路,它可以将外源刺激转入细胞内并引发细胞应答。目前C族MAPKs在甘蓝型油菜中的研究报道还很有限。本研究通过1个甘蓝型油菜的C族BnMAPK1基因的生物学进程聚类分析发现,BnMAPK1可能参与蛋白磷酸化、生长素介导的信号途径、逆境应答、细胞周期及转录等过程。BnMAPK1具有369个氨基酸残基,其相对分子质量约为42.5 kD,其可溶性蛋白可在原核系统中被诱导表达。BnMAPK1的亚细胞定位结果显示,BnMAPK1主要在细胞核内表达。Bait质粒pGBKT7-BnMAPK1在酵母双杂交系统中无毒性及自激活活性。为深入研究BnMAPK1蛋白参与的生物学进程,从甘蓝型油菜中油821苗期根、茎、叶中分别提取总RNA,分离得到mRNA,利用SMART技术合成并纯化双链cDNA后建立甘蓝型油菜混合cDNA文库。以共转化法筛选与BnMAPK1相互作用的蛋白,对其分析与鉴定显示,BnMAPK1在生长发育、非生物及生物逆境、转录、蛋白合成及代谢、翻译及翻译后修饰等过程中起作用。研究结果为MAPKs级联,尤其是C族MAPKs的研究提供了新的视野,为甘蓝型油菜抗性的机理研究及分子育种奠定了重要的理论依据。

Plant mitogen-activated protein kinases(MAPKs)cascades are common signal transduction pathways across the membrane in growth process,they are involved in transducing extracellular stimuli to induce a range of cellular responses.To date,little is known about the role of C-group MAPKs in B.napus.In this study,we analyzed biological process ontology of one C-group BnMAPK1 gene in rapeseed,indicating that BnMAPK1 may be involved in protein phosphorylation,auxin-mediated signaling,stress response,cell cycles and transcription.With 369 amino acid residues,the hydrophilic protein of BnMAPK1 could be induced to express one 42.5 kD protein in the prokaryotic system.The subcellular localization of BnMAPK1 mainly focused in the nucleus.The pGBKT7-BnMAPK1 bait plasmid without toxicity and auto-activation was constructed to perform yeast-two hybrid screening.To further study the function and mechanism of BnMAPK1,we extracted the total RNA of roots,stems,and leaves of B.napus(Zhongyou 821)at seedling stage respectively,and isolated and purified the mRNA to construct the mix cDNA library by SMART method.The BnMAPK1-interacting proteins were screened through co-transformation with bait plasmid and cDNA prey library,demonstrating that BnMAPK1 plays an important role in the growth and development processes,abiotic and biotic stresses,transcription,protein synthesis and metabolism,translation,and post-translational modification,and so on.These results provide a new perspective for the research of MAPKs cascades,especially for C-group MAPKs,and lay a theoretical foundation for the stress resistance mechanism research and molecular breeding in rapeseed.