黄瓜丝裂原活化蛋白激酶CsMPK4基因的功能鉴定

Function identification of the mitogen-activated protein kinase gene CsMPK4 in cucumber

摘要:

黄瓜是一种在全球范围内广泛栽培的蔬菜作物，高温等逆境胁迫会影响植株生长发育甚至导致其产量和品质降低。丝裂原活化蛋白激酶MAPK家族在植物逆境响应中起着至关重要的作用。为研究黄瓜MPK4基因的功能及逆境响应机制，本研究克隆了CsMPK4基因，其编码383个氨基酸；实时荧光定量聚合酶链式反应(quantitative real-time PCR,qRT-PCR)分析发现该基因在叶和花中表达量最高，根中次之，在茎和卷须中表达量最低。CsMPK4定位于细胞核和细胞质，与甜瓜CmMPK4亲缘关系最近。过表达CsMPK4的黄瓜植株矮化健壮、卷须变短变少、幼苗更耐高温，幼叶中丙二醛含量降低，过氧化物酶和超氧化物歧化酶活性增强。进一步通过酵母双杂和双分子荧光互补(bimolecular fluorescence complementation,BiFC)实验证明，黄瓜CsMPK4与缬氨酸-谷氨酰胺家族因子CsVQ10存在蛋白互作，表明CsVQ10可能协同CsMPK4参与黄瓜高温等逆境胁迫响应。本研究为深入探讨黄瓜CsMPK4逆境应答机制以及黄瓜抗逆育种等奠定了基础。

关键词:黄瓜;CsMPK4基因;高温;蛋白互作

Abstract:

Cucumber (Cucumis sativus L.) is one of the most widely cultivated vegetables in the world. High temperature and other stress conditions can affect the growth and development of this plant, even leading to the decreases in yield and quality. The mitogen-activated protein kinase (MAPK) family plays a crucial role in plant stress responses. However, the role of MPK4 in the stress response of cucumber remains to be reported. In this study, we cloned CsMPK4, which encoded 383 amino acid residues. The qRT-PCR results showed that the expression level of CsMPK4 was the highest in leaves and flowers, moderate in roots, and the lowest in stems and tendrils. CsMPK4 was located in the nucleus and cytoplasm, and it had a close relationship with CmMPK4 in muskmelon. The cucumber plants overexpressing CsMPK4 became stronger and shorter, with reduced length and quantity of tendrils. Moreover, the transgenic seedlings were more resistant to high temperatures, with decreased malondialdehyde (MDA) content and increased activities of peroxidase (POD) and superoxide dismutase (SOD) in young leaves. Furthermore, the protein-protein interaction between CsMPK4 and CsVQ10, a member of the valine-glutamine family, was confirmed by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. The results suggested that CsVQ10 cooperated with CsMPK4 in response to the high temperature stress in cucumber. This study laid a foundation for the further study on the stress response mechanism of CsMPK4 and the breeding of stress-resistant cucumber varieties.

Key words:cucumber;CsMPK4;high temperature;interaction