柱花草SgMPK6互作蛋白的筛选与验证

Screening and verification of SgMPK6-interacting proteins of Stylosanthes

丝裂原活化蛋白激酶(MAPK)级联反应在调节植物免疫中起关键作用。柱花草炭疽病是危害柱花草生产的严重病害,柱花草SgMPK6基因具有抗炭疽菌的功能。为探究响应胶孢炭疽菌侵染的柱花草SgMPK6下游互作蛋白,本研究采用酵母双杂交技术,以SgMPK6激酶结构域作为诱饵蛋白,筛选柱花草cDNA文库,共获得74个与SgMPK6激酶结构域潜在的互作蛋白,并通过酵母双杂交点对点试验,验证了候选互作蛋白SgbHLH32、SgbHLH33、SgbHLH44与SgMPK6激酶结构域间的互作关系。磷酸化位点预测显示,3个bHLH转录因子均具有MAPKs潜在的磷酸化位点。柱花草响应炭疽菌侵染的qRT-PCR分析表明,SgbHLH32、SgbHLH33、SgbHLH44转录因子均显著上调,预示互作蛋白可能作为SgMPK6的底物调控柱花草对炭疽病的抗性。本研究为进一步解析SgMPK6响应炭疽菌侵染的分子机制提供了试验依据。

The mitogen-activated protein kinase(MAPK)cascade plays a key role in regulating plant immunity.Anthracnose is a serious disease affecting the production of Stylosanthes(Stylo).The SgMPK6 gene of Stylo has been shown to function in resistance against anthracnose,which is caused by Colletotrichum gloeosporioides.To investigate the interacting proteins downstream of SgMPK6 in the response to C.gloeosporioides infection,a yeast two-hybridization technique was used to screen the cDNA library of Stylo using the SgMPK6 kinase domain as the bait.Seventy-four potential interacting proteins were obtained.The interactions between the SgMPK6 kinase domain and the bHLH transcription factors SgbHLH32,SgbHLH33,and SgbHLH44 were verified in yeast twohybrid experiments.Phosphorylation site analysis revealed that all three transcription factors contained MAPK phosphorylation sites.The results of qRT-PCR analyses showed that SgbHLH32,SgbHLH33,and SgbHLH44 were significantly up-regulated in Stylo by C.gloeosporioides infection.Together,these results suggest that SgbHLH32,SgbHLH33,and SgbHLH44 are substrates of SgMPK6 and may be involved in resistance against anthracnose.The results of this study provide a basis for further analysis of the role of SgMPK6 in resistance to C.gloeosporioides infection.