ScMYB-CC6介导细胞死亡在甘蔗抗黑穗病中的分子机制研究

The Sporisorium scitamineum infection in sugarcane would emerge a whip-shaped structure. The induction and inhibition of host programmed cell death, which is happened along with the phenotype formation, play a key role on the formation of sugarcane resistance and smut pathogenicity. A sugarcane transcription factor ScMYB-CC6, which likely involved in sugarcane defending to smut infection, was isolated in our previous study. Based on this, the bioinformatics analysis of the ScMYB-CC6 promoter sequence was proposed to obtain its upstream regulatory genes firstly. And the DNA binding sites and interaction proteins screening of ScMYB-CC6 on yeast one-two hybrid library was conducted to investigate the regulatory networks of ScMYB-CC6 during S. scitamineum infection in sugarcane. Secondly, the genetically transformation of Arabidopsis thaliana using ScMYB-CC6 and its promoter was then conducted to identify its disease resistance and tissue-specific expression. Thirdly, ScMYB-CC6 and its core up/downstream regulated genes and interaction genes in sugarcane was silenced, and the VIGS cane plants were used for smut fungus inoculation and the following integrated analysis of transcriptome and proteome. After that, the mechanism of ScMYB-CC6 involving in sugarcane defense against S. scitamineum infection by mediating cell death and its gene expression regulation network could be identified lastly. This study could provide gene resources for the breeding of smut resistant sugarcane varieties and accelerate rapid cultivation of smut-resistant sugarcane varieties through molecular techniques by breeders.

甘蔗受黑穗病菌侵染后形成“黑鞭”，该表型形成过程中伴随着寄主细胞程序性死亡的诱导与抑制，是甘蔗抗黑穗病和黑穗病菌致病性形成的关键。本项目首次筛选到一个甘蔗转录因子ScMYB-CC6，其可能通过介导细胞死亡防御黑穗病菌的侵染。基于此，首先通过ScMYB-CC6启动子序列预测其上游调控基因；通过酵母单-双杂交文库筛选其DNA结合位点及互作蛋白，探明ScMYB-CC6在甘蔗受黑穗病菌侵染过程中的表达调控网络；其次，以ScMYB-CC6基因及其启动子稳定遗传转化拟南芥，分析其抗病性和组织表达特异性；最后，在甘蔗中对ScMYB-CC6及其关键上/下游基因和互作基因进行沉默诱导，通过转录组和蛋白组整合分析，解析ScMYB-CC6基因介导细胞死亡参与甘蔗抗黑穗病的机制及其分子调控网络，为抗黑穗病育种提供基因资源，促进育种家通过分子手段培育抗黑穗病甘蔗品种。

甘蔗受黑穗病菌 (Sporisorium scitamineum) 侵染后，自顶端分生组织、于叶鞘包裹处长出的“黑鞭”是甘蔗黑穗病最为重要的表型，在此过程中寄主细胞死亡的诱导与抑制，是甘蔗抗黑穗病和黑穗病菌致病性形成的关键。项目旨在研究甘蔗ScMYB-CC6基因对细胞死亡的调控机制，可为揭示甘蔗抗黑穗病机制和获得抗黑穗病基因奠定基础，具有较为重要的理论意义和实践应用价值。主要的研究结果和结论如下：① 通过将ScMYB-CC6蛋白在烟草叶片中进行瞬时表达，初步鉴定了ScMYB-CC6的细胞致死功能。② 基于甘蔗-黑穗病菌表达谱订制芯片，构建了ScMYB-CC6-黑穗病菌共表达网络。③ 通过酵母文库筛选到39个与ScMYB-CC6互作的蛋白，主要包括转录因子，核糖体蛋白和激酶等。④ 通过DAP-seq技术，挖掘到ScMYB-CC6调控的基因多数定位于细胞膜、液泡、胞质核糖体糖蛋白等，与“黑鞭”形成过程中甘蔗细胞的形态变化相吻合，同时涉及细胞死亡信号通路。⑤ 通过酵母单杂交技术以ScMYB-CC6启动子作为诱饵筛选到19个ScMYB-CC6基因的上游调控基因。以上研究通过多种技术手段，挖掘细胞致死型ScMYB-CC6基因的上下游调控网络，为甘蔗-黑穗病菌互作分子机制奠定一定的理论基础，同时为甘蔗抗黑穗病相关基因的发掘和功能鉴定提供了研究思路。项目实施过程培养了1名硕士研究生、3名本科生，发表3篇科研论文。