苹果MdCPK2可变剪接介导抗斑点落叶病的分子机制

Alternaria blotch of apple is one of important diseases affecting apple production. In previously research, we screened a calcium independent kinase protein, MdCPK2, which was strongly induced by infection of Alternaria alternata apple pathotype and showed significantly differential expression and alternative splicing between the susceptible and resistant cultivars, suggesting that alternative splicing of MdCPK2 plays important roles in regulation of resistance to A. alternata AP. Based on the previous study, we will analyze the mechanism of MdCPK2 gene alternative splicing in response to A. alternata infection. We will further analyze the subcellular localization, kinase activity, sensitive to Ca2+ and the target proteins of MdCPK2 isoforms encoded by different transcripts. Yeast two-hybrid will be used to screen interaction proteins with MdCPK2 isoforms. The protein phosphorylation by MdCPK2 will be conducted by in gel kinase assay in vivo and in vitro. Downstream target genes which regulated by the putative MdCPK2-phosphorylated transcript factors will be screened by Chromatin immunoprecipitation (ChIP). We will also perform virus induced gene silence (VIGS) to silence or overexpress MdCPK2 transcripts by transient transfection in apple leaves to analyze the function of MdCPK2 isoforms. The phenotype of disease resistance will be analyzed. The mechanism of alternative splicing of MaCPK2 gene mediated the regulation of apple defense response and the signal transduction passway by MdCPK2 will be illustrated by the research. The results will provide the theory basis for improving apple resistance to A . alternata AP.

斑点落叶病是影响我国苹果生产主要病害之一。在前期研究中我们筛选到一个受斑点落叶病菌诱导发生可变剪接的苹果MdCPK2基因，其可变剪接方式及可变剪接转录本表达量在感、抗品种中呈显著差异，暗示MdCPK2基因可变剪接在苹果抗病反应中起重要作用。在此基础上，我们拟进一步对MdCPK2可变剪接产生的不同蛋白异构体亚细胞定位（原生质体瞬时表达）、激酶活性和Ca2+敏感性（体内体外磷酸化分析法）、作用底物和互作蛋白（酵母双杂交法）开展研究，利用反向遗传学手段，将MdCPK2不同转录本沉默或超表达，观察苹果抗病表型和抗病相关基因表达。然后通过染色质免疫共沉淀方法（CHIP）分析受MdCPK2作用的转录因子调控的下游信号，通过本项目的研究，将阐明MdCPK2可变剪接介导的苹果抗病调控机制及信号通路，为今后通过基因调控和遗传改良提高苹果抗病性提供理论依据。

苹果（Malus×domestica Borkh.）是蔷薇科（Rosaceae）苹果属（Malus Mill）植物，是世界最重要的水果之一。然而，在苹果的栽培生长过程中，常受苹果斑点落叶病(Alternaria blotch)病害侵袭，该病由链格孢属的苹果专化型(Alternaria alternata apple pathotype, AAAP)病菌引起。钙依赖蛋白激酶(Calcium-Dependent Protein Kinases, CDPKs)作为植物的一类钙信号解码蛋白, 而可变剪接(alternative splicing, AS)是一种重要的转录后调控机制，在抗病反应中发挥着重要作用。本项目通过RNA-Seq数据分析了‘红星’和‘红玉’对AAAP的响应机制，采用PacBio全长测序鉴定‘红星’响应AAAP的差异表达转录本( DETs )和响应病原菌侵染的相关基因的可变剪接事件。在抗病反应中发挥着重要作用。我们重点对MdCDPK5基因在病菌诱导条件下产生的全长转录本MdCDPK5-I和截短转录本MdCDPK5-II进行了分析。 MdCDPK5-I和MdCDPK5-II均定位在细胞核和细胞膜中。MdCDPK5-II多肽与 MdCDPK5-I蛋白互作调控钙信号下游相关基因的表达，从而调控苹果对斑点落叶病的抗性。这些结果表明，MdCDPK5基因的可变剪接是苹果调控抗病反应的一种重要机制。这一研究拓展了对苹果抗病响应机制的理解，为利用基因工程的方法进行苹果抗斑点落叶病菌防治和育种提供了新的思路。