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早衰对棉花生产危害严重。链格孢菌侵染是导致棉花早衰发生的关键成因之一。为此,发掘和利用棉花对链格孢的抗性是防控棉花早衰的重要途径。促分裂原活化蛋白激酶(Mitogen-actrvated piotein kinases,MAPK)级联途径是一种重要的信号传导系统,在植物响应各种生物和非生物胁迫的过程中起重要作用。为了揭示棉花对链格孢抗性产生的信号传导机制,本文利用已公布的陆地棉全基因组信息,筛选陆地棉MAPK和MAPKK家族的全部基因,对其进行了多序列比对和系统进化分析,并利用病毒诱导的基因沉默技术(VIGS)鉴定了这些基因在棉花抗链格孢中的作用。主要研究结果如下. 1.依据陆地棉全基因组中的基因注释信息以及多序列比对结果,筛选出了53个GhMAPK基因和24个GhMAPKK基因,并依据其同源的拟南芥MAPK和MAPKK对它们进行了命名。蛋白序列比对结果显示,大部分GhMAPK含有植物MAPK特有的T-X-Y基序,仅在Gh A07G1299(GhMAPK4-3),Gh_D07G1410(GhMAPK4-8)和Gh_A10G2117(GhMAPK17-1)这三个基因中T-X-Y变为了T-X-C,大部分的GhMAPKK含有植物MAPKK特有的保守基序D(I/L/V)K和磷酸化位点S/T-XXXXX-S/T,仅在Gh_D07G0094(GhMAPKK6-2)和Gh_A07G0085(GhMAPKK6-4)中HIK代替了DIK,在Gh_A01G1308(GhMAPKK7-1)和Gh D01G1562(GhMAPKK7-2)中S-XXXXX-N代替了S-XXXXX-S/T。与AtMAPK和AtMAPKK一起构建的系统进化树显示,GhMAPK和GhMAPKK家族的所有基因能够分别聚到AtMAPK和AtMAPKK的A、B、C、D四个族中。 2.构建GhMAPK和GhMAPKK基因的沉默载体,利用VIGS技术研究GhMAPK和GhMAPKK是否参与棉花对链格孢的抗性调控。结果发现,GhMAPK中的GhMAPK3、GhMAPK7、GhMAPK9、GhMAPK19以及GhMAPKK中的GhMAPKK2、GhMAPKK3、GhMAPKK9基因沉默后,沉默棉花植株的抗病性明显降低,病情指数显著高于野生型对照和空载体对照(P<0.05),表明这些基因正调控棉花对链格孢的抗性。 3.对于筛选出的正调控棉花对链格孢抗性的4种GhMAPK和3种GhMAPKK,进一步利用靶向性沉默策略,研究它们之中各同源基因对链格孢抗性的调控作用。结果显示,正调控棉花对链格孢的抗性的同源基因有GhMAPK3中的GhMAPK3b(GhMAPK3-2,GhMAPK3-4),GhMAPK7中的GhMAPK7b(GhMAPK7-2,GhMAPK7-5)和GhMAPK7c(GhMAPK7-3,GhMAPK7-6),GhMAPK9中的GhMAPK9a(GhMAPK9-1,GhMAPK9-3)和GhMAPK9b(GhMAPK9-2,GhMAPK9-4);GhMAPK19中的GhMAPK19b(GhMAPK19-2,GhMAPK19-5)和GhMAPK19c(GhMAPK19-3,GhMAPK19-6),GhMAPKK2中的GhMAPKK2a(GhMAPKK2-1,GhMAPKK2-4)和GhMAPKK2b(GhMAPKK2-2,GhMAPKK2-5);GhMAPKK9中GhMAPKK9a(GhMAPKK9-1,GhMAPKK9-2),而其他同源基因GhMAPK3a(GhMAPK3-1,GhMAPK3-3),GhMAPK7a(GhMAPK7-1,GhMAPK7-4),GhMAPK7d(GhMAPK7-7),GhMAPK19a(GhMAPK19-1,GhMAPK19-4),GhMAPKK2c(GhMAPKK2-3,GhMAPKK2-6),GhMAPKK9b(GhMAPKK9-3)并不参与调控棉花对链格孢的抗性。