香豆素合成途径关键酶基因Ghpal、Ghc4h和Gh4cl在棉花抗链格孢菌中的作用

链格孢菌Alternaria alternata引起的棉花轮纹斑病,是导致棉花早衰发生的主要成因之一,严重危害棉花生产。香豆素合成途径作为合成植保素的一条代谢支路,其产物能够抑制病原菌,增强植物的抗病性。为了揭示该途径在棉花抗链格孢菌中的作用,本文利用棉花皱缩病毒介导的基因沉默技术,通过对棉花香豆素合成途径中编码关键酶基因的沉默抑制,来解析其在棉花抗链格孢菌中的作用,所选择的基因有编码苯丙氨酸解氨酶(PAL)的基因Ghpal、肉桂-4-羟化酶(C4H)基因Ghc4h和4香豆酸-辅酶-A(4CL)的基因Gh4cl。结果显示,Ghpal、Ghc4h和Gh4cl基因受链格孢菌诱导转录,表明这些基因可能参与了棉花对链格孢菌的抗性反应。Ghpal、Ghc4h和Gh4cl基因分别被沉默后,结果显示各基因沉默植株对链格孢菌的抗性下降,表现为接种链格孢菌后,基因沉默植株的病情指数分别为43.36、38.27和44.78,显著高于野生型和VIGS空载体对照植株。这表明,Ghpal、Ghc4h和Gh4cl参与了棉花对链格孢菌的抗性反应。此外,离体条件下给Ghpal、Ghc4h和Gh4cl基因沉默植株叶片饲喂外源香豆素,结果发现基因沉默植株对链格孢菌的抗性得到恢复。这一结果进一步表明香豆素合成途径在棉花抗链格孢菌中起着重要作用。

Resistant roles of key enzyme encoding genes of Ghpal,Ghc4h and Gh4cl involved in cotton scopoletin biosynthesis pathway against to Alternaria alternata

Alternaria leaf spot caused by Alternaria alternata is one of the key causes of cotton premature senescence and often leads to severe loss in cotton production. Scopoletin biosynthesis pathway, a branch of phytoalexin metabolic pathway for synthesis of the scopoletin, acts the roles of growth suppression of phytopathogenic fungi and enhancement of plant disease resistance. In order to clarify the resistant roles of cotton scopoletin biosynthesis against A. alternata, cotton leaf crumple virus-induced gene silencing (VIGS) techniques was performed to silence three key enzymes encoding genes of the scopoletin biosynthesis pathway, Ghpal for phenylalanine ammonia lyase, Ghc4h for cinnamate 4-hydroxylase, and Gh4cl for 4-coumarate-CoA ligase. The results showed that the expression levels of Ghpal, Ghc4h, and Gh4cl in cotton leaves were induced by A. alternata, suggesting these genes play important roles in the early defense response against A. alternata in cotton. The results of VIGS experiments showed that disease index of the silenced Ghpal, Ghc4h, or Gh4cl plants were 43.36, 38.27, 44.78, respectively, which were significantly higher than the mock one of the wild type and the empty vector transgenic plants. These results showed that Ghpal, Ghc4h, Gh4cl in the scopoletin biosynthesis played an important role in resistance to A. alternata. Moreover, exogenous feeding of scopoletin can recover the resistance of VIGS cotton plants to A. alternata. Taken together, our study revealed that the cotton scopoletin biosynthesis played an important role in the resistance to A. alternata.