Analysis Of Key Enzyme Encoding Genes Of Ghpal、Ghc4h And Gh4cl Involved In Cotton(Gossypium Hisutum L.) Scopoletin Biosynthesis Pathway Against To Alternaria Alternata

In recent years, cotton premature senescence leads to severe losses in cotton production in China. Alternaria leaf spot caused by A. alternata is one of the key causes of cotton premature senescence. So far, there has been less research on the molecular mechanisms of cotton against to Alternaria alternata, which seriously hinders breeding resistant varieties of cotton premature senescence. Scopoletin biosynthesis pathway is a branch of phytoalexin metabolic pathway for synthesis of the scopoletin, which acts the roles of the suppression the growth of phytopathogen fungi and enhancing the plant disease resistance. In order to reveal the resistant roles of cotton scopoletin biosynthesis pathway on A. Alternata, XLZ13 was chosen to be as experimental materials. Basing on the results of microarray analysis of cotton leaves infected by A. Alternata in our laboratory, we mainly studied the resistant roles of scopoletin biosynthesis pathway against to Alternaria alternata through VIGS silencing the key enzyme genes Ghpal, Ghc4 h and Gh4 cl of scopoletin biosynthesis pathway and vitro feeding tests, etc.The main results about this study are as follows:1.Ghpal, Ghc4 h, Gh4 cl genes in cotton scopoletin biosynthesis pathway involved in the early responses to A. Alternata. qRT-PCR analysis showed that transcript levels of Ghpal, Ghc4 h and Gh4 cl genes increased significantly and reached a peak at 3dpi(day post inoculation), then began to decline and finally returned to a normal level at 12~15dpi. The results showed that transcripts of the gene Ghpal, Ghc4 h and Gh4 cl in cotton leaves were induced by A.alternata and these genes might play an important role in the early defense response against A. alternata in cotton.2. VIGS was used to silence the genes Ghpal, Ghc4 h and Gh4 cl related scopoletin biosynthesis pathway in XLZ13 via targeting the endogenous fragment of these genes. A. Alternata was used to inoculate these gene silenced cotton plants. Then the results of VIGS showed that the disease indices of silenced Ghpal, Ghc4 h or Gh4 cl gene plants were 43.36, 38.27, 44.78 respectively, which was significantly higher than the disease indices of wild type and empty vector transformed control plants. These results showed that Ghpal, Ghc4 h, Gh4 cl in scopoletin biosynthesis played an important role on the resistance of cotton to A. alternata.3. Exogenous scopoletin can help the VIGS cotton plants recover its the resistance to A. Alternata to a certain extent. The biossays for the inhibition of assay A. Alternata and feeding experiment in vitro showed that the lesion area of gene silenced cotton leaves fed with the concentration of 96 μg/mL of exogenous scopoletin for 7h reduced obviously comppared with the cotton leaves fed with sterile water. This explained that Ghpal, Ghc4 h, Gh4 cl in scopoletin biosynthesis pathway involved in the resistance of cotton to A. alternata from another perspective.4. Fluorescent substances in cottom leaves around infection induced by A. alternata and they have an relationship with the scopoletin biosynthesis and can inhibit the growth of A. alternata. Through detecting and analyzing of these fluorescent substance, we found that the synthesis of fluorescent substances in Ghpal, Ghc4 h, Gh4 cl gene silenced cotton leaves were significantly supressed, comparing with the wild-type plants and the empty vector control cotton leaves. Corresponding, the extract of fluorescent substance in these gene silenced cotton leaves could not inhibit the growth of A. Alternata, comparing with the wild-type plants and the empty vector control cotton leaves extract.The study revealed Ghpal, Ghc4 h, Gh4 cl genes of scopoletin biosynthesis pathway in cotton played an important role on the resistance to A. Alternata, which describes the resistant molecular mechanism of cotton to A. Alternata preliminaryly.