The Response Of Stylosanthes Phenylpropane Metabolic Pathway To Colletotrichum Gloeosporioides Infection

Stylo(Stylosanthes spp.)is an important legume herb in tropical regions and is widely cultivated in Hainan,Guangdong and Guangxi areas.With high crude protein content,high yield,acidic soil resistance,nitrogen fixation and other characteristics,stylo is used as pasture and green fertilizer,to improve the soil quality and increase the content of nitrogen in the soil.Anthracnose is the major threat for stylo production,causing leaf necrosis and exfoliation,inflorescence abortion and plant blight.At present,there is no effective technology and method in industry that can control the damages of Colletotrichum gloeosporioides to stylo.The genetic status of stylo can be altered by the genetic transformation by expressing the target gene(s)in stylo.Therefore,mining of disease-resistant genes and breeding of disease-resistant varieties are of great importance.In this study,the PAL enzyme activity,total phenol content,flavonoid content,total antioxidant capacity and relative expression of SgPAL genes were investigated after inoculation of Reyan NO.2 stylo with Colletotrichum gloeosporioides.In addition,metabolomics was performed to analyze the changes of flavonoid metabolites of stylo after inoculation.Furthermore,the transgenic stylo over expressing SgPAL2 was generated for functional analysis.The main research results are as follows:1.Four members of the phenylalanine ammonia-lyase gene family were cloned from stylo and named SgPAL1,SgPAL2,SgPAL3 and SgPAL4.Their nucleotide sequence lengths were 2145 bp,2073 bp,2133 bp and 2199 bp.The relative expression levels of SgPAL1,SgPAL2 and SgPAL3 were significantly increased and SgPAL4 was significantly decreased after inoculation with Colletotrichum gloeosporioides,indicating that SgPAL1,SgPAL2,SgPAL3 and SgPAL4 of stylo were responsive to the infection of Colletotrichum gloeosporioides.2.The content of total phenols,flavonoids and total antioxidant capacity were significantly increased,indicating that the accumulation of total phenols and flavonoids may play an important role in the resistance to Colletotrichum gloeosporioides.3.152 flavonoid metabolites including 16 isoflavones,19 flavonoid glycosides,22 flavonoids,36 flavonols and 59 flavonoids were detected in stylo.Post inoculation with Colletotrichum gloeosporioides,the contents of daidzein,genistein,6-hydroxydaidzein and isoflavones in rhizophorin and isoflavones were significantly increased.The results showed that the amount of rhizocutin and isoflavone metabolites in stylo were responsive to Colletotrichum gloeosporioides.4.The overexpression vector pTF101S-SgPAL2 was successfully transformed into Reyan NO.2 stylo.It was found that the relative expression of SgPAL2,PAL enzyme activity,total phenol and flavonoid content in T0 generation stylo were significantly higher than those in the wild type,indicating that overexpression of SgPAL2 in stylo promoted the synthesis of total phenols and flavonoids to some extent.