| Stylosanthes spp.is a kind of leguminous pasture plants in tropical and sub-tropical area of South China.Anthracnose,induced by Colletotrichum gloeosporioide,is the major disease in the stylo production,which can cause the decrease of nutrition value and yield of stylo.It is important to study on the physiological and biochemical response to C.gloeosporioides infection of stylo and the mining of key resistant genes for realizing resistance mechanism and cultivating resistance vatiety.In this study,the life cycle of C.gloeosporioides was observed on Reyan No.2 stylo,and the influence on antioxidant enzyme system and metabolites of stylo caused by C.gloeosporioides infection was also analyzed.The phenylalanine ammonia-lyase SgPAL1,which is a gene associated with of stylo anthrax response,was identified by RNA-seq analysis.The main conclusions are as follows:1.Lesions were observed on leaves in 36 hpi and dark brown necrotic lines appeared in 60 hpi,treated with C.gloeosporioides.The formation of large amounts of anthracnose primary hyphae in 36 hpi and secondary hyphae grew rapidly in 48-60 hpi through microscope,and then the host cell disintegrated and died.The appearance of secondary hyphae marks the transformation of anthrax from the living nutrition stage to the dead body nutrition stage.2.The production of H2O2 and O2-were detected using DAB and NBT staining respectively in the stylo leaves inoculated by C.gloeosporioides.The results demonstrated that the accumulation of active oxygen in the leaves was gradually,resulting in the cell damage.Besides,the antioxidant enzyme system and metabolites of stylo leaves were also regulated after inoculation.The activity of catalase(CAT)was rising constantly,and activity of superoxide dismutase(SOD),peroxidase(POD),the content of ascorbic acid(AsA)and glutathione(GSH)firstly increased and then decreased by the determination of the oxidase enzymes.It suggested that the increase of antioxidant enzymes activity was an adaptive response of stylo to anthracnose and played a role in coping with ROS.3.The secondary metabolites of downstream of phenylpropanoid pathway(tannins,total phenolics,anthocyanins,proanthocyanidins and flavonoids content)were significantly increased at a specific stage after C.gloeosporioides treatment.The proanthocyanidins and total flavonoids content of 96 hpi were 14 times,2 times of CK,total phenolics and anthocyanins the glycoside content in 24 hpi were increased by 70.95%and 75.61%;the tannin content increased by 69.09%in 48 hpi.The activity of PAL increased significantly in 24hpi and 48hpi,and then decreased gradually.These results suggested that PAL mediated metabolism of phenylpropane was involved in the regulation of the resistance to C.gloeosporioides.4.The differentially expressed genes of stylo leaves at CK,24 h,48 h,60 h and 96 h after inoculation with C.gloeosporioides were analyzed by RNA-Seq.The results showed that 700,643,352 clean reads,194,879 transcripts and 112,754 unigenes were obtained.In addition,largest amount of difference expression genes appeared in 60 hpi with 3831 up-regulated genes and 6596 down-regulated genes.Functional annotation analysis showed that differentially expressed genes were mainly involved in phenylpropanoid biosynthesis,plant-pathogen interaction,plant hoemone signal transduction and so on.The family members of coding genes of three key enzymes in phenylpropanoid pathway,phenylalanine ammonialyase(PAL),cinnamic acid-4-hydroxylase(C4H)and 4-coumaric acid coenzyme a ligase(4CL)were induced to enhanced expression by infection of Colletotrichum gloeoporioides.5.Three members of the SgPAL family were obtained from unigene database of stylo transcriptome,named SgPAL1?SgPAL2?SgPAL3.Their nucleotide sequence lengths were 2148 bp,2076 bp and 2136 bp,respectively encoding 715,691 and 711 amino acids.Their sequence identity was 80.51%.The expression pattern analysis by Real-time PCR showed that all three SgPALs expressed in root,stem and leaf.The expression of SgPAL1 and SgPAL2 were the highest in root,and the highest expression of SgPAL3 was in stem.The expression pattern analysis by response to infection of C.gloeosporioides indicated that the expression level of SgPAL1,SgPAL2 and SgPAL3 was higher than CK in 48 hpi,60 hpi and 90 hpi,respectively.It indicated that SgPAL1 played an important role in the SgPAL family.Moreover,analysis of subcellular localization of protoplast cells in Arabidopsis thaliana suggested that SgPAL1,SgPAL2 and SgPAL3 all located in the cytoplasm.6.SgPAL1 overexpression transgene material was successfully constructed by Agrobacterium tumefaciens mediated transformation system of stylo.The results showed that PAL enzyme,total phenolics,flavonoids and anthocyanins content of To positive line were higher than that of wild type,and lesion diameter were significantly smaller than that of wild type after C.gloeosporioides inoculation.It indicated that overexpression of SgPAL1 can significantly enhance the resistance to anthracnose of stylo.In summary,the conclusion of this study is that SgPAL1-mediated phenylpropanoid metabolic pathway could enhance the resistance to anthracnose of stylo by increasing downstream secondary metabolites(total phenolics,flavonoids and anthocyanins content).The results will provide a theoretical basis and candidate gene resources for the anthracnose resistant varieties breeding of stylo. |
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