| Common leaf spot of alfalfa, caused by Pseudopeziza medicaginis(Lib.) Sacc,has a worldwide distribution in temperate regions wherever alfalfa is grown. Common leaf spot of alfalfa is one of the most harmful diseases in alfalfa production. Infection of alfalfa by P. medicaginis can reduce the dry matter accumulation and the yield.By analyzing dynamic of disease related enzymes and transcriptome induced by P.medicaginis, Not only we can find some important genes and biological pathways which related with resistance to P. medicaginis, but also it is important for revealing the physiological, biochemical and molecular mechanism of regulating the alfalfa resistance to P. medicaginis. Two alfalfa lines we screened out: high resistant lines and high sensitive lines were used to discuss the mechanism of disease resistance P.medicaginis. Firstly, on physiological level, we determined the activity of PAO, DAO,CAT and SOD of these two lines respectively; then on molecular level, we used high-throughput sequencing to analysis the transcriptome induced by P. medicaginis.The main results are:1.High resistant lines(DR1111) and high susceptive lines of alfalfa(DS10)against common leaf spot were inoculated in lab, and then the activity of PAO, DAO,CAT and SOD was measured after the inoculation of 0, 1, 2, 3, 4, 5, 10, 15 and 20 d.The results showed that the activity of 4 antioxidant enzymes in both of DR1111 and DS10, was increased at first and then decreased in different degree and at different time. The activity of DAO was higher in DR1111 than in DS10(P<0.05); the activity of SOD in DS10 was higher in DS10 than in DR1111 after the inoculation of 3d to10d(P<0.05).The peak value of activity of PAO, DAO presented earlier in DR1111 than in DS10, and the peak value of activity of CAT presented later in DR1111 than in DS10.2.The c DNA of alfalfa leaves by infecting were subjected to Illumina Hi Seq TM2500 paired-end(PE) sequencing.By de novo assembly and data analysis, Finally, a total of 83,625 assembled transcripts and 35,061 unigenes were obtained with total residues of 100,107,179. The average length of each tanscript was 1,197.1 bps, the shortest sequence being 351 bps and the longest being 15,420 bps.3.Annotation of the transcriptome was carried out to generate a transcriptome database of alfalfa leaf. A total of 54,261 ORF(protein) were aligned to public protein databases by Blastp. Alignment of 17,164 unigenes without ORF predictions(DNA)were subjected to Blastx. The unigenes were searched against the public databases(Nr,Nt, STRING, KEGG, COG and GO). Results showed that 54,620、54,620、27,490、14,567、34,693 and 14,374 respectively were with ORF; and 15,902、9,069、1,381、520、4,707 and 1,663 without ORF. All assembled transcripts were compared against the COG database. These COG classifications were grouped into 25 function categories. The five larger categories were ‘General function prediction only’(4,651),‘Replication, recombination and repair’(2,714), ‘Transcription’(2,546), ‘Signal transduction mechanisms’(2,186) and ‘Posttranslational modification, protein turnover, chaperones’(1,400).4.The Bowtie、RSEM and edge R softwares were used to mapped and assignedthe transcripts, then, the differentially expressed genes were determined with FDR<0.05 and∣log2FC≧2∣.After induced by P. medicaginis, a total of 35,061 transcripts were identified as differentally genes, there were 331 unigenes showing up-regulated expression and 80 Unigenes showing down-regulated expression in RI vs RCK; a total of 35,061 transcripts were identified as differentially genes, 436 unigenes showing up-regulated expression and 327 unigenes showing down-regulated expression in SI vs SCK.5. By analysis of the statistically enriched GO and KEGG pathway related to the differentially expressed genes. The up-regulated transcripts in sample of RI vs RCK were grouped into 105 terms, the down-regulated transcripts were grouped into 33 terms. In SI vs SCK, the up-regulated and down-regulated transcripts were grouped into67 and 75 terms. It was revealed that the most significant and high frequently enriched terms and pathway for Plant-pathogen interaction [ko04626], Protein processing in endoplasmic reticulum [ko04141], Plant hormone signal transduction[ko04075], Phenylalanine metabolism [ko00360], Isoquinoline alkaloid biosynthesis[ko00950] and Tyrosine metabolism [ko00350].6. The 8 selected genes were validated by quantitative real-time PCR, and the results were highly accordant with the RNA-seq. And the quantitative real-time PCR was also used to analysis the expression of the 8 related resistant-genes in different time, we found that with the incubation continuing, these genes expressed differently between resistant plants and sensitive plants, and these differences of expression mod els may coursed the different resistance of different plants to P. medicaginis. |
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