Key Genes Against Altemaria Panax Whetzel Infection In Panax Notoginseng(Burk)F.H. Chen Based On Transcriptome Analysis

Black spot disease(Alternaria panax Whetz)is one of the major diseases of Panax notoginseng(Burk)F.H.Chen,and all parts of the plant can be victims.Due to the wide host range of the pathogen,the feature of it has a rapid onset,spreading fast and therefore,the yield losses of P.notoginseng parks caused by this disease usually can be more than 10%.The current study of black spot disease has focused on about the biological characteristics of pathogens,its occurrence and the chemical and biological control measures,however,the infection process and molecular interaction between P.notoginseng and pathogen are still poorly understood.In this study,RNA sequencing of seven different P.notoginseng leaves after infection pathogen was performed using next generation DNA sequencing.We observed a high expressed chitinase gene in transcriptome database.Some interesting results were obtained as follow.464 different expressed unigenes were existed in every group,which include protein kinases,cytochrome P450 family and WRKY transcription factors.1.The method of artificially inoculating black spot disease:leafs of P.notoginseng,which was pretreatment with silica sand inoculated mycelium of A.panax Whetz,in addition,keeping the environment humid.The method can quickly and efficiently make plant disease.Total RNA of the P.notoginseng root and leaf were distilled with GenStar TRIGene and tested with the agarose gel electrophoresis.The 28s,18s and 5.8S band were clear visible,brightness of 28S band was 2 times brighter than 18s.The result of ultraviolet spectrophotometer was this:the A260/280 value was between 1.8 and 2.0,A260/230 was between 0.6 and 1.5.The concentrations of total RNA was between 400?800 ng/?L.2.In this study,RNA sequencing of seven different P.notoginseng leafs was performed using Illumina Hi-Seq 4000 sequencing platform.Sequencing yielded approximately 37.65 Gb date in total.After assembling and removing redundant,finally,114 009 unigenes was obtained,total length,average length,N50 and GC content were 135607883 bp,1189 bp,1947 bp and 40.34%,respectively.Then,all-unigenes were annotated by Basic Local Alignment Search Tool(BLAST)similarity searches against public sequence databases,eventually,73870(NR:64.79%),70520(NT:61.85%),52477(Swissprot:46.03%),31240(COG:27.40%),47135(KEGG:41.34%),33569(GO:29.44%)and 53630(Interpro:47.04%)unigenes were annotated,respectively.According to the annotation results detected 72553 CDS,and unannotation unigenes obtain 4,231 CDS by ESTScan forecast.At the same time,also detected 20927 SSR distribution in 16406 Unigene;444745 SNPS were detected from seven transcript.3.A chitinase gene(Unigene19153_All)was isolated from P.notoginseng,and the full-length cDNA of this unigene was cloned with the method of rapid amplification of cDNA ends and named as PnCHI1.PnCHI1 was 1 022 bp in length and contained an intact open reading frame(ORF)of 822 bp,a 26 bp 5'-untranslated region(UTR),and a 174 bp 3'-UTR.The predicted protein of PnCHIl with 273 amino acid residues belongs to glycoside hydrolase family 19 and fell into the class IV of chitinases through phylogenetic analysis.QRT-PCR analysis showed that the expression of PnCHI1 was induced by methyl jasmonate,ethylene,H2O2,and salicylic acid.PnCHI was quickly induced after inoculation with A.panax.All the results of present study indicated that PnCHI1 was involved in defense response of P.notoginseng against the A.panax.