| Zanthoxylum issitum Hemsl,also known as single needle,is an important drug of rare forest resources in south of our country.Its roots,stems,leaves can be used as medicine,in clinically,it has the effect of dispelling wind and activating collaterals,Dispersing blood stasis and relieving pain,detumescence by detoxification.Zanthoxylum dissitum Hemsl contains dictamnine,skimmianine,ursolic acid and flavonoid glycosides and other precious medicinal ingredients,Has significant effect of anti phytopathogenic fungi activity and good treatment of high cholesterol,diabetes,cardiovascular disease and other cancers.Compared with the economic value,social value and scientific research value of Zanthoxylum dissitum Hemsl,the molecular biology research is relative lag,Restricting the study to cultivate pepper Shell,mining and key secondary metabolic mechanism of genetic resources,etc.In this article,in order to produce Zanthoxylum dissitum Hemsl seed from Hunan area of Zhangjiajie as the experimental materials,The seed soaking in the control group(0A)and treatment group(6A)GA immersion of transcriptome sequencing by Solexa sequencing technology in the second generation sequencing method,The sequencing results were analyzed systemic bioinformatics,and a PP2C gene was successfully cloned the full-length.The main conclusions are as follows:(1)the sample after extraction of RNA,cDNA library preparation,Solexa sequencing,filter unqualified sequences and assembly steps,Finally get 90,447 Zanthoxylum dissitum ALL-Unigene,the total length is 77,113,962nt,the average length is 853nt,the length of N50 is 1,407nt.(2)compared with obtained ALL-Unigene and NR,NT,Swiss-Prot,KEGG,COG,GO six database,respectively60,365,55,032,38,688,34,670,21,244,47,281 compared to the above database.By comparing with the COG database,which is divided into 25 different functional annotations,which involves the posttranslational modification,protein conversion,molecular chaperone,DNA replication,recombination and repair,signal transduction mechanism,translation,ribosomal structure and biogenesis,carbohydrate transport and metabolism function gene distribution is more.(3)Conduct the ALL-Unigene with GO and Pathway enrichment analysis,we get 55 different functional categories and 128 metabolic pathways.And further prediction of CDS,received a total of 61,885 CDS,by comparison to public databases Blastx gained 59,338 CDS,with ESTScan software predicts get 2,547 CDS.(4)Through the analysis of difference to the control group and the treatment group expression,getting 34,433 different expression genes,including 26,511 up-regulated genes,accounted for the differences in gene expression of 76.99%;7,922 down regulated genes,accounted for 23.01%,a large number of genes up-regulated occurred in the early stage of germination,display a large number of physiological activity of startup process of strong.Gene expression of GO and Pathway enrichment analysis on differences,ribosomes,the citric acid cycle(TCA cycle),glutathione metabolism significantly enriched the highest degree,display the stage of protein synthesis is exuberant,for oxidation and decomposition can be strong,antioxidation function.(5)By SSR analysis,a total of 17,262 SSR markers,the distribution of SSR type is more single base,double base type and three base type total mark 94.02%,the remaining four base type,five base type and six base type accounted for only 5.98%.(6)According to the high-throughput sequencing results of Zanthoxylum dissitum Hemsl,the PP2C gene primers were designed successfully,using RACE technology,cloned the full-length sequence of PP2C protein.The sequence and high-throughput sequencing results were compared,which has a very high similarity,shows high throughput sequencing results with high reliability. |
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