Cloning And Analysis Of Genes Involved In Saponin Biosynthesis Based On Panax Japonicus C.A.Mey Transcriptome

Panax japonicus C.A.Mey is a perennial herb of Araliaceae,and its main ch emical constituents are triterpenoid saponins,as an important medicine herb P.japo nicus is widely used in national medicine.With the development of biological scien ce,compared to the view of traditional drug chemistry,standing on the micro-mole cular perspective has become a new entry point.In this research,the Illumina Hise q 2000 sequencing platform was used to obtain P.japonicu transcriptome data,com pleted the raw data pre-treatment,metabolism and functional classification,and expl ored out the genes involved in triterpenoid saponinbiosynthesis.The molecular cloni ng method was used to restore the gene sequence from the rhizome of P.Japonicu s,and carried out the analyzes of sequence repeatability comparism,basic characteri stics of protein,multi-level structure,genetic evolution,quantitative expression.The results are as follows:The original transcriptome data of P.japonicus is 19.4Gbp,it contains 66,084,088 initial short read order,after screening for quality control,the retention52,199,221 effective short read order with an average length of 76.94 bp.Usingthe De nove method for splicing to obtain 66,403 Unigenes,there are 34,639 and 19,739 Unigen es that match the sequence information in the NR and Swiss Prot databases.There a re 26,909 and 10,638 Unigenes respectively received GO and COG information ann otations.There are 3,094 Unigenes involved in metabolic activity by KEGG,a total of 81 Unigenes were classified as saponin anabolic,contains 29 Unigenes associate d with carbon skeleton,and 52 Unigenes involved in chemical group modification.Based on the sequence information of P.japonicus transcriptome data,referenc e to triterpenoid metabolic synthesis process,the four genes of SS?SE?DSand ?-A S were selected and their full-length sequences were cloned.The full-length of ?-A Sgene was 2,716 bp,its ORF was 2,292 bp encoding 763 amino acids.The full-lengt hof DS gene was 2,556 bp,its ORF was 2,310 bp encoding 769 amino acids.The fu ll-length of SS gene was 2,104 bp,its ORF was 933 bp encoding 310 amino acids.The full-length of SE gene was 2,177 bp,its ORF was 867 bp encoding 288 amino acids.Multiple sequences were compared to the corresponding proteins of SS?SE?D S??-AS genes,to futher confirm their sequence characteristics and structural classifi cation.The results showed that four proteins were highly reproduciblewith Panax gi nseng C.A.Mey and Panax notoginseng,the homology of SS protein and Panax notoginseng(AIK21786)was 100%.The DCTAE?QW(QXXXXXW)and MWCYCR three kinds of OSC family basic sequences were screened out in ?-AS and DS prot eins.The binding sites of aspartic acid Mg2+ and phytoene synthase were screened from the SS protein.The four flavin adenine dinucleotide binding sites located at 11-119,43-124,213-281 and 222-258 amino acids,and two groups of aryl hydrocar bon hydroxylase binding domains were screened from SE protein.Real-time PCR was used to test the actual experssion of SS?SE?DS??-AS ge nes in rhizome of P.japonicus,the expression of SS gene was the highest and DS gene was the lowest,the relative expression levels of four genes may be associated with their location in the metabolic pathway.