Transcriptome Analysis And The Biosynthetic Pathway Prediction Of Steroidal Sapogenin Of Dioscorea Composita

Dioscorea composita is a perennial dioecious vine plant with high sapogenin content,high starch content,high yield and strong resistance.It is the main raw material for sapogenin extraction and the excellent raw material of bioethanol preparation.D.composita is an ideal energy and medicinal non-food plants because of the high economic value and comprehensive utilization value.Steroidal sapogenin widely existed as secondary me Tableolites in Dioscorea genus,which have pathogens preventation and physiological function of environment resistance.Steroidal sapogenin is an important raw material production of steroid hormones medicine,which has a prominent effect in the treatment of cardiovascular and cerebrovascular diseases,skin diseases,cancer and other diseases.Steroidal sapogeninexisted as aglycone of saponin.According microscopic examination,the content of diosgenin was less and it spread in compact tissue and around the vascular bundles mainly in young tubers.In old tubers,the diosgenin content was higher,and the diosgenin mainly distributed in the periderm tissue and around the vascular bundles.According determination,the sapogenin content in the tubers of male D.compostia was 10.89% higher than female.Compared with Dioscorea zingiberensis which was the main material for diosgenin production in China,the diosgenin content in the tuber of male D.compostia was 1.63 times more than it.In this paper,the transcriptome sequencing and analysis with 18 months male and female D.composita were carried out by Illumina Hiseq 2000 sequencing platform.Finally,62,341 unigenes with the length from 201 bp to 22744 bp were obtained,the average length was 724 bp and N50 was 1228 bp,the unigenes longer than 1000 bp accounted for 23.2%.With blast with Nr,Nt,Swiss Prot,KOG,GO,Pfam,KEGG da Tablease,and a total of 28,047 unigene has been annotated.Through the GO,KOG and KEGG PATHWAY classification,the number of unigenes in section of metableolic pathway,translation and signal ransduction are most.Through the calculation expression of each unigene in the male and female plants,results showed that the low expression level unigenes are the largest proportion in male and female plants,as the total number of 37~42%,while high expression level of unigenes are the smallesd.8,049 Unigenes have significant differential expression between female and male plants.There are 4,232 unigene with high expression in the female plants,and 3,817 unigene express higher in the male plants.The GO and KEGG classification of significant expression differencial gene are carried out and the result showed that female plant is faster in carbohydrate synthesis,gene replication and actin cytoskeleton regulation,while the male plant is faster in secondary me Tableolite synthesis,photosynthesis,amino acid and protein synthesis,ribosome synthsis than female plants.Within annotated Unignene,79 unigenes which encoded 35 enymes related to steroidal sapogenin biosynthesis.Diosgenin biosynthesis pathway contained sapogenin skeleton synthesis,oxidation of squalene and synthesis of steroidal sapogenin synthesis in three stages and 47,2,30 Unigenes involved respectively.The starting material for acetyl coenzyme A in the catalytic 23 kinds of enzymes,generated diphosphate farnesol after 17 step reactions,the limit step of which is mevalonate synthesis catalyzed by HMGR(hydroxyl methyl Co A reductase);In oxidosqualene biosynthesis stage,diphosphate farnesol was exchanged to 2,3-oxidation of squalene by the catalysis of 2 enzymes;The steroidal saponin synthesis stage was responsible for the synthesis of diosgenin precursor cycloartenol,and biosynthesized steroidal sapogenin under the action of various enzymes like cytochrome P450,isomerase and cyclase.the process There were about 10 kinds of enzymes involved in the third process,and the key step is the oxidosqualene cyclization catalyzied by CAS(cycloartenol synthetase).HMGR and CAS were two key enzymes in sapogenin synthesis,D.composita HMGR and CAS have coserved domains and the conserved amino acids through comparison and analysis.D.composita HMGR protein contains more alpha helies,composed of four identical subunits,positioning in the endoplasmic reticulum,is a hydrophilic protein;CAS protein also contains more alpha helies,a transmembrane helical structure located at the C terminus,positioning in the cell membrane,as a lipophilic protein.Comparion of 79 Unigenes expression in male and emale plants,the expression of 23 unigenes in the male is higher than female,3 unigenes are expressed higher in female plants than male,this result illustrates the steroidal sapogenin biosynthesis in the male plant was more exuberant than female.Reference to the me Tableolic site,sequence integrity,gene similarity and other information,10 unigenes: HMGR,MK,SQLE,FPPS,DXS,CAS,HMED,CYP51,DHCR7,DHCR24 were chosed to compare the expression in tuber,stem,leaf and seedling by the fluorescent quantitative PCR method.Ten of unigenes expressed in all selected tissues,and the expression in tuber and leaf are higher than in the stems and seedlings,this indicated that the biosynthesis of steroidal sapogenin was mainly occurred in plant tubers.The expression of unigene located in the mevalonate pathway are higher than in pyruvate pathway,this reveal that but the mevalonate pathway plays a major role in steroidal sapogenin biosynghesis on D.composita.The low express in seedling tell us the secondary me Tableolism might was not exuberant in seedling stage.According comparion,the expression of 4 genes(HMGR,CAS,SQS,SMT1)in the tuber of D.composita were 159.05,2.03,3.44,8.89 times to D.zingiberensis.Through transcriptome sequencing,this paper revealed part of gene information related to steroidal sapogenin biosynthesis and its synthetic pathway in D.composita,analyzed the biological information,structure and function of sapogenin biosynthesis key enzymes by bioinformatical methods.It laid the foundation for the future in-depth analysis on gene function,clear diosgenin synthesis pathway,the regulation of diosgenin synthesis me Tableolism andimproves the content of active ingredients.