Steroidal Saponins Biosynthesis Pathway And Functional Analysis Of Key Genes Of Downstream Path In Asparagus Officinalis L.

The garden asparagus(Asparagus officinalis L.),also called asparagus,is a perennial herbaceous plant of asparagaceae asparagus,with high nutritional and medicinal value.Asparagus is internationally recognized as the "king of vegetables" because of its richness in amino acids,vitamins,trace elements and other nutrients,and is also used in the treatment of various diseases because of its richness in steroidal saponins,polysaccharides,flavonoids and other active ingredients.Pharmacological studies have shown that the extracts of asparagus have inhibitory effects on many types of cancer cells,as well as lowering blood pressure,lowering blood lipids,anti-aging,improving immunity,diuretic and other functions.Steroidal saponins are one of the most critical material basis for the pharmacological activity of asparagus and are also important raw materials for the production of steroidal drugs.The biosynthesis of steroidal saponins has been elaborated only in a few species such as Paris polyphylla var.yunnanensis(Franch.)Hand.-Mzt.,Dioscorea zingiberensis C.H.Wright and Trigonella foenum-graecum L.,while preliminary predictions have been made in asparagus regarding the genes of the upstream synthetic pathway of steroidal saponins(i.e.,cholesterol synthesis genes),but the synthetic genes in the downstream synthetic pathway and the transcription factors related to the regulation of steroidal saponin synthesis have not been reported so far.In this study,the roots,young spear and flowers of two varieties of asparagus,green and purple,were used as materials for the study.A total of 437 metabolic compounds were detected by an extensive targeted metabolome,including 18 steroidal compounds,which can be broadly categorized into three types of steroidal saponins,spirosteroidal,isospirosteroidal and furosteroidal,based on their chemical structures;most of the steroidal metabolites had high abundance in asparagus roots.In addition,the total saponin content in each tissue of both asparagus cultivars was determined using UV spectrophotometry,and the results showed that the total saponin content in asparagus roots was significantly higher than that in stems and flowers.About 1.3 G of clean data with an average read length of 150 bp were obtained by Ilu mina high-throughput sequencing,and the average mapping rate with the asparagus referen ce genome reached 74.26 %.Through various bioinformatics approaches such as principal component analysis,differential expression analysis,weighted gene co-expression network analysis,correlation analysis,and homology search,45 genes related to asparagus steroid s aponin biosynthesis were excavated,among which 10 genes in the downstream synthesis p athway were identified for the first time,including five CYP450 genes involved in steroida l backbone hydroxylation and oxidative modifications: sterol C22-hydroxylase gene(evm.model.Asparagus V1＿03.698),sterol C22,C16-bis-oxidase gene(evm.model.Asparagus V1＿03.2424),sterol C26-hydroxylase gene(evm.model.Asparagus V1＿08.1961,evm.model.As paragus V1＿08.2077,evm.model.Asparagus V1＿02.1932,etc.),one sterol 3β-glycosyltransf erase gene(evm.model.Asparagus V1＿04.386)and three glycoside hydrolase genes(evm.m odel.Asparagus V1＿09.1129,evm.model.Asparagus V1＿09.871,evm.model.Asparagus V1＿Unassigned.906).Combining the chemical structures of the three types of steroidal saponin s detected from asparagus and the excavated steroidal saponin biosynthesis genes,the stero idal saponin biosynthesis pathway in asparagus was systematically hypothesized for the firs t time.By correlation analysis with the expression of steroid saponin synthesis pathway gen es and the interactions prediction between transcription factors and transcription factor bind ing sites,16 transcription factors that may be related to the regulation of steroid saponin sy nthesis were mined.The genes structures of all steroidal saponin synthesis and regulatory g enes were optimized by Iso-seq data.Three CYP450 genes involved in steroidal skeleton modification in asparagus(03.2424,08.1961,and 02.1932)were successfully cloned and constructed a series of heterologous expression vectors for Saccharomyces cerevisiae.The catalytic function of the three genes was preliminarily analyzed through heterologous expression in cholesterol producing engineering yeast atrain RH6829.