Analysis Of Chloroplast Genome Assembly And Study On The Transcription Factors Regulating Cucurbitadienol Synthase Gene In Siraitia Grosvenorii

Siraitia grosvenorii,an important medicinal plant,belongs to the endemic species in China,mogroside V of which fruit has the effects of cough,eliminating phlegm,antiinflammation,and it is also has been recognized a precious natural sweetener with high sweet and low calorie.However,the content of mogroside V in existing varieties is low,and its complex biosynthesis and unrecognized molecular regulation process limit the further exploitation and utilization of this precious natural.sweetener.The S.siamensis,which belongs to the same genus with S.grosvenorii,contains siamenoside which sweeter than mogroside V,and the content of siamenoside in S.siamensis is much higher than that of S.grosvenorii.While both the germplasm and resources research of S.siamensis were scarce,it is necessary to distinguish these two important medicinal and sweet plants form germplasm resources.In addition,Cucurbitadienol is a significant molecular skeleton of mogrosides and siamenosides.It was found that exogenous phytohormone could significantly promote expression of gene ecoding cucurbitadienol synthase and corresponding product content,the regulation mechanism of SgCDS expression is ambiguous,which limits the further optimization for the mogrosides biosynthesis pathway.In the first part of this project,we sequencing the DNA of S.grosvenorii and S.siamnsis,then assembled and annotated the chloroplast genomes of these two species,finally,four pairs of molecular markers were developed to distinguish the two species.In the second,we cloned the promoter of SgCDS,and use the exogenous phytohormone to induce SgCDS with high expression,then we constructed a cDNA plasmid library,and obtained candidate transcription factors that regulate SgCDS expression by Yeast one-hybrid screening.Candidate transcription factors were characterized on function,and cis-elements in SgCDS promoter interacting with the transcription factor were explored.The main findings are described as follows:1.Sequencing,splicing assembly,and annotation of the chloroplast genomes in two species of Siraitia genera were performed.We obtained 158,757 bp and 159,190 bp chloroplast genomes in S.grosvenorii and S.siamensis,respectively,which contain 138 genes,including 89 coding genes,37 tRNA and 8 rRNAs,of which 23 genes have introns.Momordica charantia was closet with the two Siraitia species in the evolutionary relationship.9 genes(accD,atpA,atpE,atpF,clpP,ndhF,psbH,rbcL and rpoC2)underwent positive pressure selection.20 fragment regions in the intergenic region of chloroplast genome have potentiality for molecular marker development.Based on nucleotide diversity,four molecular markers were designed and verified for identification of S.grosvenorii and S.siamensis.2.The effects of exogenous phytohormone drive on the growth and mogrosides biosynthesis in S.grosvenorii.It was found that the phytohormone mixture of MeJA,SA,ABA and GA can effectively promote the expression of SgCDS and can temporarily increase the accumulation of cucurbitadienol.The growth of S.grosvenorii fruit treated with CPPU was inhibited,even cracked fruit was found.It was found that the fruit epidermis exhibited lignification earlier than control.CPPU is not suitable for enlarging the S.grosvenorii fruit on 20 days after pollination.3.SgCDS gene promoter sequence was cloned and analyzed.1,258 bp length SgCDS promoter was cloned by genome walking method.the TATA-BOX and CAAT-BOX core promoter elements was predicted,as well as the response elements related to MeJA,SA,ABA and GA were contained in the promoter.Deletion of the 5' of the promoter in different lengths indicates that the promoter function is significantly enhanced in the-128 bp to-228 bp fragments,-279 to-384 fragments and-679 to-879 fragments.4.A normalizational cDNA plasmid library with high expression of SgCDS was constructed.A cDNA plasmid library pGADT7-Lib was constructed using the material of S.grosvenorii leaf treated with exogenous phytohormone.The library capacity was about 1.5×106 cfu,and the average length of the inserts was 1000 bp,the recombination rate was about 100%,and the redundancy rate was 1%.5.The screening of transcription factors that regulate the expression of SgCDS was performed.The-260?-386 fragment in SgCDS promoter was designed as the bait sequence,that used to construct the bait vector.The pGADT7-Lib library was screened by yeast one-hybrid on the plate with 120 mM 3-AT.Total of 5 candidate transcription factors(SgERF1B,SgGATA8,SgMYB1,SgbHLH92a,and SgbHLH92b)were obtained.6.Cloning,identification and functional analysis of candidate transcription factors were carried out.All candidate transcription factor genes were found high express in S.grosvenorii fruit,and their full length were cloned.Bioinformatics analysis showed that all candidate transcription factors contained conserved domains compared with homologous proteins.Yeast one-hybrid one-to-one reversion experiment confirmed that all candidate transcription factors could interact with the bait sequence,especially for SgERF1B and SgGATA8.The subcellular localization experiment verified that candidate transcription factors were localized in cell nucleus.Candidate transcription factor gene transient overexpression and silencing experiments found that both SgERF1B and SgGATA8 have positive effects on regulating SgCDS gene expression,and SgMYB1 might participate in mogrosides biosynthesis.7.Cis-elements that interact with SgERF1B and SgGATA8 in the SgCDS promoter were identified.The purified tag proteins of SgERF1B-His and SgGATA8-His were obtained in bacterial expression system.EMSA experiments proved that SgERF1B acts with the CGGCGGCGGC cis-element in SgCDS promoter.SgGATA8 can interact with the GGTGATC cis-element,but it is aspecific.To sum up,the results in these study that the chloroplast genome of two Siraitia species,and transcription factors regulating SgCDS in S.grosvenorii,which will lay a good foundation for the collection of excellent traits of Siraitia species,molecular design breeding and plant metabolic engineering research.It also provides a new insight for the optimization of secondary metabolites biosynthetic pathway in other medicinal plants.