The Metabolic And Molecular Regulating Mechanism Of Pumpkin Fruit Quality Development Using RNA-seq

Cucurbita moschata and Cucurbita maxima(2n =Zx=40)are two Cucurbita species belonging to Cucurbitaceae plant.The two species have significant differences in fruit quality.C.moschata germplasms CMO-X,CMO-E and C.maxima germplasm CMA with great difference in fruit qualities were used as object of this study.The metabolites were analyzed and quantified which were used to detect metabolites determining the sweetness,texture and flesh color difference for the three cultivars.Transcriptome technique was used to study the molecular regulation of biosynthesis of these quality related metabolites and antidiabetic compound inositol to finally obtain the key regulating enzyme genes for biosynthesis of these quality and nutritionaly metabolite.High performance liquid chromatography(HPLC)method was used to determine the sugers,carotenoid and inositol composition and contents in six developmental stages of three pumpkin germplasms.CMO-X,CMO-E and CMA with different sweetness were compared for the composition and content of glucose,fructose and sucrose.It was found that sucrose percentage and content were higher in CMO-X than the other two,indicating sucrose had the greatest contribution to the sweetness than other two sugars.The flesh colors of the three germplasms are difference as CMO-X and CMA have orange-yellow flesh,CMO-E has yellow flesh.Compared the composition and contents of carotenoids(?-carotene??-Carotene?lutein?zeaxanthin?neoxanthin and violaxanthin)of this three germplasms,we found composition instead of content determined the differences of pumpkin flesh color.High concentration of yellow color carotenoid,lutein leads to yellow flesh while high concentrations of orange color carotenoids,?-carotene and ?-Carotene lead moderate orange flesh.For inositol content,it was detected that total inositol content in CMO-E was higher than the other two.The concentrations of starch,amylopectin and amylose of three pumpkin germplasms were determined using dual wavelengh colorimetry technology and the morphology of starch were characterized using scanning electron microscopy(SEM)technology.From the results,we found pumpkin flesh powder,tight and glutinous traits were subject to the morphology of starch and the radio of amylopectin and amylose as the higher ratio of amylopectin and amylose the more glutinous taste,the smaller thestarch particles the more powder and tight taste.From this study,we frist obtained the pumpkin germplasm which have high lutein and inositol concentration and have special glutinous and delicious taste and the fruit quality related metabolites were identified.Using Illumina sequencing technology(RNA-Seq),the fruit transcritome of the three pumpkin germplasms in different developmental stages were sequenced.The two transcriptomes of C.moschata and C.maxima were assembled by De novo assembly and annotated for the first time.Using RNA-Seq,the metabolic and molecular regulating mechanism of pumpkin fruit quality were characterized.We concentrated on metabolic pathways of sugar,starch,carotenoids and inositol to explore the metabolic pathway related significantly differentially expressed genes(DEG).In sucrose and starch metabolic pathway,there were twelve DEGs.In carotenoids metabolic pathway,there were ten DEGs,In inositol metabolic pathway,there were two DEGs.The expression of CMO-X and CMO-E DEGs were confirmed by RT-PCR.The key genes that determine the difference in sucrose and starch composition and contents of CMO-X versus CMO-E were SUS,SPS,UGPase,GBSS,SS,SBE.The key genes that determine difference in carotenoid composition and contents of CMO-X versus CMO-E are PSY,BOH,EOH,LCYB and LCYE.The key genes determining difference in inositol contents of CMO-X versus CMO-E is MIPS1.Metabolome and Transcriptome technique was used to study biosynthesis of nutritionaly qualities related metabolites in two C.moschata germplasms and one C.maxima germplasm for the frist time,obtain the nutritionaly metabolites of the sweetness,texture and flesh color,and the key regulating enzyme genes for these metabolites also were obtained.The result provided the theoretical basis for further research the molecular regulation of biosynthesis of nutritionaly quality related metabolites and also served as a solid theoretical foundation for material selection and molecular marker-assisted breeding to generate high-quality pumpkin.