Cloning,Functional Analysis And Screening Of Related Molecular Markers Of Bitterness-Related Genes In Melon(Cucumis Melo L.)

Bitterness in melon fruit is main caused by the accumulation of cucurbitacins.The germplasm resources of melon are rich,and the bitterness of different varieties of melons are distinctly diverse.Although biosynthesis and regulation of bitter substance in Cucurbitaceae plants has been reported,the change pattern of bitterness during fruit development remain unknown.In this study,melon accessions Nb46(with bitter flesh)and Nb320(with non-bitter flesh)were selected to perform biosynthesis and transcription analysis.Molecular mechanisms on different stages of melons with different bitterness were analyzed,and the main results are as follows:1.Bitterness was measured for 19 melon inbred lines using tasting method.The data showed bitterness severity varied in several lines of var.chinensis during fruit development.For example,bitterness was severe at stage I,but moderate and disappeared at stage II and III in the fruit of Nb46.But there was no bitterness detected in the fruit of Nb320 during whole period of development.Furthermore,content of CuB decreased gradually in Nb46 with development,while the CuB content remained a quite low level in Nb320.Besides,the contents of total soluble sugar were measured at stage III of these melon accessions.A positive correlation(0.890**)was found between bitterness severity at stage I and sugar content at stage III,it indicated that there may was a relationship between the sugar content and the bitterness which provide insight into the mechanism of bitterness transformation during fruit development.2.The bitterness related genes CmBi and CmBr were cloned and sequenced.Then we did the phylogenetic analysis and conversed donain prediction on the CmBi by MEGA software.The data showed that CmBi with CsBi,ClBi and CPQ were clustered in the same sub-clade,which was a novel subgroup named Cucurbitadienol synthase.It consists of two conserved motif: DCTAE and QW repeat.The three-dimensional protein model of CmBi was determined using SWISS-MODEL software online,and a hybrophobic transmebrane and two barrel domains were predicted.We also predicted a five-amino acid ligand-binding site composed of Lys-783,Val-784,Tyr-785,Leu-788 and Gln-829.3.Then we investigated the expression levels of the bitterness related genes(CmBi,CmBt,Cm160,Cm170,Cm180,Cm710,Cm890,Cm490 and CmACT)in Nb46 and Nb320 during three periods of fruit development.Different expression patterns were found between Nb46 and Nb320.The expression levels of these genes were significantly higher in Nb46 than Nb320 in the early developmental stages,this correlated with higher concentration of CuB in Nb46.These results demonstrate that bitterness varies in var.chinensis during fruit development and regulation of the CuB biosynthesis related genes are critical to this process.4.One SNP locus was identified by comparing the CmBt gene of Nb46 and Nb320.Then a AS-PCR primer was designed and verified,and results showed that this primer can only identify 8 melon lines.Furthermore 39 SSR markers were tested using 34 melon linesand the results showed that the marker CMCTN7 could effectively distinguish the bitter and non-bitter phenotypes of the flesh.This marker could be used in the breeding of non-bitter melon cultivars.