Association Study Of The Variation Of Cucurbitacin E With Related Gene Expression Analysis Of Watermelon

Watermelon(Citrullus lanatus),an important fruit-based vegetable crop,occupies an important position in the horticultural industry.Bitterness is one of the most important factors affecting the quality of watermelon.During the growth of watermelon,heat,drought,insect pests and its own genetic factors cause its fruit to become bitter,which is caused by a tetracyclic triterpenoid chemical called cucurbitacin.The bitterness affects the commercial value of watermelon,but in medicine,cucurbitacin E shows good medicinal potential.Therefore,it is of great theoretical and practical importance to study the synthesis of cucurbitacin in order to improve the economic value of vegetable crops and to investigate the medical value of cucurbitacin in depth.In this study,three different bitter watermelon were used as research materials: bitter watermelon "PI 186490",sweet watermelon "COS"(Cream of Saskatchewan)and tasteless watermelon "1553".The fruit flesh of watermelon was sampled at six post-pollination periods(10,18,26,34,42 and 50 days),and the key genes of cucurbitacin E synthesis and metabolism in watermelon were identified in the Cucurbitaceae database,and the key genes related to cucurbitacin E metabolism were mined in combination with the transcriptome data constructed in the previous stage.The content of cucurbitacin E in fruits,leaves and stems of watermelon at different developmental stages was determined by high performance liquid chromatography.And q RT-PCR was used to analyze the expression patterns of the enzyme genes(Cla007077,Cla007078,Cla007079,Cla007080,Cla007081,Cla007082,Cla011508,Cla008354,Cla008355)involved in the metabolism of cucurbitacin E synthesis at different developmental periods in the fruit flesh,homology comparison with other cucurbit crops and phylogenetic tree construction,the main research contents and results are as follows:(1)The content of cucurbitacin E in watermelon fruits and leaves of bitter watermelon "PI186490" was high at the early stage of development,and decreased abruptly at the middle of development,then increased gradually until the peak during the developmental period.The key period for the accumulation of cucurbitacin E in watermelon fruit is from 26 to 34 days after pollination.(2)The gene expression level of bitter watermelon "PI 186490" was higher at the early stage of development,and was down-regulated at the middle of development,and then gradually up-regulated to the peak during the development until fruit ripening period.The gene expression level of sweet watermelon was much higher at the early stage of development than at the late stage of development.(3)The up-regulated expression of cytochrome P450 enzyme gene family,oxidized squalene cyclase gene OSC(Cla007080),transcription factor gene Cl Bt(Cla011508)and acetyltransferase gene ACT(Cla007081)were correlated with the accumulation of cucurbitacin E in watermelon fruits,and the gene expression levels were positively correlated with the change pattern of cucurbitacin E.(4)The expression levels of Cla007078 screened in the experiment were consistent with the change pattern of cucurbitacin E.The expression levels of ACT and OSC genes were higher in the critical period of cucurbitacin E accumulation in watermelon fruits than in other periods,and it was tentatively speculated that Cla007078,ACT and OSC genes were candidate genes related to cucurbitacin E anabolism in watermelon.(5)The watermelon Cla007080,pumpkin Cma Ch17G013880 and melon MELO3C022374 are on the same branch of the evolutionary tree;the watermelon Cla007081,fatmelon Bhi12G002104 and pumpkin Cma Ch08G000350 are on the same branch;the watermelon cytochrome P450 genes(Cla008354,Cla008355)are highly similar to the amino acid sequences of homologous gene proteins in melon and pumpkin;watermelon Cla007082 is the closest relative to the protein expressed in pumpkin Cma Ch08G000340.(6)Cla007077,Cla007078 and Cla008355 were not mutated in watermelon material,while a few synonymous and nonsynonymous mutations existed in Cla007080,Cla007081 and Cla007082.Cla011508 was more mutated in bitter watermelon "PI 186490",but no mutation in "COS".