Cloning And Expression Of Fuctional Genes Related To The Biosynthesis Of CuE In Citrullus Colocynthis(L.) Schrad.

Citrullus colocynthis(L.)Schrad.is rich in Cucurbitin E(CuE),which has many physiological activities and is beneficial to human health.So it attracts researchers’attention.However,the CuE synthesis pathway has not been fully defined yet,and related functional genes in the CuE synthesis pathway need to be studied in depth.In the test,the CuE and Cucurbitin E glycoside(CuE-Glu)contents were determined by HPLC from the leaves of 48 watermelon varieties and 36 watermelon varieties harvested.The measurement results show that C.cultivated watermelon resources basically contain CuE,without CuE-Glu,CuE-Glu only exists in C.colocynthis wild watermelon.In the transcriptome data of two varieties sequenced from CuE-Glu high(WM38)/CuE-Glu low(WM40),nine differentially expressed genes were screened,including 5 UDP-glycosyltransferase genes,2 Acyltransferase genes,and 1 Squalene Monooxygenase gene and a Amine Oxidase gene.Using Citrullus colocynthis(L.)Schrad.leaves as test materials,the three genes obtained in the previous research were cloned and combined with the RACE method to clone the watermelon CcSE,CcBAHD and CcVS,Bioinformatics analysis was performed;a prokaryotic expression vector was constructed for the three genes,and protein expression was induced in E.coli.Using the RACE method,a SE squalene epoxidase gene was cloned from the Citrullus colocynthis(L.)Schrad.leaves,named CcSE,with a gene length of 1876 bp,an open reading frame of 1593 bp,encoding a 530 amino acid,a molecular weight of 57.73 kDa,and an unstable protein.;Cloned and obtained the Citrullus colocynthis(L.)Schrad.BAHD acyltransferase gene,named CcBAHD,with a gene length of 1459 bp,open reading frame of 1311 bp,encoding 436 amino acids,a molecular weight of 47.68 kDa,and a stable protein;the Citrullus colocynthis(L.)Schrad.vinblastine synthetase was cloned,named CcVS,has a gene length of 1571 bp,an open reading frame of 1,314 bp,encodes 437 amino acids,has a molecular weight of 48.59 kDa,and is a stable protein.Successfully constructed the prokaryotic expression vectors pET28a-CcSE,pET28a-CcBAHD and pET28a-CcVS,and transformed them into E.coli BL21(DE3)for inducible expression.Using IPTG concentration as a variable,the best induced concentrations of CcSE protein,CcBAHD protein and CcVS were 0.8 mmol/L,1.2 mmol/L and 0.8 mmol/L,respectively.