Identification And Function Analysis Of Bf Transcriptional Factor Regulating Bitterness Biosynthesis In Cucumber (Cucumis Sativus L.)

Cucumis sativus L., a species of Cucumis (Cucurbitaceae), is one of the important worldwide vegetables. However, in the production of cucumber, genetic and cultivated factors often lead to occurrence to bitter cucumbers, which has a serious impact on the commodity quality and economic benefit. Cucumber bitterness is caused by a substance called cucurbitacin C. Our previous studies reveals that cucurbitacin C biosynthesis is controlled by a cluster consisting of a Bi gene, six P450 genes and an ACT gene (Bi gene cluster) in cucumber and Bi is the key gene controlled the first step of cucurbitacin C biosynthesis.Recently, we found a pair of bitterness cultivate ’XY-2’ and no bitterness mutant ’XY-3’ in cucumber, and searched an SNP between ’XY-2’ and ’XY-3’ using genome re-sequencing and bioinformatics comparison analysis. The SNP located on Bf gene, which encodes a bHLH family transcriptional factor (TF). Importantly, the mutant site appears on Bf intron splicing site, and it probably affects Bf expression by changing Bf intron splicing. Consequencely, we propose a hypothesis that Bf regulates biosynthesis of cucumbers bitterness substances cucurbitacin C.Due to cucumber transgenosis system is not available yet, we adopted cucumber cotyledon transient transformation methods to lead the exogenous Bf gene into Bf mutant ’XY-3’ and verifed the function of Bf transcriptional regulation to cucurbitacin C biosynthesis in cucumber. Furthermore, yeast one-hybrid, tobacco transient transgenosis system, chromatin immunoprecipitation (ChIP), and further revealed the molecular mechanism of Bf transcriptional regulation function that Bf specifically binds to Bi cluster genes promoters (BCPs) E-box to regulate the expression of Bi cluster genes, ultimately controls the biosynthesis of cucurbitacin C in cucumber. The main results were as follows:(1) Function indentification of Bf regulating cucurbitacin C biosynthesis using cucumber mutant cotyledon transient transgenosis system:Bf gene was recombined to the expression vector pCAMBIA1300 and transiently transformed into Bf mutant ’XY-3’ cucumber cotyledons by using agrobacterium infiltration so that Bf expression in’XY-3’. The results showed that Bf expression in Bf mutant ’XY-3’ induced the expression increases of Bi gene cluster, and eventually produced a complementary pheonoype that cucurbitacin C occured in Bf mutant ’XY-3’.(2) Identification of Bf transcriptional regulation function by yeast one-hybrid:Bf gene was recombined to the effect vector pGADT7-Rec2 containing the GAL4 activation domain (AD), then we ultilized yeast one-hybrid to transform Bf-pGADT7-Rec2 effect vector and pHIS2 report vector containing Bi cluster genes promoters(BCPs) into yeast, the result showed that yeast transformed Bf was obviously grown in SD-Trp-Leu-His solid culture medium with the optimum concentration of 3-AT compared to negative control. Here we verified that Bf specifically binds to BCPs to exert its function in yeast.(3) Identification of Bf positive transcriptional regulation function by tobacco transient transgenosis system:Bf gene was recombined to the effect vector pCAMBIA1300 and transformed agrobacterium, at the same time, pCAMBIA1300-LUC reporter vector containing BCPs transformed agrobacterium. Then we mixed two kinds of agrobacteriums and infiltrated into tobacco leaves. The result showed that Bf had higher activation to Luc gene in tobacco leaves compared to negative control, which was consistent with yeast one-hybrid results, collectively identified that Bf could directly bind to the BCPs and play positive transcriptional regulation function.(4) Cis-element identification of Bf specifically binds to BCPs using ChIP-qPCR:Bf gene fused 4MYC-tag was recombined to the expression vector pCAMBIA1300 and transiently transformed into Bf mutant ’XY-3’ cucumber cotyledons by using agrobacterium infiltration, so that Bf-4MYC protein expressed in ’XY-3’. We carried on ChIP with treated cotyledons, and qPCR using the E-box region of BCPs primers. The result showed that Bf TF has different binding capacity to different 8 BCPs E-box and to different E-box of an BCP, which further revealed that Bf transcriptionally regulates expression of Bi gene cluster through binding to E-box of BCPs.