| In this study,an E3 ubiquitin ligase interacting with CsHsf24.1 was preliminarily screened by yeast two-hybrid technology.The interaction between CsHsf24.1 and E3 ubiquitin ligase was further verified by bimolecular fluorescence complementation and GST-pull down experiments.The subcellular localization of E3 ubiquitin ligase and the co-expression of CsHsf24.1 and E3 ubiquitin ligase genes were analyzed.The effect of E3 ubiquitin ligase on catechin biosynthesis in tea plant was analyzed by virus-induced gene silencing(VIGS).The results further enriched the regulation theory of catechin metabolism in tea plants and laid a foundation for cultivating new varieties of high-quality tea plants.The main findings are as follows:(1)Using CsHsf24.1 as bait,a cDNA library of tea plant was screened by yeast two-hybrid technology,and a CsHsf24.1 interacting protein E3 ubiquitin ligase was obtained.Bimolecular fluorescence complementation and GST-pull down experiments verified the interaction between CsHsf24.1 and E3 ubiquitin ligase.(2)The CDS sequence of E3 ubiquitin ligase gene is 1011 bp in length,encoding336 amino acids.It contains a zf-C3HC4＿3 domain between 288 and 326 amino acids and belongs to the RING＿Ubox superfamily.The protein is a hydrophilic protein with α-helix and irregular curl as its main structure.Subcellular localization prediction and subcellular localization experiments confirmed that it was localized in the nucleus.There was a negative correlation between the expression of CsHsf24.1and E3 ubiquitin ligase genes.(3)Virus-induced gene silencing(VIGS)technology was used to silence the expression of E3 ubiquitin ligase gene in tea plant.The expression of E3 ubiquitin ligase gene was silenced in tea plant materials.The expression of CsHsf24.1 was increased,and the catechin content of tea plant leaves was significantly increased.(4)In vitro ubiquitination experiments showed that CsHsf24.1 is the substrate of E3 ubiquitin ligase,and E3 ubiquitin ligase can ubiquitinate CsHsf24.1. |
