杜仲法呢基焦磷酸合酶EuFPS1互作蛋白筛选及分析

Screening and analysis of EuFPS1 interacting proteins in Eucommia ulmoides

法呢基焦磷酸合酶(farnesyl diphosphate synthase,FPS)是杜仲胶生物合成途径中的关键酶之一,EuFPS1基因对杜仲胶积累有促进作用,但对其调控的分子机制尚不清楚。本研究采用SMART同源重组技术构建了杜仲酵母cDNA表达文库,以EuFPS1基因为诱饵筛选互作蛋白,经测序和BLAST比对初步筛选出41个互作蛋白。GO和KEGG分析结果显示,互作蛋白主要参与卟啉化合物代谢调控、紫外线与冷响应、叶片发育等生物进程,MEP代谢、卟啉代谢、泛素介导的蛋白质水解等代谢途径。选取3个互作蛋白(EuUFM1、EuLHC1、EuKS1)进一步验证互作关系,qRT-PCR分析发现EuFPS1与EuUFM1、EuLHC1、EuKS1基因在杜仲不同组织中均表达,其中EuFPS1与EuKS1的表达呈负相关。本研究为解析杜仲EuFPS1及其与互作蛋白的作用机制奠定了基础。

Farnesyl diphosphate synthase(FPS)is a crucial enzyme in the biosynthetic pathway of Eucommia ulmoides rubber.Previous studies have indicated that the EuFPS1 gene facilitates the accumulation of E.ulmoides rubber,however,the specific molecular mechanism of its regulation remains unclear.In this study,a yeast cDNA expression library of E.ulmoides was constructed using SMART homologous recombination technology,and the interacting protein was screened using EuFPS1 gene as bait.Through sequencing and BLAST comparison,41 interacting proteins were initially identified.GO and KEGG analysis showed that these intercalating proteins were mainly involved in regulating biological processes such as porphyrin compound metabolism,UV and cold response,leaf development,as well as metabolic pathways such as MEP metabolism,porphyrin metabolism,ubiquitination.Three interacting proteins(EuUFM1,EuLHC,EuKS1)were selected to further validate the interactions.qRT-PCR analysis demonstrated that EuFPS1,EuUFM1,EuLHC1,and EuKS1 genes were expressed in different tissues of E.ulmoides,with a negative correlation between the expression of EuFPS1 and EuKS1.This study serves as a foundation for the analysis of EuFPS1 and its interaction mechanisms with proteins in E.ulmoides.