中国农业科学2023，Vol.56Issue(24)：4906-4915,10.DOI:10.3864/j.issn.0578-1752.2023.24.009
枳PtMLP1启动子的克隆和表达分析
Cloning and Expression Analysis of PtMLP1 Promoter in Poncirus trifoliata
摘要
Abstract
[Objective]Genetic transformation plays a significant role in exploring gene function and improving traits in citrus.Tissue-specific promoters is a key to regulate the expression of transgenes in particular tissues.Here,expression characteristics of the PtMLP1 promoter,isolated from the root subtractive library of Poncirus trifoliata,was thoroughly examined,which could lay a foundation for the specific expression of exogenous genes in citrus root tissue.[Method]The complete sequence of PtMLP1 gene was cloned by PCR using DNA as a template.The physiochemical attributes,secondary and tertiary structures of PtMLP1 protein were predicted by ExPASy,PSIPRED,and SWISS-MODEL tools.Cis-acting elements in PtMLP1 promoter were predicted by PlantCARE.The expression pattern of PtMLP1 in P.trifoliata trees of diverse ages was examined by employing real-time qPCR.Furthermore,to investigate the tissue-specific expression of the PtMLP1 promoter in citrus,a pBI121-ProPtMLP1::GUS plasmid,in which GUS expression was controlled by the PtMLP1 promoter,was constructed and then introduced into P.trifoliata through Agrobacterium-mediated hypocotyl transformation.[Result]PtMLP1 consisted of two exons and one intron,which possessed a 471 bp open reading frame encoding a protein with 156 amino acid residues.This protein had a molecular weight of 17.63 kilodaltons with an isoelectric point of 5.49 and contained a Bet v I functional domain in its primary structure.Moreover,the secondary structure of PtMLP1 contained three α-helices and seven β-folds,while its tertiary structure had a conserved hydrophobic binding site and a cyclic domain,which was rich in glycine.The PtMLP1 promoter was 1 666 bp long.Multiple root-specific expression elements,phytohormone response elements(such as the TGACG motif,P-box,and ABRE),and the TATA box and CAAT box core elements were predicted in the promoter.Additionally,the 3-terminal untranslated region of PtMLP1 was predicted to contain a poly(A)signal AATAAA.Notably,the expression of PtMLP1 was significantly higher in the roots of 1-month,6-month,and 20-year-old P.trifoliata,with fold changes of 46.34,74.82,and 110.25,respectively,compared with those in leaves.GUS expression analysis of pBI121-ProPtMLPl::GUS transgenic plants showed that PtMLP1 promoter exhibited specific and high expression in roots,and its expression levels were 7.76 to 124.78 times of that in the leaves.[Conclusion]The sequences of the PtMLP1 gene and its promoter were successfully obtained,and the promoter demonstrated the ability to drive specific expression of GUS gene in citrus roots.关键词
枳/主要乳胶蛋白/根特异性启动子/GUSKey words
Poncirus trifoliata/major latex protein/root-specific promoter/GUS引用本文复制引用
姚利晓,苏娟,郭兴茹,李凤龙,何永睿,邹修平,陈善春..枳PtMLP1启动子的克隆和表达分析[J].中国农业科学,2023,56(24):4906-4915,10.基金项目
国家重点研发计划(2021YFD140080,2021YFD160080)、国家现代农业(柑橘)产业技术体系(CARS-26) （2021YFD140080,2021YFD160080）
