Transcriptional Regulation Analysis Of Carica Papaya Lycopene ?-cyclase Genes

Lycopene is a carotenoid that could effectively inhibit oxidation and delay aging.The content and proportion of lycopene determine the appearance quality,nutritional value and economic benefits of papaya fruit.Studying the metabolism of lycopene has theoretical implications and practical applications.Lycopene cyclase controls a key step in the carotenoid synthesis pathway.In papaya,two isoforms of lycopene cyclase are CpCYC-B and CpLCY-B.These two paralogous genes exhibit tissue-specific expression.CpCYC-B has chromoplast-specific expression,which is mainly expressed in pulp,affecting the carotenoid content and causing the color difference of papaya fruit;while CpLCY-B is highly expressed in leaves and peels of papaya.We cloned and identified the promoter sequence of CpCYC-B and CpLCY-B for further analyzing their key cis-elements and expressing activity.By transcriptome analysis of papaya pulp at different developmental stages,transcriptional factors involving fuits development could be screened.Using yeast one hybrid systems,two transcription factors binding to promoters of CpCYC-B and CpLCY-B were identified.For function analysis of those two transcription factors,dual luciferase enzyme systems experiments and light treatments were carried out.These main findings are:1.CpCYC-B/CpLCY-B promoter cloning and activity analysis.The promoter sequences of CpCYC-B and CpLCY-B were cloned from papaya and analyzed for functional cis-elements.Analysis of the full length sequence of CpCYC-B and CpLCY-B was performed on the plantcare website and the two promoter sequences contained some identical and different cis-acting elements.The full length of the CpCYC-B and CpLCY-B promoter and a series of 5'-end deletion fragments were fused upstream of the GUS reporter gene and transferred to different plants(Arabidopsis thaliana,tomato).The results showed that CpCYC-B and CpLCY-B had the strongest and weakest respectively promoter function from the translation initiation site to the upstream of 500 bp.Further deletion analysis of the promoter revealed that there is one silencer element in the 375 bp region of the CpLCY-B promoter,while the element at the 235 bp region of the CpCYC-B promoter has no specific function.2.Interaction screening of CpCYC-B/CpLCY-B promoter and candidate transcription factors.The transcriptomes of different developmental stages of two varieties were analyzed and some differentially expressed transcription factors were initially obtained.We selected eight candidate transcription factors involving in lycopene metabolism,and two bHLHs factors binding to cis-elements of CpCYC-B and CpLCY-B were identified though yeast one hybrid.3.Functional analysis of CpbHLH1 and CpbHLH2.CpbHLH1 and CpbHLH2 belong to the bHLH family involving in stress response and regulation of light signals.Transient expression analysis of CpbHLH1 and CpbHLH2 using the dual luciferase system in tabacco revealed that CpbHLH1 and CpbHLH2 inhibited and promoted CpCYC-B and CpLCY-B in papaya,respectively.Analysis of additional experimental results indicated that CpbHLH1 and CpbHLH2 were regulated by light signals during papa development,thereby regulating key gene expression on the carotenoid pathway.