| Translationally controlled tumor protein(TCTP)is a highly conserved protein,and it is ubiquitously expressed in animal,plant and yeast cells.The functions of TCTPs were systematically analyzed in animals,but the studies on plant TCTPs are very limited.Several TCTPs have been cloned from some plant species,such as alfalfa,Arabidopsis,rice,soybean,wheat,cucumber,etc.The results indicated that TCTP play a significant role in the regulation of cell growth and reproduction,stress responses(heat shock,low temperature,high salt,drought,antioxidation),and antiapoptosis.Hevea brasiliensis(rubber tree)is an important tropical and economic crop,which is the main source of natural rubber.In rubber tree,two TCTP genes(HbTCTP and HbTCTPl)were cloned,but only HbTCTP1 was reported in detail.As for HbTCTP,only gene cloning and basic expr-ession patterns were reported.In this study,we cloned HbTCTP promoter,systematically analyzed HbTCTP expression patterns in different tissues and developmental stages of leaves,TPD and healthy rubber trees,under different stresses and hormones treatments.We further analyzed the subcellular localization,interaction proteins,and antioxidant activity of HbTCTP protein.In addition,we constructed HbTCTP plant binary expression vector and obtained transgenic tobacco plants overexpressing HbTCTP.The results are as following:1.We obtained 1201 bp promoter sequence of HbTCTP,Besides the common cis-acting regulatory elements including TATA°boxes and CAAT boxes,the HbTCTP promo-ter also harbored various known cis-elements that respond to hormone/stresses,such as ethylene response boxes(ERELEE4 and LECPLEACS2),abscisic acid(ABA)response boxes(EBOXBNNAPA,MYB1AT,MYB2AT,MYB2CONSENSUSAT,SEF4MOTIFG-M7S,WBBOXPCWRKY1 and MYCCONSENSUSAT),gibberellin(GA)response boxes(GARE1OSREP1,GAREAT,PYRIMIDINEBOXHVEPB1,OXOSRAM Y1A and MYB-GAHV),salicylic acid(SA)response box(WBOXATNPR1),auxin response box MAU-X28),wounding response box(WBOXNTERF3),copper response box(CURECORECR),heat shock response box(CCAATBOX1),and salt-induced box(GT1GMSCAM4).2.HbTCTP was highly expressed in latex,with low expression levels in other tissues:HbTCTP indicated a significant change in different leaf development stages,with the hig-hest level in light young leaves and the lowest in red leaves;Under drought,low tempe-rature,high salt,and H2O2 treatments,HbTCTP transcripts steadily decreased;Under ET and Me-JA treatment,HbTCTP was totally downregulated,but indicated slight oscillations in different times.Under wounding treatment,HbTCTP transcripts were firstly decreased and then up-regulated,and the lowest and highest expression levels separately occurred at 24h and 48h.In addition,HbTCTP transcripts were higher in TPD rubber tree than healthy one.These results suggested that HbTCTP was no tissue-specific,with significantly chan-ges in different leaf developmental stages.Moreover,I-HbTCTP was regulated by ET,Me-JA and various stresses and associated with TPD in rubber tree.3.To determine subcellular localization of HbTCTP,we constructed HbTCTP:eYFP fusion protein vector and transiently expressed it in rice protoplasts.The result showed that HbTCTP located in membrane,cytosol and nucleus.4.We obtained four HbTCTP-interacting proteins with yeast two-hybrid assay.The Blastx results indicated that the four sequences encoded different proteins including rubber elongation factor protein,17.5 kDa heat shock family protein,annexin,and REF-like stress related protein 1.5.In MCO system,HbTCTP proteins indicated the protection of supercoiled DNA,suggesting that HbTCTP possessed supercoiled DNA protection activity against metal-catalyzed oxidation.6.We constructed HbTCTP binary expression vector and transformed it into tobacco via Agrobacterium-mediated plant transformation.Currently,we have obtained 17 positive transgenic plants overexpressing HbTCTP,which lays a solid foundation for further elucidating the function of HbTCTP. |
PDF Full Text Request