Cloning And Preliminary Functional Analysis Of CoWRI1, One Of WRI1-like Transcription Factor Gene From Coconut Pulp

Coconut(Cocos nueiferaL),one of the key plantion crops of the tropics. is a member of the monocotydonous family Aracaccae.Aracaccae.It is the Only species of the geous Cocos belonging to the Subfamily Cocoideac.Based on the precious construction of cDNA Library from the pulp of Coconut and EST sequence analysis,one gene likely cncoding a new kind of AP2/EREBP tanseription Cactor Was obtained. The firther investigation about its structure, expression Profiles an biological function bad been carried out.The main results are as follows:1.With the methold of bioinformatic analysis.it indicated that this gene contained an open reading frame encoding a deduced polypeptide of 342 amino acide which included two copies of pant-specific conserved AP2/ERF DNA-Binding domain.Ilomolgous analysis with some AP2 family type ramscription factors of two Copics of AP2/ERF DNA-binding domain showed that it shared high homo;uogy with the Arabidopsis AtWRII and brassica napus BNWRII.SO this transcription factor was designated as CoWRII.In addition,the compsition of amino acid sequence.secondary structure, teriary stucture and functional domains were predicted.More and more information suggested CoWRI1 played an important role as an WRII-like transcription factor in the regulation of oil accumulation in maturing seeds as well as AtWRII and BnWRI12.To investigate the dynamic expression characteristic of CoWRII during endosperm development.the.cxpression profile of CoWRII in differnet lissues of Coconut were determined using fluorescent-quantitation RT-PCR.It reveraled that the CoWRII Gone was relaticely highly expressed in fresh pulp、which was rclated to the abundant accumulalion of storage compounds in this stage.3.In orde to identify the transcriptional activation of CoWRII.the yeast two-hybrid systerm was applied.The recombinant expression vector pGBKT7 CoWRII was constructed and transformed into the yesst strain AH109,then the positive yeast transformants were Selected using the SD/-Trp/-His/-Ade selective medim. As a result, the transcriplional aclivatiod of CoWRII was confirmed.4.The yeast one-hybrid systerm was applied to identify the DNA-binding aclivalion of CoWRI1. Based on the bigh homology between CoWRII and AtWRII.itwas likely thet they had the similar DNA-binding activation,so the P580 DNA sequence of approximate 580bp from the full promoter of BCCP2 gene in Arabidopsis,was choosen as thehypothetical cis-clement for CoWRII,which il had been confirmed that A1WRI1 was able to sperifically imteract with.A recombinant reporier vector pHIS2.1-p580 with a reporter gencHIS3 was constructed. while another recombinant expression vector pGADT7 AD-CoWRI1 was constructed. After that, the two vectors were cotransformed into the yeast srrain Y187, the positive yeast transformants were selected using SD/-Trp/-Leu/-His seleclive medium with appropriate 3-AT. The result indicated that CoWRI1 owns the DNA-binding activation, it can specifically interact with the promoter sequence of BCCP2 gene from Arabidopsis.5. At the end of this study, to investigate the biological function of CoWRI1 in plant seed in which CoWRI1 was overexpressed, the seed-specific overexpression vector pCAMBIA1300S-napin-CoWRI1 in which the CoWRI1 ORF under the control of napin promoter had beend constructed successfully, the further work is carrying on.