Cloning And Functional Analysis Of CcWRI1 Gene From Carya Cathayensis Sarg.

WRI1,a transcriptional activator,plays an important role in oil biosynthesis process.In this research,after searching the transcription factor database of the embryo from Carya cathayensis Sarg.,we have cloned the CcWRI1 A and CcWRI1 B through local BLAST and Polymerase chainreaction(PCR).In order to research the molecular basis of oil synthesis regulation in Carya cathayensis Sarg.,the functions of CcWRI1 A are studyed through q-RTPCR,bioinformatics and transgenic technology.The main results are as follows:(1)CcWRI1A was cloned from Carya cathayensis Sarg.,which has 7 extrons and ORF of 1197 bp.It encodes 398 aa including two AP2 domains.It is supposed that CcWRI1 A locates in the nucleus.It is 5 helixes and a strand fewer compared with AtWRI1 in secondary structure.System evolution shows that the consequence of WRI1 cluster is the same as the family genus and the branch of CcWRI1 A is different from AtWRI1.Gene expression suggested that CcWRI1 A has a high expression level in embryo especially in the rapid oil synthesis stage,but low in stem,leaf and female flower.(2)Several genes of Carya cathayensis Sarg.involved in oil synthesis has similar expression pattern with CcWRI1 A,but CcPhoM,CcENOp,CcEAR or CcBCCP2 have mo Motifs similar to AW-Box or 15 bp element in the promoter region.What's more,the expressions of AtBCCP2?AtPhoM and AtPKp2 which are independent of CcWRI1 A in transgenic lines are similar to AtWRI1.As for the oil content,there is no difference between transgenic Arabidopsis thaliana seeds and WT seeds.CcWRI1 A plays an important role in oil synthesis in the embryo from Carya cathayensis Sarg..It is reasonable to infer that the oil synthetic regulation relying on WRI1 in Carya cathayensis Sarg.is not identical to the one in A.thaliana.Maybe the function of wri1 of Carya cathayensis Sarg.in oil synthetic regulation is different with A.thaliana,it is also possible that CcWRI1 A has disparate DNA binding specificity with AtWRI1 is one of the reason.(3)CcWRI1B has Poly(A)but no intron,these characteristics show that CcWRI1 B is one of processed pseudogenes.It is possible that as the neutral evolution occurrs,the mutations in CcWRI1 B lead to the disappearance of other sequences but coding partial of the two AP2 domain.