Cloning And Interacting Proteins Analysis Of OVATE Family Proteins In Betula Luminifera

Betula luminifera is an ideal material for forest genetic study,and improvement of wood properties is one of the important contents of its breeding nowadays.Identify and function analysis of genes related to wood properties could provide breeders key clues for breeding of B.luminifera.Therefore,in the present study,we continue to conduct the cloning and functional analysis of BlOFP gene.The main results are summarized as follows:1.On the basis of transcriptome data of Betula luminifera,10 full-length BlOFP c DNAs were isolated using RACE.The long of sequences of c DNA are ranging from 499 to 1505 bp,the identity of they are ranging from 18.9% to 65.9%.The Open Reading Frame(ORF)are ranging from 324 to 1269 bp,the predicted proteins encoded by these genes are composed of 108 to 423 amino acid residues,each of which has a OVATE domain.And their identity are ranging from 11% to 50%.The phylogenetic analysis shows that 17 BlOFP genes are distributed in four clades.The BlOFP1,BlOFP8,BlOFP16 and BlOFP17 belongs to the first clade,BlOFP2 and BlOFP10 belong to the fourth clade,BlOFP4,BlOFP6,BlOFP11,BlOFP12,BlOFP13 and BlOFP15 belong to the second clade,and the remaining belongs to the third clade.2.Expression patterns of 17 BlOFPs in 10 organs or tissues were analyzed using q RT-PCR.The results indicated that BlOFP3,BlOFP4,BlOFP6,BlOFP7,BlOFP10,BlOFP12,BlOFP13 and BlOFP15 are mainly expressed in male and female inflorescence.BlOFP1 and BlOFP9 have the similar expression patterns in the stems,their expression level increased as lignifing level improved.And they are most abundant in the inner of xylem.BlOFP2 exhibits the relatively high abundance in root,stem,inner and outer xylem.Its expression pattern in stem was similar to BlOFP1 and BlOFP9,and the expression level in inner xylem is less than that of outer xylem.This indicated that these three genes involved in secondary growth.The expressions of BlOFP5,BlOFP11,BlOFP12 and BlOFP17 have no significant difference in ten organs or tissues.BlOFP8 is strongly expressed in early male inflorescence,leaves,phloem fibers and inner xylem.BlOFP3,BlOFP6 and BlOFP10 have high expression levels in phloem,phloem fiber and xylem.Compared with the straight wood,the expressions of BlOFP1 and BlOFP2 in tension wood are significantly up-regulated,but in opposite wood were significantly down-regulated.These two genes may be play an important role during secondary cell wall thickening.The expression levels of BlOFP1,BlOFP3 and BlOFP6 are less than that in the control.Except the expression level of BlOFP3 in opposite wood is down-regulated not significantly,the expression levels of BlOFP1 and BlOFP6 are down-regulated significantly.Both tension wood section and opposite wood section,the expression level of BlOFP8 and BlOFP10 are up-regulated,the expression of BlOFP8 is significantly up-regulated,but the expression level of BlOFP8 in tension wood section is not significantly up-regulated,it is significantly up-regulated in opposite wood.3.The normalized yeast hybridization cDNA library was established using B.luminifera xylem RNA.Its titer is more than 108cfu/ml and the insert fragment length is from o.5 kb to 4.5 kb,which meets the requirement of the yeast two-hybrid library.It is convenient to explore the regulating mechanism of BlOFPs in the wood formation.4.In order to elucidate the functions of BlOFP proteins in wood formation of B.luminifera,we conducted the yeast two-hybrid with Bl BEL1-likes and yeast hybridization cDNA library,respectively.Three BlOFPs were cloned into a prey vector p GBKT7 and seven Bl BEL-likes were cloned into bait vector p GADT7.And these ten carrier vectors did not have toxicity and did not autonomously activate the reporter genes in Y2 HGold.The results of yeast two hybridizations showed that BlOFP8 could interact with Bl BLH6 and Bl BLH7 proteins.BlOFP8-BD vector was further hybridized with normalized yeast hybridization cDNA library.In total of 4 proteins were screened and validate.5.BlOFP1,BlOFP2,BlOFP3,BlOFP5 and BlOFP6 were cloned into the over-expression vector p CAMBIA13011 and were transformed into Arabidopsis thaliana.Then T0-positive plants of BlOFP1 and the T3-positive plants of BlOFP2,BlOFP3,BlOFP5 and BlOFP6 were obtained.Some plants overexpressing BlOFP genes displayed a number of abnormal phenotypes.The overexpression of BlOFP2 caused the more blades,the smaller leaf area,and bending like a bow in A.thaliana.The plants overexpressing BlOFP5 showed that width and thickenss of leaves,the POD length increased significantly.Furthermore,the edges of leaves are jagged.So these transgenic plants will provide materials for further analysis of BlOFPs functions.These above results not only laid the foundations for in-depth analysis of the OVATE family proteins but provide genetic resources for molecular breeding of B.luminifera.