Cloning And Functional Analysis Of Class Ⅲ Of HD-zip Gene In Betula Luminifera

HD-zip Ⅲ genes are plant-specifictranscription factors with crucial roles in vascular tissuesdevelopment in plants.Since their importances, it is interesting to study the fuctions of HD-zip Ⅲgene and regulation mechanism in vascular tissues development,which is meaningful toelucidation of wood formationandgenetic improvement of forest trees. Betula luminifera is aprecious timber tree asexcellent material, which grow fast and had short juvenile phase,and widelydistributed in south of China. Through cloning and functional analysis of class Ⅲ of HD-zip genein Betula luminifera,we got the following results:1. Five full-length cDNAs encoding class Ⅲ of HD-zip gene named BlHDZ1-BlHDZ5werecloned from Betula luminifera using RACE(rapid-amplification of cDNA ends) strategy. Thesequences of these genes ranged from2.5Kb to2.8Kb in length withan open reading frame (ORF)encoding approximate840aa. All of BlHD-zipⅢ genescarried conserved miR166responsiveelements, and the cleavage of these genes occurred at8th and10th nucleotide of the maturemiR166.Phylogenetic analysis indicated that they were different members of class Ⅲ of HD-zipgene family.Subcellular localization assays showed that the BlHDZ1and BlHDZ2protein werelocated in the nucleus.2. Different expression patterns of theseHD-zip Ⅲgenes were revealed in different organswith different developmental stages.High levelof BlHDZ1and BlHDZ2transcripts were showed inbracteole、female inflorescenceand shoot apical. BlHDZ3transcripts were highly expressed inshoot apical. While BlHDZ5were showed high transcriptsin root,female inflorescence and stem.Furthermore, in contrast toBlHDZ2, transcriptsof BlHDZ1、 BlHDZ3and BlHDZ4weremuchhigherin mature buds than dormant buds.While, BlHDZ5was mainly expressedindevelopingbuds and lignified stems.Detailed expression analysis instemindicatedthathighestexpression levels of BlHDZ1and BlHDZ2were detected in inner xylem,whilestrongexpression of BlHDZ3and BlHDZ5were appeared in fifthinternodsof lateral branchandinner xylem.BlHDZ4was up-regulated along with the lignification of stem.3. The relation between plant hormone and these BlHD-zip Ⅲ genes were also investigated. Except for BlHDZ1, The other BlHD-zipⅢgenes were induced by IAAin plantlets. After theapplication of IAA for2days, BlHDZ3,BlHDZ4and BlHDZ5were induced to different extent, andBlHDZ2was stronglyinduced when treatedfor8days.When treated by BRfor18days,theexpression BlHDZ1and BlHDZ2were up-regulated to3.5and2.2times thancontrol,respectively. Interestingly, the transcripts level of BlHDZ4was up-regulated to2,1.5,2.1,and2.1timeswhen exposed to GA, IAA, BR and Ethephonfor2days,suggesting thatBlHDZ4wassensitiveto these four kind hormonesin B.luminifera.4. As previous reports, GA could promote formation of tension wood, which could be seen asa model for wood formation.Similarily, under different type and concentration treatment, GA alsopromote tension wood formation in B.luminifera. Further investigations were also performed forthese BlHD-zipⅢgenes in this model.As a result, BlHDZ1was both up-regulated in TW and OWunderhigh concentration of GA. While, transcripts levelof BlHDZ2was both up-regulated in TWand OWunder lower concentration of GA. On the other hand, BlHDZ4was both down-regulated inTW and OWunder high concentration of GA.BlHDZ2and BlHDZ3wereup-regulated inTWunderhigh concentration of GAmix,suggesting these two genes may be involved in theformation of tension wood.All these results suggestedthat different members of theseBlHD-zipⅢgenesmay have different sensitivity to GA and play different roles in the woodformation.5. To further study the biological functions of these BlHD-zipⅢgenes, overexpression vectorswere constructed and transformed into Arabidopsis.Through screening of transgenicplants,transgenic lines of BlHDZ2and BlHDZ5were isolated and confirmed by PCR.Phenotypeinvestigation and functional analysis are carriedout at present.