Identification Of BHLH Gene Family And Functional Analysis Of PtrbHLH142 Gene In Populus Trichocarpa

In plants,the basic helix loop helix(bHLH)is a superfamily of transcription factors second only to MYB in number.It regulates plant growth and development,conducts signals,and participates in adversity defense.However,most bHLH transcription factor functions have been identified in herbaceous plants(eg,Arabidopsis,Rice).The information and functions of the bHLH transcription factor family in forests are rarely reported,so it is necessary to study the involvement of bHLH transcription factors in wood formation.In this study,members of the PtrbHLH transcription factor family were identified and the function of PtrbHLH142 was preliminarily analyzed.The main results obtained are as follows:Based on the amino acid sequence homology alignment of Arabidopsis bHLH members,196 bHLH members were identified in the Populus trichocarpa genome,which were named PtrbHLH1-PtrbHLH196 according to their physical locations on the chromosome.The molecular mass and isoelectric point differences between family members Larger,with only one member with a transmembrane domain.A phylogenetic tree of bHLH family members such as Populus trichocarpa and Arabidopsis was constructed,showing that bHLH falls on 20 clades and is divided into 20 subgroups.The tissue expression patterns of PtrbHLH transcription factors were analyzed by eFP and Aspwood.The eFP data showed that PtrbHLH72,PtrbHLH95,PtrbHLH126,and PtrbHLH128 were highly expressed in xylem tissues.Combined with Aspwood data,it was found that PtrbHLH72,PtrbHLH61,and PtrbHLH142 were expressed in the late secondary xylem.High abundance and specific expression.The three members were verified by semi-quantitative RTPCR,and it was found that only PtrbHLH142 was specifically expressed in the xylem,and highly abundantly expressed in the secondary xylem at the later stage of maturation.The35S::PtrbHLH142-YFP vector was constructed and transformed into the leaf protoplasts of P.trichocarpa by PEG-mediated method.The observation by laser confocal microscope showed that PtrbHLH142 was a transcription factor located in the nucleus.In order to identify the function of PtrbHLH142,the 35S::PtrbHLH142 overexpression vector was constructed in the experiment,after Agrobacterium mediated transformation of Populus trichocarpa stem segments,DNA molecular identification of antagonistic buds and transcriptional expression analysis of transferred genes,11 transgenic lines were obtained,of which 8 lines had high expression level.The 7th stem node of PtrbHLH142-overexpressing PtrbHLH142 growing upright at 2 months of age was observed by scanning electron microscope,and it was found that compared with wild-type plants,the secondary xylem fiber cell walls of PtrbHLH142 overexpressing plants developed a thicker glial layer(G layer),indicating that PtrbHLH142 is involved in the formation of the glial layer of the secondary xylem fiber cell wall of P.trichocarpa.In conclusion,this study identified and classified the bHLH transcription factor family of P.trichocarpa,and analyzed the expression patterns in each member tissue,and screened out that PtrbHLH142 was highly expressed in the secondary xylem at the later stage of maturation,and it was preliminarily identified that PtrbHLH142 was involved in formation of the glial layer of the fibrous cell wall in the secondary xylem of Populus trichocarpa.