Identification Of Gene Members Belonging To Homeobox Family In Phyllostachys Edulis And Functional Analyses Of PeKNOX9 And PeKNOX18

TheHomeobox(HB)family is an important transcription factor,and the members of several subclasses of which are closely related to the process of lignification.Therefore,it is of great reference value to systematically study the functions of theHB family gene members in the bamboo lignification process in order to artificially regulate the lignin composition and content.Moso bamboo(Phyllostachys edulis)is used as the research object,starting from the genome-wide identification of HB genes,the analysis and prediction of the genetic structure of PeHBs,basic physicochemical properties of encoding amino acids,conserved domain and GO function have been performed in this study.On the basis of the combination of the transcriptome data from different development processes and the quantitative analysis results of qRT-PCR,the expression pattern analysis of HB genes was carried out,and the preliminary function studies of PeKNOX9 and PeKNOX18 were further conducted.The main results are as follows:1.Genome-wide identification of HB family in moso bamboo.To identify and analysis theHB genes in the genome of moso bamboo by using bioinformatics methods.115 HB genes(PeHB1?PeHB115)were finally identified.According to the conserved domains and phylogenetic analysis of moso bamboo,Arabidopsis,and rice,PeHBs were divided into 13different subclasses(BEL,DDT,HD-ZIP I?IV,KNOX,NDX,PHD,PINTOX,PLINC,SAWADEE,and WOX).GO enrichment analysis showed that 19 PeHBs were annotated as related to the formation of lignification.Co-expression network analysis showed that 10 of the19 PeHBs had co-expression correlation.The analysis of PPI predicted that three PeHBs of KNOX subclass were hub proteins.2.Expression Profile Analysis of PeHBs.As the height of bamboo shoots increases,the degree of lignification gradually deepens.The qRT-PCR analysis showed that with the increase of the height of bamboo shoots of moso bamboo,except for PeHB005,the expression of the remaining 18 PeHBs genes were all up-regulated,but showed a trend of continuous rise or a rise first and then decrease.In addition,the lignin content of winter bamboo shoots increased with the extension of storage time and the degree of lignification deepened during storage,among which the expression levels of 14 PeHBs were similar to those of different heights shoots Moreover,with the lignification degree of bamboo shoots increased,the expression levels of structural genes related to lignin synthesis(Pe4CL,Pe C3H,Pe CCR and Pe COMT,containing KNOX binding motifs in their promoters)increased significantly,which was positively correlated with the expression levels of 5 PeKNOXs.This provided a strong evidence that PeHBs were involved in the regulation of lignin biosynthesis.3.The regulation of PeKNOXs verified in yeast.Analysis of autonomous transcriptional activities in yeast AH109 cells showed that PeKNOX2,PeKNOX8,PeKNOX9 and PeKNOX18had no autonomous transcriptional activity except PeKNOX1.Furthermore,based on the yeast two-hybrid experiment,preliminary judgment a weak interaction was found between Pe BELL5and PeKNOX9,Pe BELL18 and PeKNOX18,Pe BELL21 and PeKNOX2,while a strong interaction occurred between Pe BELL9 and PeKNOX9,Pe BELL21 and PeKNOX8.There was a strong interaction between Pe OFP1 and PeKNOX9.However,further experimental analysis and verification are needed.Moreover,Preliminary inference from yeast single hybridization experiment showed that the expressed PeKNOX9 could recognize the core sequence of TGACAGC-motif in yeast.PeKNOXs can not only interact with other proteins,but also participate in the regulation of lignification process by combining with specific cis-acting elements as the core protein.4.Cloning and expression analysis of PeKNOX9.The open reading frame(ORF)of PeKNOX9 was successfully cloned,which was 903 bp in length,and there were a large number of elements related to high temperature,drought and other response components in the upstream promoter region.The gene encodes a 300 aa protein with KNOX 1,KNOX 2,and homeobox-KN conserved domains,but no ELK domain.Tissue specific expression analysis showed that PeKNOX9 was expressed in the root,stem,leaf and other tissues of moso bamboo,but the expression in leaf and stem was relatively high.In addition,the expression of PeKNOX9 was induced by drought,salt,low temperature at 4?and high temperature at 42?under stress conditions,indicating that it may play an important role in stress response.The overexpression of PeKNOX9 in Arabidopsis,both of the number and leaf area of rosette leaves increased,suggesting that PeKNOX9 could promote the growth of transgenic plants.5.Cloning and expression analysis of PeKNOX18.The ORF of PeKNOX18 was also successfully isolated,which was 1038 bp and containing 2 exons and 1 intron.It can encoded a protein with 345 amino acids and containing the typical KNOX 1,KNOX 2,ELK and homeobox-KN conserved domains.Cis-element analysis showed that there had multiple hormone-related regulatory elements in the promoter sequence of PeKNOX18.Tissue specific expression analysis showed that PeKNOX18 expressed in the stems of bamboo,among which the expression level in the young stems was the highest,while the expression level in the leaves was hardly detected.These results indicated that the expression of PeKNOX18 was induced by ABA,but inhibited by GA₃.The overexpression vector of PeKNOX18 was constructed and transfected into wild-type Arabidopsis.It was found that the transgenic plants had no significant difference in phenotype from the wild type except for the appearance of twin siliques.The regulation of lignification in moso bamboo is a complex regulatory network,involving multiple TFs and structural genes.This study not only laid a foundation for revealing the function of PeHBs in bamboo lignification,but also provided a reference for a comprehensive exploration of the molecular mechanism of bamboo wood properties.