Functional Study Of B Hlh Transcription Factors QQ1 And QQ2 In Arabidopsis Thaliana

The transcription factor BZR1 play an essential role in Brassinosteroid(BR)signaling pathway.The identification and functional analysis of BZR1 target genes is essential for the further study of the BR signaling pathway and the Cross-talk between BR and other hormone signaling pathways.QQ1(unknown function,tentatively named)is one of the target genes of BZR1 and belongs to the basic helix-loop-helix(bHLH)transcription factors family.bHLH TFs function by forming homodimers or heterodimers,then bind the promoter of target genes or affect the binding with other bHLH TFs,thereby playing a regulatory role in gene transcription.Therefore,we study the function of QQ1 and its homologous gene QQ2(unknown function,tentatively named)in Arabidopsis thaliana.In this study,we analyzed the characteristics and functions of QQ1 and QQ2,the results as follow:(1)The results of bioinformatics analysis of QQ1 and QQ2 showed that they have 54%amino acid sequences identity,the conserved amino acids of HLH domain were highly conserved and the conserved sites were basically same.QQ1 and QQ2 were both atypical bHLH transcription factor,lacking the basic region which can directly bind to DNA.(2)The fusion vector of GUS gene and promoters of QQ1 and QQ2 was constructed and transformed into Arabidopsis.The results of GUS staining showed that QQ1 was abundantly expressed in flower(petal,calyx)and mature peel skin and little expression in the water hole of rosette leaves and axillary leaves.QQ2 was expressed in roots,petioles and veins at the seedling stage,with different degrees in petals,calyces,stamens and horn peels.(3)The results of subcellular localization showed that both QQ1 and QQ2 were located in the nucleu.(4)Yeast two-hybrid experiments showed that QQ1 can interact with PRE1,while can not interact with itself and QQ2.QQ2 can not interact with PRE1.(5)The over expression vector of 35S::QQ1-YFP and 35 ::QQ2-YFP was constructed and transformed into Arabidopsis.35 S :: QQ1-YFP transgenic plants showed dwarf plants,curled leaves,dark green color,short and small siliques etc.The phenotype of 35S::QQ2-YFP transgenic plants also showed phenotypes of slightly dwarfed plants,slightly curled leaves,short and small siliques.(6)The pRGEB32-QQ1 and pRGEB32-QQ2 vectors were constructed and transformed into Arabidopsis by CRISPR/Cas9 systerm.We identified a qq1 mutant line and a qq2 mutant line by PCR screening.Preliminary analysis of the mutants showed that the mutation of QQ1 and QQ2 did not affect the morphogenesis of plants,and there was no significant phenotype compared with wild-type throughout the whole growth period.(7)Preliminary analysis of RNA-seq data of QQ1 and QQ2 over expression plants found that the QQ1 and QQ2 have the same regulated mode,they may have the same regulatory network;the differentially expressed genes from QQ1 and QQ2 have the same function enrichment,mainly in terms of catalytic activity,gene binding and transcriptional regulatory activities;the differentially expressed genes from QQ1 and QQ2 are involved in the same biological pathway,mainly in terms of biological regulation,stimulating response,biological process of regulation.Therefore,we suggest that QQ1 is an atypical bHLH transcription factor,located in the nucleus,which may be involved in the development of Arabidopsis flower and siliques pods;QQ2 is also an atypical bHLH transcription factor,located in the nucleus,which may be involved in the developmental processes that may be involved in flowers,siliques pods,and also related to the vascular organization responsible for material transport in Arabidopsis.The absence of QQ1 and QQ2 did not affect plant growth and development.So we hypothesised that they might play roles with some functional redundancy in Arabidopsis.