Cloning And Function Analysis Of HubHLH1-like From Pitaya(Hylocereus Undatus)

Pitaya?Hylocereus undatus?,belonging to cactus family?Cactaceae?,is a kind of the fruit crop.Owing to its unique shape,high yield,delicious and rich in nutrition,pitaya is welcomed by consumers and possess major economic,social and commercial values.In recent years,Guizhou has marked it as an important fruit industry.However,due to the unique geographical and mountain ecological environment of karstic areas,in the process of growth and development,pitaya was frequently exposed to a series of abiotic stresses,including drought,high salt,freezing and extreme temperatures etc,leading to a serious decline on the yield and quality.Transcription factors?TFs?play an important role in regulating gene expression and were involved in many biological processes of plants.The basic helix-loop-helix proteins belong to one of the largest class of plant TFs,named after its specific HLH domain,and most plant bHLH had been identified and functionally characterized to act as important regulators in the process of growth and developments including seed germination,fruit dehiscence,secondary metabolism,and phytochrome signaling,transduction of hormone signaling and stress responses.However,none of the family members has yet been reported in pitaya so far.In the previous work,Fan et al?2014?identified several differentially expressed genes through suppression subtractive hybridization?SSH?and cDNA microarray libraries successfully in pitaya and found a bHLH transcription factor EST fragment,which was implicated in the drought responses and its expression was remarkably up-regulated under drought stress.Therefore,it is important to carry out the research of bHLH in pitaya,as well as to reveal the molecular mechanisms of regulatory roles in adversity,especially the regulation mechanism under abiotic stress,which will provide a theoretical basis for the molecular breeding and create novel germplasms with high quality and stress resistance in the future.In the present study,a full length cDNA of bHLH transcription factor genenamed HubHLH1-like was isolated from pitaya,then,its sequence features,subcellular localization,expression patterns as exposure to diverse abiotic stress and hormones were analyzed.To further validate its function,gene over-expression vector?pCAMBIA1301-HubHLH1-like?was constructed and genetically transformed into tobacco via Agrobacterium-mediated method,the main results are as follows:1.Currently,a full-length cDNA fragment of the HubHLH1-like gene was obtained with RT-PCR and the rapid amplification of complementary DNA ends?RACE?method,which was 2,343 bp and had an open reading frame?ORF?of 1,221 bp encoding 406 amino acids with a conserved HLH domain.The calculated molecular mass and isoelectric point of this TF were 45.19 kD and 8.65,respectively.Sequence analysis indicated that this protein was highly homologous with other plant bHLH protein.Phylogenetic analysis indicated that HubHLH1-like shared the highest homology with basic helix-loop-helix DNA-binding superfamily protein of Theobroma cacao?XP₀07014952.1?and has a close relationship with dicotyledonous angiosperms.2.Transient expression of recombinant plasmid pBWA?V?HS-HubHLH1-like-GFP in protoplasts of rice with the PEG-mediated method showed that HubHLH1-like had nuclear localization signal?NLS?and was distributed specifically in cell nucleus.3.Expression of HubHLH1-like under different stresses with qPCR indicated that HubHLH1-like was remarkably elevated under drought,high temperature and cold stress with a different expression patterns,the expression of HubHLH1-like at Day 5 under drought was around four folds as high as that of the minimum?Day 10?.In addition,the expression of HubHLH1-like was still very high at the 20 th day after stress.Moreover,its expression might be activated by SA,ABA as well as H2O2,and exogenous SA could also enhance its expression.4.Gene over-expression vector pCAMBIA1301-HubHLH1-like was constructed and was genetically transformed into tobacco “K326” via Agrobacterium-mediated method.Totally,10 transgenic tobacco lines were obtained by molecular detection.Meanwhile,qPCR detection showed that the expression of HubHLH1-like was thehighest on line-5 and lowest on line-3,while line-9 ranked the second,which laid a foundation for further identification of HubHLH1-like function.