Genome-wide Identification Of BHLH Transcription Factor Family In Sweet Cherry (Prunus Avium L.) And Functional Characterization In Cold Resistance

The basic helix-loop-helix(bHLH)family is one of the largest families of transcription factors(TFs)in eukaryotes.It can regulate gene expression by interacting with specific sequences in target genes.The bHLH TF not only participates in the growth,metabolism and development of plants,but also plays an important role in response of plants to abiotic stress.However,due to the complexity of the bHLH TF regulatory mechanism,the current understanding of the bHLH mechanism and related regulatory networks is still limited.Related reports are limited to Arabidopsis and other model plants.For most non-model plants,especially woody fruit trees,many biological functions of bHLH members need to be studied in depth.Based on the information of the sweet cherry(Prunus avium L.)genome database,we conducted a comprehensive analysis of the basic biological characteristics of the sweet cherry bHLH family members,and discovered candidate members that respond to low temperature stress,and then the functions of the two candidate members PavbHLH28 and PavbHLH61 were investigated.The main content and results are as follows:1.A total of 66 non-redundant bHLH family candidate genes were identified from the sweet cherry genome database.Sixty PavbHLH genes were unevenly distributed on 8 chromosomes,and 6 PavbHLH genes could not be mapped to any chromosome.The result of phylogenetic analysis indicated that these PavbHLHs could be classified into 17 subfamilies according to the classification of bHLHs in Arabidopsis thaliana.Based on the PavbHLH’s amino acid sequences,10 motifs were identified.Motif 1 and motif 2 were highly conserved among the almost all PavbHLH proteins.Exon-intron analysis indicated that proteins belonging to same subfamily exhibited a similar gene structure.Promoter cis-elements analysis found that most of the promoter regions of PavbHLHs contain low temperature response elements and other abiotic stress response elements.Additionally,protein interaction network prediction analysis found that several PavbHLHs may be involved in the regulation of cold stress response.2.The expression patterns of all PavbHLHs under low temperature stresses were analyzed by RT-PCR technology.The results showed that 62 PavbHLH genes were all expressed to varying degrees during various low-temperature treatments,of which the expression levels of approximately 20 PavbHLHs were rapidly induced in the early stages of 4 and 10 ℃ treatment,reached a peak and decreasing thereafter,while the expression continued to be upregulated at 16 ℃.Combined with the results of the qRT-PCR,it was preliminarily determined that PavbHLH1,-18,-28,-60,-61,-65 and-66 may be involved in regulating the responses to cold stress in sweet cherry.3.In order to verify the cold tolerance of PavbHLH28 and PavbHLH61,the full-length sequence of PavbHLH28 and PavbHLH61 were cloned.The CDS of PavbHLH28 is 1328 bp,encoding 435 amino acids,the protein molecule size is 46.52 kDa,and the isoelectric point is 6.30.The CDS of PavbHLH61 is 756 bp,encoding251 amino acids,with a protein molecular weight of 28.18 kDa and an isoelectric point of 7.05.GFP fusion proteins were constructed for PavbHLH28 and-61 respectively,and were introduced into Arabidopsis protoplasts and tobacco epidermal cells by transient transformation technology.The results showed that PavbHLH28 and-61 were located in the nucleus,which was consistent with the predicted results.Plant overexpression vectors containing PavbHLH28 and-61 were constructed respectively.Through Agrobacterium-mediated genetic transformation,the transgenic plants of Arabidopsis overexpressing PavbHLH28 and tobacco overexpressing PavbHLH61 were obtained respectively.4.In order to screen proteins that interact with PavbHLH61,a candidate member of the response to low temperature stress,we constructed a bait vector pGBKT7-PavbHLH61.The self-activation and toxicity tests showed that PavbHLH61 had no self-activation activity and had no toxic effect on yeast strain.The bait vector and cDNA library plasmid were co-transformed into yeast strain,and 21non-redundant proteins were found to interact with PavbHLH61 by defective culture medium screening,PCR positive identification and sequencing analysis.Among them,PavbHLH162-like and Pav HIPP46-like proteins may interact with PavbHLH61 and play an important role in the cold stress response.