Preliminary Investigation On Peanut BHLH Transcription Factor Gene AhHEGS1 In Seed Germination

bHLH（basic Helix-Loop-Helix）transcription factor is the second largest family of transcription factors in plants.It participates in many regulatory processes,such as plant growth and hormone regulation.The N-terminus of bHLH transcription factor contains DNA recognition and binding sites.About 50%of the basic amino acid region of bHLH transcription factor contains the highly conserved H5-E9-R13 sequence（His5-Glu9-Arg13）,which is an indispensable structure for DNA binding.The C-terminal of bHLH transcription factor is helix loop helix（HLH region）,which contains more than 40amino acids.It is widely believed to function as a dimer.Peanut（Arachis hypogaea）is an important cash crop and oil crop.Seed dormancy and germination are two important stages in its life cycle.Seed dormancy is a characteristic of plant adaptability to the environment.When the dormant seed meets favorable environment,it begins to germinate.Somepeanut seeds have physiological dormancy characteristics.Ifno germination-accelerating techniquesare performed,seed will germinate slowly,or even some seeds will be difficult to sprout,which affects the yield,food safety and planting benefit.Therefore,it is necessary to study peanut seed germination.According to the data of transcriptome sequencing,a gene with significant difference in expression levels at the critical stage before and after germination was screened.Because of higher expression level in the just-germinated peanut seeds,it was temporarily named AhHEGS1（High Expression in just-Germinated Seeds 1）.Our previous study found that AhHEGS1 gene has two different splicesomes,AhHEGS1.1and AhHEGS1.2.In this study,their expression patterns were clarified.AhHEGS1 gene encodes a bHLH transcription factor,and their characteristics were analyzed using subcellular localization and yeast transcriptional activation.The vector of overexpressing AhHEGS1 were constructed,and transformed into peanut and Arabidopsis（Arabidopsis thaliana）to identify its function.This study will lay a foundation for understanding the molecular mechanism of peanut germination.Through the above researches,the major results were shown as follows:（1）Sequence analysis of AhHEGS1 gene.Sequencing revealed that the c DNAs of AhHEGS1.1 and AhHEGS1.2 genes in length were 1,219 bp and 1,237 bp,respectively,and their ORFs were 1,188 bp and 1,152 bp,which encode the proteins composed of395 amino acids and 383 amino acids,respectively.AhHEGS1.2 lacks of 12 amino acids at the C-terminus than that of AhHEGS1.1.The difference between the two splices was due to the altervative splicing of the sixth intron,resulting that AhHEGS1.1deleted the larger sixth intron（358 bp）,while AhHEGS1.2 removed the smaller intron.Through homologous alignment of the amino acid sequences among AhHEGS1 and other species,the results showed that the similarity between AhHEGS1 protein and soybean Gm CIB1（Glycine max）,tribulus alfalfa MtbHLH63（Medicago truncatula）,SsbHLH63（Spatholobus suberectus）of myzula bean were more than 55%,whereas the homology between AhHEGS1 and Arabidopsis known At CIB1 was lower than those comparisons metioned above.（2）Analysis of spatial-temporal expression pattern of AhHEGS1.The expression pattern of AhHEGS1 in different tissues of FH1 was analyzed by real-time PCR.The results showed that both splicing transcripts of AhHEGS1 has the highest expression levels in flowers and the lowest expression levels in the roots.Overall,the expression of AhHEGS1.1 was higher than that of AhHEGS1.2.Taken the testa-free seeds after pegging for 10 to 70 days as samples,the gene expression level was investigated.The results showed that the expression level of AhHEGS1.1 and AhHEGS1.2 displayed a trend of"catching up with each other",and the highest expression of this gene was found in 30 to 50 days after pegging.The responding patterns of the two splicing transcripts at the different time points during the procedure of seeds imbibition was consistent,and transcripts level increased with duration of seeds imbibition,and AhHEGS1.1 always had higher expression level than that of AhHEGS1.2.It was impied that AhHEGS1 might play a role in the germination of peanut seeds.（3）Expression analysis of AhHEGS1 responding to different hormones.Analysis of AhHEGS1 promoter regulatory elements revealed that their promoter region contains many hormone-responsive elements.In our previous study,we found that the AhHEGS1expression was not regulated by exogenous ABA.In order to determine whether AhHEGS1was involved in the regulation of hormone signaling pathway further,the response of the two trancripts to Fluridone（FL）,gibberellin（GA₃）and its inhibitor PAC etc at different concentrations was analyzed by q RT-PCR.It was found that the expression of AhHEGS1.1 and AhHEGS1.2 were both induced by GA₃,and the sensitivity of AhHEGS1.1 to GA₃ was higher than that of AhHEGS1.2.The high level of endogenous ABA at the early stage of germination induced the enhanced expression of AhHEGS1.1,but had little effect on AhHEGS1.2.In short,it was indicated that GA and ABA played important roles in regulating AhHEGS1 gene expression during seed germination.The gene expression patterns under response to auxin（IAA）and 2,4-epbrassinolide（BR）and their inhibitors are under way.（4）Functional verification of the AhHEGS1 promoter.The promoter of AhHEGS1 gene was cloned.The fragment size of the promoter on chromosome 17 was2,124 bp,and that on chromosome 7 was 1,971 bp.The two GUS expression vectors driven by the AhHEGS1 promoters were constructed,and transformed into Arabidopsis.Screening of transformed plants is in progress.（5）Analysis of the transcription factor characteristics of AhHEGS1.The results of subcellular localization showed that the two fusion proteins harboring AhHEGS1splicesomes were both located in the nucleus.Yeast activation analysis assay showed that both AhHEGS1.1 and AhHEGS1.2 proteins could active the transcription of downstream genes,and their activation domains were located at the C-terminal of the proteins.（6）Functional analysis of AhHEGS1 through peanut genetic transformation.Previously,we constructed the plant overexpression vector p ROKII-AhHEGS1 and transformed it into peanut.Through PCR verification and sequencing analysis,six over-expressing peanut T2 lines with different transformation events were obtained,out of which 4 were homozygous lines.The phenotype analysis of seed germination also revealed that the speed of seed germination in transgenic plants was faster than that in FH1.The knockout vector p CAMBIA1300-cas9-AhHEGS1 was constructed by CRISPR/Cas9 strategy,and transformed into peanuts.The screening of transgenic peanuts harboring the knockout vector is ongoing.In a word,the results obtained in this study were still preliminary.Next,we will complete the screening of transgenic Arabidopsis,and observe GUS expression in transgenic Arabidopsis seeds under different germination times and different hormone treatments.Besides,after obtaining the transformed plants with AhHEGS1 knock-out vector,we are going to analyze their phenotype and response to hormone during seed germination.Furthermore,synthetically considering the results of function analysis in the overexpressing lines and the gene-editing lines,the regulatory network of this gene will be explored by means of transcriptomic analysis,Ch IP-Seq and other molecular biological methods.It will be helpful for understanding the role of AhHEGS1 during seed germination.