Cloning Of BHLH Transcription Factor Genes And Their Primary Analysis In Regulation Of Seed Dormancy And Germination In Peanut

Seed dormancy is a complex biological process that is the results of plant adaption and evolution over a long period of time for plant population survival and reproduction,which is regulated by the lots of genes and the complex environmental condition.Therefore,it has universal and important significance in biological function.However,for the pursuit of high production and efficiency,in many crops,especially peanut,during the long-term progress in domestication and cultivation,yield trait had been focused by breeders,and the seed dormancy had been ignored.Seeds with the weaker dormancy or without dormancy could cause viviparous phynotype when they are harvested delay and encounter with the successive rainy days;whereas seeds with the stronger dormant trait may not germinate in the suitable sowing time.All these elements bring about many problems in the agricultural production,such as lower yields,the decline of edible quality,and so on.Based on the transcriptome analysis of peanut varieties with the different dormant duration in the three stages from fresh harvest to just germination,we will find some regulatory factors associated with peanut seed dormancy and germination,and investigate their mechanism in these biological process.It not only provides theoretical basis on peanut planting,harvesting and storage,but also provides practical guidance for peanut breeding.In this study,two genes,that were differentially expressed between in the dried seed and in just-germinated seed significantly,were selected according to the results of transcriptome analysis.The following was the main results:1.Two bHLH transcription factor genes,comp78890_c0 and comp77639_c0,were cloned.The size of comp78890_c0 gDNA was 2066 bp,and the length of open reading frame was 1257 bp,encoding 418 amino acids.The size of comp77639_c0 gDNA was 2078 bp,and two cDNA sequences were obtained,their length of open reading frame were 1188 bp and 1152 bp,respectively,encoding 395 and 383 amino acids.According to the sequence analysis of cDNA and gDNA,it found that comp78890_c0 and comp77639_c0 all have seven exons and six introns,and comp77639_c0 genes may be spliced alternatively in the sixth intron.2.The amino acid sequences of the two genes were compared with the related sequences derived from Arabidopsis,alfalfa,soybean,rice,and many other species.The results showed that the amino acid sequence encoding by the two genes contain bHLH structure domain,especially comp77639 c0,which only fewer amino acid differences in the conservative bHLH domain compared to other corresponding sequences from other species.The similarity of the amino acid sequence between the comp78890_c0 and alfalfa MtbHLH8 or soybean GmbHLH137 was 52%and 50%,respectively.The amino acid sequence similarity between the comp77639_c0 and the related sequence AFK34964 from Lotus or soybean GmbHLH63-like reach 70%and 58%,respectively.3.The results of subcellular localization showed that the fusion protein of comp78890_c0-GFP and comp77639_c0-GFP were localized on the nucleus specifically.Yeast transcriptional activation assay showed that the comp78890_c0 had the function of transcriptional activation,and its transcriptional activation domain was located at the N-terminus and C-terminus of the protein.The comp77639_c0 also had transcriptional activation function,and its transcriptional activation domain was located at the C-terminus of the protein.4.The expression patterns of two genes were analyzed by fluorescence real-time quantitative PCR.The results showed that the expression of two genes had differential spatio-temporal profiles.The expression level of comp78890_c0 gene in roots,stems,leaves and flowers was significantly different,and the highest expression in the root,the following in the flower,and in the stem the expression level was the lowest.In the different development period of the seed and germinated embryo and cotyledons,the expression level of the comp78890_c0 gene was similar,but the expression level in the dried seed was very high,which was almost the same as that in the root.The expression level of the comp77639_c0 gene was higher in the root,and there is no significant difference in the stem,leaf and flower.The minor differences of expression level exist in the seeds of different developmental stages and dried seed.However,the comp77639_c0 gene was significantly expressed in the germinated seed,and the expression of comp77639_c0 gene in cotyledon was significantly higher than that in the embryo without cotyledon of just-germinated seed.5.Analysis of comp78890_c0 and comp77639_c0 gene responding to ABA and GA3 during seeds imbibition process,the results indicated that treatment by ABA,the expression of comp78890_c0 and comp77639_c0 gene in different treated times was not significantly different compared to that in the control,inferring that the expression of the two genes was not affected by exogenous ABA and may be associated with the change of endogenous ABA;however,treatment with GA3 or not,the expression level of comp77639_c0 increased very significantly in 12 h imbibitions seed,suggesting that this gene had a significant response to endogenous GA3,and by contrary,comp78890_c0 gene had no obvious response to GA3.6.In order to identify the function of two genes,the plant overexpression vectors,pROKII-comp78890_c0 and pROKII-comp77639_c0,were constructed and transformed into Arabidopsis and peanut.At present,14 transgenic Arabidopsis plants with comp77890_c0 gene were obtained,and 28 plants containing comp77639_c0 gene were yielded.In addition,the screening of homozygous lines for transgenic lines is underway,and the construction of the CRISPR-CAS9 edit vectors for both genes is also in progress.In the next,in order to obtain the powerful evidence that the two genes participate in the regulation of GA3 or ABA pathway,we will deeply analyze the expression patterns of the two genes which treated by GA3 inhibitor or ABA inhibitor and treated by the combination of hormone and its inhibitor.As well as,by analyzing the phenotypes of transgenic peanut harboring the constructs of over-expression and suppression expression of two genes,we will illustrate their roles in seed dormancy and germination process.