Identification And Function Analysis Of BHLH Transcription Factors In Flower Bud Of Chinese Cherry During Dormancy Release

Chinese cherry(Prunus pseudocerasus)belongs to genus Prunus of family Rosaceae.Chinese cherry is a kind of deciduous fruit tree and becomes one of the most important fruit trees due to early breaking of its bud,great taste,and high healthy value.The flower bud of Chinese cherry will be dormant in the cold winter.Low temperature can induce flower bud into dormancy,on the other side,chilling promote dormancy release.Close relationship between low temperature accumulation and flowering and fruit rate was proposed in previous studies.Therefore,it is important and practical to study how low temperature induce dormancy release of flower buds.Basic helix-loop-helix protein(bHLH)is a large class of transcription factors in plants that play a wide range of roles in growth and development as well as responding to abiotic stresses such as low temperature.In this study,bHLH genes with intact open reading frame(ORF)were screened from the transcriptomic library of P.pseudocerasus cv.'Duan bing'(accession No.SRX695147)followed by the analyses of bioinformatics properties.The qRT-PCR technique was used to analyze their expression trend in the process of flower bud dormancy release.A bHLH transcription factor with the highest up-regulated expression level during accumulation of low temperature was screened and named as PpcbHLH1.The PpcbHLH1 gene was transformed into Arabidopsis thaliana using agrobacterium-mediated method to study the effect on growth and development.The main results were summarized as followings,1.30 Unigene sequences containing complete ORF were annotated as bHLH transcription factors.Through the analysis of isoelectric point,atomic composition,instability index and fat index of amino acids,the results showed that acidic,alkaline and neutral amino acids were discovered in these found polypeptide,among them were eight alkaline amino acids[KP689412(ALN42121.1),KP689413(ALN42122.1),KP689415(ALN42124.1),KP689420(ALN42129.1),KP689423(ALN42132.1),KP689426(ALN42135.1),KP689428(ALN42137.1),KP689429(ALN42139.1)]whose isoelectric point(pI)value were larger than 8,two acidic amino acids[KP689416(ALN42125.1),KP689417(ALN42126.1)]whose pI value were smaller than 5.the pI value of others ranged from 5 to 8.It indicated that these proteins may play roles in different physiological environments.The instability index of all the 30 bHLH transcription factors were greater than 40,which indicated that these proteins were unstable.The molecular weights of amino acids were quite different from one other while atomic composition and fat index were similar.These proteins basically didn't have a transmembrane domain.The ratio of a-helix were all over 30%,indicating that a-helix was a common secondary structure in these proteins.Subcellular localization prediction revealed that these bHLH proteins were mainly located in the nucleus.Twenty-five sites with frequency of conserved amino acid beyond 50%were found by motif detection,indicating that they belong to the typical domain sequence of bHLH.Phylogenetic trees were constructed with conserved domain sequences of 30 bHLH family members from peach(P.persica)and 26 bHLH family members from Arabidopsis suggested that bHLH family of 'Duanbing'cherry could be further divided into 9 subfamilies.Among them,the third subfamily has the largest number of members.2.Flower buds were treated using low temperature with different times.Expression characteristics of the 30 PpcbHLHs genes were measured by qRT-PCR.The results showed that the expression trend of these 30 genes showed a similar up-down-up expression trend.These genes have the first highest up-regulation level at 192 h and then down-regulated.The increase times ranged from 1.5 to 21.Most genes were up-regulated again and reached the second highest level at 488 h or 576 h after the satisfication of chilling requirement.These results suggested that the PpcbHLHs genes may be involved in the dormancy release regulation of cherry flower bud by responding to low temperature.Among them,the expression of KP689428(Unigene7666)was induced to be the highest at 192 h,we named it as PpcbHLHl and did further study on the function of this gene.3.PpcbHLH1 gene was transfered into plant expression vector and transformed into Arabidopsis thaliana using flower-dipping.The growth and development characteristics of wild type(WT)and transgenic plant were analyzed.The results showed that the average number of visible leaves when the stem grew out in WT and transgenic plant were 10 and 9,respectively.The above results indicated that the over-expression of PpcbHLH1 gene can lead to early flowering of arabidopsis.The leaves of transgenic line became larger and thicker than WT,suggesting PpcbHLH1 gene could affect the leaf growth and development.In addition,the average width of stigma were 0.36 and 0.27 mm for transgenic plant and WT,respectively.Mastoid cells on the stigma of transgenic strain were more abundant than that of WT.Further observation was made on the number of seeds in young fruit pods.Seed number of WT was 28 while that of transgenic strain was 15,indicating that PpcbHLHl might affect the development of stigma and thus affect the formation of seeds.Seed germination rates of transgenic plant were 12%,27%,39%at 30 h,33 h,36 h,respectively,while that of WT were 4%,15%,32%at the same stage.Seed germination rates of overexpressing plant were higher than that of WT at all stages,which suggesting that PpcbHLHl gene could enhance seed germination of Arabidopsis thaliana.