| Chinese cherry(Prunus pseudocerasus),a deciduous fruit tree,belongs to Rosaceae family.It becomes one of the most important fruit trees due to great taste,high health value and early breaking of its bud.Similar with other deciduous fruit trees,Chinese cherry have a typical phenomena named“natural dormancy".The dormancy process and its release of flower bud are complicated and close relationship with flowering and fruit rate,which will eventually affect the yield and fruit quality.It is considered that sufficient accumulation of low temperature is one of the key factors which induce dormancy breaking.Insufficient hypothermia accumulation will affect the flower bud dormancy and dormancy breaking,so it is important to carry out low temperature accumulation induced the process.AP2/ERF gene family is a kind of transcription factors which are ubiquitous in plants.These transcription factors were considered to widely involve in growth and development,low temperature response et c.in plant In this study AP2/ERF genes with intact ORF was screened from transcriptomic library of P.pseudocerasus cv.'Duan bing'(accession No.SRX695147)followed by the analyses of bioinformatics properties.Gene expression analyses using Real-time Polymorphic Chain Reaction(PCR)discovered a dormancy-re lease-related gene,PpcERF1.Gene function of PpcERFl was studied with transforming PpcERF1 into Arabidopsis thaliana using agrobacterium-mediated transformation protocol.Further study was performed to uncover the gene expression characteristics by promoter cloning of PpcERF1.In order to deeper study,RNA sequence technique was used to analysis the early flower in Arabidopsis thaliana.The results were summarized as followings,1.21 Unigene sequences containing complete ORF were annotated as AP2/ERF transcription factors.With the evaluation of hydrophobicity,fatty index,protein secondary structurethe results showed that there are three kinds of amino acids,acidic,alkaline and neutral respectively in these found polypeptide,among them there are five alkaline amino acids(KT369533,KT369543,KT369544,KT369549,KT369553 respectively.)whose pI>8,4 acidic amino acids(KT369535,KT369536,KT369538,KT369551 respectively.)whose pI<5,the others 5<pI<8,which refers to distinct signal transduction in different environment.With the anylis of amino acids instability index,showed that except KT369536,KT369537 and KT369557 instability index is 35.14,37.39 and 39.27 respectively,the rest of the unstable index were greater than 40.The main structure of AP2/ERFs protein in P.pseudocerasus cv.'Duan bing' is hydrophilic region These transcription factors mainly distributed outside of the cell membrane,which may be beneficial to regulate the expression of downstream genes.The secondary structure of protein includes a-helix,?-turn,extension chain and random curl.In addition to KT369557,whose a-helix achieve to 42.77%,the others have the largest proportion in random curl,which were all close to 50%.Protein subcellular localization predicted KT369543 mainly located in the cytoplasm,KT369538 only distributed in nucleus and cytoplasm,KT369547 located in the nucleus,cytoplasm and cytoskeleton,while KT369535 and KT369539 loated in nucleus,cytoplasm,cytoskeleton,mitochondria and peroxisomes,indicating that AP2/ERFs are widespread in the cell.Although AP2/ERF transcription factors were found in the cytoplasm,they play roles in cell nucleus with the help of nuclear localization signal.2.The expression pattern of AP2/ERFs genes in Chinese cherry flower buds at different times were analyzed by qPCR.The expression level of 15 genes were dramatically induced by chilling treatment.KT369538(PpcERF1)was the earliest one of which induced genes.PpcERFl showed a down-regulated model during the dormancy release of flower bud,suggesting that the expression of PpcERF1 gene may be suppressed by low temperature.Hence,PpcERF1 gene was suggested to involve in dormancy breaking.Three of the 21 genes(KT369543,KT369544 and KT369545)had no response to chilling treatment,while another three genes(KT369536,KT369537 and KT369549)have moderate responce to chilling treatment3.Overexpressing PpcERFl is used to study seeds germination and other series of phenotypic traits.After 36 h cultivate in A.thaliana dedicated incubator without chilling treatment,germination percentage of seeds from the transgenic plant reached 55.94%in 36 h of after cultivation while wild type seeds was only 27.77%.These results indicated that PpcERFl should significant germination promotion.Further observation of the phenotype revealed that the overexpressing plants had a stem at the time of the average appearance of eight visible leaves,whereas the wild type had an average of 10,indicating that the transgenic one had early flower.Results obtained from promoter cloning of PpcERF1 and luciferase activity assay can induce expression ofthe promoter.The promoter of the PpcERFl gene was 1.72 times higher than that of the control.After 4 days of cryopreservation,the experimental group was 14.84 times higher than the control,while the ABA-treated group was 5.98 times of the control.Low temperature and ABA are considered to be main factors that related with dormancy and dormancy release.These results suggested that dormancy-related factors may be regulated by promoter of PpcERFl genes.Our results indicated that AP2/ERF transcription factors play inportant roles in the regulation of the dormancy release process of P.pseudocerasus cv.'Duan bing'.Nevertheless,further studies should be carried out in order to uncover the mask of dormancy breaking.4.The transcriptomic sequencing was used to study the possible causes of early flower.With multiple comparison among the differentially expressed genes,it wasfound that total clean reads was higher than 40 M,the base number is 3.6 G,which is mapped to the transcriptome total library reading about 25 M,the proportion reached 62.24%,the only match readings have 25 M,accounted for 61.42%,this results indicated that transcriptome quality is good.There were 627 up-and 364 down regulated genes were obtained from the differential gene expression data.Of these,there are 11 up-points greater than 5,66 between 2 and 5,3 downsets less than-5,40between-2 and-5.Most of the up-regulated genes were associated with CYP and JA,the most obvious one was AT3G48520.1(CYP94B3),qPCR was further validated the results.According to the above results,it could speculated that hypothermia induced Chinese cherry AP2/ERF transcription factor expression,while the AP2/ERF transcription factors and CYPs genes may have potential interactions,thus caused a series of reactions during chinese cherry flower bud dormancy breaking process. |
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