| 'Nantongxiaofangshi'(Diospyros kaki Linn.),with weak apical dominance and no obvious trunk,is a good persimmon cultivar suitable for dwarfing and closely spaced planting.Therefore,it is of great significance to study the dwarfing genes of'Nantongxiaofangshi'in dwarf breeding of fruit trees.At present,there are few studies on gibberellin-related dwarfing in persimmon plants.Based on the transcriptome data,we randomly selected 30 related genes of gibberellin signal transduction pathways and metabolism by gene function annotation.To screen out the differential genes,we analyzed their expression characteristics in different phenological periods between 'Dafangshi' and'Nantongxiaofangshi'.Meanwhile,we sprayed the leaves of both varieties during growth periods with exogenous gibberellin and PP333,and with the treatment of spraying clear water as CK.Then we measured the contents of endogenous hormones in leaves after treatment,and compared the number of internodes and annual branch internode length.Finally we analyzed the expression of the differential genes above under treatments in two species.In addition,we also assessed the stability of 13 reference genes in persimmon and selected suitable reference genes for RT-qPCR.The main results are as follows:1.Identification of reference genes for RT-qPCR normalization in persimmonIn this study,13 candidate reference genes were cloned based on the transcriptome sequencing data of persimmon.Their stabilities in persimmon were analyzed by two algorithms(geNorm and NormFinder)under abiotic stresses(heat,cold and salt)and different hormone stimulation,including gibberellin,salicylic acid and abscisic acid.As a result of the analysis,we identified suitable persimmon reference genes under specific experimental conditions.Among them,UBC and GAPDH could be considered the most suitable reference genes across all samples,whereas the commonly used persimmon reference gene ACT could not be recommended as an optimal reference gene for normalization of persimmon gene expression data.In addition,UBC combined with RPII or TUA would be appropriate for the 'abiotic stress' group and ?-TUB combined with PP2A would be appropriate for the "hormone stimuli" group.2.Screening and expression analysis of gibberellin related genesThere were 22 genes detected in 30 genes associated with gibberellin synthesis and metabolism.And we found 14 differential genes among the 22 genes in the analysis of the expression characteristics of different phenotypes.The expression levels of Unigene7595,Unigene18861,Unigene34904,Unigene11769,CL1455.Contigl and Unigene2304 in'Dafangshi'were significantly higher than those of the 'Nantongxiaofangshi'.In contrast,the expression levels of Unigene20143 and CL628.Contig2 in 'Nantongxiaofangshi' were significantly higher than those of the 'Dafangshi'.The expression of the rest genes,CL452.Contig2,CL452.Contig3,Unigene25672,CL4153.Contig2,CL4170.Contig4 and CL7393.Contig4,were with the flowering period as the turning point.Before florescence,'Dafangshi' is higher than 'Nantongxiaofangshi'.After florescence,'Nantongxiaofangshi'is higher than 'Dafangshi'.After GA3 treatment,the content of endogenous GA in two varieties increased significantly compared with the control.And the relative growth of the new shoot of'Nantongxiaofangshi' was higher than 'Dafangshi'.After PP333 treatment,tthe change of endogenous GA content in the two cultivars was still the most significant.And the content of endogenous ABA in 'Dafangshi' increased significantly.The shoot growth of two varieties was lower than the control,and the differences between the treatment group and the control group of 'Dafangshi' were significant.It is speculated that compared with the'Dafangshi',the shoot growth of 'Nantongxiaofangshi' on the GA3 response to a higher degree,while the low response to PP333.In addition,we found the expression of 13 genes in the two varieties showed differences,including Unigene7595,Unigene18861 and Unigene34904,which were homologous to KAO,KO and GA20ox seperately.And the others are GA2ox homologous genes(CL452.Contig2,CL452.Contig3 and Unigene25672),GID1 homologous gene(Unigene2304)and DELLA protein homologous genes(Unigene20143,Unigene11769,CL1455.Contigl,CL628.Contig2,CL4153.Contig2 and CL4170.Contig4). |
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