| Peach is one of the most important economic fruit trees among the world. The release and promotion of new cultivar underpin the healthy and sustainable development of peach industry. Genetic engineering is a feasible approach for cultivar breeding because of the sound technique foundation and high efficiency. But discovery of the desirable genes should be the prerequisite. Peach enjoys the unique characteristics of ripening and the accompanying biochemical mechanism has been studied intensively. Therefore, it has become the model for ripening study in fruit tree. In addition, peach is of no exception concerning stress restriction, which has provoked breeders throughout the world to create new cultivars with tolerance to multiple stresses in breeding program. In the current study, PpERS1and PpADC genes along with their respective promoters were cloned from Prunus persica and transferred into Micro-Tom tomato for functional analysis. Meanwhile, the transgenic tomato carrying PtADC were used to investigate its function under dehydration and drought stress. Main results were as follows:1. Based on the homogenous sequences, an ethylene receptor gene PpERSl was cloned from peach, containing an open reading frame (ORF) of1935bp which encodes a644amino acids polypeptide with a predicted molecular mass of72.4kDa and an isoelectric point of6.33. Analysis of the putative amino acid sequence suggests that PpERS1was clustered to into the ETR1subfamily, and showed high homology with those ethylene receptors from rosaceous plants. Bioinformatics analysis revealed that the coding region of PpERS1contained five exons interrupted by four introns, in addition, two introns located in the5'UTR of PpERSl gene, and alternative splicing occurred in the first intron of5'UTR. The expression analysis indicated that the PpERSl transcript was detected in all the tissues including root, stem, leaf, flower, fruitlet and ripening fruit. The expression of PpERSl was lowest in root and the expressions in stem, leaf, flower, fruitlet and ripening fruit were1.5,1.8,2.4,1.5and4.3fold higher to that of root, respectively. PpERSl expression was induced by injury, dehydration, ethephon, salt and peach tree gummosis, while low temperature down-regulated PpERSl transcription.2. A2798bp upstream region of PpERSl translation start codon (ATG) was cloned by genomic walking PCR.5'RACE result revealed that the transcription start site was G, located at-777bp upstream of ATG of PpERS1. In silico analysis of the PpERS1promoter revealed the presence of typical TATA box and CAAT box, ethylene response element, injury response element, enhancer element, hormone response elements including auxin, cytokinin, abscisic acid and gibberellin, and stress response elements including drought, low temperature and pathogen. We constructed vectors containing the PpERSl full-length and serial5'-deleted promoters with GUS reporter gene, and all the constructions were introduced into tomato (Lycopersicon esculentum cv. Micro-Tom) through Agrobacterium tumefaciens-mediated transformation. GUS activity analysis was carried out by histochemical staining and fluorescence detection demonstrated that the promoter activity was normal in transgenic plants and not affected by the introns contained in5'UTR. GUS activity analysis in tissue, organs and developmental stages indicated the PpERSl promoter was active in almost all tissues. Meanwhile, PpERS1promoter activity was detected in radicle, cotyledons, hypocotyls and euphylla during different development stages. Real Time PCR analysis suggested that promoter activity were induced by injury and ethephon treatment, suppressed by low temperature and immune to salt treatment.3. Based on the homogenous sequences, an arginine decarboxylase gene PpADC was cloned from peach, containing an open reading frame (ORF) of2178bp which encodes a725amino acids polypeptide with a predicted molecular mass of77.7kDa and an isoelectric point of5.19. Analysis of the putative amino acid sequence suggests that PpADC shared high homology with that from rosaceous plants. Bioinformatics analysis revealed that no intron was contained in the coding region of PpADC. The expression analysis indicated that the PpADC transcription were up-regulated by dehydration, ethephon, salt and low temperature treatment. The transgenic plants overexpressing PpADC contained higher Put, Spd and Spm level than that of the wild type (WT). Meanwhile, the transgenic plants showed dwarfism and late-flowering. However, the phenotype was rescued by gibberellin application. The expression analysis indicated that partial genes associated with gibberellin biosynthesis were suppressed in transgenic plant.4. A2043bp upstream region of PpADC translation start codon (ATG) was cloned by genomic walking and silicon cloning PCR. In silico analysis of the PpADC promoter revealed that the transcription start site was A, located at-453bp upstream of ATG of PpADC. The sequence of PpADC promoter contained the putative TATA box, stress response elements including drought, low temperature and pathogen, auxin response element, injury response element, and circadian regulation element. Vectors containing the PpADC full-length and serial5'-deleted promoters with GUS reporter gene were constructed and introduced into tomato(Lycopersicon esculentum cv. Micro-Tom) through Agrobacterium tumefaciens-mediated transformation. GUS activity analysis carried out by histochemical staining demonstrated that the promoter activity was normal in transgenic plants.5. The transgenic plants overexpressing PtADC contained higher Put and Spd level than the WT, while there was no difference in Spm level. Under dehydration, water loss of detached leaves was severer in the WT. Moreover, ion leakage of the transgenic lines was remarkably lower than that of the WT. However, chlorophyll content was higher in the WT. In addition, NBT and DAB histochemical staining revealed that WT accumulated more ROS than the transgenic lines in tomato. Therefore, drought tolerance of the transgenic lines was obviously improved. |
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