| Citrus is usually sufferd biotic and abiotic stresses in the process of growth and development, which impact on citrus industry. Therefore, in order to solve these problems, breeding of resistant cultivars must be strengthened. Due to traditional breeding with period length and larger limitation, and with the application of genetic engineering technology, transformation of resistance genes into citrus will accelerate the process of citrus breeding. Trifoliate orange(Poncirus trifoliata) is a rootstock widely used in citrus planting, which has strong cold resistance. There have been reported ABF and CDPK genes were induced expression in stress response on the previous study. In this study, we overexpressed Ptr ABF and Ptr CDPK in Poncirus trifoliata, and futher analysed their gene functions and action mechanism. The main results are as follows:1. Six positive transgenic plants overexpressing Ptr ABF were identified by PCR amplification in Poncirus trifoliata. Overexpressing of the transgene was verified in two of the selected transgenic lines(#8 and #10). The results shown overexpression of Ptr ABF in trifoliate orange significantly enhanced dehydration tolerance. The transgenic lines displayed smaller stomatal apertures and stomatal density when compared with wild type(WT) with a scanning electronic microscope. The transcript levels of three key genes( SPCH/FAMA/MUTE) associated with stomatal development were decreased in the transgenic lines. In yeast two-hybrid assay and bimolecular fluorescence complementation(Bi FC) assay, Ptr ABF was found to physically interact with Ptr ICE1, a gene critical for stomatal development.Chips analysis using uniform leaves collected from WT and one of the two transgenic lines, #10, grown under normal conditions, 70 differentially expressed genes were identified, with the cutoff/threshold of two-fold change(P ≤ 0.05), a total of 42 and 28 genes were up- and down-regulated in #10 when compared with WT. Ten up-regulated genes were randomly selected to verify the microarray results via RT-PCR amplification. Among the 42 induced DEGs, 33 can be functionally annotated. Based on the released citrus genome sequence, the presence of ABREs and CEs in the promoters of the 33 induced DEGs with annotation was investigated. The majority of them contained ABREs or CEs or both motifs in the promoter regions, while five of them did not.Chips analysis shown the transcript levels of Pt POD 和 Pt ADC gene were increased in the two transgene lines(#8 and #10). In yeast one-hybrid assay and transient expression assay, Ptr ABF was found to physically interact with Pt POD 和 Pt ADC. The POD activities in the transgenic lines were approximately two times of that in WT before and after dehydration treatment. The activities of POD, SOD and CAT in the transgenic lines were higher than that of WT after dehydration treatment, which was consisted with the transcript levels. The accumulation of H2O2 and O2- in the transgene lines significantly lower than WT, which was consisted with the histochemical staining of ROS. Consistently, the MDA levels in the two transgenic lines were substantially lower than that in WT after dehydration treatment. The three free polyamines, Put, Spd and Spm, in the transgenic lines were significantly higher than those of WT. After the transgenic lines were pretreated with different concentrations of D-arginine(D-arg), an inhibitor of ADC, the accumulation and contents of H2O2 and O2- in the transgene lines sigificantly lower than WT, which is increased with the concentration of D-arginine. After the pretreatment with different concentrations of Guazatine(an inhibitor of PAO) in the transgenic lines and WT, the accumulation and contents of H2O2 in the transgene lines sigificantly lower than WT, which is increased with the concentration of Guazatine. The activity and expression of antioxidant enzyme is sigificantly lower in in the transgene lines than WT.2. Ptr CDPK, a serenin/threonine kinase, had a complete open reading frame of 1676 bp, encoding a peptide of 558 amino acids, with calculated molecular mass of 63.25 k D and theoretical isoelectric point of 8.26. Ptr CDPK contained a conserved serenin/threonine banding domain and four EF-hand calcium-binding domains,and shared high sequence identities with CDPKs from other plants. Ptr CDPK was divided into class III compaired with Arabidopsis CDPK family by phylogenetic tree analysis. Ptr CDPK was shown to be induced by abiotic stresses, such as drought, salt, and ABA treatment. Ptr CDPK was localized in the nucleus and plasma membrane. Four positive transgenic plants overexpressing Ptr CDPK were identified by PCR amplification in Poncirus trifoliata. Overexpressing of the transgene was verified in two of the selected transgenic lines. Overexpressing of the transgene was verified in two of the selected transgenic lines(#34 and #36). Transgenic trifoliate orange plants were generated to elucidate the biological functions of Ptr CDPK. Ptr CDPK-overexpressing transgenic lines displayed enhanced tolerance to dehydration, as revealed by lowered water loss rate, electrolyte leakage and MDA content. Meanwhile, the transgenic plants accumulated lower levels of reactive oxygen species(ROS) compared with wild type under dehydration conditions, accompanied by higher activities and expression levels of three antioxidant enzymes, POD, CAT, and SOD. In addition, several phosphorylated sits, serine, threonine and lysine, were bioinformatically predicted in the coding regions of POD, CAT, SOD genes, implying that they may be targets of Ptr CDPK. Take together, Ptr CPK acts as a positive regulator of dehydration tolerance of trifoliate orange, partly by phosphorylating antioxidant enzymes to promote ROS scavenging. |
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