| Citrus is one of the most important fruits worldwide, which has important significance in promoting regional economy and human health. During the last decade, significant process has been made in citrus production. However, citrus industry is frequently affected by a variety of biotic or abiotic stresses, among which citrus canker, salinity and low temperature have caused enormous loss for citrus industry. Therefore, breeding and selection of resistant cultivars is the key point to cope with these issues. Due to limitation of classic breeding that existed in citrus, transformation of resistance genes into citrus via genetic engeneering becomes the fastest and the most effective strategy for creating novel resistant germplasm. Previous studies showed that polyamines are involved in pathogen defense, but related information is still scarce. Glycinebetaine is an important osmolyte that accumulates in plants under salt stress. ICE1 is an upstream transcription factor in cold-responsive (COR) genes, which regulates expression of a variety of COR genes. In the present study, molecular characterization and canker resistance were analyzed in'Anliucheng'sweet orange (Citrus sinensis Osbeck'Anliucheng') plants overexpressing an spermidine synthase (MdSPDS1) gene. In addition, betaine aldehyde dehydrogenase and a PtrICEl transcription factor were introduced into trifoliate orange (Poncirus trifoliata L. Raf.) and lemon (Citrus limon Burm. f.) via Agrobacterium-mediated transformation, respectively, for enhancing their salt and low temperature tolerance. The main results are as follows:1. Ten positive transgenic plants overexpressing MdSPDS1 were identified by PCR amplification in'Anliucheng'sweet orange. Overexpressing of the transgene was verified in two of the selected transgenic lines (TG4 and TG9). Southern blotting analysis showed that MdSPDS1 gene was integrated into TG9 plants as a single copy. TG4 and TG9 had significantly higher spermine (Spm) contents than the untransformed plants (WT). Pinprick inoculation of TG4, TG9 and WT with canker bacteria (Xanthomonas axonopodis pv. citri) demonstrated that the two transgenic lines exhibited significantly lower susceptibility, disease index and lesion size when compared with the WT. In addition, our data showed that canker inoculation significantly induced Spm accumulation and polyamine oxidase (PAO) activity in TG9, and TG9 showed an apparent hypersensitive response (HR) and the accumulation of more H2O2 than the WT. Pretreatment of TG9 leaves with guazatine acetate, an inhibitor of PAO, repressed PAO activity and reduced H2O2 accumulation, leading to more conspicuous disease symptoms than the H2O-treated controls. Moreover, mRNA levels of most of the defense-related genes involved in synthesis of pathogenesis-related protein and jasmonic acid were higher in TG9 than in the WT before or after inoculation.Genechip analysis using uniform TG9 and WT leaves showed that 666 differentially expressed genes were identified, among which 448 genes were up-regulated and 218 were down-regulated. Nine up-regulated and one down-regulated genes were randomly selected to verify the microarray results via RT-PCR amplification. All differentially expressed genes were functional annotated by Blast2GO software, and functional categorization also was done according to involved biological process, molecular function and cellular component, including celluar process, metabolic process, response to stimulus, localization, biological regulation, binding activity, catalytic activity, transporter activity, electron carrier activity, transcription regulator activity, organelle, macromolecular complex, extracellular region etc. In the up-regulated genes group,68 genes related to stimulus response and immune system process,12 genes related to cell wall and 13 genes related to transcriptional factor, which possibly play roles in canker resistance directly and were discussed in the current study. Moreover, expression of some important candidate genes was analyzed before or after inoculation via RT-PCR. The results showed that mRNA levels of most of the candidate genes in TG9 were higher than the WT, and canker inoculation induced these genes expression to a higher level comparing with without inoculation.2. Treatment of the trifoliate orange seedlings with 0,100 and 200 mM NaCl led to increase of the glycinebetaine (GB) content in a concentration-dependent manner. A betaine aldehyde dehydrogenase gene (AhBADH) cloned from Atriplex hortensis was introduced into the trifoliate orange by means of Agrobacterium-mediated transformation, and positive transgenic plants were obtained after PCR identification. RT-PCR analysis showed that the AhBADH gene was overexpressed in three selected transgenic lines (#48, #51 and#133), but absent in WT plants. GB levels in these three lines were higher than those in WT. Upon exposure to 200 mM NaCl, the transgenic lines showed significantly less serious leaf burning and defoliation, along with lower MDA accumulation, and higher chlorophyll contents, compared with the WT plants. Moreover, the transgenic plants accumulated lower levels of Na+ and Cl-, concurrent with increased level of K+ leading to a higher K/Na ratio. 3. An ICE1 transcription factor (PtrICE1) cloned from trifoliate orange was introduced into lemon by Agrobacterium-mediated transformation. Real-time quantitative RT-PCR analysis showed that the PtrICE1 gene was overexpressed normally in the transgenic lines, and expression level of transgene in most of the transgenic lines was significantly higher than that in the WT. Treatment at-6℃showed that two of the transgenic lines (#21 and#17) exhibited better cold tolerance. Cell death and accumulation of superoxide anion were inhibited in the transgenic plants.
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