Functional Charaterization Of Arginine Decarboxylase Gene Ptadc From Poncirus Trifolita And Indentification Of The Regulators

Trifoliate orange (Poncirus trifoliata) is wildly used as rootstock for citrus in the citrus industry, which is highly tolerant to cold but sensitive to drought. Improvement of drought tolerance in trifoliate orange is significant for the development of citrus industry. Genetic improvement mediated by modern biotechnology has been shown to be crucial in improving the drought tolerance in trifoliate orange. Based on the cloning and identification of PtADC, this study further elucidated the potential mechanism of PtADC underlying stress tolerance. In addition, we indentified the upstream regulatory factors of PtADC, which enriches the gene resource in genctic improvement of trifoliate orange and facilitates our understanding on the transcriptional regulation of polyamines (PAs) accumulation under stresses. The main results are as follows:1. Elucidation of mechanisms of PtADC underlying stress tolerance. Overexpression of PtADC in Arabidopsis mutant adc1-1led to ehanced tolerance to dehydration, drought and cold. The transgenic plants maintained higher relative water contents, but lower accumulation of H2O2and O2". Meanwhile, the roots of the transgenic plants were longer, consistent with greater extension of apical meristem and larger cell numbers.2. Isolation of the regulatory factors of PtADC. Dehydration induced the expression of PtADC, coupled with the increased accumulation of free PAs in cells. Thus we constructed the cDNA library using the trifoliate orange leaves treated with dehydration, which were then subjected to yeast one-hybrid screening. Sequencing and bioinformatic analysis showed that two transcription factors were identified, where were termed as PtABF2and PtNAC72, respectively.3. Identification and characterization of PtABF2. PtABF2contained a conserved bZIP domain, localized in the nucleus, and the expression level of PtABF2was induced by dehydration. Arabidopsis mutant abf2displayed lower expresson of ABF2and AtADC2, as well as the reduced accumulation of PAs, implying that PtABF2may positively regulate the synthesis of PAs. Both yeast one-hybrid analysis and dual luciferase assay confirmed that PtABF2can interact with the promoter of PtADC.4. Identification and characterization of PtNAC72. PtNAC72contained a conserved NAM domain, located in the nucleus. Dehydration treatment induced the transcription of PtNAC72obviously. Overexpression of PtNAC72in transgenic tobacco plants suppressed the expression of NtADC1and NtADC2, coupled with decreased levels of Put, Spd and Spm. In addition, mutation of NAC72in Arabidopsis plants resulted in elevated expression of AtADCI and AtADC2in comparison with wild type, in particularly AtADC2. It was supposed that PtNAC72acts as a negative regulator involved in the synthesis of PAs. Yeast one-hybrid assay and transient dual luciferase assay veriried the direct interaction between PtNAC72and the promoter of PtADC.5. Identification and characterization of PtsrMYB. PtsrMYB was a typical R2R3MYB transcription factor. When the transgenic plants overexpressing PtsrMYB were subjected to dehydration treatment, the accumulation of H2O2and O2were lower in transgenic plants than in wild type. qRT-PCR ananlysis revealed higher expression level of NtADC1and NtADC2in transgenic tobacco than in WT, consistent with greater levels of free PAs. The promoter of PtADC was devided into three parts based on the distribution of MYBR elements, and strong interaction between PtsrMYB and two out of the three fragments was detected by yeast one-hybrid analysis, implying that PtsrMYB might modulat the expression of PtADC and the synthesis of PAs.