Characterization Of MdSAMDC2 And MdICE1 From Apple And Their Involvement In Stress Tolerance

Abiotic stresses often adversely affect both plant growth and crop productivity. To cope with abiotic stresses, breeding new varieties which hava modified stress tolerance is one of the most important approaches. Polyamines are involved in many life processes in plant cells. Increasing evidences indicate that polyamine accumulation confers stress tolerance on plants. SAMDC is a step-limiting enzyme for polyamine biosynthesis. MdSAMDC2 encoding a SAMDC in apple is induced by low temperature, dehydration and salinity. But its biological function in plants is yet to be identified. A modified TAIL-PCR strategy was used to isolate the promoter region of MdSAMDC2 from apple, and then the ability of MdSAMDC2 promoter to drive the downstream ORF was valuated.In order to characterize the in vivo biological function of MdSAMDC2 in plant, transgenic tobaccos over-expressing MdSAMDC2 were obtained. The enhanced tolerance of transgenic lines to abiotic stresses suggests that MdSAMDC2 is a candidate gene for genetic engineering to improve plant tolerance to stresses. In addition to MdSAMDC2, we also isolated MdICE1 from apple. Functional analysis demonstrated its crucial role the responses of plant to cold stress. Over-expressing MdICE1 remarkably improved the tolerance of transgenic tobacco to low temperature. The main results of this study were summarized as follows.1) Specific primers were designed based on the cDNA sequence of MdSAMDC2. Its ORF was cloned and inserted into expression vector pMDC32 with Gateway recombination. Then the resultant construct was introduced into tobacco with Agrobacterium-mediated transformation. PCR verified the integration of MdSAMDC2 into tobacco genome. Furthermore, RT-PCR detected a high level of MdSAMDC2 transcript in transgenic tobacco. All transgenic tobaccos normally grew and developed in appearance. Polyamine titers in transgenic plants were determined with HPLC. The results showed that the titers of three polyamines increased remarkably. After treated with low temperature, 20% PEG and salt respectively, transgenic plants accumulated more Spd and Spm but less Put than non-transgenic control. Moreover, proline content and the activity of SOD and CAT increased while MDA content decreased. So over-expression of MdSAMDC2 in tobacco enhanced the tolerance of transgenic plants to stresses.2) To investigate the expression pattern of MdSAMDC2, the promoter region of MdSAMDC2 was isolated from apple with a modified TAIL-PCR approach. The promoter is 643 bp in size and contains several basic elements for promoter such as TATA-box, CAAT-box, stress-induced elements and so on. To examine its ability to drive a downstream ORF, the promoter was inserted into a plant expression vector to replace CaMV 35S promoter driving GUS gene. Transient expression of GUS in transgenic tissues was detected with histochemical GUS staining. As a result, GUS activity was detected in transgenic plants. GUS activity can be induced by low temperature and high salt.3) MDA and polyamine contents in leaves of four apple rootstocks were exmined to explore the changes of polyamines in apple during low temperature stress. The results showed that MDA content could indicate the cold tolerance of four rootstocks at time point 6h during low temperature stress. The titers of total polyamines, put, spd and spm were significantly induced by low temperature. MDA content was negatively correlated with polyamines and the changes of Spd and put content while changes of relative content of spm(Spm/PAs)and spd content(Spd/PAs)are positively correlated with (Spd+Spm)/Put. The changes in polyamines content, Put content and Spd content under low temperatures were significant and could be used as indicator of cold tolerance in apple rootstocks.4) RT-PCR and RACEs were used to isolate MdICE1 (Inducer of CBF Expression 1) from apple. The full length cDNA of MdICE1 was registered in Genebank with an accession number EF495202. MdICE1 is 1890 bp in size and encodes 531 amino acid residues. Its full-length cDNA was inserted into vector PET-30a (+) and then transferred into E. coli strain BL21. As a result, a 64.5 kDa protein was specifically induced by IPTG treatment visualized by SDS-PAGE. BLAST analysis indicates that MdICE1 is a homolog of Arabidopsis ICE1. To investigate the subcellular localization of MdICE1 in cells, a construct producing MdICE-GFP fusion protein was introduced into onion epidermal cells. GFP florescence was observed only in the nucleus, suggesting that MdICE1 was localized to nucleus.MdICE1 expression patterns under different stresses in apple were detected with Northern blotting. The result showed that the expression of MdICE1 was induced by low temperature and salt stresses. To determine the function of MdICE1, transgenic tobacco plants were created with ectopic expression of MdICE1 driven by cauliflower mosaic virus 35S promoter. After treated with low temperature and salt stresses, transgenic plants produced increased proline and activated SOD and CAT enzymes, while decreased MDA content. All evidences indicated that ectopic expression of MdICE1 in tobacco confers cold tolerance on transgenic plants.