| For high plants, Chitinase plays a significant role in disease resistance, cold tolerance and other abiotic stress conditions, but it only shows the attributes of disease-resistance and antifreeze at low temperature. The plants with transgenic antifreeze proteins (AFP) can develop cold tolerance but lose the attribute easily and unable to develop ideal cold tolerance gene if transformed into singular gene, therefore, studies on cold resistance of plant presently focus on regulation of cold resistance gene expression. Many transcription factors such as CBFs family are found to be effective in cold tolerance development.Ubiquitin promoter is applied to regulate gene expression in modifying monocotyledons.With combination of lots of methods such as RT-PCR, Gene Localization, Western blot and Genetic transformation system construction, Agrobacterium-mediated transformation and PCR-Southern, inspired by Genbank's EAPP gene, the study clones EAPP gene of Dongmu-70 rye (Secale cereale cv Dongmu-70) by RT-PCR with primers and analysis on protein function, gene sub-cellular localization and gene expression pattern under cold tolerance. With the construction of Ubi1::CBF3 plant expression vector, monocotyledon and dicotyledon transgenic forage are generated and the mechanism of how CBF3 (C-repeat/ Dehydration Binding Factor) improves cold tolerance of forage is tested. The main results are as follows:1) Full sequences of Dongmu-70 rye EAPP gene cDNA is 1085bp, which share extremely high similarity with Hordeum vulgare (95%) and Triticum aestivum (91%);2) Relative molecular weight of EAPP protein is 33.6 KD, which contains Glycoside hydrolase family 19 chitinase domain and chitin binding domain, signal peptide is at amino acid 1-20, it is a type mixed secretory Protein with isoelectric point for a PI=7.38;3) The subcelluar location in onion epidermal cell and protoplast determines EAAP is a cell membrane protein;4) Dongmu-70 rye EAPP genes express in root, stem and leaves in normal growth condition, while leaves are the highly expressed organ; its transcripts will increase in cold stress at 4℃, and reach its peak at 24hrs after treatment; 5) By agrobacterium transformation, CBF3 gene is introduced into plant. CBF3 gene is successfully inserted into the chromosome of Agrostis and Trifolium pratense by RT-PCR and PCR-Southern hybrid identification on the positive transgenic plants;6) Some seedling of transgenic lines in Agrostis show a little growth delay, but does not appear in seedling of transgenic lines in Trifolium pratense;7) CBF3 expression in Agrostis stimulates an increase of proline content of leaves, the net photosynthetic rate and transpiration rate improve 61.64%,68.85% and 46.13% respectively, more than in wild type. Plants proline content of leaves, the net photosynthetic rate and transpiration rate in Trifolium pratense transgenic lines are improved than which of the wild type by 29.99%,23.56% and 18.57%; the MDA content in Agrostis and Trifolium pratense transgenic lines decreases 41.46% and 31.29% respectively (P<0.05);8) Under 4℃cold stress, proline content of leaves, the net photosynthetic rate and transpiration rate in Agrostis and Trifolium pratense transgenic plants is higher than which of the wild type, but MDA content significantly lower than which of the wild type(P<0.05). Introduction of Ubi1::CBF3 expressed in monocotyledonous and dicotyledonous plant can significantly improve cold tolerance; But in early growth stage of the two transgenic plants, cold tolerance and photosynthetic characteristics express remarkably different in monocoty- ledons and dicotyledon. The Ubi1::CBF3 displays high expression in monocotyledon than in dicotyledon.In summary, clone and genetic analysis on EAPP gene in forage plant is helpful for understanding the mechanism of cold tolerance; the transcription factors CBF3 introduction into forage and the functional analysis on transgenic lines further enhance the feasibility of the application of transgenic forage.
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