| Potato is a vital economic crop for food, vegetable, fodder and industrial materials. Inner Mongolia is one of the main areas for seed and commercial potatoes production; however its development is severely limited by drought condition. For cultivating new varieties of drought-resistant potato, and accelerating the genetic improvement of existing main cultivars, CDPK1 gene from Arabidopsis(Col-0)was cloned with PCR and recombinant DNA technologies, and transformed the gene into poor-resistant and main planting potato cultivar ‘Favorita’ using agrobacterium-mediated method. After PEG stress, drought-resistant of the transgenic potato plants are verified by determination of physiological indicators, phenotype observation and qRT-PCR. The following results were obtained:1. We determined optimal induction medium and culture conditions of minituber development, and established tissue culture regeneration system of potato minituber.2. We predicted 1524 bp upstream of the ATG codon as promoter region of Rd29 A gene using TSSP-TCM and Plantcare software. Furthermore, the result indicated that the A was transcription initiation site, and exist the basic transcription element of promoter including TATA-box and CAAT-box and stress response elements, such as ABA, ABRE and DRE.3. The complete CDPK1 gene was cloned. The full length of the gene is 2269 bp, including 6 introns and 7exons. 1524 bp DNA upstream of the ATG codon of Rd29 A gene is cloned, and the sequence was entirely consistent with the reported sequence.4. We constructed the plant expression vectors p35S:CDPK1 and p29A:CDPK1, in which the CDPK1 gene expression was driven by Rd29 A and CaMV 35 S promoter respectively, and then the vector was transformed into potato cultivar ‘Favorita’ separately. As a result, 93 regenerated plants with resistance were obtained successfully. The PCR and Southern blotting analysis showed that the CDPK1 gene has been integrated into the potato genome, and has a single copy.5. RT-PCR analysis proved that CDPK1 gene expressed abundantly under CaMV 35 S promoter driven in transgenic plants. The expression level of CDPK1 gene driven by Rd29 A promoter was very low in transgenic plants, but after drought stress, it increased significantly.6. The average height and tuber weight of constitutive transgenic plants was significantly lower than that of non-transgenic plants 30% and 19% under normal growth conditions, respectively. After 30% PEG stress, the inducible transgenic plants have significantly higher average height and lower rate withered leaves of the plants than that of non-transgenic and constitutive transgenic plants. As the same condition, while the leaf edge has withered in the non-transgenic plants, the inducible transgenic plants may grow normally.7. After 20% PEG stress, Content of proline in two types of transgenic plants was very higher than that of the control, enhanced respectively 26% and 34%, reached an extremely significant level(P<0.05). MDA content in transgenic plants is lower than the control in the same period, while the biggest difference appeared at 24h(P<0.05), that MDA content of inducible transgenic and constitutive transgenic plants reduced 31% and 28% than control, respectively.8. qRT-PCR proves that relative expressions of P5 CS and ProDH gene both increase in early period, and then decreased after PEG stress. Relative expression of P5 CS and ProDH in transgenic plants both enhanced significantly compared with the control plants, and reached the extremely significant level(P<0.05)at 12 h after treatment, Expressions of P5 CS and ProDH in transgenic and non-transgenic plants both reduce obviously after 24 h treatment. |
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