Cloning Of The Nicotiana Protein Kinase (NPK1) And The Arabidopsis NPR1 Gene And Study The Genetic Transformation Of Capsicum Annuum L.

Pepper (Capsicum annuum L.) is one of the most important vegetables for its rich nutrient with fruit and economic value. Stresses of freezing, heat, salinity, dry and many diseases seriously affect the productivity of pepper. It has being studied to improve the stress tolerance and diseases resistance of pepper.The NPK1 gene from tobacco, which encodes a protein kinase related to MAPKKK plays important roles in embryogenesis and the oxidative stress signalling pathway. The NPR1 gene is a key regulatory component that is positioned at the crossroads of multiple defense pathways.In this research, we cloning of the Arabidopsis NPR1 gene and the tobacco NPK1 gene, study the effect of establishment of high efficiency regeneration system and determination of parameters involved in Agrobacterium mediated transformation. Screening and characterization of transforming were systematically studied. Cotyledons of pepper were successfully transformed with the Arabidopsis NPR1 gene and the tobacco NPK1 gene by Agrobacterium mediated transformation. And the tobacco NPK1 gene was transferred into Arabidopsis(Columbia-0). The results were summarized as follow:1. In vitro plant regeneration was achieved from eight chilli pepper materials (Capsicum annuum L.). The effects of genotype, various explant types (cotyledons, hypocotyls) and hormone was evaluated. It was shown that the high 6-BA/ IAA ratio was conducive to the differentiation and the low 6-BA/ IAA ratio was suitable for the elongation of the regenerative buds; different pepper materials differed greatly in their regeneration capacities. The cotyledons had stronger regenerative capacity than the hypocotyls, and the explants from 12～16 day old seedlings had a high differentiation rate. Higher generation frequency was obtained from materials of 2096, B4 and B7. Comparatively speaking , the optimal medium for the differentiation of pepper buds was Murashige and Skoog (MS)medium supplemented with B5 organic nutrient(MB), 0.8mg/L IAA , 5.0mg/L6-BA and 4.0mg/L AgNO₃. The best media for shoots elongation was MB medium with 0.8mg/L IAA , 2.0mg/L6-BA ,2.0mg/L GA₃and 4.0mg/L AgNO₃ and the rooting medium was MB medium with 0.2 mg/L IAA, 0.1 mg/L NAA.2. The efficient genetic transformation system for pepper is that cotyledon explants were preconditioned for 2 days on M1(MB+0.8mg/L IAA+5.0mg/L 6-BA +4.0mg/L AgNO₃)medium and inoculated with the cultures of Agrobacterium strain LBA4404 for 8～12 min, followed by co-cultivation with Agrobacterium tumefaciens on M1 for 2 days and delay selection on M2(MB+0.8mg/L IAA +5.0mg/L 6-BA +4.0mg/L AgNO₃+250mg/L Cef) for 2 days. The explants were then placed on M3 (MB+0.8mg/L IAA+5.0mg/L 6-BA+4.0mg/L AgNO₃+250mg/L Cef+50mg/L Km)for selection.3. We obtained 65 Km-resistant regenerated plants and DNA from the plants was subjected to PCR analysis. 9 showed PCR-positive including 4 NPR1 5 NPK1.4. Studied the stress tolerance of Arabidopsis transgenic lines, we found that the levels of MDA and Pro increased with the stress time, and the content of MDA is negative to stress tolerance, while the level of Pro is positive to resistance at the early stage of stress.