关键词: 苹果, 虾青素, bkt 基因, 遗产转化, 转化体系

Abstract: Astaxanthin is a kind of red and ketonic carotenoids which is reported to have stronger antioxidant activity against photooxidation. The present study focused on transfering β-carotene ketolase gene bkt which is the key enzyme gene of astaxanthin biosynthesis into‘Brookfield Gala’，by constructing plant expression vector pCAMBIA1301-bkt with binary gene through Agrobacterium tumefaciens. The results showed that‘Brookfield Gala’plants were sensitive to Hyg and the optimal Hyg concentration for selected-stress was 3 mg • L^-1 for leaf regeneration and 4 mg • L^-1 for proliferating shoot culture and 2 mg • L^-1 for rooting. The effect of cefotaxime（Cef）on the average regeneration bud number from the leaves was not obvious when the Cef concentration was not exceeding 400 mg • L^-1. Histochemistry GUS assay，PCR amplification and the results of PCR-Southern blotting analysis confirmed that bkt gene has been integrated into the genome of‘Brookfield Gala’and RT-PCR revealed that the bkt gene was expressed in the eight transgenic plants with pink color in phenotype. The carotenoid of transgenic plants was analysed by HPLC and results showed that novel ketocarotenoids were synthesized with accumulation of astaxanthin in the leaf up to 2.85 μg · g^-1 and canthaxanthin up to 1.79 μg · g^-1 leaf fresh weight respectively. The study is expected to change‘Brookfield Gala’apples with astaxanthin content to have stronger antioxidant ability against sunburn by regulating metabolic pathways.

Key words: apple, astaxanthin, bkt gene, genetic transformation, transformation system