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中文摘要：
虾青素是一种氧化型酮式红色类胡萝卜素，具有更强的抗光氧化能力。将β–胡萝卜素酮化酶（虾青素生物合成的关键酶）基因bkt构建入表达载体pCAMBIA1301中，获得植物表达载体p1301-bkt，转化根癌农杆菌EHA105，获得工程菌，以黄肉苹果‘Brookfield Gala’无菌试管苗叶片为受体，进行遗传转化。筛选压确定结果表明：‘Brookfield Gala’对潮霉素（Hyg）很敏感，叶片再生最佳Hyg选择压为3 mg · L-1，试管苗增殖为4 mg · L-1，生根为2 mg · L-1；头孢霉素（Cef）浓度≤400 mg · L-1时对叶片再生芽数的影响不明显。GUS染色、PCR和RT-PCR检测结果表明，有8株转基因植株整合bkt基因并获得了表达，其表型有红色产生；转基因植株类胡萝卜素的HPLC测定显示，虾青素和角黄素在叶片中的积累量达到2.85和1.79 μg · g-1。本研究结果显示有望通过调控代谢途径，在苹果中合成虾青素，提高果实自身的抗光氧化能力，防止日灼。
英文摘要：
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.
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