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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。
Astaxanthin is an oxidized keto-type red carotenoid with a greater resistance to photooxidation. The bkt gene of β-carotene keto-enzyme (the key enzyme of astaxanthin biosynthesis) was constructed into the expression vector pCAMBIA1301 to obtain the plant expression vector p1301-bkt, which was transformed into Agrobacterium tumefaciens EHA105 to obtain the engineering bacteria. ’Brookfield Gala’ sterile tube seedling leaves as a receptor for genetic transformation. The screening results showed that ’Brookfield Gala’ was sensitive to Hyg, the optimal Hyg selection pressure for leaf regeneration was 3 mg · L-1, the in vitro proliferation was 4 mg · L-1 and the rooting was 2 mg · L-1; cefotaxime (Cef) concentration ≤ 400 mg · L-1 had no significant effect on the number of regenerated shoots. GUS staining, PCR and RT-PCR results showed that 8 transgenic plants were integrated with the bkt gene and expressed, and its phenotype was red. HPLC analysis of the carotenoid in transgenic plants showed that astaxanthin and canthaxanthin Accumulation in the leaves reached 2.85 and 1.79 μg · g -1. The results of this study show that astaxanthin is expected to be synthesized in apples through the regulation of metabolic pathways, enhancing the ability of fruits to resist photooxidation and preventing sunburn.