柑橘愈伤组织RNAi沉默CCD1基因对其类胡萝卜素积累的影响

类胡萝卜素裂解双加氧酶(CCD)可催化类胡萝卜素在特定位点氧化裂解生成多种生物活性化合物,其活性差异一定程度上决定了植物体内类胡萝卜素含量和组成变化。为了探讨柑橘中CCD1蛋白对类胡萝卜素积累的影响,从红肉脐橙(Citrus sinensis L. Osbeck)果实中克隆了CCD1基因,将其特异片段通过重组反应连接到pHGRV载体中,构建CCD1的RNA干扰载体pHGRV-CCD1。利用农杆菌介导的方法将其转入‘国庆1号’蜜柑、‘伏令夏橙’和‘马叙’葡萄柚愈伤组织中,经PCR鉴定分别获得6、4、4个转基因干扰系。3种柑橘材料转基因干扰系中CCD1基因的表达水平均较野生型显著下调,其中‘马叙’葡萄柚转基因干扰系Rm2的表达水平仅为野生型的8.4%,干扰效果最佳。CCD1转基因干扰系愈伤组织外观多数呈现与野生型对照相近的色泽,仅Rm2系可见较为明显的颜色变化;综合分析3种柑橘材料转基因前后类胡萝卜素含量变化,发现CCD1基因抑制表达后,紫黄质、9–顺式–紫黄质的含量均较野生型显著增加,表明其可能是CCD1在柑橘愈伤组织体内优先选择的裂解底物。

RNAi-mediated Suppression of CCD1 Gene Impacts Carotenoid Accumulation in Citrus Calli

arotenoid cleavage dioxygenase（CCD）can catalyze the oxidative cleavage of carotenoidsa t specific sites to generate a variety of bioactive compounds. To some extent，their activities determine thearotenoid content and composition in plants. In this study，the CCD1 gene was cloned from fruits of CaraCara navel orange（Citrus sinensis L. Osbeck）. The RNAi construct，pHGRV-CCD1，was constructed byinserting CCD1 specific fragments into plasmid pHGRV via recombination，and then introduced into calliof Satsuma mandarin Guoqing 1，Valencia sweet orange and Marsh grapefruit by Agrobacterium-mediatedtransformation. As a result，6，4 and 4 transgenic lines were obtained respectively. Compared with the wild-type，expression level of the transgenic lines were significantly downregulated to variousdegrees，among which the CCD1 expression of Marsh grapefruit transgenic line Rm2 was only 8.4% ofcontrol as the greatest interference effect. Most transformed calli with CCD1 RNAi construct exhibitedsimilar color phenotype to wild-type controls except for Rm2 line（macroscopic color changes）. Comparedwith the wild-type，the inhibition of CCD1 gene expression resulted in a significant increase ofviolaxanthin and 9-cis-violaxanthin content，suggesting these two compounds might be the preferred invivo cleavage substrates of CCD1 in citrus calli.