雨生红球藻是一种淡水浮游单细胞绿藻, 逆境条件下可积累大量的类胡萝卜素。番茄红素是类胡萝卜素中的一种, 是类胡萝卜素合成代谢中的一个重要中间产物。番茄红素β-环化酶(LycB)是催化番茄红素形成β-胡萝卜素的关键酶。文章以杜氏盐藻lycB基因为干扰序列, 构建了含卡那霉素与阿特拉津双抗性的RNAi载体p1301-BS-RNAi。将其电转化进雨生红球藻细胞, 经抗性筛选、基因组PCR及RT-PCR筛选, 获得了16个独立的干扰株系。选取生长良好的7个进行高光诱导, 发现其番茄红素含量增加了99.4%, β-胡萝卜素含量减少了48.4%, 即通过异源的lycB-RNAi基因沉默可抑制番茄红素向β-胡萝卜素的转化。对比分析发现, 番茄红素增加量仅是β-胡萝卜素减少量的5%, 表明因lycB-RNAi抑制而产生的番茄红素的95%又被其他通路转换成了其他代谢产物, 因此要实现雨生红球藻番茄红素含量的大幅增长, 需协同调控其他代谢通路。
Haematococcus pluvialis is a freshwater planktonic single-cell microalgae. It will accumulate high amount of carotenoids under unfavorable environmental conditions. As one of carotenoids, lycopene is an important intermediate in the carotenoid biosynthesis pathway. Lycopene β-cyclase (LycB) is the key enzyme that catalyzes the circularization of lycopene to form β-carotene. In this study, we constructed a p1301-BS-RNAi vector using lycB from Dunaliella salina as the interference sequence with kanamycin and atrazine resistance marker, and then transformed it into H. pluvialis by electroporation. Sixteen independent transgenic lines were obtained after resistance selection, genome PCR, and RT-PCR analyses. Seven well-grown lines were selected to determine the contents of carotenoids by HPLC analysis after inducing by high light. The lycopene content in these lines was increased by 99.4% while the β-carotene content was decreased by 48.4%, indicating that the interference by heterogenous lycB could inhibit the conversion of lycopene into β-carotene. The amount of increase in lycopene was only 5% of the amount of decrease in β-carotene, indicating that 95% of the decreased β-carotene was converted into other metabolites. Therefore, in order to largely increase the lycopene content in H. pluvialis, it is necessary to coordinatively regulate other metabolic pathways.
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