雨生红球藻Rubisco在虾青素积累过程中的表达分析

虾青素因其具有极强的抗氧化性及优越的着色作用，被广泛应用于营养保健和水产养殖中，备受国内外研究者的关注。红球藻是目前虾青素的生物来源中较有优势的一条途径。 我们选取雨生红球藻作为研究材料，收集其生长过程中四个不同的阶段，分别为绿色游动细胞阶段，绿色不动细胞阶段，绿褐色不动细胞阶段（开始积累虾青素），以及红色不动孢子阶段。本研究中提取了核酮糖-1,5-二磷酸羧化/加氧酶（Rubisco）的粗提液，并测定了酶活。其编码基因rbcL的mRNA表达量也被测定。另外，光合速率与呼吸速率的比值（P/R）通过测定与计算得出，各个阶段藻细胞中虾青素的含量由分析得出。本研究还应用了叶绿素荧光测定方法，确定了光系统II潜在最大活性（Fv / Fm），光系统II实际活性（ΦPSII），电子传递速率（ETR）和非光化学淬灭参数（NPQ）。 结果表明，绿色游动细胞的生长状态最佳，其P/R、Fv / Fm、ΦPSII均为最大，NPQ为最小。这说明在此状态的细胞中，光系统II的活性最强；但是其Rubisco活性与rbcL表达量均为最小。相比之下，在绿褐色不动细胞中，P/R和NPQ的值较低，Fv / Fm、ΦPSII和ETR值都最小，但Rubisco活性与rbcL表达量均为最高。 结合工业生产虾青素的方法，我们认为，Rubisco或许参与了虾青素的合成，而非Calvin循环为色素合成提供前体和能量。因此，在生产过程中适当加入碳源，比如CO2，可以有效增大虾青素的产量。

There is more and more concentration on astaxanthin by many researchers in the world, due to its effective coloration in aquaculture and anti-oxidation in nutracetical. Haematococcus is one of the most effective natural sources of astaxanthin. Cells of Haematococcus Pluvialis Flot. et Will in four different growth phases were collected, which are green vegetative cells, green unmoving cells, orange unmoving cells and red cysts. Ribulose-1,5-bisphosphate carboxylase (Rubisco) activity in crude extracts and large-subunit ribulose-1,5-bisphosphate caboxylase/oxygenase (rbcL) mRNA was quantified. Meanwhile, the ratios of photosynthesis rate to respiration rate (P/R), the maximal effective quantum yield of photosystem II (Fv/Fm), electron transport rate (ETR), actual photochemical efficiency of PSⅡ in the light (ΦPSⅡ) and non-photochemical quenching (NPQ) were measured. Moreover, the content of astaxanthin was determined. It was showed that green vegetative cells were in the most active state with relatively higher P/R and they also displayed the lowest value of NPQ and the highest value of Fv / Fm, ETR and ΦPSⅡ, indicating the most effective PSⅡ, but both Rubisco activity and rbcL mRNA expression were the lowest. However, in orange resting cysts with relatively lower P/R and NPQ, Rubisco activity and rbcL expression reached the peak, while Fv / Fm, ETR and ΦPSⅡ was the lowest. Together with the methods of astaxanthin induction in industry, it was suggested that Rubisco possibly participated in astaxanthin accumulation rather than Calvin cycle in orange resting cells; therefore, some carbon source such as CO2 should be sufficiently supplied in a large scale of astaxanthin production in Haematococcus pluvialis.