缺氮介导的莱茵衣藻油脂合成过程的内参及标志物蛋白质的筛选鉴定

Identification of Reference and Biomarker Proteins in Nitrogen Depletion-mediated Triacylglycerol Biosynthesis in Chlamydomonas reinhardtii

微藻被认为是最有潜力的生物能源原料之一,了解油脂合成机理、提升油脂合成的效率是重要的生物学问题.莱茵衣藻缺氮胁迫是油脂合成机理研究的模式系统,组学研究已经积累了大量的数据,但针对莱茵衣藻缺氮介导的油脂合成过程的内参及标志物蛋白质还鲜有报道.本研究对莱茵衣藻进行了对照和缺氮胁迫培养,比较了多个时间点(0、1、2、4和6d)在2种处理条件下的培养物表型、细胞密度、油脂含量以及总蛋白质含量的变化等特征.结果显示:莱茵衣藻细胞在受到缺氮胁迫后表现为培养物颜色由绿变黄;A750和细胞计数结果显示细胞生长趋于停滞;尼罗红染色定量实验鉴定到油脂含量的显著升高;考马斯亮蓝染色实验检测到总蛋白质含量降低.以20个莱茵衣藻蛋白质为候选,利用蛋白质印迹技术(Western blot,WB)检测了其在不同处理和不同时间点的表达特征变化,通过计算总蛋白质和候选蛋白质含量的皮尔森相关系数(Pearson’s correlation coefficient, PCC)筛选了莱茵衣藻缺氮胁迫的内参蛋白质,发现Histone H3、 RBCL(ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit)和BCR1(biotin carboxylase,ACCase complex 1)在对照和缺氮胁迫条件下均与总蛋白质含量变化呈现极显著或显著正相关,所以被选作内参蛋白质.进而通过比较候选蛋白质的平均相对倍率变化(average relative fold change, ARF),鉴定了莱茵衣藻缺氮胁迫的标志物蛋白质,发现ATPs-β(ATP synthase CF1beta subunit)、 GAP2(glyceraldehyde 3-phosphate dehydratase 2)和RMT1(rubisco large subunit N-methyltransferase 1)蛋白质的ARF值分别为180.59、52.90和12.48,明显高出其他蛋白质,由此把它们选做缺氮胁迫的标志物蛋白质.接下来,对缺氮胁迫早期(0、2、4、8、12、18、24和48 h)的样品进行蛋白质印迹法分析,发现可检测的ATPs-β、GAP2和RMT1的缺氮诱导条带出现的时间分别是8、18和12 h.综上可以认为,在所有候选蛋白质中,ATPs-β是出现最早且变化幅度最大的缺氮处理标志物蛋白质.本研究鉴定的内参和标志物蛋白质对了解缺氮应答及油脂合成机理会有所帮助,所积累的蛋白质表达信息可供研究同行参考.

Due to the relatively high lipid productivity, microalgae are promising raw material for biofuel production.The understanding for the mechanism of lipid biosynthesis is an important concern which may contribute to the increase of lipid production.Chlamydomonas reinhardtii under nitrogen depletion condition is a model system to investigate the pathway of lipid biosynthesis.Substantial amount of data have been accumulated using “omics” approaches recently, however, the identification of reference and biomarker proteins in nitrogen depletion-mediated triacylglycerol biosynthesis in C.reinhardtii is limited.In this study, C.reinhardtii CC-124 grown in control and nitrogen depleted medium were surveyed to compare the morphology, cell density, lipid content, and total protein content at multiple time points(0,1,2,4 and 6 days).Under nitrogen depletion treatment, the culture turned yellow from green and the A750 and cell number was decreased, indicated that the cell growth was retarted.Furthermore, the concentration of neutral lipid was up-regulated based on nile red staining assay, while the coomassie brilliant blue staining assay indicated that the concentration of total protein was decreased.Western blot(WB)was performed to detect the expression patterns for 20 candidate C.reinhardtii proteins at different time points.To identify the reference proteins under nitrogen depleted condition, the Pearson’s correlation coefficient(PCC)between the content of total proteins and candidate proteins was calculated.Three proteins(Histone H3, RBCL(ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit)and BCR1(biotin carboxylase, ACCase complex 1))were selected due to their significant positive correlations(P < 0.01)in both nitrogen depleted and control conditions.Comparison of average relative fold change(ARF)of the candidate proteins indicated that the top 3 proteins are ATPs-β(ATP synthase CF1 beta subunit), GAP2(glyceraldehyde 3-phosphate dehydroase 2)and RMT1(rubisco large subunit N-methyltransferase 1), their ARFs are 180.59, 52.90 and 12.48, respectively, which were then chosen as biomarkers.Futhermore, to compare the earliest time point at which biomarker band can be detectable, protein samples at early stage(0, 2, 4, 8, 12, 18, 24 and 48 h)at nitrogen depletion treatment were collected and analysed by WB, it was showed that the induction bands of ATPs-β, GAP2 and RMT1 appeared at 8, 18 and 12 h, respectively.Thus, ATPs-β was the most appreciate biomarker since it is the earliest showed and the largest degree varied among candidates proteins tested.The reference and biomarker proteins identified in this study will provide help for the mechanism investigation of nitrogen depletion responses and lipid biosynthesis in C.reinhardtii, the expression profiling acculumated for candidate proteins can be refered by the reseach community.