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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
史佳楠 , 杜铁民 , 陈悦 , 周艳 , 杨亚茹 , 李莉云 , 窦世娟 , 刘丽娟 , 刘国振     
河北农业大学生命科学学院,保定 071000
摘要: 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 CF1 beta 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.
关键词: 莱茵衣藻     缺氮胁迫     油脂合成     内参蛋白质     标志物蛋白质     免疫印迹    
Identification of Reference and Biomarker Proteins in Nitrogen Depletion-mediated Triacylglycerol Biosynthesis in Chlamydomonas reinhardtii
SHI Jia-Nan , DU Tie-Min , CHEN Yue , ZHOU Yan , YANG Ya-Ru , LI Li-Yun , DOU Shi-Juan , LIU Li-Juan , LIU Guo-Zhen     
College of Life Sciences, Hebei Agricultral University, Baoding 071001, China
*This work was supported by grants from Key Discipline of Botany in Hebei Province and Key Laboratory of Hebei Province for Molecular Plant-Microbe Interaction
** Corresponding author: LIU Guo-Zhen, Tel: 0312-7528787, E-mail: gzhliu@hebau.edu.cn
Received: August 22, 2018 Accepted: November 6, 2018
Abstract: 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.
Key words: Chlamydomoans reinhardtii     nitrogen depletion     lipid biosynthesis     reference protein     biomarker protein     Western blot    

由于微藻具有生长速度快、油脂产量高、不与人争粮、不与粮争地等特点, 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.莱茵衣藻是微藻生物学研究的模式生物, 广泛应用于胁迫应答、光合作用、呼吸过程、油脂合成及鞭毛运动等重要生物学过程的机理研究.在缺氮胁迫培养条件下, 莱茵衣藻和多种微藻都会表现油脂含量升高.多年来, 莱茵衣藻缺氮胁迫诱导油脂合成的途径一直是人们研究的热点, 了解缺氮胁迫介导的相关基因的表达变化是油脂合成机理研究的重要内容.除了油脂含量升高这一典型特征外, 在缺氮胁迫条件下, 莱茵衣藻细胞还表现为叶绿体呼吸增强、类囊体成分改变[1]、营养细胞分化为生殖细胞[2]、细胞增殖受到强烈抑制、细胞颜色由绿变黄及蛋白质合成速率下降等特征.以莱茵衣藻CW-15藻株为对象, 对缺氮胁迫24 h和48 h的样品进行转录组分析, 发现缺氮胁迫激活了配子发育相关基因, 而光合作用及蛋白质合成相关的基因表达下调, 同时碳的利用方式由参与细胞组成转为脂类合成[3].以莱茵衣藻CC-400藻株为对象, 缺氮胁迫24 h, 并对8个时间点的样品(0、0.5、1、2、4、6、12和24 h)进行多组学分析, 分别在转录组、蛋白质组和代谢组水平上鉴定到多个差异表达的基因、蛋白质以及含量发生变化的三酰甘油(triacylglycerol, TAG)、氨基酸等有机小分子[4].分析莱茵衣藻CC-503在缺氮胁迫下的多个生理指标, 以及蛋白质组、代谢组以及磷酸化蛋白质组的变化, 将细胞应答缺氮胁迫分为3个阶段, 依次为淀粉积累、叶绿体降解伴随细胞自噬和油脂合成.在补充氮源后, 叶绿体重新生成、细胞生长被激活[5].在莱茵衣藻缺氮胁迫应答机理研究中, 已经积累了大量的"组"学数据, 今后应加强针对特定蛋白质的研究, 以便对油脂合成机理进行深入的了解.

Knepper等[6]于2011年最早提出了基于抗体的蛋白质组学策略, 继而应用于人和动物的蛋白质组学研究[7-8].这一策略具有靶向性强、灵敏度高、结果直观、操作简便等特点, 但需要借助大量有效的抗体资源.本实验室曾筛选鉴定了水稻的内参蛋白质[9]和病程相关蛋白质标志物[10-11], 其中水稻热激蛋白质HSP82(heat shock protein 82)在多个生长时期的多数组织中都稳定表达, 现广泛用作水稻蛋白质研究的内参[9].莱茵衣藻研究中也报道了一些内参蛋白质, 如线粒体ATP合成酶β亚基(ATP synthase subunit β, ATPB)作为内参评价捕光复合物相关(light-harvesting complex stress-related, LHCSR)蛋白质的表达状况[12]; 类鸟苷酸结合蛋白质β亚基(chlamydomonas beta subunit-like polypeptide, CBLP)的mRNA在细胞周期和鞭毛再生过程中稳定表达[13], 其蛋白质与水稻RWD(rice protein containing the WD-40 repeat)蛋白质同源, 亦呈组成型表达[14].光合系统Ⅱ中的核心蛋白质D1(photosystem Ⅱ protein D1)在高光/低光处理条件下表达稳定, 可用于标定不同光照强度处理的样品上样量[15].通常条件下α-Tubulin的丰度变动不大, 该蛋白质常用于标定对照和突变体材料的上样量[16].在叶绿体中大量存在的Rubisco大亚基(ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit, RBCL)可用做内参蛋白质, 标定高光/低光处理的材料上样量[17].微丝动力蛋白质亚基IC2(flagellar outer dynein arm intermediate chain 2)主要存在于莱茵衣藻细胞的鞭毛中, 可用于标定野生型和突变体的上样量[18].本实验室曾对冷、热、盐、暗、糖等处理条件下的莱茵衣藻内参蛋白质进行了鉴定, 发现组蛋白H3亚基(histone H3)、微管蛋白质(β-tubulin 1, TUB1)、核酮糖-1, 5-二磷酸羧化酶大亚基RBCL和线粒体ATP合酶亚基(mitochondrial F1FO ATP synthase subunit 6, ATPs-6)等在多种非生物胁迫下表达量与总蛋白质含量显著正相关, 适用于蛋白质样品的定量[19].标志物蛋白质是生物体在特定条件下, 表达特征发生显著变化的蛋白质, 这种变化包括但不限于表达丰度、修饰、分子质量变化等.蛋白质标志物既可作为蛋白质定量分析的参照, 还可用于判断某种刺激/胁迫是否发生以及发生的程度的指标.如HSP基因的上调是生物对热胁迫应答的标志物[20]、HSP70B是莱茵衣藻氧胁迫应答的标志物[21]、病程相关蛋白质的上调多作为植物发生抗病反应的标志[22].此外, 细胞器标志物在蛋白质亚细胞定位研究中也发挥重要的作用, KARI(keto-acid reductase isomerase)[23]和PFO(pyruvate ferredoxin oxidoreductase)[24]是叶绿体标志物、COX2b(cytochrome c oxidase 2b)蛋白质[23]是线粒体标志物、IC2蛋白质在莱茵衣藻鞭毛中大量表达[25].筛选、鉴定和应用生物标志物, 是蛋白质功能研究的基础.鉴定标志物蛋白质不但可以作为生物发生抗逆应答的重要指标, 对研究抗逆机理也有重要意义.本实验室前期鉴定并报道了莱茵衣藻中的HSP90B、FAP127(flagellar associated protein 127)和ATPs-A(chloroplast ATP synthase CFO A subunit)是广谱的胁迫应答标志物[19].

尽管越来越多的内参蛋白质及标志物蛋白质被鉴定, 但针对莱茵衣藻缺氮胁迫内参及标志物蛋白质的了解还很有限.本研究对莱茵衣藻进行了正常和缺氮胁迫培养实验, 记录比较了多个时间点在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, 鉴定到在缺氮胁迫过程中伴随着油脂含量的显著升高.用20个莱茵衣藻蛋白质为候选, 利用蛋白质印迹(Western blot, WB)技术检测了其表达特征的变化、通过计算总蛋白质和靶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、通过比较候选蛋白质的平均相对变化倍率(average relative fold change, ARF), 鉴定了莱茵衣藻缺氮胁迫的标志物蛋白质.这些结果可用于莱茵衣藻缺氮应答机理研究, 也可供研究同行在莱茵衣藻油脂合成机理研究中应用.

1 材料与方法 1.1 藻种与莱茵衣藻的培养

莱茵衣藻CC-124藻种购于美国莱茵衣藻中心(http://www.chlamycollection.org/), 在Tris-acetate-phosphate (TAP)[26]固体培养基上划线获得单克隆, 取单克隆藻细胞接种于含200 ml TAP液体培养基的500 ml三角瓶中悬浮振荡培养(120 r/min), 培养温度25℃, 24 h持续光照, 光照强度为100 μmol·m-2·s-1.预培养3d, A750达到0.2~0.3后, 离心(800 g, 5 min)收集微藻, 分别用TAP正常或缺氮(培养基中不添加NH4Cl成分)液体培养基重悬浮, 漂洗2次后, 将莱茵衣藻在200 ml正常或缺氮培养基培养, 分别于0、1、2、4和6d取材, 拍照记录表型, 光学显微镜下用血球计数板计数, 取30 ml培养物经2 000 g离心10 min, 收集至2 ml Eppendorf管中, -70℃冻存待用.所用试剂均购于上海生工生物工程有限公司.

1.2 莱茵衣藻培养的生长曲线

用紫外/可见分光光度计(Unico WFJ 7200)测量培养物在波长750 nm的吸光度值(A750), 绘制生长曲线, 每个实验重复3次并计算平均值和方差.

1.3 油脂含量测定

莱茵衣藻三酰甘油(TAG)含量的测定方法参照文献[27], 主要步骤为:将5 ml藻培养物经800 g, 5 min离心收集, 重悬浮于等体积PBS缓冲液(2.7 mol/L NaCl, 8.1 mmol/L Na2HPO4, 1.5 mmol/L KH2PO4, 2.7 mmol/L KCl, pH 7.4)中, 加入终浓度为20%的DMSO和尼罗红染料(终浓度1 mg/L), 震荡混匀, 40℃温浴10 min, 用荧光分光光度计(Shimadzu RF-6000)在激发波长为485 nm, 发射波长580 nm处检测得到中性脂的相对荧光强度, 根据标准曲线计算TAG含量.每个实验重复3次, 计算平均值和方差.

1.4 总蛋白质提取及定量

莱茵衣藻总蛋白质的提取方法按参考文献[27], 主要步骤为:将冻存的莱茵衣藻沉淀用1 ml蛋白质提取缓冲液(60 mmol/L DTT, 60 mmol/L Na2CO3, 2% SDS(w/v)和12%蔗糖(w/v))重悬浮, 加入总体积约100 μl的氧化锆珠, 在高通量组织研磨仪(鼎昊源TL2010)中剧烈震荡20 min, 4℃, 10 000 g离心, 收集上清, 按照1:4加入5×上样缓冲液(250 mmol/L Tris-HCl(pH 6.8), 10% SDS(w/v), 0.5%溴酚蓝(w/v), 50%甘油(v/v)和5% β-巯基乙醇(w/v)), 沸水浴10 min, -20℃冻存待用.蛋白质相对定量采用SDS-PAGE分离全蛋白质, 凝胶浓度10%, 上样量20 μl, 电压160 V, 电泳时间1 h, 具体步骤参考文献[9].电泳结束后将凝胶浸泡于新鲜配制的考马斯亮兰染色液(1 L:Coomassie Brilliant Blue R-250(生工生物工程(上海)股份有限公司) 2.5 g, 甲醇450 ml, 冰醋酸100 ml, 蒸馏水450 ml)中, 染色2 h后转移至脱色液(1 L:冰醋酸130 ml, 甲醇300 ml, 蒸馏水570 ml)中, 在脱色摇床中脱色至条带和背景清晰、用Minichem 610凝胶/化学发光成像仪(北京赛智生物技术公司)拍照, 使用Image J软件[28]采集信号进行相对比较.

1.5 候选蛋白质及抗体的选择

20个候选蛋白质及抗体信息, 包括每个蛋白质的Au10_locus ID、预测的亚细胞定位、注释及分子质量、制备抗体所用的免疫原、免疫多肽的序列等见文献[19].

1.6 蛋白质印迹检测

蛋白质印迹(WB)实验步骤参照文献[9].SDS-PAGE分离全蛋白质, 上样量10 μl, 电泳条件160 V, 时间1~1.5 h; 采用湿转方法将蛋白质转移至PVDF膜上, 转膜条件100V恒压1 h.使用5%脱脂牛奶的TTBS溶液(20 mmol/L Tris-HCl(pH 7.5), 500 mmol/L NaCl, 0.05% Tween 20)封闭1 h; 一抗(北京华大蛋白质研发中心有限公司制备)按1:5 000稀释于5%脱脂牛奶中, 孵育3 h, TTBS洗膜3次, 每次5 min; 辣根过氧化物酶标记的羊抗兔二抗(北京华大蛋白质研发中心有限公司)按1:15 000稀释于5%脱脂牛奶中, 孵育1 h, TTBS洗膜3次, 每次5 min; 将600 μl发光液(康为世纪)均匀滴加在膜上, 用Minichem 610凝胶/化学发光成像仪(北京赛智生物技术公司)检测信号.对于分子质量小于30 ku的蛋白质, 采用Tricine-SDS-PAGE[29-30]分离全蛋白质, 80 V电泳30 min, 160 V电泳2.5 h, 后续转膜、孵育等步骤与普通SDS-PAGE一致.

1.7 蛋白质表达丰度相关性分析

用Image J软件采集获得总蛋白质、目标蛋白质信号强度, 用IBM SPSS V24 (IBM公司, Armonk, 纽约, 美国)软件计算总蛋白质含量和候选蛋白质丰度之间的皮尔森相关系数(PCC).为了便于比较, 对蛋白质印迹信号进行了归一化处理, 即将0时间点的信号强度定义为1, 据此计算其他时间点的相对信号值, 用归一化后的蛋白质印迹信号代表候选蛋白质的表达丰度.

1.8 平均相对倍率变化的计算

平均相对倍率变化(ARF)的计算按如下公式:

其中, I(Treatment)为缺氮胁迫第1、2、4或6 d归一化后的蛋白质印迹信号强度, I(CK)为正常培养样品中归一化后的蛋白质印迹信号强度.

2 结果与分析 2.1 莱茵衣藻缺氮胁迫后表型特征

正常培养及缺氮胁迫的莱茵衣藻在不同时间点的外观形态见图 1a, A750生长曲线见图 1b, 细胞数的变化曲线见图 1c, 油脂含量变化见图 1d.正常培养的莱茵衣藻在6 d的生长过程中细胞逐渐增多, 颜色由浅绿逐渐变为深绿, A750值从0.21上升至1.21, 细胞数从0.7×106/ml上升到3.3×106/ml.缺氮胁迫条件下培养物颜色由浅绿色变化黄绿色, A750从0.21缓慢升至0.41, 细胞数从0.7×109/L上升到1.5×109/L.由此可见, 缺氮胁迫后细胞数增长几乎停滞, 细胞内叶绿素的合成也受到强烈抑制.同时, 计算单位细胞中的中性脂相对含量(图 1d)可知, 正常培养条件下, 由于细胞数的增加, 单位细胞内中性脂相对含量下降36%, 而缺氮胁迫6 d, 单位细胞内中性脂含量升高2.37倍, 所以缺氮胁迫显著提高了莱茵衣藻的油脂含量.

Fig. 1 The influence of nitrogen (N) depletion stress on the growth of C. reinhardtii cells (a) Morphology alterations of C. reinhardtii grown in N-depleted medium.Each samples were collected at the set time points in the experiment, the photos were taken with digital camera Canon A100.+N: control; -N: N-depleted stress.d0, d1, d2, d4 and d6 are culture time (days). (b) The influence of N-depletion stress on the absorbance (A) of C. reinhardtii.The absorbance at 750 nm (A750) of the culture were determined by the ultraviolet-visible spectrophotometer, the bar graph was drawn with Microsoft Excel 2016.X-and Y-axes on the graph correspond to culture days and A750, respectively.Black bar, control; blank bar, N-depleted stress.Data are x±s of three independent experiments. (c) The influence of N-depletion stress on the cell number of C. reinhardtii.Cell number of batch culture was counted using hemocytometer under microscope, the bar graph was drawn with Microsoft Excel 2016. X-and Y-axes on the graph correspond to culture days and cell number, respectively.Black bar, control; blank bar, N-depleted stress.Data are x±s of three independent experiments. (d) The influence of N-depletion stress on the content of triacylglycerol (TAG) of C. reinhardtii.The intracellular TAG was stained with Nile red and quantified using spectrophotometer, TAG content per cell was calculated, the bar graph was drawn with Microsoft Excel 2016. X-and Y-axes on the graph correspond to culture days and fold change of TAG content per cell.Black bar, control; blank bar, N-depleted stress.Data are x±s of three independent experiments.
2.2 缺氮胁迫对莱茵衣藻总蛋白质含量的影响

氮是植物生长发育必需的大量营养元素之一, 主要参与蛋白质和叶绿素的合成.在培养的不同时间点提取莱茵衣藻的总蛋白质, 通过SDS-PAGE分离后考马斯亮蓝染色(图 2a), 用Lane 1D软件采集考染信号并进行定量比较(图 2b), 由图 2可见, 在正常培养条件下, 总蛋白质含量随时间延长逐渐升高, 培养6 d单位体积内总蛋白质含量升高到2.69倍, 在缺氮胁迫条件下, 总蛋白质含量下降到54%.

Fig. 2 The influence of nitrogen (N) depletion stress on the total protein content of C. reinhardtii (a) Extracted total protein was separated by SDS-PAGE and stained with Coomassie brilliant blue.+N: control; -N: N-depleted stress.d0, d1, d2, d4 and d6 are culture time (days). (b) Quantitative comparison of total protein content of C. reinhardtii determined by Coomassie brilliant blue.Signal intensities were extracted by ImageJ software, and showed as relative protein abundance.The intensity of 0 day was set to 1, the fold change of 1, 2, 4 and 6 day to 0 day were calculated. X-and Y-axes on the graph correspond to culture days and fold change of total protein content, respectively.Black bar, control (+N); blank bar, N-depleted stress (-N). Data are x±s of three independent experiments.
2.3 缺氮胁迫条件下候选蛋白质的表达特征

用20个候选蛋白质的特异抗体, 对正常培养和缺氮胁迫共10份蛋白质样品进行总计200个泳道的蛋白质印迹分析(图 3).由图 3可见, 蛋白质IC2和FKBP12(peptidyl-prolyl cis-trans isomerase FKBP-type)未检测到信号, 其他18个候选蛋白质均检测到清晰的信号条带.在正常培养的材料中, 多数检测到候选蛋白质条带的信号增加, 在缺氮胁迫的样品中, 大部分蛋白质条带的信号下降, 但有3个蛋白质(ATPs-β(ATP synthase CF1 beta subunit)、GAP2(glyceraldehyde 3-phosphate dehydrogenase 2)和RMT1(rubisco large subunit N-methyltransferase 1))的条带信号缺氮胁迫上调.另外, 在ATPs-6的蛋白质印迹检测结果中, 有分子质量不同的缺氮诱导条带.13个蛋白质检测到单一主带, 5个蛋白质检测到2条主带, 其中TUB1、FAB2(plastid acyl-ACP desaturase 2)和MPC1(mitochondrial phosphate carrier protein 1)的蛋白质印迹结果中有分子质量不同但平行变化的2条主带.

Fig. 3 Western blot detection of candidate proteins in C. reinhardtii cells C. reinhardtii cells cultured under control and N-depleted stress condition were collected at five time points. Total proteins were extracted and separated by SDS-PAGE.PVDF-membrane-immobilized proteins were detected by antibodies against each 20 candidate proteins. Culture conditions and time points were labeled on the top of each Western blot results, and the name of proteins were labeled under the Western blot results. +N: control; -N: N-depleted stress. 0, 1, 2, 4 and 6 are culture time (days). The arrows indicate the bands of candidate proteins.
2.4 内参蛋白质的鉴定

内参蛋白质是在特定条件下蛋白质丰度保持稳定的蛋白质, 在多个时间点的样品中, 应该是与总蛋白质丰度变化相关性高的蛋白质.为了鉴定莱茵衣藻缺氮胁迫的内参蛋白质, 本研究计算了候选蛋白质丰度变化与总蛋白质含量之间的皮尔森相关系数(PCC)(表 1).由表 1可见, 在正常培养条件下, Histone H3、RBCL和BCR1(biotin carboxylase 1, ACCase complex 1)蛋白质的表达丰度与总蛋白质呈现极显著正相关, 可作为正常培养条件下的内参蛋白质首选.在缺氮胁迫条件下, Histone H3、HSP70A和FAB2蛋白质的表达丰度与总蛋白质含量呈现极显著正相关, 可作为缺氮胁迫的内参蛋白质.Histone H3、RBCL和BCR1在正常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, 可作为2种条件下的内参蛋白质.

Table 1 Correlation analyses between abundance of candidate proteins and total protein content of C. reinhardtii cells
2.5 标志物蛋白质的鉴定

标志物蛋白质是在特定条件下变化倍率最大的蛋白质, 在多个时间点的样品中, 则是以平均相对变化倍率(ARF)作为筛选的指标, 图 4展示了每个候选蛋白质的ARF数值.由图 4可见, 在缺氮胁迫条件下, ATPs-β、GAP2和RMT1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, 由此根据缺氮胁迫条件下蛋白质的丰度变化幅度, 可把ATPs-β、GAP2和RMT1选做缺氮胁迫的标志物蛋白质.

Fig. 4 The identification of biomarker proteins under N-depleted condition According to the formula (1) described in the method section 1. 8, Average relative fold change (ARF) was calculated as follows: Western blot signal intensities of 4 time points (day 1, 2, 4 and 6) were normalized based on the signal on day 0, which was set to 1. Relative fold change were calculated with (I(Treatment)-I(CK))/I(CK), where I(Treatment) and I(CK) are the normalized Western blot signal in N-depleted and control samples, respectively. The average relative fold change at 4 time points were calculated and then the bar graph was drawn. X-and Y-axes on the graph correspond to protein names and ARF, respectively.
2.6 标志物蛋白质在缺氮胁迫早期的表达特征

为了比较标志物蛋白质在缺氮胁迫过程中出现的时间, 采集了缺氮胁迫早期(48 h内)的正常培养和缺氮胁迫的莱茵衣藻样品, 提取蛋白质后进行了蛋白质印迹分析(图 5).由图 5可见, 肉眼可区分的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, 可以认为, ATPs-β是出现最早的缺氮诱导标志物蛋白质.

Fig. 5 The abundance alteration of candidate biomarker proteins at the early stage of N-depleted stress Upper panel: Bar graph. Fold change of abundance alteration of candidate biomarker proteins. Western blot signals intensities were extracted by ImageJ software, the fold change at each time points compared to the signal at day 0 were calculated (Y-axes). Data are x±s of three independent experiments. Lower panel: Western blot results. The abundance of candidate biomarker proteins detected by Western blot (ATPs-β, GAP2 and RMT1) at the early stage of N-depleted stress and control. 0, 2, 4, 8, 12, 24 and 48 are culture time (h). Each bar corresponds to one Western blot lane.
3 讨论

氮是植物生长必需的大量营养元素之一, 缺氮会严重影响植物的生长[31].为应对缺氮胁迫, 保证个体在不良环境下存活, 植物会做出应答反应, 如诱导应答基因的转录或转录后调控、降低新陈代谢水平等[4].在莱茵衣藻等微藻中, 缺氮胁迫往往也伴随着油脂含量的升高.本研究比较了正常培养和缺氮胁迫条件下莱茵衣藻的生长指标、总蛋白质含量及油脂含量等特征, 采集了不同时间点的样品, 提取蛋白质后进行了20个候选蛋白质的免疫印迹分析, 获得了这些蛋白质的丰度变化信息, 通过计算候选蛋白质丰度与总蛋白质含量之间的相关性, 提出了推荐的内参蛋白质, 通过计算缺氮胁迫条件下候选蛋白质丰度变化的平均相对变化倍率, 鉴定到缺氮胁迫的蛋白质标志物, 为进一步开展莱茵衣藻缺氮胁迫应答过程中蛋白质的功能研究提供了便利, 也将为缺氮介导的油脂合成机理研究提供一个观察指标.

根据候选蛋白质的预测功能, 可将20个候选蛋白质分为结构、分子伴侣、光合作用、代谢相关和其他5类.结构类蛋白质中, Histone H3和FAP127分别参与染色体的组装和鞭毛的生长[32-33], 这两个蛋白质在正常培养条件下表达丰度增加, 1 d后趋于稳定表达, 在缺氮胁迫条件下, 蛋白质的表达明显受到抑制, 丰度迅速下降, 说明缺氮胁迫可能加速这两个蛋白质的降解.TUB1在正常条件下表达稳定, 在缺氮条件下并未受到明显抑制, 而是维持在一个相对稳定的水平, 说明TUB1对缺氮胁迫并不敏感.分子伴侣和光合作用类蛋白质在正常生长条件下均有不同幅度的积累, 在缺氮胁迫条件下合成均受到抑制.代谢类蛋白质中, ATPs-6和ATPs-β属ATP合成酶的亚基[4, 34], 检测到了缺氮诱导上调的条带, 提示这两个蛋白质可能通过调节ATP水平参与莱茵衣藻缺氮胁迫应答, 且对缺氮应答发挥正调控作用.ATPs-A和BCR1的合成受缺氮胁迫所抑制.在"其他"类蛋白质中, 甘油醛- 3-磷酸脱氢酶GAP2和核酮糖-1, 5-二磷酸羧化酶转甲基酶RMT1蛋白质均检测到缺氮诱导上调的条带, 提示这两个酶可能在缺氮胁迫应答中也发挥正调控作用.在FAB2和MPC1的蛋白质印迹结果中, 除检测到符合理论分子质量的条带外, 在更高分子质量位置也检测到清晰的条带, 提示这两个蛋白质可能存在修饰或二聚体现象.值得注意的是, ATPs-6和GAP2的缺氮诱导条带与正常培养材料中的条带相比, 发生了分子质量的变化, 说明这两个蛋白质可能是以某种修饰形式参与到缺氮胁迫应答过程中的.

内参蛋白质是指在特定条件下表达量稳定或者说与总蛋白质含量相关的蛋白质, 本研究中鉴定到Histone H3、RBCL和BCR1在正常和缺氮胁迫条件下与总蛋白质含量变化呈现极显著或显著正相关, 被选做两种条件下的内参蛋白质.以前我们曾报道在多种非生物逆境胁迫下, TUB-1、RBCL和ATPs-6被选做内参蛋白质, 比较可见RBCL可以是共同的内参蛋白质, 这个内参蛋白质具有相当广泛的适应性, 两种条件下内参蛋白质并不完全重合, 说明内参蛋白质是条件依赖的, 不同的处理应该采用不同的内参蛋白质.

本研究鉴定的缺氮胁迫标志物蛋白质是ATPs-β、GAP2和RMT1, 我们曾报道的非生物逆境胁迫的标志物蛋白质有HSP90B、FAP127和ATPs-A等, 它们适合多种胁迫处理条件, 具体到每种胁迫处理的标志物蛋白质为:RCK1是暗处理特异的标志物, BCR1是冷处理特异的标志物, MPC1是热处理特异的标志物, RMT1在添加葡萄糖的处理中显著升高[19].比较可见, 缺氮胁迫的标志物是独特的, 实际上培养基中添加葡萄糖会增加油脂的含量, 所以RMT1在糖处理中显著升高与本实验结果是吻合的, 实验结果为RMT1在油脂合成途径中发挥作用提供了又一个独立的证据.ATPs-β和GAP2作为缺氮胁迫标志物都是特异的, 且这两个蛋白质在6 d时的变化倍率分别为180.59和52.90倍, 远高于RMT1(12.48倍).另外, 比较变化倍率可见缺氮胁迫的3种标志物的变化倍率大于非生物逆境胁迫, 从一个侧面说明了这些标志物的可靠性.这3个蛋白质标志物在缺氮胁迫中显著诱导上调, 说明它们发挥一定作用, 有进一步探究其功能的价值.从缺氮胁迫早期各标志物的表现来看, 在处理后8 h, 即可从蛋白质印迹信号中检测到ATPs-β蛋白质的变化, 所以从变化倍率的幅度和出现变化的时间两方面来讲, ATPs-β蛋白质都应该是最优先推荐的缺氮胁迫标志物蛋白质.

ATPs-β蛋白质参与组装线粒体F1FO ATP合成酶(复合体V), 对维持呼吸作用效率、ATP合成以及线粒体嵴的正常结构起重要作用[35], 与位于叶绿体和液泡中的ATP合成酶亚基相比, 线粒体中的ATP合成酶亚基对光、碳源和氮源的变化更敏感[36].质谱数据显示, 在莱茵衣藻CC-125中, 与正常氮源(400 mg/L NH4Cl)相比, 低浓度氮源(200 mg/L和100 mg/L NH4Cl)可诱导ATPs-β蛋白质表达上调[37]; 利用iTRAQ技术调查莱茵衣藻CC-400缺氮0、0.5、1、2、4、6、12和24 h的蛋白质组变化, 发现ATPs-β在缺氮胁迫24 h后可被检测到上调信号[38].Goold等[39]利用高强度光照诱导莱茵衣藻细胞中油脂积累, 并分离了油体(lipid droplets, LD)进行蛋白质组学分析, 鉴定到大量的ATPs-β, 提示该蛋白质很有可能参与油脂合成过程.在本研究中, 蛋白质印迹实验在缺氮胁迫8 h检测到ATPs-β的诱导表达, 所以该蛋白质不但是缺氮胁迫的标志物, 还可能从某种程度上与油脂含量的升高直接相关.

在本研究中, IC2和FKBP12这两个蛋白质未检测到可见信号, 另外在暗、冷、热、盐和糖等处理中也没有检测到它们的表达信号[19].IC2是在鞭毛中特异表达的蛋白质, 在全蛋白质样品中可能相对含量较低, 导致蛋白质印迹信号不明显[18].FKBP12是雷帕霉素的靶蛋白质, 可能其本底表达较低, 或许需要某种条件的诱导[39].当然, 期望所有抗体都检测到清晰的信号也是不太现实的.本文中有一些蛋白质印迹检测到多个条带, 如ATPs-6、FAB2和GAP2等, 产生这种现象的原因比较复杂, 如可能的修饰、蛋白质的聚合体、蛋白质的降解产物或不同拼接体等, 甚至仅仅是非特异的杂交条带.后续研究中可以对这些条带信息进行深入挖掘.

植物处于逆境胁迫时, 通过积累中性脂, 即三酰甘油(TAG)的方式存储碳源和能量, 避免细胞受到氧化损伤[40].在这个过程中, 甘油醛三磷酸脱氢酶(glyceraldehyde 3-phosphate dehydrogenase, GAPDH)催化合成三磷酸甘油, 为三酰甘油的合成提供甘油骨架[41].对等鞭金藻(Isochrysis galbana) IOAC724S[42]和小球藻(Chlorella) UTEX29[41]进行缺氮胁迫, 均可检测GAPDH的上调表达.在莱茵衣藻中, GAPDH家族共有4个成员, 分别为GAP1(Cre12.g485150)、GAP2(Cre07.g354200)、GAP3(Cre01.g010900)和GAP4(Cre12.g556600)[34], 在糖类代谢和脂类代谢之间起枢纽作用, 质谱分析结果显示GAP4受缺氮诱导上调表达[4].在本研究中, GAP2的免疫印迹结果表现为在缺氮18 h检测到可见信号, 且表达上升明显, 为揭示该蛋白质参与中性脂的合成提供了证据.

RMT1为Rubisco大亚基甲基转移酶1, 催化合成大亚基中的三甲基赖氨酸[43].莱茵衣藻Sta 6-1在缺氮胁迫应答过程中, 叶绿体内的Rubisco参与合成磷酸丙糖, 磷酸丙糖在磷酸丙糖异构酶的作用下首先形成二羟丙酮磷酸, 继而形成三磷酸甘油, 三磷酸甘油离开叶绿体后, 在细胞质中参与合成油体(lipid bodies, LB)[44].本研究中, RMT1蛋白质在缺氮胁迫12 h检测到诱导条带, 表明该蛋白质可能对缺氮诱导的油脂合成过程起正调控作用.

综上, 本研究对莱茵衣藻在正常和缺氮培养条件下培养物的表型、细胞密度、叶绿素含量、油脂含量和总蛋白质含量进行了检测, 鉴定到胁迫过程中伴随着油脂含量的显著升高, 进而采用基于抗体的蛋白质组学策略, 通过多个时间点样品的系统蛋白质印迹分析, 从20个候选蛋白质中筛选鉴定了缺氮胁迫的内参和标志物蛋白质, 所积累的抗体资源和蛋白质表达信息可供研究同行参考, 所采用的研究策略也具有更为广泛的应用, 获得的内参和标志物蛋白质对了解缺氮应答及油脂合成机理可能提供帮助.

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中国科学院生物物理研究所和中国生物物理学会共同主办
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文章信息

史佳楠, 杜铁民, 陈悦, 周艳, 杨亚茹, 李莉云, 窦世娟, 刘丽娟, 刘国振
SHI Jia-Nan, DU Tie-Min, CHEN Yue, ZHOU Yan, YANG Ya-Ru, LI Li-Yun, DOU Shi-Juan, LIU Li-Juan, LIU Guo-Zhen
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
生物化学与生物物理进展, 2018, 45(12): 1268-1279
Progress in Biochemistry and Biophysics, 2018, 45(12): 1268-1279
http://dx.doi.org/10.16476/j.pibb.2018.0228

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收稿日期: 2018-08-22
接受日期: 2018-11-06

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