CN113189214B - 一种大黄鱼增殖放流的分子标志物及其筛选方法 - Google Patents
一种大黄鱼增殖放流的分子标志物及其筛选方法 Download PDFInfo
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Abstract
本发明一种大黄鱼增殖放流的分子标志物及其筛选方法，特点是包括3,4‑二羟基杏仁酸、对羟基苯基乙酰硫代氢氧酸酯、丙基戊二酸、皮质醇、麦角硫茵氨基酸、(R)‑甲羟戊酸、循环去氧黄嘌呤氟他洛辛、甲羟戊酸、生物素、8‑氨基‑7‑氧代壬酸酯、N‑花生四烯多巴胺、肾上腺素、δ1‑哌啶‑2‑羧酸酯、肌酸和卡利车霉素和T0 L‑2‑氨基己二酸酯6‑半醛等，筛选方法步骤如下：取暂养不同时间的大黄鱼肌肉样本，采用LC‑MS/MS定性及定量的确定大黄鱼肌肉组织的小分子代谢物，通过火山图和主成分分析确定大黄鱼鱼苗适应放流水域的时间，通过偏最小二乘判别分析和维恩图筛选出，优点是降低大黄鱼暂养成本及提高增殖放流成活率。
Description
技术领域
本发明属于分子生物学以及代谢组学领域，尤其是涉及一种大黄鱼增殖放流的分子标志物及其基于液相-串联质谱技术的筛选方法。
背景技术
大黄鱼(Larimichthys crocea)俗称黄花鱼，其主要生活在我国黄海中南部至琼州海峡的近岸海域，是我国四大海洋渔业品种之一。但由于过度捕捞和栖息环境恶化而导致我国大黄鱼野生资源量急剧衰退甚至于枯竭。针对目前大黄鱼资源衰退的现象，实施增殖放流是最直接、最根本的资源恢复措施。自1998年开始，大黄鱼增殖放流工作对浙东沿海等海域大黄鱼资源的恢复性增长起到了积极的促进作用，深受沿海渔民的欢迎，取得了良好的生态、社会和经济效益。但是，放流工作技术上仍旧存在很多不足，其中选择放流对象、苗种规格、数量和合理配比投放等方面存在一定盲目性，特别是增殖放流前鱼苗暂养工作缺乏科学的评估标准。
大黄鱼鱼苗暂养是在放流前将鱼苗从养殖车间移至与放流水域相同的环境中进行适应性短期饲养，然而，在暂养过程中缺乏对大黄鱼放流水域生态环境的调查与研究，忽视大黄鱼放流海域的环境与工厂养殖生活环境的差异。同时，对于暂养时间的长短，也缺乏科学的指导。暂养时间太长，则导致鱼苗暂养成本增加；暂养时间过短，则造成放流的鱼苗对环境适应性差、成活率低等。因此，大黄鱼增殖放流前鱼苗暂养时间的科学评估和分子标志物的筛选显得尤为重要。
代谢组学(Metabolomics)技术是观察生物体细胞经过刺激或者影响后，生物体细胞中的代谢产物随和时间波动变化的学科，广泛应用于生物标志物的发现、临床诊断、药效与药毒评估、环境暴露等研究。其主要原理为利用液相色谱质谱联用(LC-MS)、气相色谱质谱联用(GC-MS)、核磁共振(NMR)技术进行检测，获得代谢物的定性定量信息。通过比较不同组别样品中代谢物的含量变化，寻找差异代谢物，并探索差异代谢物相关的代谢通路。不同于基因组学和蛋白组学反映的生物体内在差异，代谢组学的研究领域扩展到了机体与环境之间的相互影响和作用。小分子代谢物不仅是机体生命活动、生化代谢的物质基础，还体现了某些外来因素对体内代谢环境的改变，因而某些独特代谢物的浓度在不同个体间的差异事实上反映了生物体对外界环境的反应。代谢组学所得到的信息与生物的表现型最为接近，对代谢终产物的研究，可以揭示生物系统受环境影响后的变化规律和相关机制。有研究发现，对暴露于双酚A下的斑马鱼体内脂肪酸和氨基酸产物进行代谢成分分析，结果表明苯丙氨酸和赖氨酸的表达量显著上升，脂肪酸中的十四烷酸表达量也明显上升。另外有研究通过代谢组学技术分析发现，菲律宾蛤仔在铜污染的环境中，体内柠檬酸代谢途径的中间产物琥珀酸以及丙氨酸表达量显著下降。此外，水环境中汞污染导致菲律宾白色蛤仔肌肉代谢产物发生明显变化，被用作为环境中汞含量的指标生物。目前，代谢组学技术在水生生物中得到了广泛的应用，特别是在评估水生生物的环境适应性研究中发挥重要的作用。
发明内容
本发明所要解决的技术问题是提供一种降低大黄鱼暂养成本及提高增殖放流成活率的大黄鱼增殖放流的分子标志物及其基于LC-MS/MS技术的筛选方法。
本发明解决上述技术问题所采用的技术方案为：
1、一种大黄鱼增殖放流的分子标志物，包括显著上调的为3,4-二羟基杏仁酸(3,4-Dihydroxymandelic acid)、对羟基苯基乙酰硫代氢氧酸酯(p-Hydroxyphenylacetothiohydroximate)、丙基戊二酸(2-Propylglutaric acid)、皮质醇(Cortisol)、麦角硫茵氨基酸(Ergothioneine)、(R)-甲羟戊酸((R)-Mevalonate)、循环去氧黄嘌呤氟他洛辛(Cyclicdehypoxanthine futalosine)、甲羟戊酸(Mevalonic acid)、生物素(Biotin)、8-氨基-7-氧代壬酸酯(8-Amino-7-oxononanoate)、N-花生四烯多巴胺(N-Arachidonyl dopamine)、肾上腺素(Adrenosterone)；显著下调的为δ1-哌啶-2-羧酸酯(Delta1-Piperideine-2-carboxylate)、肌酸(Creatine)、卡利车霉素T0(Calicheamicin T0)、L-2-氨基己二酸酯6-半醛(L-2-Aminoadipate 6-semialdehyde)、α-1，6半乳三糖(D-Gal alpha 1->6D-Galalpha 1->6D-Glucose)、丁醛(Butanal)、脂酰AMP(Lipoyl-AMP)、乙酰假藤碱(Acetylpseudotropine)。
2、一种大黄鱼增殖放流的分子标志物基于液相-串联质谱(LC-MS/MS)技术的筛选方法，包括以下步骤：取暂养不同时间的大黄鱼肌肉样本，采用LC-MS/MS定性及定量的确定大黄鱼肌肉组织的小分子代谢物，通过火山图和主成分分析确定大黄鱼鱼苗适应放流水域的时间，通过偏最小二乘判别分析和维恩图筛选出可以指示大黄鱼已经适应放流环境的分子标志物。
具体步骤如下：
(1)从转移至暂养水域第一天开始，每隔4天采集6只大黄鱼鱼苗并提取肌肉组织样品，采用有机溶剂沉淀蛋白法对大黄鱼肌肉样本进行提取，提取液经过滤得待测液；
(2)大黄鱼样品的检测分析：将待测液进行液相色谱分离后，将色谱柱上洗脱下来的小分子，利用高分辨串联质谱分别进行正负离子模式采集，通过商业软件Progenesis QI实现峰提取，基于数据库KEGG进行代谢物鉴定；
(3)统计学分析：利用代谢组学R软件包metaX对质谱数据进行统计分析，通过火山图和主成分分析确定大黄鱼鱼苗适应放流水域的时间为16天，通过偏最小二乘判别分析和维恩图筛选出可以指示大黄鱼已经适应放流环境的分子标志物，其中显著上调的为3,4-二羟基杏仁酸(3,4-Dihydroxymandelic acid)、对羟基苯基乙酰硫代氢氧酸酯(p-Hydroxyphenylacetothiohydroximate)、丙基戊二酸(2-Propylglutaric acid)、皮质醇(Cortisol)、麦角硫茵氨基酸(Ergothioneine)、(R)-甲羟戊酸((R)-Mevalonate)、循环去氧黄嘌呤氟他洛辛(Cyclic dehypoxanthine futalosine)、甲羟戊酸(Mevalonic acid)、生物素(Biotin)、8-氨基-7-氧代壬酸酯(8-Amino-7-oxononanoate)、N-花生四烯多巴胺(N-Arachidonyl dopamine)、肾上腺素(Adrenosterone)；显著下调的为δ1-哌啶-2-羧酸酯(Delta1-Piperideine-2-carboxylate)、肌酸(Creatine)、卡利车霉素T0(CalicheamicinT0)、L-2-氨基己二酸酯6-半醛(L-2-Aminoadipate 6-semialdehyde)、α-1，6半乳三糖(D-Gal alpha 1->6D-Gal alpha 1->6D-Glucose)、丁醛(Butanal)、脂酰AMP(Lipoyl-AMP)、乙酰假藤碱(Acetyl pseudotropine)。
优选的，步骤(2)中液相色谱分离条件为采用ACQUITY UPLC HSS T3 column进行色谱分离，色谱柱的柱温为50℃，流速为0.4ml/min，其中A流动相为含有0.1％v/v甲酸的水溶液，B流动相为含0.1％v/v甲酸的甲醇溶液，对代谢物采用以下梯度进行洗脱：0-2min，100％流动相A；2-11min，0-100％流动相B；11-13min，100％流动相B；13-15min则为0-100％流动相A，每个样本的上样体积为5μL。
优选的，步骤(2)中高分辨串联质谱分别进行正负离子模式采集具体如下：正离子模式下，毛细管电压和锥孔电压分别为3.0kV和40.0V；负离子模式下，毛细管电压及锥孔电压分别为2.0kV和40.0V，采用MSE模式进行centroid数据采集，一级扫描范围为50-1200Da，扫描时间为0.2s，对所有母离子按照20到40eV的能量进行碎裂，采集所有的碎片信息，扫描时间为0.2s。
与现有技术相比，本发明的优点在于：本发明首次公开了一种大黄鱼增殖放流的分子标志物及其基于液相-串联质谱技术的筛选方法，利用基于LC-MS/MS技术的代谢组学方法分析放流水域暂养对大黄鱼体内代谢产物的影响，对暂养不同时间的大黄鱼肌肉组织进行检测，得到相应代谢物定性及定量数据，并对数据进行Volcano plot、PCA、OPLS-DA、维恩图建模，确定放流前大黄鱼鱼苗合适暂养时间，并且根据差异代谢物，得到指示大黄鱼已经适应放流水域的分子标志物，通过该方法所得分子标志物对于指示水生生物是否适应新环境具有良好的灵敏性和特异性，尤其适合于水生生物放流前的暂养工作，对于降低大黄鱼暂养成本及提高增殖放流成活率具有重要意义。
附图说明
图1为大黄鱼代谢标志物筛选的信息分析流程示意图；
图2为本发明QC样本总离子流图，即TIC重叠图；
图3为本发明QC样本PCA分析；
图4为本发明5-23号样本与其他暂养天数样本进行单变量分析的火山图；
图5为本发明所有暂养天数样本的PCA分析图；
图6为本发明6-08号样本与6-12号样本的PCA分析图；
图7为本发明大黄鱼鱼苗已经适应放流水域分子标志物的维恩图分析。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
具体实施例一
1、应用LC-MS/MS技术的大黄鱼肌肉非靶向代谢组学分析
1.1大黄鱼肌肉样本代谢物提取
大黄鱼鱼苗来自宁德市富发水产有限公司，体长为4.2±0.5cm，转移到三沙湾水域暂养。从转移至暂养水域第一天开始(2020-5-23)，每隔4天采集6只大黄鱼鱼苗并提取肌肉组织样品，采用有机溶剂沉淀蛋白法对大黄鱼肌肉样本进行代谢物提取，提取液经过滤处理，得待测液；同时制备20个质控(QC)样本(取等量制备好的分析样本混合而成)，对所提取的样本进行上机检测，具体操作如下：
(1)将每个样品取100μL加新的1.5mlEP管中；
(2)加冷甲醇300μL，震荡混匀，使用TissueLyser(频率调至50HZ，时间5min)研磨破碎，-20℃放置2h；
(3)25000rpm 4℃离心15min，取上清350μL，置于新的EP管中，25000rpm 4℃离心15min；
(4)每个样品取50μL上清混合QC，用微孔滤膜(0.22μm)过滤样品，转入新的1.5mlEP管，上机检测。
1.2大黄鱼肌肉组织非靶向代谢检测
将处理后的不同天数取得的所有大黄鱼肌肉样本作为分析样本，打乱顺序后随机化排序进样，以排除进样顺序带来的偏倚。先用10个QC样本来平衡仪器(监测液相色谱-质谱检测过程中仪器的状态)，每上10个检测样本穿插一个QC样本，最后上三个QC样本结束实验。QC样本的设计可以评估样本数据的采集质量。所用液相色谱、质谱参数如下：
液相色谱参数：采用ACQUITY UPLC HSS T3 column(100mm*2.1mm，1.8μm，Waters，UK)进行色谱分离，色谱柱的柱温为50℃，流速为0.4ml/min，其中A流动相为水和0.1％v/v甲酸，B流动相为甲醇和0.1％v/v甲酸。对代谢物采用以下梯度进行洗脱：0-2min，100％流动相A；2-11min，0-100％流动相B；11-13min，100％流动相B；13-15min则为0-100％流动相A。每个样本的上样体积为5μL。
质谱参数：对从色谱柱上洗脱下来的小分子，利用高分辨串联质谱Xevo G2-XSQTOF(Waters，UK)分别进行正负离子模式采集。正离子模式下，毛细管电压和锥孔电压分别为3.0kV和40.0V。负离子模式下，毛细管电压及锥孔电压分别为2.0kV和40.0V。采用MSE模式进行centroid数据采集，一级扫描范围为50-1200Da，扫描时间为0.2s，对所有母离子按照20到40eV的能量进行碎裂，采集所有的碎片信息，扫描时间为0.2s。在数据采集过程中，对LE信号每3s进行实时质量校正。按照上述色谱质谱条件对样本进行分析，获得所有样本的原始数据。
2.大黄鱼代谢标志物筛选
2.1数据预处理
在获得原始数据后，依次根据信息分析流程步骤对下机数据进行处理，流程如图1所示。将质谱下机原始数据导入商业软件Progenesis QI(version 2.2，以下简称QI)进行峰提取，获得代谢物相关的质荷比、保留时间和离子面积等信息。QI的工作流程主要包括以下几个步骤：峰对齐，峰提取以及峰鉴定。数据预处理使用metaX软件完成，包括对提取出来的数据使用KNN方法进行缺失值填充、去低质量离子(将QC样本中缺失超过50％，或者实际样本中缺失超过80％的离子去除)，以及采用QC-RSC(Quality control-based robustLOESS signal correction)方法进行校正。对校正后的数据进行过滤，即将所有QC样品中相对标准偏差(RSD)＞30％的离子过滤掉(RSD＞30％的离子在实验过程中波动较大，不纳入下游统计学分析)，最后得到的离子统计信息如下：
表1离子数目统计
| 模式 | 离子总数 | RSD<＝30％离子数 | 一级鉴定 | 二级鉴定 |
| 正离子 | 12067 | 9919 | 4366 | 2233 |
| 负离子 | 14417 | 12581 | 5291 | 3150 |
。
2.2大黄鱼LC-MS/MS实验质控分析
(1)QC样本TIC重叠图
进行大黄鱼LC-MS/MS非靶向代谢组学分析时，将制备的QC样品按每10个分析样本安排一个QC样品的顺序均匀地插入分析样本中，用于实时监测分析样本从进样预处理到分析检测过程中的质量控制情况。以时间点为横坐标，以每个时间点质谱图中所有离子的强度加和为纵坐标，连续描绘得到正离子(QC-pos)和负离子(QC-neg)的总离子流图，又叫TIC重叠图(图2)。图2表明不同批次的QC样本中正离子和负离子模式下各色谱峰的响应程度和保留时间基本重叠，说明在整个实验过程中仪器误差引起的变异较小。
(2)QC样本PCA分析
和TIC重叠图同理，质控样本QC可以相对聚集在一起，聚集得越好表明仪器越稳定，采集的数据质量越好。X轴表示第一个主成分，Y轴表示第二个主成分。括号里的数字表示该主成分能综合原始信息的比例(图3)。图3表明正离子(QC-pos)和负离子(QC-neg)模式下QC样本
紧密聚集在一起，并与其他样本明显分离，表明本项目的实验重复性较好，数据质量较高。
2.3单变量分析
本研究采用的是T检验和变异倍数分析(Fold change analysis，FC analysis)。在统计分析过程中，进一步对统计检验产生的p-value进行FDR校正得到q-value。最终结果以火山图(Volcano plot)形式呈现差异倍数(Fold change)和q-value两个指标，通常以差异倍数≥1.2或≤0.8333，q-value值小于0.05作为筛选差异代谢物的条件。通过单变量分析，最终得到如图4所示，在正离子和负离子模式下，大黄鱼5-23号样本的代谢物含量与5-27号样本、5-31号样本、6-04号样本、6-08号样本、6-12号样本都存在非常大的差异，具有显著变化的代谢物占所检测代谢物总数的70％以上，这表明放流前的大黄鱼暂养工作是十分有必要的。
2.4PCA分析
PCA分析是一种通过降维技术把多个变量转化成少数几个重要变量(主成分)的多元统计分析方法。通过少数几个主成分来代表原始数据的整体情况。本研究中PCA分析前对数据进行了log2转换和比例调整(scaling)，采用Pareto调整法(Pareto scaling)。通过PCA分析最终得到如图5所示，在正离子和负离子模式下，6-08号样本
与6-12号样本
在模型中基本拟合。此外，对6-08号样本和6-12号样本进行单独的PCA分析发现，如图6所示两样本在模型中并没有分离的趋势。因此，这两个结果表明6-08号(即暂养16天后)大黄鱼已经适应新环境。
2.5大黄鱼代谢标志物的筛选及鉴定
首先采用PLS-DA分析法，即对数据进行偏最小二乘变换PLS，然后再做线性判别分析LDA。本研究中，在做PLS-DA模型之前进行了log2转换及采用Pareto scaling的方法对数据进行比例调整(scaling)。然后采用多变量PLS-DA模型前两个主成分的变量投影重要度值(Variable Important for the Projection，VIP)，结合单变量分析差异倍数(fold-change)和q-value值来筛选差异表达的代谢物。筛选条件：(1)VIP≥1；(2)fold-change≥1.2或者≤0.8333；(3)q-value<0.05，三者取交集，得到共有的离子即差异离子。随后，在利用维恩图筛选出6-08号样本与5-27、5-31、6-04样本的共有差异离子，即得到大黄鱼已经适应放流水域环境的代谢标志物(图7)。差异离子鉴定采用的软件是Progenesis QI(version2.2)。
根据上述方法，我们鉴定出正离子模式下13个，负离子模式下7个，共20个大黄鱼的代谢物作为指示于大黄鱼已经适应放流水域环境的标志物，见表2。
表2大黄鱼代谢标志物
上述说明并非对本发明的限制，本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内，做出的变化、改型、添加或替换，也应属于本发明的保护范围。
Claims (4)
1.一种分子标志物用于大黄鱼增殖放流的应用，其特征在于所述的分子标志物包括3,4-二羟基杏仁酸、对羟基苯基乙酰硫代氢氧酸酯、丙基戊二酸、皮质醇、麦角硫茵氨基酸、(R)-甲羟戊酸、循环去氧黄嘌呤氟他洛辛、甲羟戊酸、生物素、8-氨基-7-氧代壬酸酯、N-花生四烯多巴胺、肾上腺素、δ1-哌啶-2-羧酸酯、肌酸、卡利车霉素T0、L-2-氨基己二酸酯6-半醛、α-1，6半乳三糖、丁醛、脂酰AMP和乙酰假藤碱，其中显著上调的为3,4-二羟基杏仁酸、对羟基苯基乙酰硫代氢氧酸酯、丙基戊二酸、皮质醇、麦角硫茵氨基酸、(R)-甲羟戊酸、循环去氧黄嘌呤氟他洛辛、甲羟戊酸、生物素、8-氨基-7-氧代壬酸酯、N-花生四烯多巴胺、肾上腺素；显著下调的为δ1-哌啶-2-羧酸酯、肌酸、卡利车霉素T0、L-2-氨基己二酸酯6-半醛、α-1，6半乳三糖、丁醛、脂酰AMP、乙酰假藤碱。
2.一种大黄鱼增殖放流的分子标志物基于LC-MS/MS技术的筛选方法，其特征在于包括以下步骤：取暂养不同时间的大黄鱼肌肉样本，采用LC-MS/MS定性及定量的确定大黄鱼肌肉组织的小分子代谢物，通过火山图和主成分分析确定大黄鱼鱼苗适应放流水域的时间，通过偏最小二乘判别分析和维恩图筛选出可以指示大黄鱼已经适应放流环境的分子标志物，具体步骤如下：
（1）从转移至暂养水域第一天开始，每隔4天采集6只大黄鱼鱼苗并提取肌肉组织样品，采用有机溶剂沉淀蛋白法对大黄鱼肌肉样本进行提取，提取液经过滤得待测液；
（2）大黄鱼样品的检测分析：将待测液进行液相色谱分离后，将色谱柱上洗脱下来的小分子，利用高分辨串联质谱分别进行正负离子模式采集，通过商业软件Progenesis QI实现峰提取，基于数据库KEGG进行代谢物鉴定；
（3）统计学分析：利用代谢组学R软件包metaX对质谱数据进行统计分析，通过火山图和主成分分析确定大黄鱼鱼苗适应放流水域的时间为16天，通过偏最小二乘判别分析和维恩图筛选出可以指示大黄鱼已经适应放流环境的分子标志物，其中显著上调的为3,4-二羟基杏仁酸(3,4-Dihydroxymandelic acid)、对羟基苯基乙酰硫代氢氧酸酯(p-Hydroxyphenylacetothiohydroximate)、丙基戊二酸(2-Propylglutaric acid)、皮质醇(Cortisol)、麦角硫茵氨基酸(Ergothioneine)、(R)-甲羟戊酸((R)-Mevalonate)、循环去氧黄嘌呤氟他洛辛(Cyclic dehypoxanthine futalosine)、甲羟戊酸(Mevalonic acid)、生物素(Biotin)、8-氨基-7-氧代壬酸酯(8-Amino-7-oxononanoate)、N-花生四烯多巴胺(N-Arachidonyldopamine)、肾上腺素(Adrenosterone)；显著下调的为δ1-哌啶-2-羧酸酯(Delta1-Piperideine-2-carboxylate)、肌酸(Creatine)、卡利车霉素T0(Calicheamicin T0)、L-2-氨基己二酸酯6-半醛(L-2-Aminoadipate 6-semialdehyde)、α-1，6半乳三糖(D-Gal alpha1->6D-Gal alpha 1->6D-Glucose)、丁醛(Butanal)、脂酰AMP(Lipoyl-AMP)、乙酰假藤碱(Acetyl pseudotropine)。
3.根据权利要求2所述的一种大黄鱼增殖放流的分子标志物基于LC-MS/MS技术的筛选方法，其特征在于步骤（2）中液相色谱分离条件为采用ACQUITY UPLC HSS T3 色谱柱进行色谱分离，色谱柱的柱温为50℃，流速为0.4 ml/min，其中A流动相为含有0.1%v/v甲酸的水溶液，B流动相为含0.1%v/v甲酸的甲醇溶液，对代谢物采用以下梯度进行洗脱：0-2 min，100%流动相A；2-11 min，0-100%流动相B；11-13 min，100%流动相B；13-15 min则为0-100%流动相A，每个样本的上样体积为5 µL。
4.根据权利要求2所述的一种大黄鱼增殖放流的分子标志物基于LC-MS/MS技术的筛选方法，其特征在于步骤（2）中高分辨串联质谱分别进行正负离子模式采集具体如下：正离子模式下，毛细管电压和锥孔电压分别为3.0 kV 和40.0 V；负离子模式下，毛细管电压及锥孔电压分别为2.0 kV和40.0 V，采用MSE模式进行centroid数据采集，一级扫描范围为50-1200 Da，扫描时间为0.2s，对所有母离子按照20到40 eV的能量进行碎裂，采集所有的碎片信息，扫描时间为0.2 s。
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