CN114045348A - 一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法 - Google Patents
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Abstract
本发明公开了一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法，首次通过转录组水平分析了四指马鲅低温胁迫下的关键基因和代谢途径，为四指马鲅的抗逆性研究提供了重要数据；低温胁迫不同时间：NTvsLT2，LT2vsLT6，LT6vsLT12的差异表达基因分别为：3296，2134，890个；差异表达基因富集分析发现：四指马鲅低温胁迫诱导的差异基因极显著富集在生物节律、类固醇合成、免疫反应、代谢反应、信号转导和DNA复制等信号通路中，为了解四指马鲅低温适应机制提供重要依据。
Description
技术领域
本发明属于鱼类养殖的技术领域，特别是涉及一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法。
背景技术
鱼类的地域分布、生活习性和繁殖条件等受到环境温度的影响；每一种鱼对温度变化都有其特定的适应机制，在适合鱼类生存温度范围内，外界水体温度的骤变，鱼类的免疫系统、生长状况、摄食、新陈代谢以及心血管系统等都会受到不同程度影响；鳃是鱼类重要的呼吸器官和免疫器官，在气体交换、离子调节和酸碱平衡等生理活动中起关键作用，转录组测序能够快速获得较全面的转录本信息，在生态学、遗传学以及环境科学等学科研究中，采用该技术来解释分析生物对环境变化适应的分子机制；
目前，四指马鲅的养殖技术虽然趋于成熟，并开展大规模的养殖，但在养殖过程中仍有很多需要注意的问题，四指马鲅属于暖水性鱼类，不耐低温，温度低于14℃，就会造成养殖鱼类大量死亡。
技术方案
为实现以上目的，本发明对在低温水体胁迫下四指马鲅鳃组织组织进行了转录组测序，通过分析其转录组的特征及差异表达基因，找到与温度变化相关的功能基因，促进四指马鲅养殖产业健康发展，通过以下技术方案予以实现：一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法，包括如下步骤：
(1)捕捉一定的四指马鲅，经培育后，随机挑选体色正常、体格均匀健壮、活力强的个体进行实验；
(2)设计低温胁迫组和常温组两个实验组，所述低温胁迫组和常温组分别设三个平行，每个平行放30尾鱼，所述低温胁迫组通过制冷水槽实现温度控制，取低温胁迫前的样品作为0h采样点，实验开始后，每个温度组分别于第2、6、12h取样，每个节点分别取3尾，活体解剖取鳃组织，存放至液氮中备用；
(3)按照Trizol方法对步骤(2)中所得的鳃组织进行总RNA提取，RNA的降解程度和纯度用1％的琼脂糖凝胶电泳和Nanodrop检测；
(4)将所述步骤(3)中提取的总RNA采用DNase I处理，处理后进行富集，富集后加入打断试剂，再将一链和二链cDNA合成，处理后进行PCR扩增富集，富集后进行测序，为保证信息分析的质量，将测序得到的原始测序序列进行过滤；
(5)将经过所述步骤(4)过滤后的原始测序序列进行组装，对组装的序列进行去除冗余和拼接，并对Unigene做统计和质控；
(6)将所述过滤后的原始测序序列和Unigene进行比对，然后计算比对上每个Unigene的reads数，结合Unigene的有效长度以及总比对上的reads数，标准化Unigene的表达量；
(7)筛选差异基因然后对差异表达基因进行GO和KEGG富集分析；
(8)使用SSR分析软件，对Unigene进行检测。
优选的，所述步骤(1)中的四指马鲅培育条件为先暂养7d，盐度为9.5-10.5‰，温度为26-28℃，连续24h充氧，每天换水一次，每次换1/3；挑选条件为体长为4.5-5.5cm，体重1.45-1.55g。
优选的，所述步骤(2)的实验周期为12h，所述低温胁迫组的温度范围控制在15.5℃-16.5℃。所述常温组的温度范围控制在常温组24.5℃-25.5℃。
优选的，所述步骤(3)中的样品RNAOD260/OD280在1.8-2.2之间，OD260/OD230值不低于2.0。
优选的，所述步骤(4)中的测序采用Illumina HiSeqTM 2000测序平台进行测序。
优选的，所述步骤(5)中用De novo组装软件Trinity进行组装，通过软件TGICL对组装的序列进行去除冗余和拼接，并对Unigene做统计和质控
有益效果
本发明提供了一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法。具备以下有益效果：对在低温水体胁迫下四指马鲅鳃组织组织进行了转录组测序，通过分析其转录组的特征及差异表达基因，找到与温度变化相关的功能基因，为进一步探究四指马鲅的耐低温分子机制提供理论依据，促进四指马鲅养殖产业健康发展。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供一种技术方案：一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法，包括如下步骤：
(1)捕捉一定的四指马鲅，经培育后，随机挑选体色正常、体格均匀健壮、活力强的个体进行实验；
(2)设计低温胁迫组和常温组两个实验组，所述低温胁迫组和常温组分别设三个平行，每个平行放30尾鱼，所述低温胁迫组通过制冷水槽实现温度控制，取低温胁迫前的样品作为0h采样点，实验开始后，每个温度组分别于第2、6、12h取样，每个节点分别取3尾，活体解剖取鳃组织，存放至液氮中备用；
(3)按照Trizol方法对步骤(2)中所得的鳃组织进行总RNA提取，RNA的降解程度和纯度用1％的琼脂糖凝胶电泳和Nanodrop检测；
(4)将所述步骤(3)中提取的总RNA采用DNase I处理，处理后进行富集，富集后加入打断试剂，再将一链和二链cDNA合成，处理后进行PCR扩增富集，富集后进行测序，为保证信息分析的质量，将测序得到的原始测序序列进行过滤；
(5)将经过所述步骤(4)过滤后的原始测序序列进行组装，对组装的序列进行去除冗余和拼接，并对Unigene做统计和质控；
(6)将所述过滤后的原始测序序列和Unigene进行比对，然后计算比对上每个Unigene的reads数，结合Unigene的有效长度以及总比对上的reads数，标准化Unigene的表达量；
(7)筛选差异基因然后对差异表达基因进行GO和KEGG富集分析；
(8)使用SSR分析软件，对Unigene进行检测。
原始数据质控：
对四指马鲅鳃组织进行总RNA提取，通过检测，转录组测序和抽提之后进行数据质控，具体结果如表1：
表1
注：Q20、Q30分别表示数值大于20、30的碱基占总体碱基的百分比；GC表示计算碱基G和C的数量总和占总的碱基数量的百分比。
通过分析四指马鲅鳃在低温胁迫下KEGG的富集结果，筛选出四指马鲅低温胁迫过程中差异表达基因极显著富集的信号通路。
低温胁迫相关候选基因分析：
低温胁迫中生物节律(如昼夜节律、昼夜节律-果蝇)，显著表达基因主要有PER2、Cry1、ARNTL2；类固醇生物合成，差异表达基因有LSS、DHCR24、CYP51A1；免疫通路(如单纯疱疹感染、化学致癌作用、核因子-κB、肿瘤坏死因子、破骨细胞分化)，涉及的差异表达基因主要有TRAF3、TRAF2、USP7、MYD88、IL1R1、CD40、BCL2L1、CCL4、CCL8、CXCL10、FOS、RELB、Fosl2等；代谢通路(细胞色素P450对外源物质的代谢、过氧化物酶体增殖物激活受体)，差异表达的基因有UGT3、mGST3、CYP1A1、PLEKHA1、PPARG、ACSL1；信号转导通路(剪接体)，代表性差异基因有SRSF2、Ddx5、SRSF3；DNA复制通路中，差异表达的基因主要有Rnaseh1、RFC4、mcm5、fen1等受到影响。具体结果如表2：
表2
本研究对Illumina HiSeqTM 2000测序得到的原始数据进行组装，结果共得到108697条转录本(Transcripts)，平均长度为1394.19bp，N50为2657bp。同时得到76878条基因(unigenes)，平均长度为820.80bp，N50为1333bp。长度大于2000bp的有26026条,约占总基因(unigene)的33.8％。并成功注释了77393条基因信息。本研究结果丰富了四指马鲅基因资源库信息，为其种质资源保护和利用提供支持。
在低温应激下，鱼体通过调整脂肪酸组成来适应寒冷环境。类固醇激素是一类脂溶性的激素，对脊椎动物的生长、发育以及体内稳态调节等方面发挥重要的作用。本研究中，与NT相比，当低温胁迫2h时，类固醇合成相关的基因LSS、DHCR24、CYP51A1都发生显著变化。DHCR24和CYP具有相同的功能，主要是合成胆固醇。鲤在低温胁迫下参与脂肪酸和胆固醇合成的基因增加。斑马鱼仔鱼和幼体大海马(Hippocampus kuda)在温度胁迫下类固醇生物合成都发生显著富集。本研究中低温胁迫导致类固醇相关基因差异表达，表明类固醇合成通路对于四指马鲅幼鱼抵抗低温应激有重要作用。
鳃是鱼类重要呼吸器官，在免疫系统中发挥重要的物理屏障作用。本研究结果表明，低温胁迫过程差异表达基因富集到多条免疫通路中，表明低温对四指马鲅鳃免疫功能产生影响。TRAFs能参与多个信号通路和信号转导途径。鳃结构的损伤容易造做成机体感染，低温胁迫过程中Herpes simplex infection通路的显著富集也正说明这一点。本研究筛选的免疫相关代谢通路之间相关联系，其中TRAF3基因参与多条信号通路。本研究中TRAF3、TRAF2、USP7、MYD88、IL1R1、CD40、BCL2L1、CCL4、CCL8、CXCL10、FOS、RELB、Fosl2等基因均发生上调，说明在低温胁迫下，四指马鲅机体通过启动相关免疫通路以抵抗低温伤害。
本研究首次通过转录组水平分析了四指马鲅低温胁迫下的关键基因和代谢途径，为四指马鲅的抗逆性研究提供了重要数据。低温胁迫不同时间：NT vs LT2，LT2 vs LT6，LT6 vs LT12的差异表达基因分别为：3296，2134，890个。差异表达基因富集分析发现：四指马鲅低温胁迫诱导的差异基因极显著富集在生物节律、类固醇合成、免疫反应、代谢反应、信号转导和DNA复制等信号通路中，为了解四指马鲅低温适应机制提供重要依据。
Claims (6)
1.一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法，包括如下步骤：
(1)捕捉一定的四指马鲅，经培育后，随机挑选体色正常、体格均匀健壮、活力强的个体进行实验；
(2)设计低温胁迫组和常温组两个实验组，所述低温胁迫组和常温组分别设三个平行，每个平行放30尾鱼，所述低温胁迫组通过制冷水槽实现温度控制，取低温胁迫前的样品作为0h采样点，实验开始后，每个温度组分别于第2、6、12h取样，每个节点分别取3尾，活体解剖取鳃组织，存放至液氮中备用；
(3)按照Trizol方法对步骤(2)中所得的鳃组织进行总RNA提取，RNA的降解程度和纯度用1％的琼脂糖凝胶电泳和Nanodrop检测；
(4)将所述步骤(3)中提取的总RNA采用DNase I处理，处理后进行富集，富集后加入打断试剂，再将一链和二链cDNA合成，处理后进行PCR扩增富集，富集后进行测序，为保证信息分析的质量，将测序得到的原始测序序列进行过滤；
(5)将经过所述步骤(4)过滤后的原始测序序列进行组装，对组装的序列进行去除冗余和拼接，并对Unigene做统计和质控；
(6)将所述过滤后的原始测序序列和Unigene进行比对，然后计算比对上每个Unigene的reads数，结合Unigene的有效长度以及总比对上的reads数，标准化Unigene的表达量；
(7)筛选差异基因然后对差异表达基因进行GO和KEGG富集分析；
(8)使用SSR分析软件，对Unigene进行检测。
2.根据权利要求1所述的一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法，其特征在于：所述步骤(1)中的四指马鲅培育条件为先暂养7d，盐度为9.5-10.5‰，温度为26-28℃，连续24h充氧，每天换水一次，每次换1/3；挑选条件为体长为4.5-5.5cm，体重1.45-1.55g。
3.根据权利要求1所述的一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法，其特征在于：所述步骤(2)的实验周期为12h，所述低温胁迫组的温度范围控制在15.5℃-16.5℃。所述常温组的温度范围控制在常温组24.5℃-25.5℃。
4.根据权利要求1所述的一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法，其特征在于：所述步骤(3)中的样品RNAOD260/OD280在1.8-2.2之间，OD260/OD230值不低于2.0。
5.根据权利要求1所述的一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法，其特征在于：所述步骤(4)中的测序采用Illumina HiSeqTM 2000测序平台进行测序。
6.根据权利要求1所述的一种低温胁迫条件下四指马鲅转录组的差异表达基因和分析方法，其特征在于：所述步骤(5)中用De novo组装软件Trinity进行组装，通过软件TGICL对组装的序列进行去除冗余和拼接，并对Unigene做统计和质控。
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