技术领域technical field
本发明涉及基因调控网络技术领域，特别涉及一种热应激条件下奶牛乳腺差异表达基因调控网络的构建方法。The invention relates to the technical field of gene regulation network, in particular to a method for constructing a gene regulation network of differentially expressed milk cow mammary gland under heat stress conditions.
背景技术Background technique
随着全球气候变暖，热应激对畜牧业的影响越来越严重。而我国幅员辽阔，每年夏季大片区域受热应激影响，导致畜禽代谢紊乱、生产性能和繁殖性能降低，造成严重的经济损失。热应激反应是动物机体一种自我保护机制，相关基因和蛋白质表达发生改变。研究表明，热应激对奶牛的多数免疫指标有重要影响。多数有关缓解热应激发生的研究措施主要是从营养角度出发。关于体外分子诊断的研究方法却较少。As the global climate warms, the impact of heat stress on the livestock industry is becoming more and more severe. However, my country has a vast territory, and a large area is affected by heat stress every summer, which leads to metabolic disorders of livestock and poultry, reduced production performance and reproductive performance, and causes serious economic losses. Heat stress response is a self-protection mechanism of the animal body, and the expression of related genes and proteins changes. Studies have shown that heat stress has an important impact on most immune indicators of dairy cows. Most of the research measures to alleviate the occurrence of heat stress are mainly from the perspective of nutrition. Research methods on in vitro molecular diagnostics are less.
微小RNA(microRNA，即miRNA)通过与靶mRNA特异性的碱基配对，引起靶mRNA的降解或者抑制其翻译，从而发挥基因转录后调节功能。miRNA分子广泛参与到调控细胞生长、发育、分化及凋亡的各个环节，并与动物生理状态的改变以及疾病的发生、发展密切相关。miRNA在哺乳动物泌乳过程中也起着重要的作用。MicroRNA (microRNA, ie miRNA) causes the degradation of the target mRNA or inhibits its translation through specific base pairing with the target mRNA, thereby exerting the post-transcriptional regulation function of the gene. miRNA molecules are widely involved in the regulation of cell growth, development, differentiation and apoptosis, and are closely related to changes in animal physiological states and the occurrence and development of diseases. miRNAs also play an important role in mammalian lactation.
miRNAs作为一种转录后水平的调控因子，在哺乳动物基因的转录水平中只有1％～5％被编码，却调控超过60％的基因。miRNAs作为一种转录后调控机制，在细胞生长、凋亡，机体免疫和抗应激反应、生长性能和繁殖性能等多方面发挥重要的调控作用。As a post-transcriptional regulatory factor, miRNAs are encoded in only 1% to 5% of mammalian gene transcription levels, but regulate more than 60% of genes. As a post-transcriptional regulatory mechanism, miRNAs play an important regulatory role in many aspects such as cell growth, apoptosis, immune and anti-stress responses, growth performance and reproductive performance.
发明内容Contents of the invention
本发明的目的是提供一种热应激条件下奶牛乳腺差异表达基因调控网络的构建方法。The purpose of the present invention is to provide a method for constructing a regulatory network of differentially expressed genes in dairy cow mammary glands under heat stress conditions.
一种热应激条件下奶牛乳腺差异表达基因调控网络的构建方法，该方法包括以下步骤：A method for constructing a regulatory network of differentially expressed genes in dairy cow mammary glands under heat stress conditions, the method comprising the following steps:
步骤一、热应激奶牛与非热应激奶牛乳腺组织样本采集；Step 1. Collection of mammary gland tissue samples from heat-stressed dairy cows and non-heat-stressed dairy cows;
步骤二、Solexa高通量测序鉴定参与奶牛热应激反应的miRNA，寻找差异表达miRNA；Step 2: Solexa high-throughput sequencing identifies miRNAs involved in the heat stress response of dairy cows, and searches for differentially expressed miRNAs;
步骤三、差异表达显著的miRNA靶基因预测；Step 3. Prediction of miRNA target genes with significant differential expression;
步骤四、差异表达显著的miRNA调控网络构建。Step 4, constructing the regulatory network of miRNAs with significant differential expression.
进一步的，所述的热应激条件下奶牛乳腺差异表达基因调控网络的构建方法，步骤一中，热应激奶牛与非热应激奶牛乳腺组织样本采集，根据系谱资料、奶牛生产性能测定体系DHI数据和牛场记录数据，选取性别相同，饲养环境、年龄及胎次相近的4头热应激奶牛和4头非热应激奶牛作为实验牛，采集乳腺组织样本放入液氮中，到实验室转入-70℃冰箱保存备用。Further, in the method for constructing the regulatory network of differentially expressed genes in dairy cow mammary glands under heat stress conditions, in step 1, the mammary gland tissue samples of heat-stressed dairy cows and non-heat-stressed dairy cows are collected, and according to the pedigree data and dairy cow production performance measurement system DHI data and cattle farm record data, select 4 heat-stressed dairy cows and 4 non-heat-stressed dairy cows with the same gender, similar feeding environment, age and parity as experimental cows, collect mammary gland tissue samples and put them in liquid nitrogen. Store in a -70°C freezer for later use.
进一步的，所述的热应激条件下奶牛乳腺差异表达基因调控网络的构建方法，步骤二包括如下步骤：Further, in the method for constructing a regulatory network of differentially expressed genes in dairy cow mammary glands under heat stress conditions, step 2 includes the following steps:
(a)用Trizol试剂提取热应激期和非热应激期奶牛乳腺组织总RNA并鉴定；(a) Extract and identify the total RNA of dairy cow mammary gland tissue during heat stress period and non-heat stress period with Trizol reagent;
(b)采用TA克隆和传统Sanger测序对所提总RNA鉴定是否可用于Solexa测序，建立小RNA文库，并进行测序分析验证；(b) Use TA cloning and traditional Sanger sequencing to identify whether the proposed total RNA can be used for Solexa sequencing, establish a small RNA library, and perform sequencing analysis verification;
(c)分离长度18-30nt范围的小RNA，两端分别加上特定接头后体外反转录成cDNA，经RT-PCR扩增后用Solexa测序仪对DNA片段进行单向末端直接测序；(c) Isolate small RNAs with a length of 18-30 nt, add specific adapters at both ends and reverse transcribe them into cDNA in vitro, and perform unidirectional direct sequencing on the DNA fragments with a Solexa sequencer after RT-PCR amplification;
(d)对所测的原始序列进行处理与统计；(d) processing and counting the measured original sequence;
(e)将步骤(d)处理过的序列与已知库中序列进行比对分析并分类注释；(e) comparing and analyzing the sequence processed in step (d) with the sequence in the known library, and classifying and annotating;
(f)鉴定已知miRNA和预测新miRNA；(f) identifying known miRNAs and predicting new miRNAs;
(g)寻找差异表达miRNA。(g) Search for differentially expressed miRNAs.
进一步的，所述的热应激条件下奶牛乳腺差异表达基因调控网络的构建方法，步骤二第(a)条包括以下内容：Further, in the method for constructing a regulatory network of differentially expressed genes in dairy cow mammary glands under heat stress conditions, step 2 (a) includes the following content:
①取-70℃冻存组织约100mg在液氮中研磨后加入1ml Trizol试剂混匀；①Take about 100mg of tissue frozen at -70°C, grind it in liquid nitrogen, add 1ml Trizol reagent and mix well;
②样品中加入Trizol试剂后反复吹打几次，使样本充分裂解，室温放置5分钟，使蛋白核酸复合物完全分离；② Add Trizol reagent to the sample and pipette several times repeatedly to fully lyse the sample, and place it at room temperature for 5 minutes to completely separate the protein-nucleic acid complex;
③向以上溶液中加入0.2ml氯仿，盖好管盖，剧烈振荡15秒，室温放置2-3分钟；③ Add 0.2ml chloroform to the above solution, cover the tube cap, shake vigorously for 15 seconds, and place at room temperature for 2-3 minutes;
④4℃12000rpm离心15分钟，此时样品分成三层：红色有机相，中间层和上层无色水相，RNA主要在水相中，把水相(约600μl)转移到一个新的RNase-Free离心管中；④ Centrifuge at 12000rpm at 4°C for 15 minutes. At this time, the sample is divided into three layers: the red organic phase, the middle layer and the upper colorless aqueous phase. RNA is mainly in the aqueous phase. Transfer the aqueous phase (about 600 μl) to a new RNase-Free centrifuge tube;
⑤在得到的水相溶液中加入等体积异丙醇，颠倒混匀，室温放置10分钟；⑤ Add an equal volume of isopropanol to the obtained aqueous phase solution, mix it upside down, and place it at room temperature for 10 minutes;
⑥4℃12000rpm离心10分钟，弃上清；⑥Centrifuge at 12000rpm at 4°C for 10 minutes, discard the supernatant;
⑦加入1ml 75％乙醇(用无RNase的水配制)洗涤沉淀；⑦Add 1ml of 75% ethanol (prepared with RNase-free water) to wash the precipitate;
⑧4℃12000rpm离心3分钟，小心吸弃上清，注意不要吸弃RNA沉淀；⑧Centrifuge at 12000rpm at 4°C for 3 minutes, carefully discard the supernatant, and be careful not to discard the RNA precipitate;
⑨室温放置2-3分钟，晾干。加入30μL无RNase的水，充分溶解RNA，得到的RNA保存在-70℃，防止降解；⑨Leave at room temperature for 2-3 minutes and dry. Add 30 μL of RNase-free water to fully dissolve the RNA, and store the obtained RNA at -70°C to prevent degradation;
⑩由1％琼脂糖凝胶电泳鉴定总RNA提取结果，用生物分析仪分析RNA的完整性和浓度。⑩ The total RNA extraction results were identified by 1% agarose gel electrophoresis, and the integrity and concentration of RNA were analyzed with a bioanalyzer.
进一步的，所述的热应激条件下奶牛乳腺差异表达基因调控网络的构建方法，步骤二第(d)条包括以下内容：Further, in the method for constructing the regulatory network of differentially expressed genes in dairy cow mammary glands under heat stress conditions, step 2 (d) includes the following content:
通过去接头、去污染、统计序列长度分布等过程，获得去杂质的“干净”序列(Clean序列)。Through processes such as joint removal, decontamination, and statistical sequence length distribution, a "clean" sequence (Clean sequence) with no impurities is obtained.
进一步的，所述的热应激条件下奶牛乳腺差异表达基因调控网络的构建方法，步骤二第(e)条包括以下内容：Further, in the method for constructing a regulatory network of differentially expressed genes in dairy cow mammary glands under heat stress conditions, step 2 (e) includes the following content:
将Clean序列通过与NCBI Genbank(http:www.ncbi.nlm.nih.gov/)中rRNA、tRNA、snRNA、snoRNA、repeat、exon、intron和miRNA等库中序列进行比对分析并分类注释。The Clean sequence was compared with the sequences in the rRNA, tRNA, snRNA, snoRNA, repeat, exon, intron, and miRNA libraries in NCBI Genbank (http:www.ncbi.nlm.nih.gov/) and classified and annotated.
进一步的，所述的热应激条件下奶牛乳腺差异表达基因调控网络的构建方法，步骤二第(f)条包括以下内容：Further, in the method for constructing the regulatory network of differentially expressed genes in dairy cow mammary glands under heat stress conditions, step 2 (f) includes the following content:
根据牛、人、猪、犬、大猩猩和老鼠等哺乳动物已经注册的miRNA分子信息，用同源搜索的计算分析方法预测牛新的候选miRNA，通过碱基错配、二级结构、A+U含量、自由能大小等分析候选序列特征，鉴定已知miRNA和预测新miRNA。According to the registered miRNA molecular information of mammals such as cattle, humans, pigs, dogs, gorillas and mice, the computational analysis method of homology search is used to predict new candidate miRNAs of cattle, based on base mismatch, secondary structure, A+ Analyze candidate sequence characteristics such as U content and free energy, identify known miRNAs and predict new miRNAs.
进一步的，所述的热应激条件下奶牛乳腺差异表达基因调控网络的构建方法，步骤二第(g)条包括以下内容：Further, in the method for constructing a regulatory network of differentially expressed genes in dairy cow mammary glands under heat stress conditions, step 2 (g) includes the following content:
将测序样品测序量归一化到同一个量级，使用标准化后的结果统计倍数变化和P值，定义差异表达miRNA的P值＜0.01、FDR＜0.05，寻找差异表达miRNA。Normalize the sequencing volume of the sequencing samples to the same magnitude, use the normalized results to count the fold change and P value, define the P value of differentially expressed miRNAs <0.01, and FDR <0.05, and search for differentially expressed miRNAs.
进一步的，所述的热应激条件下奶牛乳腺差异表达基因调控网络的构建方法，步骤三包括以下内容：Further, in the method for constructing the regulatory network of differentially expressed genes in dairy cow mammary glands under heat stress conditions, step 3 includes the following contents:
对差异表达显著的miRNA，通过应用含有预编译的Miranda算法结果的数据库MicroCosm(http:www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5)以及预测软件TargetScan 6.0(http://www.Targetscan.org/)分别获得2个预测软件的靶基因集，并求取2个靶基因集的交集作为差异miRNA调控的全体靶基因。For miRNAs with significant differential expression, the database MicroCosm (http:www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5) containing pre-compiled Miranda algorithm results and the prediction software TargetScan 6.0 (http: //www.Targetscan.org/) respectively obtained the target gene sets of the two prediction software, and calculated the intersection of the two target gene sets as the overall target genes regulated by differential miRNAs.
进一步的，所述的热应激条件下奶牛乳腺差异表达基因调控网络的构建方法，步骤四包括以下内容：Further, in the method for constructing the regulatory network of differentially expressed genes in dairy cow mammary glands under heat stress conditions, step 4 includes the following contents:
应用DAVID(database for annotation,visualization and integrateddiscovery)网站(http://david.abcc.ncifcrf.gov/home.jsp)对差异miRNA调控的差异靶基因进行GO分析和Pathway分析，确定预测靶基因参与的生物过程及最主要生化代谢途径和信号转导途径等生物学功能，构建基因调控网络，以P＜0.01、FDR＜0.05为筛选标准。Using the DAVID (database for annotation, visualization and integrated discovery) website (http://david.abcc.ncifcrf.gov/home.jsp) to conduct GO analysis and Pathway analysis on the differential target genes regulated by differential miRNAs, and determine the factors involved in the prediction of target genes. Biological processes and biological functions such as the most important biochemical metabolic pathways and signal transduction pathways, construct a gene regulatory network, and use P<0.01, FDR<0.05 as screening criteria.
本发明以热应激奶牛乳腺和正常奶牛乳腺为试验材料，采用高通量测序技术筛选出两种乳腺中差异表达的miRNA，并对差异表达的miRNAs进一步验证分析，利用生物信息学方法预测奶牛热应激反应中差异表达miRNA靶基因并分析其信号通路，构建miRNA介导的调控网络。揭示高温环境下miRNA的调控作用及其机制，为调控或减弱奶牛的热应激反应，以及提高产奶量提供新的试验数据。同时，对开发热应激蛋白质饲料、调控奶牛环境等方面也具有非常重要的意义。The present invention uses heat-stressed dairy cow mammary glands and normal dairy cow mammary glands as test materials, uses high-throughput sequencing technology to screen out differentially expressed miRNAs in the two mammary glands, and further verifies and analyzes the differentially expressed miRNAs, using bioinformatics methods to predict dairy cows Differential expression of miRNA target genes in response to heat stress and analysis of their signaling pathways to construct a miRNA-mediated regulatory network. To reveal the regulatory role and mechanism of miRNA in high temperature environment, and to provide new experimental data for regulating or weakening the heat stress response of dairy cows and improving milk production. At the same time, it is also of great significance to the development of heat stress protein feed and the regulation of the environment of dairy cows.
附图说明Description of drawings
图1为RNA的Agilent 2100生物分析仪检测结果Fig. 1 is the detection result of Agilent 2100 bioanalyzer of RNA
图2为差异表达的已知miRNA的靶基因位点统计图Figure 2 is a statistical map of the target gene loci of known miRNAs that are differentially expressed
具体实施方式detailed description
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.
一种热应激条件下奶牛乳腺差异表达miRNAs基因调控网络构建，包括如下步骤：The construction of a regulatory network of differentially expressed miRNAs genes in dairy cow mammary glands under heat stress conditions comprises the following steps:
步骤一、热应激奶牛与非热应激奶牛乳腺组织样本采集。Step 1: Collection of mammary gland tissue samples from heat-stressed dairy cows and non-heat-stressed dairy cows.
根据系谱资料、DHI数据和牛场记录数据，选取性别相同，饲养环境、年龄及胎次相近的4头热应激奶牛(夏季，8月)和4头非热应激奶牛(春季，3月)作为实验牛，采集乳腺组织样本放入液氮中，到实验室转入-70℃冰箱保存备用。According to the pedigree data, DHI data and cattle farm record data, select 4 heat-stressed dairy cows (summer, August) and 4 non-heat-stressed dairy cows (spring, March) with the same gender and similar feeding environment, age and parity As an experimental cow, mammary gland tissue samples were collected and placed in liquid nitrogen, and then transferred to the laboratory and stored in a -70°C refrigerator for later use.
步骤二、Solexa高通量测序鉴定参与奶牛热应激反应的miRNA，寻找差异表达miRNA。Step 2: Solexa high-throughput sequencing identifies miRNAs involved in the heat stress response of dairy cows, and searches for differentially expressed miRNAs.
(a)用Trizol试剂提取热应激和非热应激期奶牛乳腺组织总RNA并鉴定。提取步骤如下：(a) Total RNA was extracted and identified from cow mammary gland tissues during heat stress and non-heat stress periods with Trizol reagent. The extraction steps are as follows:
①取-70℃冻存组织约100mg在液氮中研磨后加入1ml Trizol试剂混匀。①Take about 100 mg of tissue frozen at -70°C, grind it in liquid nitrogen, add 1 ml of Trizol reagent and mix well.
②样品中加入Trizol试剂后反复吹打几次，使样本充分裂解。室温放置5分钟，使蛋白核酸复合物完全分离。② After adding Trizol reagent to the sample, pipette repeatedly several times to fully lyse the sample. Leave at room temperature for 5 minutes to completely separate the protein-nucleic acid complex.
③向以上溶液中加入0.2ml氯仿，盖好管盖，剧烈振荡15秒，室温放置2-3分钟。③ Add 0.2ml of chloroform to the above solution, cover the tube cap, shake vigorously for 15 seconds, and place at room temperature for 2-3 minutes.
④4℃12000rpm离心15分钟，此时样品分成三层：红色有机相，中间层和上层无色水相，RNA主要在水相中，把水相(约600μl)转移到一个新的RNase-Free离心管中。④ Centrifuge at 12000rpm at 4°C for 15 minutes. At this time, the sample is divided into three layers: the red organic phase, the middle layer and the upper colorless aqueous phase. RNA is mainly in the aqueous phase. Transfer the aqueous phase (about 600 μl) to a new RNase-Free centrifuge tube.
⑤在得到的水相溶液中加入等体积异丙醇，颠倒混匀，室温放置10分钟。⑤ Add an equal volume of isopropanol to the obtained aqueous phase solution, mix by inverting, and place at room temperature for 10 minutes.
⑥4℃12000rpm离心10分钟，弃上清。⑥Centrifuge at 12,000 rpm at 4°C for 10 minutes, and discard the supernatant.
⑦加入1ml 75％乙醇(用无RNase的水配制)洗涤沉淀。⑦ Add 1ml of 75% ethanol (prepared with RNase-free water) to wash the precipitate.
⑧4℃12000rpm离心3分钟，小心吸弃上清，注意不要吸弃RNA沉淀。⑧Centrifuge at 12,000 rpm at 4°C for 3 minutes, carefully discard the supernatant, and be careful not to discard the RNA precipitate.
⑨室温放置2-3分钟，晾干。加入30μL无RNase的水，充分溶解RNA，得到的RNA保存在-70℃，防止降解。⑨Leave at room temperature for 2-3 minutes and dry. Add 30 μL of RNase-free water to fully dissolve the RNA, and store the obtained RNA at -70°C to prevent degradation.
⑩由1％琼脂糖凝胶电泳鉴定总RNA提取结果，用生物分析仪分析RNA的完整性和浓度。由图1可知，样品总RNA浓度为235ng/μL，28S:18S约为2.0，RNA完整性(RIN)为8.4，达到了文库构建的要求，可以进行后续的试验。⑩ The total RNA extraction results were identified by 1% agarose gel electrophoresis, and the integrity and concentration of RNA were analyzed with a bioanalyzer. It can be seen from Figure 1 that the total RNA concentration of the sample is 235ng/μL, the 28S:18S is about 2.0, and the RNA integrity (RIN) is 8.4, meeting the requirements for library construction, and subsequent experiments can be carried out.
(b)建立小RNA文库，并进行测序分析验证。(b) Establish a small RNA library and perform sequencing analysis verification.
采用TA克隆和传统Sanger测序对所提总RNA鉴定是否可用于Solexa测序。建立了小RNA文库，并进行了测序分析。TA cloning and traditional Sanger sequencing were used to identify whether the extracted total RNA could be used for Solexa sequencing. A small RNA library was established and sequenced for analysis.
(c)用Solexa测序仪对DNA片段进行单向末端直接测序。(c) DNA fragments were subjected to unidirectional direct sequencing using a Solexa sequencer.
分离长度18-30nt范围的小RNA，两端分别加上特定接头后体外反转录成cDNA，经RT-PCR扩增后用Solexa测序仪对DNA片段进行单向末端直接测序。Small RNAs with a length of 18-30 nt were isolated, reverse-transcribed into cDNA in vitro after adding specific adapters at both ends, and after RT-PCR amplification, the DNA fragments were directly sequenced with a Solexa sequencer.
(d)对所测的原始序列进行处理与统计；(d) processing and counting the measured original sequence;
共获得21,684,303个高质量序列，通过去接头、去污染、统计序列长度分布等过程，获得去杂质的“干净”序列(Clean序列)21,042,828个，占所有小RNA的97.04％。A total of 21,684,303 high-quality sequences were obtained, and 21,042,828 "clean" sequences (Clean sequences) were obtained by removing adapters, decontamination, and statistical sequence length distribution, accounting for 97.04% of all small RNAs.
(e)将步骤(d)处理过的序列与已知库中序列进行比对分析并分类注释；(e) comparing and analyzing the sequence processed in step (d) with the sequence in the known library, and classifying and annotating;
将Clean序列通过与NCBI Genbank(http:www.ncbi.nlm.nih.gov/)中rRNA、tRNA、snRNA、snoRNA、repeat、exon、intron和miRNA等库进行比对和分类注释。87.62％为已知的miRNA，只有3.73％为tRNAs、snRNAs、rRNA和snoRNAs。The Clean sequence was compared and classified with the rRNA, tRNA, snRNA, snoRNA, repeat, exon, intron and miRNA libraries in NCBI Genbank (http:www.ncbi.nlm.nih.gov/). 87.62% were known miRNAs, and only 3.73% were tRNAs, snRNAs, rRNAs and snoRNAs.
(f)鉴定已知miRNA和预测新miRNA。(f) Identification of known miRNAs and prediction of new miRNAs.
根据牛、人、猪、犬、大猩猩和老鼠等哺乳动物已经注册的miRNA分子信息，用同源搜索的计算分析方法预测牛新的候选miRNA，通过碱基错配、二级结构、A+U含量、自由能大小等分析候选序列特征。发现已知miRNAs 483个，新miRNAs 139个。According to the registered miRNA molecular information of mammals such as cattle, humans, pigs, dogs, gorillas and mice, the computational analysis method of homology search is used to predict new candidate miRNAs of cattle, based on base mismatch, secondary structure, A+ U content, free energy, etc. to analyze the characteristics of candidate sequences. 483 known miRNAs and 139 new miRNAs were found.
(g)寻找差异表达miRNA。(g) Search for differentially expressed miRNAs.
将测序样品测序量归一化到同一个量级，使用标准化后的结果统计倍数变化和P值。定义差异表达miRNA的P值＜0.01、FDR＜0.05。发现差异表达miRNA27个，其中已知miRNA24个，新miRNA 3个(如表1所示)；上调miRNA20个，下调miRNA7个，丰富了奶牛miRNA数据库。Normalize the sequencing volume of the sequencing samples to the same magnitude, and use the normalized results to calculate the fold change and P value. Define the P value<0.01 and FDR<0.05 of differentially expressed miRNA. A total of 27 differentially expressed miRNAs were found, including 24 known miRNAs and 3 new miRNAs (as shown in Table 1); 20 up-regulated miRNAs and 7 down-regulated miRNAs enriched the dairy miRNA database.
表1 差异表达新miRNATable 1 Differential expression of new miRNAs
步骤三、差异表达显著的miRNA靶基因预测。Step 3. Prediction of miRNA target genes with significant differential expression.
对差异表达显著的miRNA，通过应用含有预编译的miRanda算法结果的数据库MicroCosm(http:www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5)以及预测软件TargetScan 6.0(http://www.Targetscan.org/)分别获得2个预测软件的靶基因集，并求取2个靶基因集的交集作为差异miRNA调控的全体靶基因，24个差异表达的miRNA共有65625个靶基因，靶基因位点数为293949个，如图2所示。For miRNAs with significant differential expression, the database MicroCosm (http:www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5) and the prediction software TargetScan 6.0 (http: //www.Targetscan.org/) obtained target gene sets of 2 prediction software respectively, and calculated the intersection of the 2 target gene sets as the overall target genes regulated by differential miRNAs. The 24 differentially expressed miRNAs had a total of 65,625 target genes , the number of target gene loci is 293949, as shown in Figure 2.
新发现的novel-miR-278在热应激组奶牛乳腺组织中表达下降，其预测靶基因HSP40作为HSP70蛋白的一种共伴侣分子，调节HSP70的ATP酶活性促进HSP70蛋白完成许多细胞过程，包括新生蛋白质的折叠、错误折叠蛋白质的解折叠、蛋白质的转运、组装和解离过程。novel-miR-278靶向调节的另一个基因是IFNAR2(干扰素α/β受体β链)。干扰素(Interferon,IFN)是一种具有抑制细胞分裂、调节免疫和促进细胞凋亡等多种生物学活性的细胞因子。干扰素受体与之结合可转换成细胞内信号,从而启动细胞核内基因的转录。以上结果表明，这个新发现的miRNAs在热应激条件下可能通过调控靶基因表达，参与机体免疫和抗应激反应。The expression of the newly discovered novel-miR-278 decreased in the mammary gland tissue of cows in the heat stress group, and its predicted target gene HSP40, as a co-chaperone molecule of HSP70 protein, regulates the ATPase activity of HSP70 and promotes HSP70 protein to complete many cellular processes, including Folding of nascent proteins, unfolding of misfolded proteins, protein transport, assembly and dissociation processes. Another gene targeted and regulated by novel-miR-278 is IFNAR2 (interferon α/β receptor β chain). Interferon (Interferon, IFN) is a cytokine with various biological activities such as inhibiting cell division, regulating immunity and promoting apoptosis. Binding to interferon receptors can be converted into intracellular signals, thereby initiating the transcription of genes in the nucleus. The above results indicated that the newly discovered miRNAs may participate in the immune and anti-stress responses of the body by regulating the expression of target genes under heat stress conditions.
步骤四、差异表达显著的miRNA调控网络构建。Step 4, constructing the regulatory network of miRNAs with significant differential expression.
为了进一步研究组织差异表达miRNAs的生物学功能，应用DAVID(database forannotation,visualization and integrated discovery)网站(http://david.abcc.ncifcrf.gov/home.jsp)对差异miRNA调控的差异靶基因进行GO分析和Pathway分析，确定预测靶基因参与的生物过程及最主要生化代谢途径和信号转导途径等生物学功能。以P＜0.01、FDR＜0.05为筛选标准。In order to further study the biological functions of differentially expressed miRNAs in tissues, the DAVID (database for annotation, visualization and integrated discovery) website (http://david.abcc.ncifcrf.gov/home.jsp) was used to analyze the differential target genes regulated by differential miRNAs. GO analysis and Pathway analysis determine the biological processes involved in the predicted target genes and the biological functions such as the most important biochemical metabolic pathways and signal transduction pathways. The selection criteria were P<0.01 and FDR<0.05.
表2 miRNA差异表达靶基因GO富集分析(靶基因数>6000)Table 2 GO enrichment analysis of miRNA differentially expressed target genes (number of target genes >6000)
表3 miRNA靶基因的KEGG通路分析Table 3 KEGG pathway analysis of miRNA target genes
通过对奶牛热应激基因的调控网络分析发现，Wnt、TGF-β、MAPK、Notch和JAK-STAT信号通路等广泛参与了奶牛热应激。大多情况下MAPK通常作为执行者发挥主要调控作用，TGF-β作为激活其他信号通路的领导者，Wnt信号通路起协调作用，而Notch和JAK-STAT信号通路起微调作用。我们对预测的靶基因进行KEGG分析中这六个信号通路都有发现，其中MAPK信号通路中相关的预测的靶基因占0.23％，Wnt信号通路占1.65％，TGF-β信号通路占0.71％，Notch信号通路占0.58％，JAK-STAT信号通路占1.22％。这些靶基因所对应的miRNA是研究奶牛热应激反应中的重要候选miRNA。Through the analysis of regulatory network of cow heat stress genes, it was found that Wnt, TGF-β, MAPK, Notch and JAK-STAT signaling pathways were widely involved in cow heat stress. In most cases, MAPK usually plays a major regulatory role as an executor, TGF-β acts as a leader in activating other signaling pathways, Wnt signaling pathway plays a coordinating role, and Notch and JAK-STAT signaling pathways play a fine-tuning role. These six signaling pathways were found in the KEGG analysis of the predicted target genes, among which the predicted target genes related to the MAPK signaling pathway accounted for 0.23%, the Wnt signaling pathway accounted for 1.65%, and the TGF-β signaling pathway accounted for 0.71%. Notch signaling pathway accounted for 0.58%, JAK-STAT signaling pathway accounted for 1.22%. The miRNAs corresponding to these target genes are important candidate miRNAs in the study of cow heat stress response.
SEQUENCE LISTINGSEQUENCE LISTING
<110> 廊坊师范学院<110> Langfang Teachers College
<120> 一种热应激条件下奶牛乳腺差异表达基因调控网络的构建方法 <120> A method for constructing regulatory network of differentially expressed genes in dairy cow mammary gland under heat stress conditions
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