李广栋¹, 吕东颖¹, 田秀芝², 姬鹏云¹, 郭江鹏³, 路永强³, 刘国世^,11 中国农业大学动物科学技术学院,北京 100193
2 中国农业科学院北京畜牧兽医研究所,北京 100193
3 北京市畜牧总站,北京 100101

Research Progress of Omics Technologies in Cow Mastitis

LI GuangDong¹, Lü DongYing¹, TIAN XiuZhi², JI PengYun¹, GUO JiangPeng³, LU YongQiang³, LIU GuoShi^,1 1 College of Animal Science and Technology,China Agriculture University,Beijing 100193
2 Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193
3 Beijing Animal Husbandry Station,Beijing 100101

通讯作者: 刘国世,E-mail： gshliu@cau.edu.cn

责任编辑: 林鉴非
收稿日期:2017-12-5接受日期:2017-12-22网络出版日期:2019-01-16

基金资助:

转基因生物新品种培育重大专项.2014ZX0800802B 北京市奶牛创新团队.BAIC06-2017

Received:2017-12-5Accepted:2017-12-22Online:2019-01-16
作者简介 About authors
李广栋,E-mail：15600911225@cau.edu.cn。









摘要
奶牛乳房炎发病率较高、病因复杂,是影响世界奶牛业发展的主要常见疾病之一。由金黄色葡萄球菌、大肠杆菌、链球菌等病原体引起的临床和隐形乳房炎对动物性食品安全及畜牧业的正常发展构成巨大安全隐患,全球每年因奶牛乳房炎导致的经济损失多达数十亿美元。近年来随着测序技术的不断突破和测序成本的不断降低,生命科学的研究进入了多组学时代,其在畜牧业中的应用也越来越广泛。对奶牛乳房炎来说,传统的组织病理学筛查、体细胞计数、牛乳pH检测、牛乳电导率检测、酶活检验、红外热显影等诊断技术由于其自身的局限性难以充分全面地阐明其发病机理,已不能满足科研人员的需求。组学技术即Omics,主要包括基因组学技术、蛋白质组学技术和代谢组学技术等。通过基因组学研究不仅能从转录层面上揭示奶牛乳房炎复杂性状的表型变异及遗传基础,还能从转录后调控（miRNAs、LncRNAs等）和表观遗传学修饰（DNA甲基化、组蛋白修饰等）层面挖掘出相关的DNA和RNA变化及多分子间的相互作用规律,能够帮助我们更好地了解奶牛乳腺组织对病原体的免疫应答机制,筛选鉴定出乳房炎抗性的信号通路及关键候选基因,从而提高基因组预测或选择的准确性。利用蛋白质组学技术能够对不同环境不同状态的牛乳及乳腺组织的蛋白质种类、表达丰度、蛋白互作、翻译后修饰等进行比较分析,对差异表达的蛋白质经过COG功能注释、数据库比对、GO和Pathway富集分析,可以从蛋白水平揭示乳房炎发生及防御过程中的复杂调控机制,同时还能发现乳房炎诊断的标记分子,进而为乳房炎治疗药物的研发提供潜在的精准靶点。代谢组学是系统生物学的重要组成部分。通过代谢组学研究,能够同时对机体在内、外环境等复杂因素作用下及特定生理时期内所有低分子量代谢产物（如氨基酸、脂类、碳水化合物等）进行精准、高效的定性和定量分析,从而阐明相关的代谢途径;其作为基因表达的最下游,能使基因和蛋白质表达的微小变化在代谢物水平上得到放大,进而可以更充分地反映细胞功能。将代谢组学技术应用到奶牛乳房炎研究中,能够分析出差异代谢物、鉴定出相关的生物标志物,进而揭示奶牛乳腺的生理及病理变化过程。总之,将各组学技术及多组学整合关联分析应用到奶牛乳房炎研究中可以更深入地揭示其病原防御机制,进而为乳房炎的预测、诊断和治疗提供有价值的参考和借鉴。本文就最近几年组学技术在奶牛乳房炎领域的研究进展进行综述,以期为我国奶牛健康及奶业安全发展提供指导。
关键词： 组学技术;奶牛;乳房炎

Abstract
Dairy mastitis, a common and complex disease with a high incidence, takes its toll on the development of world dairy industry, brings economic losses of billions of dollars per year. Clinical and subclinical mastitis, caused by pathogens such as Staphylococcus aureus, Escherichia coli and Streptococcus agalactiae, posed a huge security risk to milk industry. In recent years, with the continuous breakthrough of sequencing technology and decline of sequencing cost, the research of life science has entered into the Omics era. The traditional methods such as histopathological screening, somatic cell counting, milk PH value detection, detection of milk conductivity, enzyme activity test, infrared thermal imaging can be employed for clinical diagnosis of dairy cow mastitis, but these methods are not powerful enough to elucidate the pathogenesis in a cellular or molecular view. Omics technologies are mainly composed of genomics, proteomics and metabolomics. Genomics can not only reveal the phenotypic variation and genetic basis of the complex trait of dairy mastitis at the transcriptional level, but also reveal the molecular patterns of the mastitis from the aspects of transcriptional regulation (miRNAs, LncRNAs, etc.) and epigenetic modification (DNA methylation, histone modification, etc.). Genomic analysis of mastitis can also dig out the related changes of DNA, RNA and the rules of multi-molecule interaction, which accounts for a better understanding of the immune mechanism of the host against the pathogen, so as to screen and identify the signal pathways and key candidate genes of mastitis resistance, thus improving the accuracy of genome prediction or selection. Proteomics can not only compare milk protein type and abundance but also analyze protein interaction and post-translational modification in breast tissues under different states and environments. The differentially expressed proteins are annotated by COG (Cluster of Orthologous Groups of protein) function followed by database comparison, GO and Pathway enrichment analysis, which help bring to light the complex regulatory mechanism of mastitis occurrence and defense process at protein level. Proteomic analysis can also be used to find molecular marker of mastitis diagnosis, which will provide a potential precise target for the development of therapeutic drugs. Metabolomics, an important part of the system biology, can detect metabolites of low molecular weight (such as amino acids, lipids, carbohydrates, etc.) of the specific tissues or organs in specific environment or specific physiological states. Efficient qualitative and quantitative analysis will elucidate the relevant metabolic pathways. As the ultimate downstream of gene expression, metabolomics technology enables small changes in gene expression and protein synthesis to be amplified at metabolite levels to fully reflect cellular functions, whose application in dairy mastitis will be able to identify related biomarkers and reveal the physiological and pathological changes of dairy breasts. In conclusion, applying Omics or multi-Omics association analysis techniques to mastitis can further reveal the pathogenic defense mechanism, which will provide valuable reference for disease prediction, diagnosis and treatment. This paper reviews the latest research progress about application of Omics in the field of cow mastitis, aiming to provide solid theoretical bases and practical references for cow health and safety of dairy industry in China.
Keywords：Omics;cow;mastitis


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本文引用格式
李广栋, 吕东颖, 田秀芝, 姬鹏云, 郭江鹏, 路永强, 刘国世. 组学技术在奶牛乳房炎上应用的相关研究进展[J]. 中国农业科学, 2019, 52(2): 350-358 doi:10.3864/j.issn.0578-1752.2019.02.013
LI GuangDong, Lü DongYing, TIAN XiuZhi, JI PengYun, GUO JiangPeng, LU YongQiang, LIU GuoShi. Research Progress of Omics Technologies in Cow Mastitis[J]. Scientia Acricultura Sinica, 2019, 52(2): 350-358 doi:10.3864/j.issn.0578-1752.2019.02.013


奶牛乳房炎是乳腺组织发生的一种炎症性反应,诱发因素较多,主要为微生物感染（如细菌、真菌、支原体、病毒等）、环境因素（如卫生条件、温度湿度、饲料等）、人为因素（如机械性损伤、挤奶应激、饲养管理不当等）及牛只自身因素（如年龄、胎次、产奶量、泌乳阶段等）^[1,2,3,4]。乳房炎的特点为乳腺组织发生或轻或重的病理学变化,乳汁中的体细胞数增多,乳品质异常^[5]。该病是世界范围内奶牛养殖业中治疗成本最为昂贵的感染疾病,仅对美国畜牧业造成的影响而言,每年由于牛奶产量及品质下降、兽医治疗成本飙升及牧场管理费用增加等方面造成的损失高达数十亿美元^[6]。根据乳房及乳汁有无肉眼可见的变化,研究人员通常将奶牛乳房炎分为临床型乳房炎和亚临床乳房炎即隐性乳房炎^[7,8]。引起奶牛乳房炎的微生物种类繁多,据报道有137种,较为常见的有20多种^[9,10]。其中金黄色葡萄球菌、大肠杆菌和链球菌在检出的致病菌中占有较高比例,为最常见的乳房炎致病菌类型^[11,12,13]。代谢组学、蛋白质组学和基因组学是系统生物学的重要组成部分^{[14,15,16,17,18,19,20]},是近年来发展十分迅速的学科,其不仅在人类医学领域占用重要地位,而且也不断在畜牧业中的相关研究中崭露头角^[21,22,23],一代又一代组学技术的变革让人们得以探究微观世界的真理并从分子水平上解析生命的奥秘。在奶牛乳房炎的相关研究中,以往的生物技术因其自身的局限性已经不能满足科研人员的需求,而组学技术的出现恰恰能够从多个角度深入的解析出奶牛乳房炎的复杂发病机理,进而能够筛选出有效的生物学标记从而进行及时准确的预防,同时其还能为相关治疗药物的研发提供精准的靶点,最终达到防治结合的预期结果。因此,本文从代谢组学、蛋白质组学和基因组学三个方面阐述了组学技术在奶牛乳房炎领域的研究进展,希望能够为后续的奶牛健康及奶业安全的相关研究提供新的思路。

1 代谢组学

随着代谢组学相关仪器和分析技术的不断完善与提高,其在奶牛业样品分析中的应用研究越来越 多^[24,25,26],牛奶代谢组学一般是通过检测牛奶中的代谢产物来研究乳品质、乳成分等,进而从侧面研究牛只的健康状况,因此,利用代谢组学的技术可以很好的揭示出奶牛乳房炎的病理代谢机制^[27,28]。代谢组学的过程通常包括制备和收集奶样,利用质谱、气相色谱-质谱联用、液相色谱-质谱联用、核磁共振等手段进行检测,最后对获得的原始数据进行生物信息学分析找出差异标志物,再通过比对相关数据库进行代谢通路的分析,最终明确代谢产物之间的互作关系。

越来越多的专家****利用代谢组学的方法寻找与奶牛乳房炎相关的活性标记物。THOMAS等^[29]以由链球菌特异性诱导的奶牛乳房炎作为实验模型,将采集的奶样通过液相色谱和质谱联用特异性分析了奶样的代谢组学,结果获得了3 000个色谱峰,层次聚类分析和主成分分析显示在诱导乳房炎的81 h后奶样中的代谢产物变化最大,312 h后才恢复到正常水平,代谢通路分析表明链球菌刺激后的前81 h内碳水化合物和核苷酸代谢物多数呈减少趋势而脂代谢物和二、三和四肽却截然相反,另外还发现胆汁酸-核受体FXR信号通路显著上调,这提示胆汁酸有可能参与了乳腺的炎症反应,这也可以对乳腺组织在应答外界感染时的反应有了更好的认识。HETTINGA等^[30]采用两种不同的顶空气相色质谱法（headspace gas chromatography-mass spectrometry, GC-MS）对大多数病原菌导致的临床乳房炎的奶样进行了代谢组学分析,并成功的对奶中的挥发性代谢物进行了定量,而且还绘制出了特定挥发性代谢物的表达谱,解决了传统手段无法解决的难题。SUNDEKILDE等^[31]利用核磁共振光谱（nuclear magnetic resonance spectroscopy, NMR）法分析了脱脂牛奶中的代谢组学,结果发现低、高体细胞数的样本之间的代谢产物差异显著,在高体细胞数的样品中乳酸、丁酸、异亮氨酸、乙酸和β-羟基丁酸酯的含量显著增加,而马尿酸和富马酸含量则降低,最终确定了丁酸、β-羟基丁酸酯、异亮氨酸、马尿酸和富马酸可以作为牛奶中高体细胞数的新的标志物,而高体细胞数也正是隐性乳房炎和临床乳房炎的典型特征,因此这些代谢物可以间接的反应牛只是否患了乳房炎。而DERVISHI等^[32]通过气相色谱-质谱联用的手段对围产期患隐性乳房炎的荷斯坦奶牛进行了氨基酸、碳水化合物和脂类代谢有关的代谢组分析,确定了缬氨酸、丝氨酸、酪氨酸和苯丙氨酸可以作为产前4到8周的奶牛隐性乳房炎患病与否的标记物,而缬氨酸、异亮氨酸、丝氨酸和脯氨酸则可作为产后4—8周泌乳期的诊断标记物,因此,可以通过氨基酸的代谢变化来预测围产期奶牛隐性乳房炎的患病风险。XI^[33]等则利用新型的超高效液相色谱四极杆飞行时间质谱（UPLC-Q-TOF-MSE）技术对健康组、临床乳房炎组和隐性乳房炎组的奶牛乳样进行了代谢组分析,结果发现和健康组相比,临床乳房炎组的葡萄糖、一磷酸甘油、4-羟基苯乳酸、左旋肉碱、甘油- 3 -磷酸胆碱、柠檬酸和马尿酸显著减少,而在隐性乳房炎组一磷酸甘油、苯甲酸、左旋肉碱和顺乌头酸显著减少,同时,精氨酸和亮氨酸含量在隐性乳房炎组中显著增加,该结果又为乳房炎的诊断提供了更多的标记物。综上所述,代谢组学不论在隐性乳房炎还是在临床乳房炎中的应用都取得了理想的效果,为该病的诊断提供了更多的依据。

2 蛋白质组学

传统的蛋白质组学主要包括二维凝胶电泳、质谱等技术,目前,二维毛细管电泳（2D-CE）、二维色谱（2D-LC）、液相色谱–毛细管电泳（LC-CE）、电喷雾质谱（ESI- MS）和基质辅助激光解吸电离-飞行时间质谱（MALDI-TOF-MS）等新技术异军突起。目前,随着科技的发展和成本的降低,蛋白质组学在动物疾病相关领域的研究越来越广泛^[34],畜牧业上对于奶牛乳房炎的蛋白质组学研究也逐渐增多^[35,36]。

MANSOR等^[37]利用毛细管电泳质谱法（CE-MS）对健康奶牛和患临床乳房炎的奶牛乳样进行了蛋白质组学分析,结果显示和对照组相比,患病组的β-乳球蛋白、α_S1-酪蛋白、β-酪蛋白、乳过氧化物酶、骨桥蛋白、白细胞介素4受体、成纤维细胞生长因子结合蛋白和糖基化依赖性细胞粘附分子-1差异显著,另外还发现α_S1-酪蛋白、β酪蛋白和微管α-1C链蛋白可以作为区分由革兰氏阴性菌（如大肠杆菌等）和革兰氏阳性菌（如金黄色葡萄球菌等）所致奶牛乳房炎的生物标记物。ZHAO等^[38]则利用二维凝胶电泳和无标记定量分析技术对正常奶牛和由大肠杆菌诱导的患乳房炎的奶牛乳腺组织进行了比较蛋白质组分析,通过绘制差异蛋白互作网络发现了波形蛋白和α-烯醇化酶为蛋白调控网络的中心,进而成功揭示了机体在应对大肠杆菌入侵乳腺时的防御机制。JACOB等^[39]则利用液相色谱-质谱联用（LC-MS）结合二维凝胶电泳和Western Blot技术对隐性乳房炎奶牛和健康奶牛的乳样中乳清蛋白成分进行了组学分析,结果发现在乳房炎早期?蛋白胨-3前体、胰蛋白酶前体、补体成分-c3、免疫球蛋白重链前体、C型凝集素等差异十分显著,并且确定了补体C3a可以作为诊断奶牛隐性乳房炎的潜在标记物。而HUANG等^[40]利用同位素标记相对绝对定量（isobaric tags for relative and absolute quantification,iTRAQ）技术和二维液相色谱-串联质谱法（2D-LC- MS/MS）并结合生物信息学分析了由金黄色葡萄球菌导致的奶牛乳房炎感染盛期乳腺组织的蛋白组,结果发现和对照组相比,I型胶原-α1（COL1A1）和间α-球蛋白抑制剂H4（ITIH4）在感染后的乳腺组织中显著上调,并且最终通过免疫印迹和免疫组化得到了证实,这为奶牛乳房炎的精准医疗提供了新的靶点。综上所述,蛋白质作为中心法则的重要核心,无论是DNA还是RNA最终都要通过蛋白质来行使其功能,因此,在奶牛乳房炎中蛋白质组学的应用可以更加直观的描绘出相关的抗病机制。

3 基因组学

基因组学是人类医学、动植物遗传育种和进化研究中的重要组成部分,随着高通量深度测序技术的不断突破和测序平台的升级换代,基因组学的应用越来越广泛。复杂性状的表型变异通常被认为受到许多微效基因和环境因素的影响,通过评估基因组特征中所有遗传标记对复杂性状的整体效应,可以揭示出复杂性状的遗传基础,从而提高基因组预测或选择的准确性。将相关的基因组学技术运用到奶牛乳房炎的研究中能够发现奶牛病理组织和正常组织中的差异表达基因和相关的生物学通路,进而可以鉴定出乳房炎的关键候选基因及遗传标记^[41,42]。该技术的运用不仅可以从转录水平上反应出功能基因的变化,还能从转录后调控（如基因调控原件miRNA）及表观遗传学修饰（如DNA甲基化和组蛋白修饰）两个方面揭示出更深层的互作关系。

3.1 基因组

TIEZZI等^[43]利用Illumina BovineSNP50芯片对103 585头患临床乳房炎且处于第一个泌乳期的荷斯坦奶牛和1 361头公牛进行了全基因组关联分析（genome wide association study, GWAS）,通过单步基因组BLUP法发现临床乳房炎具有多基因遗传效应,并且其性状和遗传变异密切相关,还发现在2号（IFIH1, LY75, ITGB6, NR4A2 and DPP4）、14号（LY6K, LY6D, LYNX1, LYPD2, SLURP1, PSCA）、20号（GHR, OXCT1, C6, C7, C9, C1QTNF3, DAB2, OSMR, PRLR）染色体上的QTL影响临床乳房炎的遗传变异,这些候选QTL在免疫反应中起着十分重要的作用;在8、11、16、19和24号染色体上还发现了未经注释的基因,这些基因能够作为临床乳房炎的潜在候选基因,该研究确定的基因组区域可作为预测荷斯坦奶牛临床乳房炎抗性的遗传学依据。WANG等^[44]将2 093头中国荷斯坦奶牛体细胞数估计育种值（SCC EBVs）作为表型性状,利用GWAS和 MMRA 分析鉴定了与奶牛乳房炎抗性及易感性相关的SNPs和候选基因。结果发现48个SNPs与奶牛乳房炎抗性显著相关并且大多数定位在14号染色体上,有6 个显著的SNPs 被注释在TRAPPC9 和ARHGAP39 基因中,其可作为奶牛乳房炎易感性/抗性候选基因。而BRAND等^[45]利用高乳房炎易感性牛和低乳房炎易感性牛的乳腺组织为试验材料通过分子标记辅助选择结合微阵列表达芯片（marker assisted selection-Microarray chip,MAS）技术发现了与奶牛金黄色葡萄球菌乳房炎密切相关的候选基因RELB,还发现与奶牛金黄色葡萄球菌乳房炎易感性相关的候选基因可能潜藏着QTL效应,该结果为金葡菌乳房炎抗性基因的筛选提供了更多的选择。IM等^[46]则以经过金黄色葡萄球菌细胞壁成分脂磷壁酸和肽聚糖处理的奶牛乳腺上皮细胞为材料,利用Affymetrix芯片技术检测了基因的表达谱,共筛选到2 019个差异表达基因,其中801个上调基因,1 218个下调基因;在上调基因中有14个与炎症调控相关的基因、22个细胞内分子信号通路相关的基因还有15 个与转录因子相关的基因;而下调基因中有17 个与炎症相关。最后还通过qPCR对18个差异极显著的基因进行了验证,这为金葡菌感染奶牛乳房炎的病理学研究提供了参考。以上的研究表明,尽管奶牛乳房炎性状存在复杂的遗传变异效应,但也有一定规律可寻,显著差异表达基因的发现为相关的预测和选择提供了可能。

3.2 转录组

XIU等^[47]使用金黄色葡萄球菌（S108）、大肠杆菌（E23）及克雷白氏杆菌（K96）分别感染奶牛的乳腺上皮细胞,并对感染后的细胞通过Solexa系统进行了转录组测序。GO分析显示三种病原菌感染组的差异表达基因分别集中在细胞代谢,细胞凋亡和胚胎发育上,同源蛋白的聚类分析结果表明它们均参与了翻译,核糖体生物合成和修复等生物过程,而KEGG分析表明三者分别在氧化磷酸化通路、NOD样受体信号通路和凋亡信号通路显著富集,还发现了NRF1、IL8、CXCL5、IL1α、PDCD2L、RAB3A、RAB1B 等基因可作为金葡菌乳房炎抗性候选基因。WANG等^[48]利用Illumina系统的Paired-End技术对健康牛和金葡菌乳房炎牛的乳腺组织进行了转录组测序,结果筛选到1 352 个差异表达基因,其中一些免疫相关基因ITGB6、MYD88、ADA、ACKR1、TNFRSF1B 与金葡菌乳房炎密切相关,可作为金葡菌乳房炎抗性候选基因,另外还发现在受感染的乳腺组织中CCL5、Colec2、LTF、CD46和NCF1等基因存在复杂的可变剪接。WANG等^[49]则对经过S56、S178和S36三种金黄色葡萄球菌诱导的奶牛乳腺上皮组织进行了转录组测序,分别筛选到1 720, 427和219个差异表达基因,GO和Pathway分析显示这些基因显著地参与炎症反应、代谢转化、细胞增殖和凋亡信号通路,IL-1α、TNF、EFNB1、IL-8 和EGR1 等促炎因子显著上调。而PU等^[50]对经过无乳链球菌诱导的患乳房炎和健康的中国荷斯坦牛乳腺组织进行了miRNA测序,结果发现和对照组相比,乳房炎组有35个差异表达的miRNA,其中有10个显著上调（miR-223最高）,25个显著下调（miR-26a最低）,这些miRNA的靶基因主要富集在RIG-I-like受体信号通路、胞质DNA传感通路和Notch信号通路上,该研究为miRNA参与无乳链球菌感染奶牛乳房炎的发病调控提供了有力的证据。FANG等^[51,52]对经过高、低浓度金葡菌攻毒24h后的奶牛乳腺组织进行了RNA-seq和miRNA-seq,结果鉴定出194个差异表达基因与高浓度的金葡菌感染有关,这些基因主要参与了先天性免疫反应过程;转录组和QTL数据库的联合分析发现了28 个与奶牛金葡菌乳房炎抗性相关的候选基因（如SLC4A11等）;他们还发现高浓度金葡菌感染组β-mir-223和β-mir-21-3p显著上调,互作分析显示这两个miRNA通过抑制CXCL14和KIT来抵抗病原入侵,这些结果从转录调控和转录后调控的两个角度综合分析了金葡菌入侵时的机体免疫机制,具有一定的借鉴意义。JIN等^[53]则对经金葡菌和大肠杆菌感染的牛乳腺上皮细胞（Mac-T）进行了RNA-Seq和miRNA-Seq,结果发现两种菌感染牛乳腺细胞后共有17个miRNA显著差异,其中金葡菌感染细胞后特有的差异表达miRNA 有4个（bta-miR-2339, miR-499, miR-23a 和miR- 99b）,而大肠杆菌感染细胞后的差异表达miRNA 有5 个（bta-miR-184, miR-24-3p, miR-148, miR-486 和let-7a-5p）,靶基因预测显示主要富集在细胞增殖和凋亡生物过程中。以上研究为奶牛乳房炎相关基因的转录、转录后调控及宿主细胞对病原菌的免疫应答等研究提供了参考,这些新发现的潜在的靶基因和miRNA可作为奶牛隐性和临床乳房炎诊断和预防的生物学标记。

3.3 表观组

目前,灵长类及模式动物的表观遗传学研究如火如荼,尤其是疾病相关领域的研究工作甚多,但在家养动物中相关的研究开展相对较晚,奶牛乳房炎因受病原菌和环境共同影响,若单从病原角度或单从遗传学角度均难以有效实现对奶牛乳房炎的防治,因此,表观遗传学的出现对奶牛乳房炎的研究是一个很好的补充。

VANSELOW等^[54]研究发现,在大肠杆菌性乳房炎中酪蛋白的表达受到αS1-酪蛋白基因（CSN1S1）远端启动子区的DNA 甲基化调控,这说明CpG岛甲基化变化可能与奶牛乳房炎的发病有重要关系。WANG等^[55]则通过重亚硫酸盐-焦磷酸测序技术定量检测了中国健康荷斯坦牛与乳房炎牛CD4基因的启动子CpG甲基化水平,结果发现临床乳房炎牛CD4基因启动子区的甲基化水平显著高于健康奶牛,从而导致该基因的表达水平下降,这表明CD4 基因启动子区的高DNA甲基化水平可作为奶牛乳房炎易感性的分子标记。而SONG等^[56]通过DNA甲基化免疫共沉淀-芯片（MeDIP-chip）结合亚硫酸氢盐测序（BSP）技术首次获得了金葡菌隐性乳房炎牛外周血淋巴细胞的全基因组DNA甲基化图谱,结果分析鉴定出的58个差异甲基化表达基因中有20.7%的超甲基化基因显著下调,14.3%显著上调;KEGG分析表明这些基因参与炎症反应、ErbB信号通路和DNA错配修复等;最终获得了三个新的DNA 甲基化修饰靶基因MST1、NRG1和NAT9,它们可作为潜在的金葡菌隐性乳房炎抗性生物学标记,该研究为奶牛乳房炎易感性的表观遗传学研究提供了新的依据。HE等^[57]则采用染色质免疫共沉淀（ChIP-seq）和数字基因表达谱（DGE-seq）技术对隐性乳房炎奶牛的淋巴细胞进行了测序,获得了奶牛组蛋白修饰H3K27me3 的全基因组表达谱及金葡菌乳房炎抗性相关的靶基因,发现隐性乳房炎组的H3K27me3在沉默基因中的表达水平显著高于健康牛,还确定了金葡菌乳房炎抗性重要候选基因PTX3、IL10等,该研究表明奶牛的金葡菌乳房炎抗性与组蛋白的甲基化调控密切相关。以上研究结果表明DNA、组蛋白甲基化等表观遗传因素对于奶牛乳房炎的发生、发展和维持等具有重要影响。奶牛产奶性状的遗传力属于中等遗传力,而奶牛乳房炎遗传力非常低,因此乳房炎相关基因更易受到环境影响而使DNA甲基化程度发生改变。

4 展望

中国是畜牧业大国,畜牧业的可持续发展是社会稳定的基础,奶牛在畜牧业中占用重要的地位,乳房炎是制约奶牛健康及乳品安全的关键,三大组学技术从蛋白质、氨基酸、代谢物等深入到基因的转录表达、转录后调控、表观修饰、信号通路等,从微观和分子角度极大地丰富了对于奶牛乳房炎的研究。与此同时,也应当清醒的意识到三大组学技术也存在一定的局限性,比如对于基因组测序而言,仍然是以Illumina等为首的国外公司独占鳌头,完全具有自主知识产权的国内测序平台仍然寥寥无几,而蛋白质组学和代谢组学相对高昂的成本也并非每个实验室都能够负担的起,因此,组学的应用推广工作任重而道远,需要每一位科研工作者的不断努力。

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