丙酸对奶牛肝细胞糖异生的影响及其转录组学分析
丙酸是奶牛肝脏糖异生的主要前体物质，但是其对奶牛肝细胞糖异生的调控机制尚不清晰。为探究丙酸对奶牛肝细胞糖异生的影响并解析其调控的分子机制，本研究选择3.75 mmol·L−1的丙酸钠处理奶牛肝细胞，并收集细胞进行转录组测序，分析差异mRNA基因及其潜在的调控机制并通过qRT-PCR进行验证。结果表明，相比对照组，丙酸钠显著增加奶牛肝细胞代谢葡萄糖浓度(P < 0.05)。丙酸处理组和对照组共筛选331个差异表达基因(FDR < 0.05) (上调222个，下调109个)，上调基因包含炎症反应相关基因CYP1A1、CYP1B1和ARRB1等，下调基因包含氧化还原相关基因GPX2和GPX7等；筛选出ALDOC、JAK3和ANGPTL8等与糖代谢调节有关的基因。qRT-PCR验证了随机选取的10个差异基因的表达量与RNA-seq的分析结果一致。GO富集分析显示差异基因共富集至115 条，其中主要集中在催化活性和结合等功能。KEGG分析显示差异基因富集到12条代谢通路(P < 0.05)，主要包括胰岛素分泌和MAPK信号通路等。本研究筛选了丙酸调控奶牛肝细胞糖异生的相关基因与通路，为低值粗饲料的高效利用提供了新的思路和靶点。
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对于泌乳动物来说，葡萄糖作为乳糖合成的底物，是影响牛奶产量和奶品质的重要因素[1]。与单胃动物不同，肝脏糖异生作用所合成的内源性葡萄糖是反刍动物体内所需葡萄糖的主要来源(80%～90%) [2]。目前，优质牧草短缺是导致我国奶牛单产水平低、奶品质差的主要原因之一，致使近年来国外优质苜蓿的进口量剧增[3]。饲喂低值饲料稻草时，奶牛肝脏丙酮酸羧化酶、线粒体磷酸烯醇式丙酮酸羧激酶和磷酸果糖激酶的mRNA相对丰度降低[4]，肝脏糖异生通路丰度与功能均显著下调，葡萄糖生成量较少[5]，导致乳腺葡萄糖供给量和摄取量降低，最终降低了产奶量[6]。可见，肝脏糖异生功能下降是限制奶牛利用低值粗饲料的主要原因之一。因此，深入探究奶牛肝脏糖异生的代谢机制，有助于实现对其有效调控，利于提高低值粗饲料的利用效率，从而达到改善奶牛机体健康并提高生产性能的重要目的。
与单胃动物不同，奶牛瘤胃发酵生成的丙酸是其肝脏糖异生的主要前体物，可为糖异生提供 60%以上的碳源[7]。Zhang等[8]研究报道，灌注1.68 mol的丙酸可促进泌乳期奶牛肝脏糖异生进而提高血糖浓度。通过营养调控增加丙酸的生成量可增加肝脏糖异生，进而提高奶产量[9]。这些研究表明丙酸可有效调节奶牛肝脏糖异生。近年来，已有少量报道初步探讨丙酸影响奶牛肝脏糖异生的分子机制，但大都停留在调控已报到的糖异生关键酶基因mRNA的表达研究[10-11]。丙酸调控肝脏细胞糖异生是否存在其他潜在的分子靶点以及调控的信号通路均未见报道，需要进一步探究。
迄今，针对丙酸对奶牛肝细胞转录组学的影响尚未见报道，本研究利用RNA-seq技术，分析丙酸处理对奶牛肝细胞基因表达的影响，发掘潜在调控糖异生的靶点，为更精准、更科学调控奶牛肝脏糖异生提供理论依据，也为奶牛高效养殖提供新思路，还可以为低值粗饲料的高效利用提供新的思路和靶点。
1. 材料与方法
1.1 试验设计
采用改良的胶原酶法，对犊牛肝细胞进行分离培养，具体步骤可参照实验室前期研究报道[12]。培养细胞至第11代，72 h后分别用添加0 (对照组)和3.75 mmol·L−1 (处理组)丙酸钠(浓度根据前期预试验结果选择)的培养基培养12 h，收集细胞，每个处理3次重复。
1.2 试验材料
DMEM培养基、Ⅳ型胶原酶和胎牛血清(Gibco，美国)、青霉素和链霉素(Zeta life公司)。葡萄糖检测试剂盒(南京建成生物工程研究所，A154-1-1)，RNA提取与纯化试剂盒(TaKaRa公司，9767)、反转录酶和荧光定量PCR试剂盒(RR037A，RR086A，TaKaRa公司)。
1.3 葡萄糖检测
取肝细胞培养物，800 g离心5 min，收集细胞培养上清液，用葡萄糖检测试剂盒(南京建成生物工程研究所，A154-1-1)检测葡萄糖浓度。
1.4 奶牛肝细胞转录组学的测序分析
奶牛肝细胞总 RNA 提取与质量检测：使用Trizol法进行奶牛肝细胞总RNA的提取，分别利用NanoDrop和Qubit2.0检测RNA的纯度及浓度。Agilent 2100分析所提RNA样品的完整性，所有RNA样品提取并检验合格后，−80 ℃保存。
cDNA文库制备及质量控制：RNA质量检测合格后，室温变性打开其二级结构，使用适温变性打开其二级结构，使用oligo (dT)磁珠富集mRNA；以mRNA片段为模板，利用缓冲液、六碱基随机引物(random hexamers)、dNTPs和DNA polymeraseⅠ合成cDNA，AM-pure XP beads 纯化双链cDNA，再在3′末端加上A碱基，使接头与cDNA连接；最后进行PCR富集得到链特异性cDNA文库。
测序数据分析与差异表达基因(DEGs)的筛选 文库构建完成后，利用DNBSEQ (深圳华大基因)进行测序分析，原始数据使用过滤软件SOAPnuke (v1.5.2)去除低质量reads和测序接头，获得高质量的clean reads [13]。利用HISAT (v2.1.0) 软件与牛的参考基因组(GCF_002263795.1_ARS-UCD1.2)进行序列比对，获得奶牛肝细胞样本特异序列信息，进行基因组的定位分析。以FDR < 0.05且|log2FC| > 1为阈值筛选DEGs，其中FC (fold change)代表两组之间基因表达的差异倍数。
差异基因的实时荧光定量PCR (qRT-PCR)验证：为了验证RNA-Seq测定结果，随机挑选果糖二磷酸醛缩酶C (fructose-bisphosphate aldolase C，ALDOC)、胶原α-1 (I)链 (collagen alpha-1 (I) chain， COL1A1)、酪氨酸蛋白激酶A3受体(ephrin-A3，EFNA3)、整合素亚单位β4 (integrin subunit beta 4，ITGB4)、整合素亚单位β6 (integrin subunit beta 6，ITGB6)、酪氨酸蛋白激酶 (tyrosine-protein kinase JAK3)、Lamin亚单位α-1 (laminin subunit alpha-1，LAMA1)、转录因子SOX9 (transcription factor，SOX9)、高流动性组蛋白2 (high mobility group protein 2，HMGA2)以及叉头框蛋白P3 (forkhead box protein P3，FOXP3)共10个DEGs，利用Primer5.0软件设计扩增引物(表1)，采用qRT-PCR的方法验证其表达水平。将各处理的样品总RNA反转录合成cDNA，qRT-PCR 的反应体系、反应程序以及基因相对表达量的计算方法均参照 Li等[14]。每个样品3个重复，采用2−ΔΔct法计算DEGs相对定量的结果。
表 1 qRT-PCR反应所用引物Table 1. Forward and reverse primers used for qRT-PCR序号
No.基因
Gene登录号
GenBank ID引物序列
Primer sequences (5′-3′)产物大小
Product size/bp退火温度
Annealing temperature/℃1 ALDOC NM_001097984.2 F: GCCCAGGGCAAATATGAAGG
R: CGTGGTTGGCAATGTAGAGG73 65.5 2 COL1A1 NM_001034039.2 F: CCACCTCAAGAGAAGGCTCA
R: ACCACATTGGCATCATCAGC62 65.0 3 EFNA3 NM_001105429.1 F: CCACCAAGTAGGGCACTGTA
R: CATTACCAGAGCCACAAGGC77 65.6 4 ITGB4 NM_001193257.2 F: GGAGCGAGAGGGAATCATCA
R: GAGCCACTTGCTTCCAGATG192 66.0 5 ITGB6 NM_ 174698.2 F: TCGCCAGACTGAGGATTACC
R: GGGAGATGGGTTTCTCCACA176 66.0 6 JAK3 XM_010806605.3 F: TGTCATCCGTGACCTGAACA
R: TTGCCCAGCTGTGAGATGTA174 63.5 7 LAMA1 XM_024984606.1 F: AGTCCTTCGACTTCAGCACA
R: GATGGTTCCCAACTGAGGGA105 65.0 8 SOX9 XM_024981096.1 F: CCGGCTCCGACACCGAGAACA
R: CCAGCGTCCAGTCGTAGCCCT150 71.0 9 HMGA2 XM_002704288 F: TTATCCGCCCACGATTAGAG
R: TTGAGTGTGTGTGTGCTTGG72 63.5 10 FOXP3 NM_001045933.1 F: ACCTGGAAGAATGCCATCCGCC
R: TGTGGGGTTGGAACACCTGCTG147 69.0 11 β-actin NM_ 173979.3 F: TGTGCTGTCCCTGTATGCCTCTG
R: TTGGGAATGCTCGATCCAACCG910 68.0 差异表达基因的生物信息分析：对DEGs进Gene Ontology (GO) (http://www.geneontology.org/)和KEGG (https://www.kegg.jp/)富集分析，P ≤ 0.05为显著富集[11]。利用STRING数据库的蛋白质互作信息构建丙酸调节奶牛肝细胞糖异生相关差异表达基因的蛋白质互作网络(Protein-Protein Interaction Network) (置信度分值 > 0.7) [10]。
1.5 数据统计分析
葡萄糖浓度数据使用SPSS 22.0中的Student’s-ttest进行统计分析，P < 0.05为差异显著，采用GraphPad Prism 8软件进行作图。
2. 结果与分析
2.1 奶牛肝细胞代谢的葡萄糖浓度
对照组葡萄糖浓度为3.04 mmol·L−1，而处理组葡萄糖浓度为4.57 mmol·L−1，该结果表明丙酸的添加显著增加了奶牛肝细胞代谢葡萄糖浓度(P < 0.05)。
2.2 测序数据质控结果
本研究采用DNBSEQ平台对两种不同浓度丙酸(0和3.75 mmol·L−1)处理的奶牛肝细胞进行RNA测序。测序结果显示，共获得273 420万条reads，过滤后获得 268 070万条高质量数据(clean reads)，样本过滤之后clean reads比例均在97%以上，表明样品构建文库质量较好；同时 clean reads Q30比例均在93%以上，说明测序结果可靠，可用于下一步分析。
2.3 差异表达基因分析
对两个试验组的基因表达进行分析，以|log2FC| > 1、Q < 0.05为筛选条件，共筛选331个DEGs。其中，与对照组相比，丙酸处理组上调的基因222个，包括JAK3、ALDOC、趋化因子受体1型(chemokine receptor type 1，CXCR1)等；下调的基因109个，血管生成素样蛋白8 (angiopoietin-like protein 8，ANGPTL8)、NADPH氧化酶激活剂1 (NADPH oxidase activator 1，NOXA1)及FOXP3等，部分结果如表2所列。
表 2 转录组测序前20个上下调基因Table 2. The top 20 up- and down-regulated genes between the two groups基因名称
Gene name基因描述
Gene descriptionlog2FC CXCR1 趋化因子受体1型 Chemokine receptor type 1 4.88 SLC17A7 谷氨酸细胞转运体1 Vesicular glutamate transporter 1 3.94 TEX13B 睾丸表达蛋白13B Testis-expressed protein 13B 3.67 ASIC1 酸感应离子通道1 Acid-sensing ion channel 1 3.57 DES 肌间线蛋白 Desmin 3.56 CACNA1S 电压依赖性L型钙通道亚单位α-1S Voltage-dependent L-type calcium channel subunit alpha-1S 3.36 GGT1 谷胱甘肽水解酶 Glutathione hydrolase 3.33 CYP1A1 细胞色素P450 1A Cytochrome P450 1A 3.19 HSD11B2 羟类固醇11-β脱氢酶2 Hydroxysteroid 11-beta dehydrogenase 2 3.18 PPP1R1B 蛋白磷酸酶1调节抑制器亚单位1B Protein phosphatase 1 regulatory inhibitor subunit 1B 3.12 GRIN3B 谷氨酸电离性受体 Glutamate receptor ionotropic 3.00 ADGRG5 粘附性G-蛋白偶联受体G5 Adhesion G-protein coupled receptor G5 2.75 SAT2 三甘氨酸N-ε-乙酰转移酶Triglycine N-epsilon-acetyltransferase 2.74 TRPV2 瞬时受体电位阳离子通道亚家族V成员2
Transient receptor potential cation channel subfamily V member 22.68 TAGLN3 转基因蛋白-3 Transgelin-3 2.65 SBK2 丝氨酸/苏氨酸蛋白激酶 Serine/threonine-protein kinase 2.61 TSPAN1 四萜苷Tetraspanin 2.59 APOL3 载脂蛋白L3 Apolipoprotein L3 2.51 CCDC116 含有螺旋结构域的蛋白116 Coiled-coil domain-containing protein 116 2.49 TRIM55 含有蛋白质55的三元基序 Tripartite motif-containing protein 55 2.49 STRC 立体纤毛蛋白 Stereocilin −6.29 COCH 科克林 Cochlin −5.79 FAM71A 具有序列相似性的家族71，成员A Family with sequence similarity 71, member A −4.30 RGL3 鸟嘌呤核苷酸解离刺激因子样3 Ral guanine nucleotide dissociation stimulator-like 3 −4.29 HEPACAM 肝细胞粘附分子 Hepatocyte cell adhesion molecule −3.41 ANGPTL8 血管生成素样蛋白8 Angiopoietin-like protein 8 −2.67 ATP2B2 钙运输ATP酶 Calcium-transporting ATPase −2.47 CCDC151 含有卷曲结构域的151 Coiled-coil domain containing 151 −2.41 NOXA1 NADPH氧化酶激活剂1 NADPH oxidase activator 1 −2.40 FBLL1 rRNA/tRNA 2'-O-甲基转移酶 纤维素样蛋白1
rRNA/tRNA 2'-O-methyltransferase fibrillarin-like protein 1−2.33 NECAB3 N端钙结合蛋白3 N-terminal EF-hand calcium-binding protein 3 −2.28 SOX9 转录因子SOX-9 Transcription factor SOX-9 −2.00 PDK2 蛋白−丝氨酸/苏氨酸激酶 Protein-serine/threonine kinase −1.94 FOXP3 叉头框蛋白P3 Fork head box protein P3 −1.93 CARMIL3 盖帽蛋白调节器和肌球蛋白1连接器3 Capping protein regulator and myosin 1 linker 3 −1.92 NLGN3 神经元-3 Neuroligin-3 −1.89 CCDC85B 含有卷曲结构域的85B Coiled-coil domain containing 85B −1.86 CNTNAP1 接触素相关蛋白1 Contacting associated protein 1 −1.79 HMGA2 高流动性组蛋白 High mobility group protein −1.79 2.4 qRT-PCR验证
随机选取了10个基因进行qRT-PCR扩增以验证RNA-seq的准确性，所选的10个基因中，在丙酸处理下7个上调，3个下调，qRT-PCR扩增结果表达趋势与RNA-seq一致，表明本研究RNA-seq测序结果可靠(图1)。
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图 1 qRT-PCR与RNA-seq基因表达水平比较Figure 1. Comparison of the quantitative results for selected genes from the RNA-seq and qRT-PCR analyses2.5 差异表达基因的富集分析
为了进一步了解DEGs的功能，对331个DEGs进行GO功能注释(表3)，共发现574条显著富集的GO条目(P < 0.05)。经过GO富集分析，细胞组分(cellular component，CC)共富集到53个条目，其中质膜的内在成分和受体复合体占比最高；分子功能(molecular function，MF)富集到115个条目，其中钙通道活性和蛋白酪氨酸激酶活性占比较高；生物过程(biological process，BP)共富集到406个条目，其中中胚层细胞分化占比最高。
表 3 差异表达基因的前10 GO分析Table 3. GO analysis of the top 10 differentially expressed genes分类
ClassificationGO编号
GO number条目
TermP 基因数量
Gene number生物学过程
Biological
processGO:0007165 信号转导 Signal transduction 0.002 450 GO:0045662 成肌细胞分化的负调控 Negative regulation of myoblast differentiation 0.003 20 GO:0032720 肿瘤坏死因子产生的负调控 Negative regulation of tumor necrosis factor production 0.004 42 GO:0071260 细胞对机械刺激的反应 Cellular response to mechanical stimulus 0.010 47 GO:0032689 干扰素γ产生的负调控 Negative regulation of interferon-gamma production 0.010 26 GO:0048333 中胚层细胞分化 Mesodermal cell differentiation 0.010 9 细胞组分
Cellular
componentGO:0005887 质膜的组成部分 Integral component of plasma membrane 0.002 870 GO:0098978 谷氨酸能突触 Glutamatergic synapse 0.003 224 GO:0005737 细胞质 Cytoplasm 0.004 3 788 GO:0043235 受体复合物 Receptor complex 0.010 186 GO:0031982 囊泡 Vesicle 0.010 69 GO:0031226 质膜固有成分 Intrinsic component of plasma membrane 0.010 14 GO:0005604 基底膜 Basement membrane 0.010 72 GO:0005783 内质网 Endoplasmic reticulum 0.020 686 GO:0005576 胞外区 Extracellular region 0.020 821 GO:0009925 细胞基膜 Basal plasma membrane 0.030 21 分子功能
Molecular
functionGO:0015631 微管蛋白结合 Tubulin binding 0.020 44 GO:0005518 胶原结合 Collagen binding 0.030 49 GO:0004714 跨膜受体蛋白酪氨酸激酶活性
Transmembrane receptor protein tyrosine kinase activity0.034 89 GO:0004114 3′, 5′-环核苷酸磷酸二酯酶活性
3′, 5′-cyclic-nucleotide phosphodiesterase activity0.040 22 GO:0101020 雌激素16α-羟化酶活性 Estrogen 16-alpha-hydroxylase activity 0.040 3 GO:0001228 转录激活剂活性，RNA聚合酶
Transcriptional activator activity, RNA polymerase II
transcription regulatory region sequence-specific binding0.040 401 2.6 差异表达基因KEGG分析
KEGG通路分析发现，DEGs共显著富集到12条通路，包括：胰岛素分泌(insulin secretion)、有丝分裂活化蛋白激酶通路(MAPK)、糖磷脂生物合成−乳酸和新乳酸系列(glycosphingolipid biosynthesis-lacto and neolacto series)、花生四烯酸代谢(arachidonic acid metabolism)、细胞凋亡(apoptosis)和cAMP信号通路(cAMP signaling pathway)等(表4)。
表 4 差异表达基因的KEGG分析Table 4. KEGG analysis of differentially expressed genes编号
No.通路名称
Pathway name基因数量
Gene numberP 富集率
Rich ratio4060 细胞因子−细胞因子受体的相互作用 Cytokine-cytokine receptor interaction 12 0.01 0.04 4010 MAPK信号传导途径 Mitogen-activated protein kinase signaling pathway 11 0.01 0.04 601 糖磷脂生物合成−乳酸和新乳酸系列
Glycosphingolipid biosynthesis−lactose and neglect series3 0.01 0.10 590 花生四烯酸代谢 Arachidonic acid metabolism 5 0.01 0.06 4512 ECM与受体的相互作用 ECM-receptor interaction 5 0.02 0.06 4810 肌动蛋白细胞骨架调控 Regulation of actin cytoskeleton 8 0.03 0.04 140 类固醇激素的生物合成 Steroid hormone biosynthesis 4 0.03 0.06 4210 细胞凋亡 Apoptosis 6 0.03 0.04 910 氮代谢 Nitrogen metabolism 2 0.03 0.12 4918 甲状腺激素的合成 Thyroid hormone synthesis 4 0.04 0.05 4024 cAMP信号传导途径 cAMP signaling pathway 8 0.04 0.03 4911 胰岛素分泌 Insulin secretion 4 0.04 0.05 2.7 差异基因的蛋白互作网络分析
蛋白质互作网络分析结果发现，在331个差异表达基因中，有55个基因之间存在较强的互作关系。越靠近互作图的中心位置，表示该基因可能发挥的作用越大。 BMP4、GPX2、JAK3、COL1A1以及ARRB1等与其他基因有较强的互作关系(图2)。
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图 2 差异表达基因的PPI分析图中基因简称参见附件1。Figure 2. PPI analysis of the proteins encoded by the differentially expressed genesRefor to annex 1 for gene abbreviations in the figure.3. 讨论
Rathert-Williams等[15]研究报道饲粮添加丙酸钙可显著提高肉牛血糖含量。奶牛肝脏糖异生的合成量与丙酸的供给量正相关[16]。Zhang等[8] 进一步研究报道奶牛体内灌注丙酸可增加PEPCK2和G6PC的mRNA表达，从而促进奶牛的肝脏糖异生。上述研究结果表明丙酸可通过调控奶牛糖异生的关键酶基因表达进而调节糖异生。本研究同样发现丙酸的添加可显著增加奶牛肝细胞代谢葡萄糖浓度，因此借助转录组学技术，以期筛选出丙酸调节奶牛肝脏糖异生的潜在基因和通路。
本研究共筛选出331个差异基因，并对其中10个基因进行了qRT-PCR验证，验证结果与测序结果一致，表明测序结果可靠。丙酸的添加显著增加了ALDOC和JAK3表达量，降低了ANGPTL8基因表达量。JAK3是Janus激酶家族的一个成员，广泛表达于各种器官[17]，Mishra等[18]研究发现敲除 JAK3基因后血糖动态平衡被破坏，可见JAK3在调节血糖动态平衡中起到重要作用。ALDO家族包括3种亚型，其中ALDOC参与到糖代谢通路中，是糖酵解中的关键酶[19]。血管生成素样蛋白(ANGPTLs)，具有8种分泌蛋白，其中ANGPTL8又被称为促代谢因子，在肝脏和直肠中大量表达，与糖异生作用等促能量消耗等过程负相关[20]。Guo等[21]研究报道，ANGPTL8通过AKT-GSK3b 或 AKT-FoxO1分支增强胰岛素信号通路的降糖作用，ANGPTL8可以磷酸化AKT的丝氨酸473，进而允许糖原合成并抑制糖异生。可见，ALDOC、JAK3以及ANGPTL8基因均参与糖的代谢调节中，极有可能作为丙酸调控奶牛肝细胞糖异生的分子靶点，其具体调控机制需进一步深入探究。
本研究通过KEGG富集分析发现与类固醇代谢相关的MAPK信号通路以及与血糖调节相关的胰岛素分泌通路得到显著富集。上调基因PLA2G4E富集于MAPK信号通路。PLA2G4E属于胞浆型的磷脂酶A2，是由10组亚型组成的一组膜磷脂选择性水解酶，研究发现PLA2G4E与胰岛素敏感性显著相关[22]。上调基因CACNA1S和CACNA1F基因富集于胰岛素分泌通路。CACNA1S和CACNA1F均是离子通道蛋白，其表达量增加可激活内质网中钙释放通道的开放，增加细胞质中钙离子浓度[23-24]，而适当的钙离子浓度可促进胰岛素分泌[25]。这表明丙酸的添加对激活胰岛素分泌通路有促进作用，可能是由于葡萄糖合成量增加所致。
谷胱甘肽过氧化酶能够催化还原过氧化氢和有机过氧化物，将其转变为水和相应的有机醇，起着抗氧化和维持细胞内氧化还原平衡的作用，在哺乳动物中已经鉴定出8个成员(GPX1～GPX8) [26]。细胞色素P450是多种酶组成的亚铁血红素蛋白族，细胞色素P4501A1 (CYP1A1)和细胞色素P4501B1 (CYP1B1)是肝外酶，是P450亚族CYP1酶的成员，能参与多种内、外源化合物的氧化/还原反应，其活性与炎症的发展密切相关[27]。通过PPI互作图发现，ARRB1与其他基因有较强的互作关系。ARRB1是抑制蛋白β家族重要一员，具有分子支架样的结构与其他伴侣蛋白相互作用来调节细胞的功能，参与免疫应答和炎症反应等多个生理过程[28]。本研究中，丙酸的添加显著降低了GPX2和GPX7基因表达，增加了CYP1A1、CYP1B1和ARRB1基因表达，表明丙酸的添加可能对奶牛肝细胞氧化还原能力有影响。
4. 结论
综上所述，丙酸可通过调节关键基因的表达调控奶牛肝细胞糖异生；ALDOC、JAK3和ANGPTL8基因可作为丙酸调控奶牛肝细胞糖异生的潜在靶基因，丙酸的添加激活胰岛素相关信号通路；丙酸降低了奶牛肝细胞氧化还原基因表达，增加炎症反应相关基因表达，提示丙酸可能影响了肝细胞氧化还原能力。本研究筛选了丙酸调控奶牛肝细胞糖异生的相关基因与通路，为低值粗饲料的高效利用提供新的思路和靶点。
参考文献
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图 1 qRT-PCR与RNA-seq基因表达水平比较
Figure 1. Comparison of the quantitative results for selected genes from the RNA-seq and qRT-PCR analyses
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图 2 差异表达基因的PPI分析
图中基因简称参见附件1。
Figure 2. PPI analysis of the proteins encoded by the differentially expressed genes
Refor to annex 1 for gene abbreviations in the figure.
表 1 qRT-PCR反应所用引物
Table 1 Forward and reverse primers used for qRT-PCR
序号
No.基因
Gene登录号
GenBank ID引物序列
Primer sequences (5′-3′)产物大小
Product size/bp退火温度
Annealing temperature/℃1 ALDOC NM_001097984.2 F: GCCCAGGGCAAATATGAAGG
R: CGTGGTTGGCAATGTAGAGG73 65.5 2 COL1A1 NM_001034039.2 F: CCACCTCAAGAGAAGGCTCA
R: ACCACATTGGCATCATCAGC62 65.0 3 EFNA3 NM_001105429.1 F: CCACCAAGTAGGGCACTGTA
R: CATTACCAGAGCCACAAGGC77 65.6 4 ITGB4 NM_001193257.2 F: GGAGCGAGAGGGAATCATCA
R: GAGCCACTTGCTTCCAGATG192 66.0 5 ITGB6 NM_ 174698.2 F: TCGCCAGACTGAGGATTACC
R: GGGAGATGGGTTTCTCCACA176 66.0 6 JAK3 XM_010806605.3 F: TGTCATCCGTGACCTGAACA
R: TTGCCCAGCTGTGAGATGTA174 63.5 7 LAMA1 XM_024984606.1 F: AGTCCTTCGACTTCAGCACA
R: GATGGTTCCCAACTGAGGGA105 65.0 8 SOX9 XM_024981096.1 F: CCGGCTCCGACACCGAGAACA
R: CCAGCGTCCAGTCGTAGCCCT150 71.0 9 HMGA2 XM_002704288 F: TTATCCGCCCACGATTAGAG
R: TTGAGTGTGTGTGTGCTTGG72 63.5 10 FOXP3 NM_001045933.1 F: ACCTGGAAGAATGCCATCCGCC
R: TGTGGGGTTGGAACACCTGCTG147 69.0 11 β-actin NM_ 173979.3 F: TGTGCTGTCCCTGTATGCCTCTG
R: TTGGGAATGCTCGATCCAACCG910 68.0 
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表 2 转录组测序前20个上下调基因
Table 2 The top 20 up- and down-regulated genes between the two groups
基因名称
Gene name基因描述
Gene descriptionlog2FC CXCR1 趋化因子受体1型 Chemokine receptor type 1 4.88 SLC17A7 谷氨酸细胞转运体1 Vesicular glutamate transporter 1 3.94 TEX13B 睾丸表达蛋白13B Testis-expressed protein 13B 3.67 ASIC1 酸感应离子通道1 Acid-sensing ion channel 1 3.57 DES 肌间线蛋白 Desmin 3.56 CACNA1S 电压依赖性L型钙通道亚单位α-1S Voltage-dependent L-type calcium channel subunit alpha-1S 3.36 GGT1 谷胱甘肽水解酶 Glutathione hydrolase 3.33 CYP1A1 细胞色素P450 1A Cytochrome P450 1A 3.19 HSD11B2 羟类固醇11-β脱氢酶2 Hydroxysteroid 11-beta dehydrogenase 2 3.18 PPP1R1B 蛋白磷酸酶1调节抑制器亚单位1B Protein phosphatase 1 regulatory inhibitor subunit 1B 3.12 GRIN3B 谷氨酸电离性受体 Glutamate receptor ionotropic 3.00 ADGRG5 粘附性G-蛋白偶联受体G5 Adhesion G-protein coupled receptor G5 2.75 SAT2 三甘氨酸N-ε-乙酰转移酶Triglycine N-epsilon-acetyltransferase 2.74 TRPV2 瞬时受体电位阳离子通道亚家族V成员2
Transient receptor potential cation channel subfamily V member 22.68 TAGLN3 转基因蛋白-3 Transgelin-3 2.65 SBK2 丝氨酸/苏氨酸蛋白激酶 Serine/threonine-protein kinase 2.61 TSPAN1 四萜苷Tetraspanin 2.59 APOL3 载脂蛋白L3 Apolipoprotein L3 2.51 CCDC116 含有螺旋结构域的蛋白116 Coiled-coil domain-containing protein 116 2.49 TRIM55 含有蛋白质55的三元基序 Tripartite motif-containing protein 55 2.49 STRC 立体纤毛蛋白 Stereocilin −6.29 COCH 科克林 Cochlin −5.79 FAM71A 具有序列相似性的家族71，成员A Family with sequence similarity 71, member A −4.30 RGL3 鸟嘌呤核苷酸解离刺激因子样3 Ral guanine nucleotide dissociation stimulator-like 3 −4.29 HEPACAM 肝细胞粘附分子 Hepatocyte cell adhesion molecule −3.41 ANGPTL8 血管生成素样蛋白8 Angiopoietin-like protein 8 −2.67 ATP2B2 钙运输ATP酶 Calcium-transporting ATPase −2.47 CCDC151 含有卷曲结构域的151 Coiled-coil domain containing 151 −2.41 NOXA1 NADPH氧化酶激活剂1 NADPH oxidase activator 1 −2.40 FBLL1 rRNA/tRNA 2'-O-甲基转移酶 纤维素样蛋白1
rRNA/tRNA 2'-O-methyltransferase fibrillarin-like protein 1−2.33 NECAB3 N端钙结合蛋白3 N-terminal EF-hand calcium-binding protein 3 −2.28 SOX9 转录因子SOX-9 Transcription factor SOX-9 −2.00 PDK2 蛋白−丝氨酸/苏氨酸激酶 Protein-serine/threonine kinase −1.94 FOXP3 叉头框蛋白P3 Fork head box protein P3 −1.93 CARMIL3 盖帽蛋白调节器和肌球蛋白1连接器3 Capping protein regulator and myosin 1 linker 3 −1.92 NLGN3 神经元-3 Neuroligin-3 −1.89 CCDC85B 含有卷曲结构域的85B Coiled-coil domain containing 85B −1.86 CNTNAP1 接触素相关蛋白1 Contacting associated protein 1 −1.79 HMGA2 高流动性组蛋白 High mobility group protein −1.79
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表 3 差异表达基因的前10 GO分析
Table 3 GO analysis of the top 10 differentially expressed genes
分类
ClassificationGO编号
GO number条目
TermP 基因数量
Gene number生物学过程
Biological
processGO:0007165 信号转导 Signal transduction 0.002 450 GO:0045662 成肌细胞分化的负调控 Negative regulation of myoblast differentiation 0.003 20 GO:0032720 肿瘤坏死因子产生的负调控 Negative regulation of tumor necrosis factor production 0.004 42 GO:0071260 细胞对机械刺激的反应 Cellular response to mechanical stimulus 0.010 47 GO:0032689 干扰素γ产生的负调控 Negative regulation of interferon-gamma production 0.010 26 GO:0048333 中胚层细胞分化 Mesodermal cell differentiation 0.010 9 细胞组分
Cellular
componentGO:0005887 质膜的组成部分 Integral component of plasma membrane 0.002 870 GO:0098978 谷氨酸能突触 Glutamatergic synapse 0.003 224 GO:0005737 细胞质 Cytoplasm 0.004 3 788 GO:0043235 受体复合物 Receptor complex 0.010 186 GO:0031982 囊泡 Vesicle 0.010 69 GO:0031226 质膜固有成分 Intrinsic component of plasma membrane 0.010 14 GO:0005604 基底膜 Basement membrane 0.010 72 GO:0005783 内质网 Endoplasmic reticulum 0.020 686 GO:0005576 胞外区 Extracellular region 0.020 821 GO:0009925 细胞基膜 Basal plasma membrane 0.030 21 分子功能
Molecular
functionGO:0015631 微管蛋白结合 Tubulin binding 0.020 44 GO:0005518 胶原结合 Collagen binding 0.030 49 GO:0004714 跨膜受体蛋白酪氨酸激酶活性
Transmembrane receptor protein tyrosine kinase activity0.034 89 GO:0004114 3′, 5′-环核苷酸磷酸二酯酶活性
3′, 5′-cyclic-nucleotide phosphodiesterase activity0.040 22 GO:0101020 雌激素16α-羟化酶活性 Estrogen 16-alpha-hydroxylase activity 0.040 3 GO:0001228 转录激活剂活性，RNA聚合酶
Transcriptional activator activity, RNA polymerase II
transcription regulatory region sequence-specific binding0.040 401
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表 4 差异表达基因的KEGG分析
Table 4 KEGG analysis of differentially expressed genes
编号
No.通路名称
Pathway name基因数量
Gene numberP 富集率
Rich ratio4060 细胞因子−细胞因子受体的相互作用 Cytokine-cytokine receptor interaction 12 0.01 0.04 4010 MAPK信号传导途径 Mitogen-activated protein kinase signaling pathway 11 0.01 0.04 601 糖磷脂生物合成−乳酸和新乳酸系列
Glycosphingolipid biosynthesis−lactose and neglect series3 0.01 0.10 590 花生四烯酸代谢 Arachidonic acid metabolism 5 0.01 0.06 4512 ECM与受体的相互作用 ECM-receptor interaction 5 0.02 0.06 4810 肌动蛋白细胞骨架调控 Regulation of actin cytoskeleton 8 0.03 0.04 140 类固醇激素的生物合成 Steroid hormone biosynthesis 4 0.03 0.06 4210 细胞凋亡 Apoptosis 6 0.03 0.04 910 氮代谢 Nitrogen metabolism 2 0.03 0.12 4918 甲状腺激素的合成 Thyroid hormone synthesis 4 0.04 0.05 4024 cAMP信号传导途径 cAMP signaling pathway 8 0.04 0.03 4911 胰岛素分泌 Insulin secretion 4 0.04 0.05
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[1] ZHAO F Q. Biology of glucose transport in the mammary gland. Journal of Mammary Gland Biology and Neoplasia, 2014, 19: 3-17. doi: 10.1007/s10911-013-9310-8
[2] ASCHENBACH J R, KRISTENSEN N B, DONKIN S S, HAMMON H M, PENNER G B. Gluconeogenesis in dairy cows: The secret of making sweet milk from sour dough. IUBMB Life, 2010, 62(12): 869-877. doi: 10.1002/iub.400
[3] 张英俊. 饲草间混套作在农区的应用前景. 中国乳业, 2021(8): 33. ZHANG Y J. The application prospect of forage intercropping in the agricultural region. China Dairy, 2021(8): 33.
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