奶牛乳脂代谢关键候选基因的鉴定与功能分析

doi:10.3969/j.issn.1004-1524.20221710

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (12): 2794-2808.DOI: 10.3969/j.issn.1004-1524.20221710

奶牛乳脂代谢关键候选基因的鉴定与功能分析

李艳艳¹(), 卜建华², 韩丽云¹, 王川川³, 母童³^,^*()

1.宁夏农垦贺兰山奶业有限公司,宁夏银川 750205
2.宁夏农垦农林牧技术推广服务中心,宁夏银川 750024
3.宁夏大学农学院,宁夏银川 750021

收稿日期:2022-11-29 出版日期:2023-12-25 发布日期:2023-12-27
作者简介:李艳艳(1976—),女,宁夏银川人,学士,高级畜牧师,研究方向为动物繁育。E-mail: 13909513189@163.com
通讯作者: *母童,E-mail: mtlv3h@163.com
基金资助:
宁夏回族自治区育种专项优质高产奶牛选育资助项目(2019NYYZ0501)

Identification and functional analysis of key candidate genes for milk fat metabolism in dairy cattle

LI Yanyan¹(), BU Jianhua², HAN Liyun¹, WANG Chuanchuan³, MU Tong³^,^*()

1. Ningxia Agricultural Reclamation Helan Mountain Dairy Co., Ltd., Yinchuan 750205, China
2. Ningxia Agriculture and Forestry and Animal Husbandry Technology Extension Service Center, Yinchuan 750024, China
3. Agricultural College, Ningxia University, Yinchuan 750021, China

Received:2022-11-29 Online:2023-12-25 Published:2023-12-27

摘要/Abstract

摘要：

为深入挖掘影响荷斯坦奶牛乳脂代谢的关键候选基因,利用实时荧光定量聚合酶链式反应(qRT-PCR)技术检测课题组前期通过转录组和加权基因共表达网络分析(WGCNA)得到的15个候选差异基因的组织表达谱。确定乳脂代谢关键候选基因后检测其在原代奶牛乳腺上皮细胞(BMECs)中的定位,并进行克隆测序,以及结构和功能分析。结果表明,ENPP2、PI4K2A、CTSH和PTPRR基因在乳腺组织中的表达水平均高于其他组织,同时PI4K2A和CTSH在乳腺组织和原代BMECs中的表达相对于其他基因处于较高水平。通过qRT-PCR结合前期转录组测序的结果,最终确定PI4K2A为调控奶牛乳脂合成的关键候选基因,其主要在BMECs的细胞质中表达。克隆测序发现,奶牛PI4K2A基因编码区(CDS)全长1 440 bp,编码479个氨基酸。结构和功能分析表明,PI4K2A为非分泌型且不稳定的亲水蛋白,含有40个磷酸化位点,翻译后的修饰作用非常丰富。PI4K2A蛋白的二级和三级结构均以无规则卷曲为主,α螺旋次之,且在不同物种中具有较高的保守性。本研究确定PI4K2A为奶牛乳脂代谢的重要候选基因,研究结果可以为奶牛乳脂代谢的分子调控机制研究提供理论依据。

关键词: 荷斯坦奶牛, 候选基因, 乳脂, PI4K2A, 实时荧光定量聚合酶链式反应

Abstract:

In order to deeply explore the key candidate genes affecting milk fat metabolism in Holstein cows, this study used real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) to examine the tissue expression profiles of 15 candidate differential genes obtained by transcriptome and weighted gene co-expression network analysis (WGCNA) in the previous phase. After the key candidate genes for milk fat metabolism were identified, their localization in primary dairy mammary epithelial cells (BMECs) was examined, and clonal sequencing and functional analysis were performed. Results showed that the expression levels of ENPP2, PI4K2A, CTSH and PTPRR genes were higher in breast tissue than in other tissues, respectively, while the expression of PI4K2A and CTSH were at higher levels in both breast tissue and primary BMECs compared to other genes. The results of qRT-PCR combined with sequencing of the transcriptome finally identified PI4K2A as a key candidate gene regulating milk fat synthesis in dairy cows, mainly expressed in the cytoplasm of BMECs. Clone sequencing study revealed that the CDS (coding sequence) region of the cow PI4K2A gene was 1 440 bp in length and encoded 479 amino acids. Structural and functional analysis showed that PI4K2A was a non-secretory and unstable hydrophilic protein containing 40 phosphorylation sites with abundant post-translational modifications. Both the secondary and tertiary structure of PI4K2A protein were predominantly random coil, followed by α-helix, and was highly conserved across species. This study identified PI4K2A as an important candidate gene for milk fat metabolism in dairy cow, providing theoretical basis for the study of the molecular regulation mechanism of milk fat metabolism in dairy cow.

Key words: Holstein dairy cattle, candidate gene, milk fat, PI4K2A, real-time fluorescence quantitative polymerase chain reaction

中图分类号:

S823.91

李艳艳, 卜建华, 韩丽云, 王川川, 母童. 奶牛乳脂代谢关键候选基因的鉴定与功能分析[J]. 浙江农业学报, 2023, 35(12): 2794-2808.

LI Yanyan, BU Jianhua, HAN Liyun, WANG Chuanchuan, MU Tong. Identification and functional analysis of key candidate genes for milk fat metabolism in dairy cattle[J]. Acta Agriculturae Zhejiangensis, 2023, 35(12): 2794-2808.

图/表 15

表1 引物序列与退火温度

Table 1 Primer sequence and annealing temperature

基因登录号 GenBank ID	基因 Gene	上游引物序列 Forward primer sequence(5’→3’)	下游引物序列 Reverse primer sequence(5’→3’)	产物长度 Product length/bp	退火温度 Annealing temperature/℃
NM_001034034.2	GAPDH	TCGGAGTGAACGGATTCGG	TGATGACGAGCTTCCCGTTC	192	60.0
NM_173893.3	B2M	ACACCCACCAGAAGATGGAAAG	CAGGTCTGACTGCTCCGATTTA	124	60.0
NR_036642.1	18S RNA	ACTTTCGATGGTAGTCGCTGTGC	TCCTTGGATGTGGTAGCCGTTT	105	60.0
NM_001167834.1	CES4A	TTTGAGCACTACGCTCCTGG	CCACCATTGGGGTTTCCTGT	188	58.2
NM_001163802.3	ATP8A2	GCCCACAGCTGGAGAAGATA	GTACTTGGCCGTGCTGATCT	189	59.4
NM_001034385.2	CTSH	CGCCCAGAACTTCAACAACC	TACTTGCAGTCACCATCCTGG	132	59.4
NM_001100297.1	APOL3	GCAGACTCCTGGGGTGAAAC	TGGACAAATCAGCCTCGGTC	247	58.2
NM_001205544.1	DKK1	ATTGACAACCACCAGCCGTA	AGAAGGCATGCATATCCCGT	189	59.4
NM_001080293.1	ENPP2	TCACTTTTGCCGTCGGTGTCAA	AATCAGGGGGTCCAGCCTCTTG	174	58.0
NM_001083644.1	BCAT1	TGTGTTGTTTGCCCTGTTTC	GTCGCTCTCTTCTCTTCCGT	138	59.5
NM_001113261.1	PTPRR	TGAGGACAAGACAGCCAACAG	AAAGGAGAAGGGCAGACAGAG	129	56.4
NM_001075477	U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT	132	58.2
NM_173979.3	β-actin	CATCGGCAATGAGCGGTTCC	ACCGTGTTGGCGTAGAGGTC	80	60.0
NM_001083706.1	PDGFD	GTGAAAAAGTACCACGAGGTGT	TAAGTTCGGTTGCTGGTGGG	237	58.2
NM_174680.2	KCNMA1	CTAACCTGGAGCTGGAAGCCT	ACGCATCTGCTGACTCTATCTTGA	117	58.2
XM_005208272.4	ZFYVE28	ACCGCCTGTTTGTCTGTATCTC	TTCCTTGTCGTCCGTCTCTG	128	58.2
NM_001100316.1	PI4K2A	CGGAACCCCTTCCTGAGAAC	CCAGTCTGTGTCCCGAGAGT	161	58.2
NM_001100316.1	PI4K2A(CDS)	ATGGACGAGACGAGCCCACT	CTACCACCAGGAGAAGAAGGG	1 440	58.0
NM_001097568.2	ID1	CTGGGATCTGGAGTTGGAGC	GGAACACACGCCGCCTCT	135	58.2
NM_001101043.2	VEGFD	CCACTCGCAGGAATGGAAGATCAC	GAAAGGGGCATCTGTCCTCACA	238	58.2
NM_001037319.1	SLC16A1	TGGCAGCACCTTTATCCTCTAC	ACTCCACAATGGTCACCAATCC	160	58.2

表1 引物序列与退火温度

Table 1 Primer sequence and annealing temperature

基因登录号 GenBank ID	基因 Gene	上游引物序列 Forward primer sequence(5’→3’)	下游引物序列 Reverse primer sequence(5’→3’)	产物长度 Product length/bp	退火温度 Annealing temperature/℃
NM_001034034.2	GAPDH	TCGGAGTGAACGGATTCGG	TGATGACGAGCTTCCCGTTC	192	60.0
NM_173893.3	B2M	ACACCCACCAGAAGATGGAAAG	CAGGTCTGACTGCTCCGATTTA	124	60.0
NR_036642.1	18S RNA	ACTTTCGATGGTAGTCGCTGTGC	TCCTTGGATGTGGTAGCCGTTT	105	60.0
NM_001167834.1	CES4A	TTTGAGCACTACGCTCCTGG	CCACCATTGGGGTTTCCTGT	188	58.2
NM_001163802.3	ATP8A2	GCCCACAGCTGGAGAAGATA	GTACTTGGCCGTGCTGATCT	189	59.4
NM_001034385.2	CTSH	CGCCCAGAACTTCAACAACC	TACTTGCAGTCACCATCCTGG	132	59.4
NM_001100297.1	APOL3	GCAGACTCCTGGGGTGAAAC	TGGACAAATCAGCCTCGGTC	247	58.2
NM_001205544.1	DKK1	ATTGACAACCACCAGCCGTA	AGAAGGCATGCATATCCCGT	189	59.4
NM_001080293.1	ENPP2	TCACTTTTGCCGTCGGTGTCAA	AATCAGGGGGTCCAGCCTCTTG	174	58.0
NM_001083644.1	BCAT1	TGTGTTGTTTGCCCTGTTTC	GTCGCTCTCTTCTCTTCCGT	138	59.5
NM_001113261.1	PTPRR	TGAGGACAAGACAGCCAACAG	AAAGGAGAAGGGCAGACAGAG	129	56.4
NM_001075477	U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT	132	58.2
NM_173979.3	β-actin	CATCGGCAATGAGCGGTTCC	ACCGTGTTGGCGTAGAGGTC	80	60.0
NM_001083706.1	PDGFD	GTGAAAAAGTACCACGAGGTGT	TAAGTTCGGTTGCTGGTGGG	237	58.2
NM_174680.2	KCNMA1	CTAACCTGGAGCTGGAAGCCT	ACGCATCTGCTGACTCTATCTTGA	117	58.2
XM_005208272.4	ZFYVE28	ACCGCCTGTTTGTCTGTATCTC	TTCCTTGTCGTCCGTCTCTG	128	58.2
NM_001100316.1	PI4K2A	CGGAACCCCTTCCTGAGAAC	CCAGTCTGTGTCCCGAGAGT	161	58.2
NM_001100316.1	PI4K2A(CDS)	ATGGACGAGACGAGCCCACT	CTACCACCAGGAGAAGAAGGG	1 440	58.0
NM_001097568.2	ID1	CTGGGATCTGGAGTTGGAGC	GGAACACACGCCGCCTCT	135	58.2
NM_001101043.2	VEGFD	CCACTCGCAGGAATGGAAGATCAC	GAAAGGGGCATCTGTCCTCACA	238	58.2
NM_001037319.1	SLC16A1	TGGCAGCACCTTTATCCTCTAC	ACTCCACAATGGTCACCAATCC	160	58.2

图1 BMECs复苏培养 A,复苏培养24 h的BMECs,绿色箭头指向分泌的乳滴;B,复苏培养3 d的BMECs,绿色箭头指向细胞接触后的隆起。

Fig.1 Resuscitation culture of BMECs A, BMECs after 24 h of resuscitation culture, the green arrow points to the secreted milk droplet; B, BMECs after 3 d of resuscitation culture, with green arrows pointing to the bulge after cell contact.

图2 奶牛不同组织和BMECs的总RNA提取结果 A,1~7分别为奶牛小肠、肝、肾、子宫、心脏、卵巢和乳腺的总RNA提取结果;B,1和2是BMECs总RNA提取结果。M为DL 2000 DNA分子量标准。

Fig.2 Results of total RNA extraction from different tissues and BMECs of dairy cow A, 1-7 are the results of total RNA extraction from the small intestine, liver, kidney, uterus, heart, ovary and mammary gland of cow, respectively; B, 1 and 2 are the results of total RNA extraction from BMECs. M is DL 2000 DNA marker.

图3 不同内参基因的稳定性 A,不同内参基因Cq值的标准偏差和变异系数;B,不同内参基因Cq值的分布。

Fig.3 Stability of different internal reference genes A, Standard deviation and coefficient of variation of Cq values of different internal reference genes; B, Cq values of different internal reference genes.

图4 乳脂代谢候选差异基因在奶牛不同组织中的相对表达水平柱上无相同小写字母表示差异显著(P<0.05)。下同。

Fig.4 Relative expression levels of candidate differential genes of milk fat metabolism in different tissues of dairy cow The bars marked without the same lowercase letter indicated significant differences at P<0.05. The same as below.

图5 乳脂代谢候选差异基因在乳腺(A)和BMECs(B)中的表达差异 *表示与其他基因之间差异显著。

Fig.5 The expression differences of candidate differential genes of milk fat metabolism in mammary gland (A) and BMECs (B) * represented significant difference compared with other genes.

图6 牛PI4K2A基因的结构示意图

Fig.6 Schematic diagram of the structure of bovine PI4K2A gene

图7 PI4K2A基因克隆 A,PI4K2A基因CDS区PCR扩增结果,1和2为扩增产物;B,扩增产物回收纯化结果,1和2为回收产物;C,PI4K2A基因的菌液扩增结果,1~8为扩增产物。M为DL2000 DNA分子量标准。

Fig.7 Cloning of PI4K2A gene A, Results of PCR amplification of CDS region of PI4K2A gene, 1 and 2 are amplification products; B, Results of amplification product recovery and purification, 1 and 2 are recovered products; C, Results of bacteria amplification of PI4K2A gene, 1-8 are amplification products. M is DL2000 DNA marker.

图8 PI4K2A蛋白的氨基酸组成

Fig.8 Amino acid composition of PI4K2A protein

图9 PI4K2A蛋白功能预测 A,疏水性/亲水性预测;B,信号肽预测;C,跨膜结构预测;D,磷酸化位点预测。

Fig.9 Function prediction of PI4K2A protein A, Hydrophobicity/hydrophilicity prediction; B, Signal peptide prediction; C, Transmembrane structure prediction; D, Phosphorylation site prediction.

表2 PI4K2A蛋白的亚细胞定位预测

Table 2 Subcellular localization prediction of PI4K2A protein

预测软件 Prediction software	预测含量Prediction content/%
预测软件 Prediction software	细胞质 Cytoplasm	细胞核 Cell nucleus	线粒体 Mitochondria	液泡 Vacuole	高尔基体 Golgi apparatus	细胞骨架 Cytoskeleton
Euk-mPLOC 2.0	100	0	0	0	0	0
YLoc	87.1	12.8	0.1	0	0	0
MultiLoc2	86.0	13.0	1.0	0	0	0
PSORT II Prediction	34.8	26.1	21.8	8.7	4.3	4.3

图10 BMECs的细胞核、细胞质总RNA提取与PI4K2A mRNA的亚细胞定位 A,BMECs的核、质总RNA提取结果;B,PI4K2A在BMECs中的分布情况。

Fig.10 Total RNA extraction from nucleus and plasma of BMECs and subcellular localization of PI4K2A mRNA A, Results of nuclear and plasma total RNA extraction from BMECs; B, The distribution of PI4K2A in BMECs.

图11 不同物种PI4K2A蛋白的系统进化树分析

Fig.11 Phylogenetic tree analysis of PI4K2A proteins of different species

表3 不同物种PI4K2A蛋白二级结构预测

Table 3 Predicted secondary structure of PI4K2A protein in different species

物种 Species	占比Percentage/%
物种 Species	α螺旋 α-Helix	延伸链 Extended chain	β转角 β-Turn	无规则卷曲 Random coil
牛Bos taurus	34.66	14.61	6.47	44.26
绵羊Ovis aries	32.57	14.61	6.26	46.56
山羊Capra hircus	32.99	14.61	6.26	46.14
水牛	33.40	15.24	6.89	44.47
Bubalus bubalis
人Homo sapiens	35.07	14.41	6.05	44.47
猪Sus scrofa	34.86	14.61	5.01	45.51
小鼠Mus musculus	32.36	15.45	5.43	46.76
大鼠	33.26	15.27	6.49	44.98
Rattus norvegicus
原鸡Gallus gallus	34.12	14.59	6.01	45.28

图12 不同物种PI4K2A蛋白空间结构相似性分析红色箭头指向表示相对于牛而言,其他物种PI4K2A蛋白空间结构发生变化的位置。

Fig.12 Similarity analysis of the spatial structure of PI4K2A protein in different species Red arrows indicate the locations where the spatial structure of PI4K2A protein changes in other species relative to that of cattle.

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奶牛乳脂代谢关键候选基因的鉴定与功能分析

Identification and functional analysis of key candidate genes for milk fat metabolism in dairy cattle

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