刘莉莉¹, 郭爱伟¹, 吴培福¹, 陈粉粉¹, 杨亚晋¹, 张勤^,2 1. 西南林业大学生命科学学院,昆明 650224
2. 中国农业大学动物科技学院,畜禽育种国家工程实验室,北京 1000193

Regulation of VPS28 gene knockdown on the milk fat synthesis in Chinese Holstein dairy

Lili Liu¹, Aiwei Guo¹, Peifu Wu¹, Fenfen Chen¹, Yajin Yang¹, Qin Zhang^,2 1. College of Life Science, Southwest Forestry University, Kunming 650224, China
2. Key Laboratory of Animal Genetics and Breeding of Ministry of Agriculture,College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

通讯作者: 张勤,博士,教授,研究方向：动物遗传育种。E-mail: qzhang@cau.edu.cn

编委: 蒋思文
收稿日期:2018-05-15修回日期:2018-07-31网络出版日期:2018-12-20

基金资助:

国家自然科学基金项目.3120772 国家转基因育种发展专项.2014ZX0800953B 教育部重点学科生物学一级学科建设项目.51400666 云南省教育厅科学研究基金教师类项目.2018JS333

Received:2018-05-15Revised:2018-07-31Online:2018-12-20

Fund supported:

Supported by the National Natural Science Foundation of China.3120772 the National Major Development Program of Transgenic Breeding.2014ZX0800953B Ministry of education key subject biology level discipline construction project.51400666 Yunnan Provincial Department of Education Fund for Scientific Research.2018JS333

作者简介 About authors
刘莉莉,博士,讲师,研究方向：动物遗传育种E-mail:liulily0518@163.com。








摘要
本课题组前期通过GWAS研究,发现VPS28基因在荷斯坦奶牛乳腺组织中特异性高表达,且其5′-UTR的突变位点-58C>T与乳脂性状关联,但其对乳脂性状的调控机理尚未明确。本研究为了明确VPS28基因及其突变位点-58C>T对乳脂的调控机理,首先利用启动子活性分析检测突变位点-58C>T对VPS28基因的影响,发现该突变位点显著降低VPS28基因启动子活性;然后利用RNA干扰技术敲降奶牛原代乳腺上皮细胞中VPS28基因表达量,检测VPS28通路和乳脂合成相关基因mRNA表达量以及细胞中脂肪滴形态,分析结果发现敲降VPS28基因可降低泛素化-溶酶体和泛素化-蛋白酶体通路基因和乳脂合成相关基因的表达量,并提高细胞中甘油三酯的合成,预示VPS28基因可能通过泛素化-溶酶体和泛素化-蛋白酶体途径调控乳脂生成。本研究结果在转录组水平揭示VPS28基因对乳脂合成的调控机制,为奶牛乳脂性状的分子育种研究提供参考依据。
关键词： 中国荷斯坦奶牛;VPS28基因;单核苷酸多态性;乳脂合成;调控

Abstract
In a previous genome-wide association study on milk production traits in Chinese Holstein population, we discovered VPS28 gene was highly expressed in mammary gland tissue. Further, a -58C>T mutantion at the 5°-UTR of the gene was significantly associated with milk fat content traits. However, its regulation on milk fat content is still uncertain. In this study, we explored the effect of this -58C>T mutation on VPS28, and found that it could significantly reduce promoter activity of VPS28 gene. To identify the potential function of this SNP, we performed RNAi experiments to knockdown VPS28 gene in BMECs and examined the general effects of VPS28 knockdown on BMECs. The results showed that VPS28 knockdown could decrease the mRNA expression of genes in ubiquitination-lysosome and ubiquitination- proteasome pathways, increase the mRNA expression of genes in milk fat synthesis pathway and promote triglyceride synthesis in BMECs. Our data indicate that VPS28 gene could regulate milk fat synthesis pathway; and promote triglyceride synthesis in BMECs. Our data indicate that VPS28 gene could regulate milk fat synthesis through modulating the ubiquitination-lysosome and ubiquitination-proteasome systems. Our results demonstrate the molecular mechanism of VPS28 on regulation of milk synthesis in dairy cattle through the ubiquitination signaling pathway, thereby supporting a relationship between milk fat synthesis and ubiquitination and laying the molecular foundation in breeding of dairy cattle in the future.
Keywords：Chinese Holstein dairy;VPS28 gene;single nucleotide polymorphism;milk fat related genes;transcriptional regulation


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本文引用格式
刘莉莉, 郭爱伟, 吴培福, 陈粉粉, 杨亚晋, 张勤. 敲降VPS28基因对中国荷斯坦奶牛乳脂合成的调控[J]. 遗传, 2018, 40(12): 1092-1100 doi:10.16288/j.yczz.18-134
Lili Liu, Aiwei Guo, Peifu Wu, Fenfen Chen, Yajin Yang, Qin Zhang. Regulation of VPS28 gene knockdown on the milk fat synthesis in Chinese Holstein dairy[J]. Hereditas(Beijing), 2018, 40(12): 1092-1100 doi:10.16288/j.yczz.18-134


牛奶中乳脂含量约3%~5%,其含量高低直接影响了牛奶的营养价值和口味,因此乳脂含量是衡量牛奶品质的重要指标。如何提高牛奶中的乳脂水平并改善牛奶品质成为学术界关注的焦点,也是生产者、消费者和乳品加工企业关注的热点。虽然可以通过生鲜奶后加工而提高乳脂含量,但人工处理过程会导致牛奶中其他重要营养成分损失,甚至生成其他有害物质,同时额外增加了牛奶的生产成本。因此,通过分子遗传标记辅助选育等现代分子育种手段选育提高乳脂含量一直是奶牛育种的主要育种目标之一。

乳脂的主要成分是甘油三酯(triglyceride, TG),约占99%,由磷脂膜包被形成乳脂肪球从奶牛乳腺组织的乳腺上皮细胞释放到牛奶中^[1]。乳脂的合成途径主要有两种：一是直接利用血液中游离的长链脂肪酸(18~24个碳原子)进行合成;二是利用前体小分子从头合成短链和中链脂肪酸(4~14个碳原子)再合成乳脂。游离的长链脂肪酸以及从头合成短/中链脂肪酸都需要逐步经过激活、合成、转运等酶类作用合成甘油三酯,并在脂蛋白的协助下被释放到细胞外形成乳脂^[2,3,4,5]。由此可见,乳脂合成过程中有关酶类发挥了关键作用。

在前期奶牛产奶性状全基因组关联分析研究中,本课题组发现VPS28(vacuolar protein sorting 28)基因5°-UTR (5°-untranslated region)存在一个突变位点-58C>T与奶牛乳脂率存在关联(P=7.32E-60),是与乳脂率关联最强的SNP (single nucleotide polymorphism)之一,对该基因的mRNA表达研究表明,该基因在奶牛乳腺组织中特异性高表达^[6],但是其具体调控机理尚未明确,在畜禽中也尚未见到关于该基因的报道。因此,本研究以VPS28基因为奶牛乳脂性状的重要候选基因,采用RNA干扰技术(RNA interference, RNAi)对牛原代乳腺上皮细胞中的VPS28进行敲降,检测重要乳脂合成相关基因的表达变化,为阐明VPS28基因调控乳脂合成的分子机制提供理论基础。

1 材料和方法

1.1 材料

牛原代乳腺上皮细胞(bovine primary mammary epithelial cells, BMECs)、人胚肾293T细胞系(human embryo kidney 293T, HEK293T)和牛基因组DNA为本实验室冻存;DMEM/F12培养基、DMEM培养基、Opti-MEM培养基、胎牛血清、双抗(Penicillin-Streptomycin Solution)购自美国Gibco公司;限制性内切酶KpnⅠ、BglⅡ和T4 DNA连接酶购自美国NEB公司;DNA纯化回收及质粒制备试剂盒购自北京全式金生物科技有限公司;大肠杆菌感受态细胞DH5α购自北京天根生化科技有限公司;干扰片段siRNA合成于上海吉玛制药技术有限公司;双荧光素酶报告基因检测试剂盒(Dual-Luciferase Reporter Assay System)、萤光素酶报告基因载体(pGL4.14)、海肾荧光素酶报告基因载体(pRL-TK)购自美国Promega公司;转染试剂Lipofectamin 2000、TRIzol购自美国Invitrogen公司;转染试剂X-treme GENE siRNA Transfection Reagent、SYBR Green Mix购自美国Roche公司。

1.2 启动子报告基因载体的构建

根据VPS28基因(GenBank登录号：AC_000171.1)及萤光素酶报告基因载体pGL4.14的序列信息设计PCR引物,并在上、下游引物的5′端加入酶切位点Kpn I和BglⅡ(引物信息见表1)。然后以奶牛基因组DNA为模板,通过PCR扩增得到CC型和TT型VPS28基因5′UTR区域并将其克隆到pUCm-T载体上。测序无误后,经双酶切及胶纯化回收,分别将CC型和TT型VPS28基因5′UTR区域插入pGL4.14荧光素酶报告基因载体上,分别获得报告基因载体质粒pGL4.14-CC和pGL4.14-TT。

Table 1
表1
表1 牛VPS28基因启动子区扩增引物
Table 1 Primers of bovine VPS28 gene promoter

引物名称	引物序列(5'→3')	引物长度(bp)	酶切位点	产物大小(bp)
VPS28-F	CGGAggtaccTGCACCAGGATAAGCCCAGA	30	KpnⅠ	335
VPS28-R	TTTTagatctGAGTGGCTGGGATCCCGTGA	30	BglⅡ

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1.3 VPS28基因的启动子活性分析

将HEK293T细胞系培养于DMEM全培养基中(含有10%胎牛血清,100 U/mL青霉素-链霉素),并将其置于37℃、5% CO₂浓度以及湿度为90%的细胞培养箱中。当细胞汇合度达到90%时,用0.05%胰酶进行消化并将HEK293T细胞以2×10⁵/孔传代接种至24孔板中。待细胞汇合度达到70%时,分别取将450 ng 报告基因载体质粒pGL4.14-CC、pGL4.14- TT及pGL4.14与50 ng RPL-TK 内参质粒混合,使用LTX转染试剂共转染HEK293T细胞系,6~8 h后更换完全培养基。转染48 h后,收集细胞并制备细胞裂解液,然后按双荧光素酶报告基因检测试剂盒说明书(Promega,美国)进行操作,分别测定各组的萤火虫荧光素酶及海肾荧光素酶活性,通过二者比值获取启动子活性。

1.4 BMECs中VPS28基因的敲降

将BMECs培养于DMEM/F12全培养基中(含有10%胎牛血清,100 U/mL青霉素-链霉素),并将其置于温度37℃,CO₂浓度5%,湿度90%的细胞培养箱中。当细胞汇合度达到100%时,用0.25%胰酶进行消化并将BMECs以2.5×10⁵/孔传代接种至6孔板中。培养24 h后汇合度达80%时,将2 μg siRNA溶解于Opti-MEM中,使用转染试剂X-treme GENE siRNA Transfection Reagent转染BMECs,6~8 h后更换完全培养基。转染24 h后在荧光显微镜下检测转染效率,转染72 h后收集细胞并在每孔细胞中加入1 mL TRIzol裂解进行总RNA提取。将提取的总RNA进行1%琼脂糖凝胶电泳,检测RNA的完成性及有无DNA污染。采用Nanodrop检测总RNA的浓度和质量,保证样品浓度≥200 ng/μL,然后按照TaKaRa反转录试剂盒说明书进行反转录。

1.5 VPS28基因及乳脂合成相关基因表达量的检测

结合KEGG网站(KEGG-Table of Contents, http:// www.genome.jp/kegg/kegg2.html),本研究选择脂类合成、泛素化-溶酶体和泛素化-蛋白酶体通路中38个基因作为候选基因,采用qRT-PCR方法检测这38个相关基因mRNA表达量的变化(具体基因和引物信息见表2)。以GAPDH为内参基因,采用比较Ct值法即2^-△Ct (△Ct =目的基因Ct值-内参基因Ct值)表示目的基因的相对表达量。采用ROCHE公司SYBR Green Mix进行定量表达检测。反应体系及反应程序参照SYBR Green Master Mix说明书,在Roche Light Cycler 480荧光定量PCR仪上进行扩增。

Table 2
表2
表2 候选基因的引物序列及其相对表达量
Table 2 Primers of the candidate genes for qRT-PCR and their relative expression

基因	引物序列 (5'→3')	相对表达量	基因	引物序列 (5'→3')	相对表达量
GAPDH	AGATGGTGAAGGTCGGAGTG	/	HERC3	CTCGAGGGCCTAGCTGTCT	0.73*
	CGTTCTCTGCCTTGACTGTG			TTTGTCAGAAGGGTCTGGCG
VPS28	GGAAACAAGCCGGAGCTGTA	0.28*	VPS45	CCCCAAAGATGCTGTGGCTA	0.42*
	CTGGATCTCGTCCATGGCTC			AGTGTGCTGGGGCCTAGATA
LPL	AGCTCCAAGTCGCCTTTCTC	0.48*	CHMP2B	ACGAGGTACACAGAGGGCTA	0.38*
	TCCTGGTTGGAAAGTGCCTC			AGCTGTTTGGCTAAAACTCTGC
LDLR	TGTTGGACACACGTACCCAG	2.72*	CHMP3	GTTTGAAATCACCGCAGGGG	0.77*
	AAGGTCGCGACTTGTCTCAG			CTAAAGGTTCAGGCTCCGGG
CD36	GACGGATGTACAGCGGTGAT	16.00**	PIP5K	CTCAGCACCTGGAAGAGCAA	1.48*
	GAAAAAGTGCAAGGCCACCA			TTCTTCTTTCCCCGAGCCAC
ACSL1	GGGCCTGCGGAGGAGA	3.76*	CYHR1	GCCAACCTGCTTTTGGGAAG	1.21*
	GGCAGCCGAAAGTACTGGAA			GGTTGTGAAAACGGCCACAA
FABP3	ACGCGTTCTCTGTCGTCTTT	3.79*	CP	CATGGTGGCCAAAGGTGTTG	1.5*
	AACCGACACCGAGTGACTTC			CATCTGCTGGAGATTTTTGGCA
ACACA	AGTGTTCTGATCAGGTCTTCTTGT	0.67*	PSMC1	GGTACGACTCCAACTCAGGC	2.90*
	GGGAGGCAAAAACCTCCAGA			ATCCGGTTTGTGGCCATGAT
ACBP	TGGAATCTTTGCAACACCGC	0.83*	PSMC3	TGAACAAGACGCTGCCGTAT	1.76*
	TGTCACCCACAGTTGCTTGT			TGCCGCGTAGAGGTTTTGAT
FASN	AGGCGTGCGTGACACTT	6.85*	PSMC5	CTCTGCACAAGATCCTGCCT	3.27*
	AATACAGTTGGCCGTCACCA			ATGCTTCACAGGCAGCTCAA
SCD	TCCTGATCATTGGCAACACCA	1.48*	PSMD12	ATACGTCAGGCATCTCGCAG	0.37*
	CCAACCCACGTGAGAGAAGAA			GGCCATGTTGTAGGGGACAA
DGAT1	TACCCCGACAACCTGACCTA	2.06*	UBB	TGGCATTGTTGGGTTCCTGT	0.56*
	GGGAAGTTGAGCTCGTAGCA			CGAAGATCTGCATTTTGACCTG
LPIN1	CTTCGATTCCCAAACCGGGA	2.35*	UBC	GACCGGGAGTTCAGTCTTCG	1.28*
	TCACAGTGACGAACACCTGG			TTTACCAGTGAGGGTCTTCACAA
ADFP	GCGTCTGCTGGCTGATTTC	2.95*	UBA52	GCCCAGTGACACCATTGAGA	1.28*
	AGCCGAGGAGACCAGATCATA			GCAGGGTGGACTCTTTCTGG
APOE	CATGCTGGGCCAGTCTACC	0.48*	UBE2	CTGGCACAGTATATGAAGACCTGA	1.09*
	CTTCTTCAGGTCGTCAGCGTTGGAA			GGTAGCAGGGTGTGAGGAAC
PRKCA	GACTTCGGGATGTGCAAGGA	2.40*	TUBA	TGTCTACTCCTGTTGCCTGC	0.5*
	CGTACGGCTGATAGGCGATT			AGGCATTGCCGATCTGGAC
MAPK1	AACAAAGTCCGAGTCGCCAT	2.54*	ISG15	CCATCCTGGTGAGGAACGAC	19**
	CGATGGTCGGTGCTCGAATA			GTCTGCTTGTACACGCTCCT
ARF6	AACTGGTATGTGTCAGCCCTC	4.68*	MX1	TGCCAACTAGTCAGCACTACATT	1.08*
	GAAAGAGGTGATGGTGGCGA			TGTACAGGTTGCTCTTGGACTC
STAM1	CCTGGTACTGCGGCTAACAA	1.23*	SPP1	TCCGCCCTTCCAGTTAAACC	3.2*
	ACGAACTTTCCGGCCTTCAT			GCTTCTGAGATGGGTCAGGC
EEA1	CAGGCCCAGGACAGCTTAAA	7.68*	RPS27A	TTTCGTGAAGACCCTGACGG	2.13*
	GCAAGTTCCTGTGCTGCTTG			GTCTTTGCTGGTCAGGAGGAA

*表示P<0.05,差异显著;**表示P<0.01,差异极显著。

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1.6 BMECs中脂肪滴的电镜检测

收集足量敲降VPS28基因前后的BEMC,然后依次对其进行固定、包埋、切片和染色。将处理好的切片置于JEM-1400透射电子显微镜(日本电子公司)下观察。

1.7 数据与分析

所有实验数据均为3次独立重复实验结果,以均数±标准差表示,两组数据差异使用T检验进行统计分析,对3组或以上实验数据采用单因素方差分析(one-way analysis of variance, ANOVA)结合Bonferroni或者Duncan多重比较方法进行统计分析,P<0.05时认为具有统计学意义。

2 结果与分析

2.1 VPS28基因多态位点对该基因的表达调控

本课题组前期研究发现VPS28基因5′-UTR存在-58C>T突变位点,该位点与奶牛乳脂率存在极强关联^[6]。为了进一步验证该突变位点对VPS28基因的表达调控,本研究克隆了野生型和突变型牛VPS28基因5′-UTR片段,并构建了启动子报告基因载体pGL4.14-CC和pGL4.14-TT。将所构建的重组载体转染HEK293T细胞系,48 h后检测报告基因活性。结果如图1所示,与pGL4.14载体相比,两重组载体均显示了较强的荧光素酶信号,但突变型重组载体pGL4.14-TT的活性显著低于野生型重组载体pGL4.14-CC (P<0.05)。该结果表明,VPS28基因5′-UTR的-58C>T突变位点可以降低该基因的启动子活性而降低其mRNA表达量。

图1

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图1VPS28基因不同基因型启动子荧光素酶表达载体转染293T细胞的相对荧光值

pGL4.14-CC：野生型启动子片段重组载体;pGL4.14-TT：突变型启动子片段重组载体。每组数据均通过3次重复计算平均数±标准差;*表示P<0.05,数据间差异显著。
Fig. 1Relative fluorescence values of different genotype promoters of bovine VPS28 gene in 293T cells

2.2 BMECs中VPS28基因的敲降

本研究采用RNAi技术对BMECs中VPS28基因进行敲降。首先基于VPS28基因的mRNA序列(GenBank登录号：NM_001035504.2)设计3条RNA干扰片段(图2A),然后分别转染单片段siRNA (siRNA1、siRNA2、siRNA3)及串联片段siRNAs (siRNA12、siRNA13、siRNA23、siRNA123)敲降VPS28基因。结果表明(图2,B和C),在干扰片段siRNA连续作用72 h后,串联片段siRNA23显示了最高的干扰效率,达71%。因此,本研究将串联干扰片段siRNA23作为敲降VPS28基因的干扰片段。

图2

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图2BMECs中VPS28基因的干扰

A：VPB28基因siRNA干扰片段;B：细胞中VPS28基因串联干扰片段siRNA23的转染效率;C：细胞中VPS28基因干扰片段siRNAs的干扰效率。每组数据均通过3次重复计算平均数±标准差。
Fig. 2The VPS28 knockdown with siRNAs in BMECs

2.3 差异表达基因的效应分析

以GAPDH 为内参基因,采用qRT-PCR 方法检测38个候选基因在敲降VPS28基因后的表达趋势,结果如表2所示。在乳脂合成过程中,相关酶类基因DGAT1、FASN、SCD、GPI、ACSL1、FABP3、ADFP和CD36 mRNA表达量上调(P<0.05),而ACACA mRNA表达量下调(P<0.05);包含VPS28基因在内的内体蛋白分选转运装置超级复合体ESCRTs (endosomalsorting complexes required for transport)的亚基ARF6、EEA1、STAM、VPS45、CHMP2B、CHMP3基因mRNA表达量显著下调(P<0.05);蛋白酶体系统的亚基PSMC1、PSMC3和PSMC5基因显著上调(P<0.05),而PSMD12基因mRNA表达量下调(P<0.05);泛素化途径相关基因UBB和TUBA mRNA表达量下调(P<0.05),ISG15、UBE2L6、UBC、SPP1和MX1基因mRNA表达量上调(P<0.05)。

为明确这些候选基因间的联系及其对乳脂合成的作用,本研究利用String在线软件(STRING: functional protein association networks, http://string- db.org/)对其进行综合分析并绘制了相应的通路图(图3)。结合38个候选基因所在通路的生物学功能,本研究发现,VPS28基因被敲降可以直接下调细胞中ESCRTs基因的表达。这表明VPS28基因可通过ESCRTs下调泛素化-溶酶体和泛素化-蛋白酶体通路基因的表达来影响乳脂合成过程中酶类的泛素化降解,进而调控乳脂的生成。

图3

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图3BMECs中VPS28基因被敲降后乳脂合成及VPS28所在通路中相关基因通路图

Fig. 3Possible signal network and distinct pathway related to the effect of VPS28 knockdown on milk fat synthesis in BMECs

2.4 BMECs中脂肪滴的检测

为了更直观地检测VPS28基因敲降对BMECs中乳脂合成的影响,本研究使用电子显微镜直接观察VPS28基因被敲降前后细胞中脂肪滴的变化。结果如图4所示,与正常BMECs相比,敲降VPS28基因的细胞中能直观地观察到大量且体积较大的脂肪滴储存在细胞质中。因此,VPS28基因被敲降可直接导致BMECs中脂肪滴的合成增加。

图4

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图4BMECs中脂肪滴电镜扫描图

A：VPS28^+/+ BMECs;B：VPS28^-/- BMECs。
Fig. 4Electron micrographs of lipid droplets in BMECs

3 讨 论

本研究将VPS28基因作为影响奶牛乳脂性状的重要功能候选基因,选择该基因的突变位点-58C>T作为重要突变位点,对该基因和突变位点进行了功能验证,试图揭示VPS28基因及其突变位点-58C>T 对乳脂合成的调控作用,为VPS28基因的功能研究以及奶牛乳脂性状相关分子遗传标记的筛选提供科学依据。

人(Homo sapiens)和小鼠(Mus musculus)中有关VPS28的研究表明,VPS28是真核细胞中内体蛋白分选转运装置复合体ESCRT-I的一个亚单位,ESCRT-Ⅰ与ESCRT-0、ESCRT-Ⅱ和ESCRT-Ⅲ共同组成超级复合体ESCRTs^[7,8,9]。VPS28通过与其他复合体亚基相互作用保证了对其他复合体的召集和连接过程,因此VPS28与ESCRTs的稳定性有关^[10]。ESCRTs是真核细胞中由囊泡分拣蛋白组成的一种重要复合体,通过与多囊体共同作用对泛素化膜蛋白进行识别分选,并将其转运至溶酶体中降解,这种泛素-溶酶体系统是细胞中多种膜结构受体下调的主要途径^[11,12]。泛素化是一种重要的蛋白质翻译后修饰,指泛素蛋白在一系列酶的催化作用下共价结合到靶蛋白的过程。泛素化信号通路直接介导了真核生物细胞内蛋白质的降解,影响蛋白质的活性和定位,调控包括细胞周期、细胞凋亡、转录调控、DNA损伤修复以及免疫应答等在内的多种细胞活动^[13,14]。研究发现,ESCRTs中VPS28突变可以导致胚胎细胞中囊泡数量显著增多,说明VPS28可以通过ESCRTs对细胞中膜结构内陷和分泌过程产生影响^[15]。另有研究表明ECSRTs可以通过泛素化作用参与蛋白酶体对胞质中泛素化蛋白质的降解^[16]。蛋白酶体是存在于哺乳动物细胞中具有蛋白水解酶作用的巨型蛋白质复合物,与泛素化信号系统一起构成泛素-蛋白酶体系统,对细胞中胞质蛋白进行选择性降解。泛素-蛋白酶体系统介导的蛋白质降解通路不仅可以清除细胞内功能异常及变性蛋白质,还可以通过降解不同途径活化物或抑制物而调控特定蛋白质的表达^[17]。因此,泛素-蛋白酶体系统是调节多种细胞生物学过程如基因转录调控、细胞信号转导和转运特殊细胞内蛋白的关键机制。有研究表明泛素-蛋白酶体系统通过调控蛋白质降解影响细胞中脂类合成过程,我们在前期研究中也同样证实泛素-蛋白酶体系统在高糖调控乳脂合成过程汇总起到了重要作用^[18]。综合以上研究,我们推测VPS28可能通过ESCRTs参与泛素-溶酶体和泛素-蛋白酶体系统影响奶牛乳脂合成过程中膜受体蛋白和胞质蛋白的泛素化降解和乳脂肪球的形成分泌,进而调控乳脂的合成。

为阐明VPS28基因及其突变位点对奶牛乳脂合成的调控机理,本研究首先对VPS28基因5'-UTR的-58C>T突变位点进行启动子活性分析,结果发现VPS28基因的该突变位点确实可导致其基因mRNA表达量显著下降,预示了该突变位点可能通过下调基因表达量而影响乳脂生成;然后,进一步利用RNAi干扰技术敲降奶牛原代乳腺上皮细胞中VPS28基因,并检测泛素化-溶酶体、泛素-蛋白酶体和乳脂合成途径中的相关基因mRNA表达量,试图在RNA水平上分析VPS28基因低表达对乳脂合成的影响。结果发现,ESCRT-0亚基的基因(ARF6、EEA1和STAM)mRNA表达量上调,ESCRT-Ⅱ亚基的基因(VPS45、CHMP2B和CHMP3) mRNA表达量下调,ESCRTs亚基的基因mRNA表达量的不平衡说明VPS28基因敲降确实造成了ESCRTs稳定性受到了影响;泛素-蛋白酶体系统的大部分相关基因(SG15、UBE2L6、UBC、SPP1、MX1、PSMC1、PSMC3和PSMC5) mRNA表达量上调,说明细胞中泛素化水平上升。因此,该结果从RNA水平证明了VPS28基因被敲降确实可以通过抑制细胞中ESCRTs的功能而增加泛素-溶酶体和泛素-蛋白酶体系统在细胞中泛素化作用,从而造成细胞中泛素化水平上升。

本研究通过检测乳脂合成相关基因的mRNA表达量也证实了该结果：乳脂合成过程的合成酶相关基因FASN、ASCL1、SCD和DGAT1及转运酶相关基因FABP3、ADFP和CD36的 mRNA表达量均显著上调。乳脂的主要成分是甘油三酯,约占99%,由磷脂膜包被形成乳脂肪球从奶牛乳腺组织的乳腺上皮细胞释放到牛奶中^[1]。乳脂的合成途径主要有两种：一种直接利用血液中游离的长链脂肪酸(18~24个碳原子)进行合成;另一种是利用乙酸、丙酸等前体小分子从头合成短链和中链脂肪酸(4~14个碳原子)再合成乳脂。游离的长链脂肪酸需要与细胞膜上受体作用通过转运蛋白CD36进入细胞中,前体小分子物质则可以直接进入细胞中并在合成酶FASN等酶类作用下从头合成短链和中链脂肪酸;这些脂肪酸在激活酶和结合酶ASCL1、FABP3等酶类作用下被转运至内质网中,再逐步通过内质网中的甘油二酯酰基转移酶DGAT1和酰基辅酶A去饱和酶SCD等酶类作用下合成甘油三酯;甘油三酯在内质网小叶中形成并累积成脂肪滴,在脂蛋白ADFP等协助下被分泌到腺泡中,最后通过与细胞膜上受体相结合使得脂肪滴通过细胞的顶浆膜释放到细胞外形成乳脂^[2,4,5,19]。CD36是细胞膜上的转运膜蛋白,通过翻转作用将细胞外的游离脂肪酸转运进细胞中,并且研究发现CD36直接受泛素化调控^[20]。本研究发现VPS28基因被敲降可显著增加脂肪酸转运蛋白CD36基因的mRNA表达量,这就表明乳腺上皮细胞中VPS28基因被敲降可以通过增加CD36表达量而提高细胞转运脂肪酸的能力。脂肪酸合成酶FASN和酯酰辅酶A合成酶ASCL1是前体分子合成脂肪酸过程中的关键酶^[21,22],本研究发现VPS28基因被敲降可显著增加脂肪酸合成酶FASN和酯酰辅酶A合成酶ASCL1的mRNA表达量,这就表明乳腺上皮细胞中VPS28基因被敲降可以提高细胞中前体分子合成脂肪酸的能力。甘油二酯酰基转移酶DGAT1和酰基辅酶A去饱和酶SCD是细胞中合成甘油三酯的限速酶,可以被甘油二酯特异激活,DGAT1和SCD基因高表达可以使细胞中甘油三酯合成增加^[4,23]。脂肪分化蛋白ADFP是细胞中脂肪滴表面蛋白,是脂质蓄积的特异性标志^[24,25],本研究发现VPS28基因被敲降可以显著增加DGAT1、SCD和ADFP基因的mRNA表达量,这就从RNA水平表明VPS28被敲降可以增加细胞中甘油三酯的合成。同时,本研究通过电子电子显微镜的观察,确实在VPS28基因敲降的奶牛乳腺上皮细胞中观察到大量蓄积的脂肪滴,这就再次证实了VPS28基因被敲降可以增加乳脂的合成。综上所述,本研究从RNA水平证明了VPS28基因5°-UTR的突变位点-58C>T可以降低其mRNA表达量,影响ESCRTs的稳定性而阻碍细胞中泛素-溶酶体系统对泛素化膜蛋白的降解以及泛素-蛋白酶体系统对泛素化胞质蛋白的降解,进而增加了细胞转运游离脂肪酸和从头合成脂肪酸的能力,导致细胞中乳脂的合成增加。

综上所述,本文在前期研究基础上,选择VPS28基因作为影响奶牛乳脂性状的重要候选基因,选择该基因的突变位点-58C>T作为重要突变位点,并对该基因和该突变位点进行了RNA水平的功能验证,发现VPS28基因及其突变位点可以通过泛素化信号通路调控乳脂的合成,为VPS28基因的功能研究以及奶牛乳脂性状相关分子遗传标记的筛选提供科学依据。此外,泛素化信号通路在细胞中参与多种信号通路,本研究结果有望揭示泛素化信号通路调控乳脂合成的新机制,并为泛素化信号通路在其他奶牛生产性状或其他畜禽生产性状的作用机制研究提供参考。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。

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Lipid droplets are the least characterized of cellular organelles. Long considered simple lipid storage depots, these dynamic and remarkable organelles have recently been implicated in many biological processes, and we are only now beginning to gain insights into their fascinating lives in cells. Here we examine what we know of the life of lipid droplets. We review emerging data concerning their cellular biology and present our thoughts on some of the most salient questions for investigation.

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The cream or fat fraction of milk consists of fat droplets composed primarily of triacylglycerols that are surrounded by cellular membranes. In this review we discuss what is known about how these droplets are formed in and secreted by mammary epithelial cells during lactation. This secretion mechanism, which appears to be unique, is unlike the exocytotic mechanism used by other cell types to secrete lipids. Milk fat globules originate as small, triacylglycerol-rich, droplets that are formed on or in endoplasmic reticulum membranes. These droplets are released from endoplasmic reticulum into the cytosol as microlipid droplets coated by proteins and polar lipids. Microlipid droplets can fuse with each other to form larger cytoplasmic lipid droplets. Droplets of all sizes appear to be unidirectionally transported to apical cell regions by as yet unknown mechanisms that may involve cytoskeletal elements. These lipid droplets appear to be secreted from the cell in which they were formed by being progressively enveloped in differentiated regions of apical plasma membrane. While plasma membrane envelopment appears to be the primary mechanism by which lipid droplets are released from the cell, a mechanism involving exocytosis of lipid droplets from cytoplasmic vacuoles also has been described. As discussed herein, while we have a general overview of the steps leading to the fat globules of milk, virtually nothing is known about the molecular mechanisms involved in milk fat globule formation, intracellular transit, and secretion.

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The mammary gland has an incredible level of organization and a remarkable ability to convert circulating nutrients into milk components. This review highlights four areas of high interest in the biology of milk synthesis where advances over the last quarter-century have resulted in new understanding or revealed new opportunities. First, advances in our understanding of the mechanisms of milk secretion has led to a substantial increase in our knowledge of the intracellular origin of lipid droplets and the identity and potential function of milk fat globule membrane proteins in milk-lipid secretion. Second, recent breakthroughs have advanced our understanding of the nutritional regulation of milk fat and highlighted the interrelations between dietary components, digestive processes in the rumen, and the regulation of mammary synthesis of milk fat. Third, nutritional quality is becoming increasingly important in food choices because of consumer awareness of the links between diet and health. The traditional nutritional value of milk and dairy products is well established, but recent discoveries have identified a number of “bioactive” components in milk with potential to improve human health. Finally, the concept of genetic engineering and the use of animals as “bioreactors” and the “pharming” of proteins not normally found in milk have gained recognition, with the dairy industry ideally suited to take advantage of advances in these areas.

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The molecular events associated with regulation of milk fat synthesis in the bovine mammary gland remain largely unknown. Our objective was to study mammary tissue mRNA expression via quantitative PCR of 45 genes associated with lipid synthesis (triacylglycerol and phospholipids) and secretion from the late pre-partum/non-lactating period through the end of subsequent lactation. mRNA expression was coupled with milk fatty acid (FA) composition and calculated indexes of FA desaturation and de novosynthesis by the mammary gland. Marked up-regulation and/or % relative mRNA abundance during lactation were observed for genes associated with mammary FA uptake from blood (LPL, CD36), intracellular FA trafficking (FABP3), long-chain (ACSL1) and short-chain (ACSS2) intracellular FA activation, de novoFA synthesis (ACACA, FASN), desaturation (SCD, FADS1), triacylglycerol synthesis (AGPAT6, GPAM, LPIN1), lipid droplet formation (BTN1A1, XDH), ketone body utilization (BDH1), and transcription regulation (INSIG1, PPARG, PPARGC1A). Change in SREBF1mRNA expression during lactation, thought to be central for milk fat synthesis regulation, was 2-fold in magnitude, while expression of INSIG1, which negatively regulates SREBP activation, was >12-fold and had a parallel pattern of expression to PPARGC1A. Genes involved in phospholipid synthesis had moderate up-regulation in expression and % relative mRNA abundance. The mRNA abundance and up-regulation in expression of ABCG2during lactation was markedly high, suggesting a biological role of this gene in milk synthesis/secretion. Weak correlations were observed between both milk FA composition and desaturase indexes (i.e., apparent SCD activity) with mRNA expression pattern of genes measured. A network of genes participates in coordinating milk fat synthesis and secretion. Results challenge the proposal that SREBF1is central for milk fat synthesis regulation and highlight a pivotal role for a concerted action among PPARG, PPARGC1A, and INSIG1. Expression of SCD, the most abundant gene measured, appears to be key during milk fat synthesis. The lack of correlation between gene expression and calculated desaturase indexes does not support their use to infer mRNA expression or enzyme activity (e.g., SCD). Longitudinal mRNA expression allowed development of transcriptional regulation networks and an updated model of milk fat synthesis regulation.

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Mammary epithelial cells secrete lipids by an envelopment process that produces lipid droplets coated by membranes derived from the plasma membrane and possibly secretory vesicles. This secretion process, which resembles viral budding, is hypothesized to be mediated by specific interactions between molecules on the surface of intracellular lipids and membrane elements of the cell. Multiple lines of evidence indicate that milk lipid secretion occurs through a tripartite complex between the integral transmembrane protein, butyrophilin (BTN); the soluble metabolic enzyme, xanthine oxidoreductase (XOR); and the lipid droplet surface protein, adipophilin (ADPH). However, topological evidence from freeze-fracture replica immunolabelling (FRIL) challenge this model and suggests that milk lipid secretion is mediated by butyrophilin alone. Advances in our understanding of the molecular, structural, and functional properties of these proteins now make it possible to understand the physiological functions of each of these molecules in detail and to identify the specific molecular determinants that mediate milk lipid secretion.

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Background Genome wide association study (GWAS) has been proven to be a powerful tool for detecting genomic variants associated with complex traits. However, the specific genes and causal variants underlying these traits remain unclear. Results Here, we used target-enrichment strategy coupled with next generation sequencing technique to study target regions which were found to be associated with milk production traits in dairy cattle in our previous GWAS. Among the large amount of novel variants detected by targeted resequencing, we selected 200 SNPs for further association study in a population consisting of 2634 cows. Sixty six SNPs distributed in 53 genes were identified to be associated significantly with on milk production traits. Of the 53 genes, 26 were consistent with our previous GWAS results. We further chose 20 significant genes to analyze their mRNA expression in different tissues of lactating cows, of which 15 were specificly highly expressed in mammary gland. Conclusions Our study illustrates the potential for identifying causal mutations for milk production traits using target-enrichment resequencing and extends the results of GWAS by discovering new and potentially functional mutations.

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[9] Agromayor M, Soler N, Caballe A, Kueck T, Freund SM, Allen MD, Bycroft M, Perisic O, Ye Y , McDonald B,Scheel H, Hofmann K, Neil SJ, Martin-Serrano J, Williams RL. The UBAP1 subunit of ESCRT-I interacts with ubiquitin via a SOUBA domain
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78 ESCRT-I subunit UBAP1 is essential for degradation of antiviral protein tetherin 78 UBAP1 has a domain consisting of a solenoid of overlapping UBAs (SOUBA) 78 Each of the three UBAs in the SOUBA binds monoubiquitin

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The ubiquitin system is a network of proteins dedicated to the ubiquitylation of cellular targets and the subsequent control of numerous cellular functions. The deregulation of components of this elaborate network leads to human pathogenesis, including the development of many types of tumour. Alterations in the ubiquitin system that occur during the initiation and progression of cancer are now being uncovered, and this knowledge is starting to be exploited for both molecular diagnostics and the development of novel strategies to combat cancer.

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The profile of protein sorting into multivesicular bodies (MVBs) has risen recently with the identification of three heteromeric complexes known as ESCRT-I,-II, II (Endosomal Sorting Complex Required for Transport). Genetic analyses in yeast have identified up to 15 soluble class E VPS (vacuolar protein sorting) proteins that have been assigned to the ESCRT machinery and function in cargo recognition and sorting, complex assembly, vesicle formation and dissociation. Despite their functional importance in yeast and mammalian cells, little is known about their presence and function in other organisms including plants. We have made use of the fully sequenced genomes of Arabidopsis thaliana and Oryza sativa, Drosophila melanogaster and Caenorhabditis elegans to explore the identity, structural characteristics and phylogenetic relationships of proteins assigned to the ESCRT machinery.

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Trends Biochem Sci, 2007,32(12):561-573.
URLPMID:17988873 [本文引用: 1]
The endosomal sorting complex required for transport (ESCRT) machinery is highly conserved and its components have been found in all five major supergroups of eukaryotes. The three ESCRT complexes and associated proteins play critical roles in receptor downregulation, retroviral budding, and other normal and pathological cellular processes. Besides monoubiquitin-dependent protein cargo recognition and sorting, the ESCRT machinery also appears to drive the formation of multivesicular bodies (MVBs). Recent advances in the determination of the function and structure of the ESCRT complexes have improved our understanding of the molecular details underlying the assembly and regulation of the ESCRT machinery.

[13] Hirsch C, Gauss R, Horn SC, Neuber O, Sommer T . The ubiquitylation machinery of the endoplasmic reticulum
Nature, 2009,458(7237):453-460.
URL [本文引用: 1]

[14] Katzmann DJ, Babst M, Emr SD . Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I
Cell, 2001,106(2):145-155.
URLPMID:11511343 [本文引用: 1]
The multivesicular body (MVB) pathway is responsible for both the biosynthetic delivery of lysosomal hydrolases and the downregulation of numerous activated cell surface receptors which are degraded in the lysosome. We demonstrate that ubiquitination serves as a signal for sorting into the MVB pathway. In addition, we characterize a 350 kDa complex, ESCRT-I (composed of Vps23, Vps28, and Vps37), that recognizes ubiquitinated MVB cargo and whose function is required for sorting into MVB vesicles. This recognition event depends on a conserved UBC-like domain in Vps23. We propose that ESCRT-I represents a conserved component of the endosomal sorting machinery that functions in both yeast and mammalian cells to couple ubiquitin modification to protein sorting and receptor downregulation in the MVB pathway.

[15] Sevrioukov EA, Moghrabi N, Kuhn M, Krämer H . A mutation in dVps28 reveals a link between a subunit of the endosomal sorting complex required for transport-I complex and the actin cytoskeleton in Drosophila
Mol Biol Cell, 2005,16(5):2301-2312.
URLPMID:1087236 [本文引用: 1]
Abstract Proteins that constitute the endosomal sorting complex required for transport (ESCRT) are necessary for the sorting of proteins into multivesicular bodies (MVBs) and the budding of several enveloped viruses, including HIV-1. The first of these complexes, ESCRT-I, consists of three proteins: Vps28p, Vps37p, and Vps23p or Tsg101 in mammals. Here, we characterize a mutation in the Drosophila homolog of vps28. The dVps28 gene is essential: homozygous mutants die at the transition from the first to second instar. Removal of maternally contributed dVps28 causes early embryonic lethality. In such embryos lacking dVps28, several processes that require the actin cytoskeleton are perturbed, including axial migration of nuclei, formation of transient furrows during cortical divisions in syncytial embryos, and the subsequent cellularization. Defects in actin cytoskeleton organization also become apparent during sperm individualization in dVps28 mutant testis. Because dVps28 mutant cells contained MVBs, these defects are unlikely to be a secondary consequence of disrupted MVB formation and suggest an interaction between the actin cytoskeleton and endosomal membranes in Drosophila embryos earlier than previously appreciated.

[16] Ciechanover A . The ubiquitin-proteasome proteolytic pathway
Cell, 1994,79(1):13-21.
URLPMID:7923371 [本文引用: 1]
Cell. 1994 Oct 7;79(1):13-21. Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Review

[17] da Fonseca PC, He J, Morris EP . Molecular model of the human 26S proteasome
Mol Cell, 2012,46(1):54-66.
URLPMID:22500737 [本文引用: 1]
78 Cryo-EM structure of human 26S proteasome at 7–90203 resolution 78 Molecular model of all the major subunits of the 26S proteasome 78 The locations of 19S-RP subunits are revised 78 The 20S core subunits undergo rearrangement, but the axial gate is not fully open

[18] Liu L, Jiang L, Ding XD, Liu JF, Zhang Q . The regulation of glucose on milk fat synthesis is mediated by the ubiquitin- proteasome system in bovine mammary epithelial cells
Biochem Biophys Res Commun, 2015,465(1):59-63.
URLPMID:26231798 [本文引用: 1]
Glucose as one of the nutrition factors plays a vital role in the regulation of milk fat synthesis. Ubiquitin-proteasome system (UPS) is a vital proteolytic pathway in all eukaryotic cells through timely marking, recognizing and degrading the poly-ubiquitinated protein substrates. Previous studies indicated that UPS plays a considerable role in controlling the triglyceride (TG) synthesis. Therefore, the aim of this study is to confirm the link between high-glucose and UPS and its regulation mechanism on milk fat synthesis in BMEC (bovine mammary epithelial cells). We incubated BMEC with normal (17.5 mm/L) and high-glucose (25 mm/L) with and without proteasome inhibitor epoxomicin and found that, compared with the control (normal glucose and without proteasome inhibitor), both high-glucose concentration and proteasome inhibitor epoxomicin could increase the accumulation of TG and poly-ubiquitinated proteins, and reduce significantly three proteasome activities (chymotrypsin-like, caspase-like, and trypsin-like). In addition, high-glucose concentration combined with proteasome inhibitor further enhanced the increase of the poly-ubiquitinated protein level and the decrease of proteasome activities. Our results suggest that the regulation of high-glucose on milk fat synthesis is mediated by UPS in BMEC, and high-glucose exposure could lead to a hypersensitization of BMEC to UPS inhibition which in turn results in increased milk fat synthesis.

[19] Reinhardt TA, Lippolis JD . Bovine milk fat globule membrane proteome
J Dairy Res, 2006,73(4):406-416.
URLPMID:16834814 [本文引用: 1]
Abstract Milk fat globule membranes (MFGM) were isolated from the milk of mid-lactation Holstein cows. The purified MFGM were fractionated using 1-dimensional SDS gels. Tryptic peptides from gel slices were further fractionated on a micro-capillary high performance liquid chromatograph connected to a nanospray-tandem mass spectrometer. Analysis of the data resulted in 120 proteins being identified by two or more unique peptide sequences. Of these 120 proteins, 71% are membrane associated proteins with the remainder being cytoplasmic or secreted proteins. Only 15 of the proteins identified in the cow MFGM were the same as proteins identified in previous mouse or human MFGM proteomic studies. Thus, the bulk of the proteins identified are new for bovine MFGM proteomics. The proteins identified were associated with membrane/protein trafficking (23%), cell signalling (23%), unknown functions (21%), fat transport/metabolism (11%), transport (9%), protein synthesis/folding (7%), immune proteins (4%) and milk proteins (2%). The proteins associated with cell signalling or membrane/protein trafficking may provide insights into MFGM secretion mechanisms. The finding of CD14, toll like receptor (TLR2), and TLR4 on MFGM suggests a direct role for the mammary gland in detecting an infection.

[20] Smith J, Su X, El-Maghrabi R, Stahl PD, Abumrad NA . Opposite regulation of CD36 ubiquitination by fatty acids and insulin: effects on fatty acid uptake
J Biol Chem, 2008,283(20):13578-13585.
URL [本文引用: 1]

[21] Roy R, Ordovas L, Zaragoza P, Romero A, Moreno C, Altarriba J, Rodellar C . Association of polymorphisms in the bovine FASN gene with milk-fat content
Anim Genet, 2006,37(3):215-218.
URLPMID:16734679 [本文引用: 1]
Fatty acid synthase (FASN) is a multifunctional protein that carries out the synthesis of fatty acids so it plays a central role in de novo lipogenesis in mammals. Previously, we defined the genetic structure and expression of the bovine FASN gene. Our mapping studies placed FASN on BTA19 (19q22) where several quantitative trait loci (QTL) affecting milk-fat content and related traits have been described. This study was conducted to identify polymorphisms in the bovine FASN gene and to study their association with milk-fat content. The bovine FASN gene was screened for polymorphisms in two cattle breeds. Sequence analysis revealed several single nucleotide polymorphisms (SNPs), and two of them were analysed: a G&gt;C substitution in the untranslated exon 1 (g.763G&gt;C), altering a potential Sp1 transcription factor-binding site, and an A&gt;G substitution in exon 34 (g.16009A&gt;G), which determines a non-conservative substitution of threonine by alanine. Allele-specific amplification of the SNPs in FASN revealed significant frequency differences for both polymorphisms in Holsteins with high and low breeding values for milk-fat content. The intragenic haplotypes comprising exon 1 (alleles G and C ) and exon 34 (alleles A and G ) polymorphisms were studied, and the existence of linkage disequilibrium between these SNPs was found ( D CG = 0.048, P &lt; 0.001). Our results suggest that the FASN gene polymorphisms contribute to variation in milk-fat content. We propose that the bovine FASN gene is a candidate gene for a milk-fat content QTL.

[22] Bionaz M, Loor JJ . ACSL1, AGPAT6, FABP3, LPIN1, and SLC27A6 are the most abundant isoforms in bovine mammary tissue and their expression is affected by stage of lactation
J Nutr, 2008,138(6):1019-1024.
URLPMID:18492828 [本文引用: 1]
The lactating bovine mammary gland is a formidable triacylglycerol-synthesizing machine and, as such, represents an ideal model for studying putative functions of distinct isoforms of solute carrier family 27 transporters [(SLC27A) 1, 2, 3, 5, 6], long chain acyl-CoA synthetases [(ACSL) 1, 3, 4, 5, 6], fatty acid binding proteins [(FABP) 1, 3, 4, 5, 6], 1-acylglycerol-3-phosphate O-acyltransferases [(AGPAT) 1, 2, 3, 4, 5, 6, 7, 8], and lipins [(LPIN) 1, 2, 3]. The relative percentage of mRNA abundance and fold-changes in the expression of isoforms in mammary tissue from 6 cows each at -15, 15, 60, and 240 d relative to parturition were analyzed using quantitative PCR. Transcripts of FABP isoforms were most abundant, accounting for 78% of the 28 genes measured, and SLC27A isoforms were least abundant (< 0.5% of genes measured). mRNA of AGPAT, ACSL, and LPIN accounted for approximately 12, 7, or approximately 2%, respectively, of all genes measured. The mRNA abundance at 60 d postpartum for FABP3, ACSL1,AGPAT6, and LPIN1 was 80-, 7-, 15-, and 20-fold greater relative to -15 d. Transcripts of these isoforms constituted the most abundant within each specific gene family. SLC27A2, SLC27A5, and SLC27A6 had peak expression at 240, 240, or 15 d relative to parturition, respectively. Results suggest that SLC27A6, ACSL1, FABP3, AGPAT6, and LPIN1 coordinately regulate the channeling of fatty acids toward copious milk fat synthesis in bovine mammary.

[23] Tao H, Chang GJ, Xu TL, Zhao HJ, Zhang K, Shen XZ . Feeding a high concentrate diet down-regulates expression of ACACA, LPL and SCD and modifies milk composition in lactating goats
PLoS One, 2015,10(6):e0130525.
URLPMID:26086219 [本文引用: 1]
Abstract High concentrate diets are fed to early and mid-lactation stages dairy ruminants to meet the energy demands for high milk production in modern milk industry. The present study evaluated the effects of a high concentrate diet on milk fat and milk composition, especially, cis-9, trans-11 CLA content in milk and gene expression of lactating goats. Eight mid-lactating goats with rumen fistula were randomly assigned into a high concentrate diet (HCD) group and low concentrate diet (LCD) group. High concentrate diet feeding significantly increased lipopolysaccharides (LPS) in plasma and decreased milk fat content, vaccenic acid (VA) and cis-9, trans-11 CLA in milk of the lactating goats. The mRNA expression levels of sterol regulatory element binding protein B 1c (SREBP1c), lipoprotein lipase (LPL), fatty acid synthetase (FASN) and acetyl-CoA carboxylase (ACACA, ACC ) involving in lipid metabolism were analyzed, and ACACA and LPL all decreased in their expression level in the mammary glands of goats fed a high concentrate diet. DNA methylation rate of stearoyl-CoA desaturase (SCD) was elevated and decreased, and SCD mRNA and protein expression was reduced significantly in the mammary glands of goats fed a high concentrate diet. In conclusion, feeding a high concentrate diet to lactating goats decreases milk fat and reduced expression of SCD in the mammary gland, which finally induced cis-9, trans-11 CLA content in milk.

[24] McManaman JL, Zabaronick W, Schaack J, Orlicky DJ . Lipid droplet targeting domains of adipophilin
J Lipid Res, 2003,44(4):668-673.
URLPMID:12562852 [本文引用: 1]
Abstract Adipophilin (ADPH), a prominent protein component of lipid storage droplets (LSDs), is postulated to be necessary for the formation and cellular function of these structures. The presence of significant sequence similarities within an approximately 100 amino acid region of the N-terminal portions of ADPH and related LSD binding proteins, perilipin and TIP47, has implicated this region, known as the "PAT" domain, in LSD targeting. Here we investigate the role of the PAT domain in targeting ADPH to LSDs by expressing this region, as well as selected N- and C-terminal truncations of mouse ADPH in COS7 cells as epitope-tagged fusion proteins. Our studies show that truncations lacking either the PAT domain or the C-terminal half of ADPH both correctly targeted LSDs and increased the LSD content of transfected cells. Neither the PAT domain nor the C-terminal half of ADPH appeared to target LSDs or affect the LSD number. Instead, targeting fragments encompassed a putative alpha-helical region between amino acids 189 and 205, implicating this region in both LSD targeting and regulation of LSD formation.

[25] Heid HW, Moll R, Schwetlick I, Rackwitz HR, Keenan TW . Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases
Cell Tissue Res, 1998,294(2):309-321.
URLPMID:9799447 [本文引用: 1]
We report the human DNA and protein sequence of adipophilin and its association with the surface of lipid droplets. The amino acid sequence of human adipophilin has been determined by using cDNA clones from several tissues and confirmed by the reverse transcription/polymerase chain reaction method and Edman sequencing. The open reading frame of adipophilin encodes a polypeptide with a calculated molecular weight of 48.1 kDa and an isoelectric point of 6.72. By immunofluorescence and electron-microscopic localization with newly raised specific poly- and monoclonal antibodies, we show that this protein is not restricted to adipocytes as previously indicated by studies of the mouse homologous protein, adipose-differentiation-related protein. Adipophilin occurs in a wide range of cultured cell lines, including fibroblasts and endothelial and epithelial cells. In tissues, however, expression of adipophilin is restricted to certain cell types, such as lactating mammary epithelial cells, adrenal cortex cells, Sertoli and Leydig cells of the male reproductive system, and steatosis or fatty change hepatocytes in alcoholic liver cirrhosis. Our results reveal adipophilin as a possible new marker for the identification of specialized differentiated cells containing lipid droplets and for diseases associated with fat-accumulating cells.