奶山羊<i>SIRT7</i>基因序列特征、组织表达及其对乳脂合成的影响

doi:10.7668/hbnxb.20195461

摘要/Abstract

摘要：

旨在克隆奶山羊沉默信息调节因子2相关酶7基因(SIRT7)并进行生物信息学分析,检测该基因在奶山羊不同组织中的表达规律及其对奶山羊乳腺上皮细胞乳脂合成的影响。结果表明,通过设计SIRT7基因引物,成功克隆奶山羊SIRT7基因序列1 376 bp,编码区长1 203 bp,编码400个氨基酸,与绵羊同源性最高。SIRT7是亲水性蛋白,不含信号肽和跨膜结构,二级结构有无规则卷曲、α-螺旋、延伸链和β-转角。qRT-PCR结果显示,SIRT7基因在奶山羊肝脏组织中的相对表达量最高,在其他组织中也有一定的表达量。构建SIRT7基因过表达载体pcDNA3.1-SIRT7,将其转染至奶山羊乳腺上皮细胞中,与空载体对照组相比,过表达组SIRT7基因mRNA水平上调约33倍。SIRT7基因过表达后,乳脂代谢相关基因中硬脂酰辅酶A去饱和酶基因(SCD1)表达量显著降低,抗原分化簇36基因(CD36)、二酰基甘油酰基转移酶1基因(DGAT1)、固醇调节元件结合蛋白1基因(SREBP1)的表达量极显著降低;细胞中甘油三酯含量显著降低;细胞核周脂滴聚集量明显减少。因此,SIRT7基因抑制奶山羊乳腺上皮细胞的脂质代谢,为进一步探索SIRT7在奶山羊乳腺上皮细胞中的调控机制奠定基础。

关键词: 奶山羊, SIRT7, 基因克隆, 组织表达, 乳脂合成

Abstract:

The aim of this study was to clone the silent mating type information regulator 2-related enzyme 7(SIRT7)gene,detect its expression pattern in different tissues and investigate its effect on milk fat synthesis in mammary epithelial cells of dairy goats.The results showed that by designing the primers of SIRT7 gene,the sequence of SIRT7 gene in dairy goat was successfully cloned,with 1 376 bp and a coding region of 1 203 bp,encoding 400 amino acids and showing the highest homology with sheep.SIRT7 was a hydrophilic protein,free of signal peptides and transmembrane structures,with secondary structures such as irregular coils,α-helices,extended chains and β-turns.The relative expression of SIRT7 gene was the highest in the liver tissues of dairy goats,and it also had certain expression levels in other tissues by qRT-PCR.The pcDNA3.1-SIRT7 overexpression vector was successfully constructed and transfected into mammary epithelial cells of dairy goats.Compared with the control group,the mRNA level of SIRT7 gene in the overexpression group was up-regulated about 33 times.After SIRT7 gene overexpression,the expression of stearoyl-CoA desaturase-1(SCD1)gene was significantly decreased,and the expression levels of cluster of differentiation 36(CD36),diacylglycerol acyltransferase 1(DGAT1),sterol regulatory element binding protein 1(SREBP1)genes extremely significantly reduced.The content of triglyceride in cells was significantly decreased.The results of oil red O staining showed that the accumulation of lipid droplets around the nucleus was significantly reduced.Thus, SIRT7 gene inhibited lipid metabolism in mammary epithelial cells of dairy goats,which laid a foundation for further exploration of the regulatory mechanism of SIRT7 in mammary epithelial cells of dairy goats.

Key words: Dairy goat, SIRT7, Gene cloning, Tissue expression, Milk fat synthesis

中图分类号:

S827

李忠莹, 郭震楠, 朱洪林, 李佳琳, 施会彬, 韩浩园, 李君, 权凯. 奶山羊SIRT7基因序列特征、组织表达及其对乳脂合成的影响[J]. 华北农学报, 2025, 40(3): 199-206. doi: 10.7668/hbnxb.20195461.

LI Zhongying, GUO Zhennan, ZHU Honglin, LI Jialin, SHI Huibin, HAN Haoyuan, LI Jun, QUAN Kai. Sequence Characteristics,Tissue Expression and Effect on Milk Fat Synthesis of SIRT7 Gene in Dairy Goats[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 199-206. doi: 10.7668/hbnxb.20195461.

http://www.hbnxb.net/CN/Y2025/V40/I3/199

图/表 15

表1 引物序列信息

Tab.1 Primer sequence information

基因 Gene	引物序列(5'—3') Primer sequence (5'—3')	退火温度/℃ Annealing temperature	用途 Application
SIRT7	F:GTCTCCTCAGGAGCGATG	53.5	PCR
	R:CACGAGGACAAATGGCTA
SIRT7	F:GATTACCGGGGCCCTAATGG	60.0	qRT-PCR
	R:TCGCAGTTCTGAGACACCAC
SREBP1	F:ACGCCATCGAGAAACGCTAC	60.0	qRT-PCR
	R:GTGCGCAGACTCAGGTTCTC
SCD1	F:CCATCGCCTGTGGAGTCAC	60.0	qRT-PCR
	R:GTCGGATAAATCTAGCGTAGCA
FADS2	F:GGGCTCCACCACCGTGTTCCA	60.0	qRT-PCR
	R:GCTCTGCTGGGCCTGCAGCTG
CD36	F:GTACAGATGCAGCCTCATTTCC	60.0	qRT-PCR
	R:TGGACCTGCAAATATCAGAGGA
DGAT1	F:AAGAGGCTCACCCTGTCGCA	60.0	qRT-PCR
	R:ACTGTTGTTGTCCATGGACG
ATGL	F:GGAGCTTATCCAGGCCAATG	60.0	qRT-PCR
	R:TGCGGGCAGATGTCACTCT
UXT	F:TGTGGCCCTTGGATATGGTT	60.0	qRT-PCR
	R:GGTTGTCGCTGAGCTCTGTG

表2 生物信息学分析工具

Tab.2 Bioinformatics analysis tools

分析工具 Analysis tool	网址 Website	功能 Function
Blast	https: //blast.ncbi.nlm.nih.gov/Blast.cgi	同源性比对分析
ORF Finder	https: //www.ncbi.nlm.nih.gov/orffinder/	开放阅读框分析
ExPASy	https://www.expasy.org	蛋白质理化性质分析
ProScale	https: //web.expasy.org/protscale	蛋白质亲疏水性分析
SignalP	http: //www.cbs.dtu.dk/services/SignalP/	蛋白质信号肽分析
TMHMM 2.0	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0	蛋白质跨膜结构预测
SOPMA	https://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html	蛋白质二级结构预测
SWISS-MODEL	http: //swissmodel.expasy.org/	三级结构预测

图1 奶山羊SIRT7基因PCR扩增结果 M.DL5000 Marker;1.PCR扩增条带。

Fig.1 Results of PCR amplification of SIRT7 gene M.DL5000 Marker;1.PCR amplified strips.

图2 SIRT7基因系统进化树

Fig.2 Phylogenetic tree of SIRT7 gene

表3 奶山羊SIRT7蛋白氨基酸组成

Tab.3 Amino acid composition of dairy goat SIRT7 protein

氨基酸 Amino acid	数量/个 Amount	含量/% Content	氨基酸 Amino acid	数量/个 Amount	含量/% Content
Arg	46	11.5	Gln	17	4.2
Leu	44	11.0	Asp	16	4.0
Ala	32	8.0	His	12	3.0
Glu	32	8.0	Cys	11	2.8
Gly	31	7.8	Ile	11	2.8
Ser	29	7.2	Tyr	8	2.0
Thr	23	5.8	Asn	7	1.8
Lys	22	5.5	Met	7	1.8
Val	22	5.5	Trp	7	1.8
Pro	19	4.8	Phe	4	1.0

图3 SIRT7蛋白亲疏水性预测

Fig.3 Prediction of SIRT7 protein hydrophilicity and hydrophobicity

图4 SIRT7蛋白跨膜结构预测

Fig.4 Transmembrane prediction of SIRT7 protein

图5 SIRT7蛋白二级结构预测 h.α-螺旋;e.延伸链;t.β-转角;c.无规则卷曲。

Fig.5 Secondary structure prediction of SIRT7 protein h.Alpha-helix;e.Extended chain;t.Beta-turn;c.Irregular curl.

图6 SIRT7蛋白三级结构预测

Fig.6 Tertiary structure prediction of SIRT7 protein

图7 SIRT7基因在各组织中的相对mRNA表达量 A.脾脏;B.肝脏;C.肾脏;D.瘤胃;E.小肠;F.背最长肌;G.尾脂;H.乳腺。不同小写字母表示差异显著(P<0.05)。图10,12同。

Fig.7 Relative mRNA expression of SIRT7 gene in each tissue A.Spleen;B.Liver;C.Kidney;D.Rumen;E.Small intestine;F.Longest dorsal muscle;G.Caudal fat;H.Mammary gland.Different lowercase letters indicate significant difference(P<0.05).The same as Fig.10,12.

图8 pcDNA3.1-SIRT7重组质粒酶切鉴定 M.DL15000 Marker;1.pcDNA3.1-SIRT7质粒酶切条带。

Fig.8 Enzymatic digestion of pcDNA3.1-SIRT7 recombinant plasmid M.DL15000 Marker;1.Double enzyme cut strips of pcDNA3.1-SIRT7 plasmid.

图9 SIRT7过表达载体转染效果检测不同大写字母表示差异极显著(P<0.01)。图12同。

Fig.9 Detection of SIRT7 overexpression vector transfection effect Different capital letters indicate highly significant difference(P<0.01).The same as Fig.12.

图10 SIRT7过表达对细胞中甘油三酯的影响

Fig.10 Effect of SIRT7 overexpression on triglycerides in cells

图11 油红O染色结果

Fig.11 Oil red O staining results

图12 SIRT7过表达对脂质代谢相关基因mRNA表达量的影响

Fig.12 Effect of SIRT7 overexpression on mRNA expression of lipid metabolism related genes

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