山羊ZNF32的克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0098

摘要/Abstract

摘要：

以成年健康简州大耳羊为实验动物，利用RT-PCR技术克隆山羊ZNF32基因。利用生物信息学方法对其进行表达特性分析，并利用实时荧光定量PCR（real-time quantitative PCR，qPCR）技术检测ZNF32在山羊各组织中和诱导分化不同阶段的皮下脂肪细胞中的表达水平。同时构建过表达载体并利用qPCR技术检测过表达或干扰ZNF32对增殖抑制相关基因表达的影响，探究其对山羊皮下前体脂肪细胞增殖的作用。结果显示，克隆所得山羊ZNF32核苷酸序列长度为1 049 bp，其中CDS区长为822 bp，编码273个氨基酸；ZNF32分子量为30 983.03 Da，理论等电点为9.52，亲水性平均值为-0.813；在氨基酸序列组成中，丝氨酸含量为9.2%，占比最高；蛋白质二级结构预测显示，149个氨基酸形成无规卷曲，含量最高；氨基酸同源性比对结果显示，简州大耳羊与绵羊氨基酸序列相似性最高且在进化上亲缘关系最近；组织表达谱表明，ZNF32基因在山羊各个组织中广泛表达，其中在大肠中表达量最高（P<0.01）；时序表达谱表明，ZNF32基因表达水平呈上升趋势，在96 h表达量达到最高，极显著高于分化前（P<0.01）；qPCR技术检测结果表明，过表达ZNF32基因抑制增殖抑制相关基因p27和p57的表达，干扰ZNF32基因促进p21、p27、p53及p57的表达。以上结果表明，ZNF32基因可能是山羊皮下脂肪细胞分化和增殖的正调控因子。

关键词: 山羊, ZNF32, 基因克隆, 表达分析, 分化, 增殖

Abstract:

Having adult healthy Jianzhou big-ear goat as experimental animals，RT-PCR technology was used to clone the goat ZNF32 gene. The bioinformatics analysis was used to analyze the expression characteristics，and real-time quantitative PCR（qPCR）was used to detect the expressions of ZNF32 in goat tissues and inducing differentiation of subcutaneous fat cells at different stages. Meanwhile，the overexpression vector was constructed and qPCR was to detect the effects of overexpression or interference of ZNF32 on the expressions of genes related to proliferation inhibition，and its effect on the proliferation of subcutaneous precursor adipocytes was explored. The results showed that the length of the cloned goats ZNF32 nucleotide sequence was 1 049 bp，of which the CDS area length was 822 bp and 273 amino acids were encoded. ZNF32 molecular weight was 30 983.03 Da，the theoretical isoelectric point was 9.52，and the average hydrophilic value was -0.813. In the composition of amino acid sequences，serine content was 9.2%，accounting for the highest proportion. The secondary structure prediction of proteins demonstrated that 149 amino acids formed irregular curls and the highest content. The results of amino acid homology alignment revealed that the Jianzhou big-ear goat shared the highest similarity with sheep，and they were in the closest genetic relationship. Tissue expression spectrum showed that the ZNF32 gene was widely expressed in various tissues of the goat. Among them，the expression in the large intestine was the highest（P<0.01）. The timing expression spectrum showed that the expression level of the ZNF32 gene was on the rise，reached the highest expression at 96 h，which was significantly higher than that before differentiation（P<0.01）. The detection results of qPCR technology suggested that the overexpression of the ZNF32 gene inhibited the expression of proliferative inhibition-related genes p27 and p57，while the interference of ZNF32 promoted the expression of p21，p27，p53 and p57. The above results show that the ZNF32 gene may be a positive regulatory factor for subcutaneous fat cell differentiation and proliferation.

Key words: goat, ZNF32, gene cloning, expression analysis, differentiation, proliferation

盛雪晴, 赵楠, 林亚秋, 陈定双, 王瑞龙, 李傲, 王永, 李艳艳. 山羊ZNF32的克隆及表达分析[J]. 生物技术通报, 2022, 38(12): 300-311.

SHENG Xue-qing, ZHAO Nan, LIN Ya-qiu, CHEN Ding-shuang, WANG Rui-long, LI Ao, WANG Yong, LI Yan-yan. Cloning and Expression Analysis of ZNF32 Gene in Goat[J]. Biotechnology Bulletin, 2022, 38(12): 300-311.

图/表 14

表1 引物信息

Table 1 Primers information

目的基因 Target gene	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	产物长度 Product length/bp	用途 Purpose
ZNF32	S：CCGCAAGGGTCTAGCTG	58	1 049	克隆
	A：TGGATAGGCTGTTTAATCTCAGATG			Clone
ZNF32	S：TGGAAGATATGCCCAGAATGTAG	60	293	qPCR
	A：CACAATGGGTACACTCGGG
ZNF32	S：CCGGAATTCCATGTTTGGATTTCCAACAGCTACCC	58	822	过表达载体构建
	A：CCGCTCGAGACTTACAGGGTGAGCCTTTGTGAGC			Overexpression plasmid construction
Si-NC	S：UUCUCCGAACGUGUCACGUTT	60		干扰
	A：ACGUGACACGUUCGGAGAATT			Interference
Si-ZNF32	S：GGGUAGUCUAACAUUACAUTT	60		干扰
	A：AUGUAAUGUUAGACUACCCTT			Interference
TBP	S：AACAGCCTCCCACCTTATGC	60	155	内参
	A：TGCTGCTCCTCCTAGAC			Reference
UXT	S：GCAAGTGGATTTGGGCTGTAAC	60	180	内参
	A：ATGGAGTCCTTGGTGAGGTTGT			Reference

表 2 生物信息学分析内容及分析工具

Table 2 Analytical contents and analysis tools for bioinfo-rmatics

分析内容 Analytical content	分析软件或在线工具 Analytical software or online tools
氨基酸序列比对 Amino acid sequences alignment	DNAMAN
氨基酸序列翻译、同源性比对 Amino acid sequence translation and homology comparison	ORF Finder、Blast（NCBI）
蛋白质理化性质分析Protein physicochemical properties analysis	ExPASy ProtParam
蛋白质磷酸化位点分析 Protein phosphorylation site analysis	NetPhos 3.1
蛋白质功能结构域分析 Protein functional domain analysis	Conserved Domain
蛋白质二级结构预测 Protein secondary structure prediction	Npsa-prabi
蛋白质三级结构预测 Protein tertiary structure prediction	SWISS-MODEL
蛋白质相互作用预测 Interaction protein prediction	STRING
系统进化树构建 Phylogenetic tree construction	MEGA 5.0

表3 增殖抑制相关基因引物信息

Table 3 Primers information of genes associated with pro-liferation inhibition

目的基因 Target gene	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	用途 Purpose
P21	S：AGGGCACGTCTCAGGAGGA A：CAGTCTGCGTTTGGAGTGGTAG	60	qPCR
P27	S：CGGCGGTGCCTTTACTT A：GCAGGTCGCTTCCTTATCC	60	qPCR
P53	S：CGCCTCAGCACCTTATCCG A：GGCACCACGCACCTCA	60	qPCR
P57	S：CAGCCAGAGCATTGGCAATG A：CTGTCCACCTCGGTCCACT	60	qPCR

图1 山羊ZNF32基因扩增结果 M：DNA相对分子质量标准DL2000；1-2：山羊ZNF32基因目的条带

Fig. 1 Amplification of ZNF32 gene in goat M：DL2000 DNA marker. 1-2：Target strip of ZNF32 gene in goat

图2 山羊ZNF32基因的ORF序列及推断的氨基酸序列星形：丝氨酸磷酸化位点；四边形：苏氨酸磷酸化位点；圆形：酪氨酸磷酸化位点；阴影框：C2H2锌指结构域；ATG：起始密码子；TAA：终止密码子

Fig. 2 Sequences of ORF and deduced amino acids of ZNF32 gene in goat Stars：Serine phosphorylation sites. Quadrilaterals：Threonine phosphorylation sites. Circles：Tyrosine phosphorylation sites. Shadow boxes：C2H2 zinc finger domains. ATG：The start codon. TAA：The stop codon

图3 山羊ZNF32蛋白疏水性及氨基酸组成分析 A：山羊ZNF32蛋白疏水性分析；B：山羊ZNF32蛋白氨基酸组成分析

Fig. 3 Analysis of hydrophobicity and amino acid compos-ition of ZNF32 protein in goat A：Hydrophobicity analysis of ZNF32 protein in goat. B：Analysis of amino acid composition of ZNF32 protein in goat

图4 山羊ZNF32蛋白质结构及相互作用预测 A：山羊ZNF32蛋白质二级结构预测；B：山羊ZNF32蛋白质三级结构预测；C：ZNF32蛋白质互作网络

Fig. 4 Protein structure and interaction prediction of goat ZNF32 A：Prediction of secondary structure of goat ZNF32 protein. B：Prediction of tertiary structure of goat ZNF32 protein. C：ZNF32 protein interaction network

图5 ZNF32氨基酸序列比对及蛋白系统进化树 A：不同物种ZNF32氨基酸序列比对；B：系统进化树

Fig. 5 Amino acid sequence alignment and protein phylogenetic tree of goat ZNF32 gene A：Alignment of amino acid sequence of ZNF32 in different species. B：Phylogenetic tree

图6 山羊ZNF32组织表达谱 1：背最长肌；2：心；3：肝；4：瘤胃；5：肺；6：臂三头肌；7：股二头肌；8：肾；9：皮下脂肪；10：胰腺；11：小肠；12：腹间脂肪；13：脾；14：大肠。不同大写字母表示差异极显著（P<0.01）；不同小写字母表示差异显著（P<0.05）；相同字母表示差异不显著（P>0.05）.下同

Fig. 6 Expression profile of ZNF32 gene in different tissues of goat 1：Longissimus dorsi；2：heart；3：liver；4：rumen；5：lung；6：triceps brachii；7：biceps femoris；8：kidney；9：subcutaneous；10：pancreas；11：small intestine；12：abdominal fat；13：spleen；14：large intestine. Different capital letters indicate significant differences（P<0.01）. Different lowercase letters showed significant differences（P<0.05）. The same letters indicate insignificant differences（P>0.05）. The same below

图7 ZNF32基因在山羊皮下脂肪细胞不同分化阶段的相对表达水平

Fig. 7 Relative expressions of ZNF32 gene in goat during different stages of differentiation

图8 山羊ZNF32基因CDS区扩增结果 M：DNA相对分子质量标准DL2000；1-2：山羊ZNF32基因CDS区扩增结果

Fig. 8 CDS amplification results of goat ZNF32 gene M：DL2000 DNA marker. 1-2：Target strip in the CDS region of goat ZNF32 gene

图9 山羊ZNF32基因过表达载体测序结果

Fig. 9 Sequencing results of goat ZNF32 gene overexpression vector

图10 山羊ZNF32基因表达效率及增殖抑制相关基因表达检测 A：过表达ZNF32效率检测；B：过表达ZNF32后增殖抑制相关基因表达检测；C：干扰ZNF32效率检测；D：干扰ZNF32后增殖抑制相关基因检测。*：P<0.05，* *：P<0.01

Fig. 10 Detection of ZNF32 gene expression efficiency and the expressions of genes related to proliferation inhibition in goats A：Efficiency detection of ZNF32 overexpression. B：Expression detection of genes associated with proliferation inhibition after the overexpression of ZNF32. C：Efficiency detection after the interference of ZNF32. D：Detection of genes associated with proliferation inhibition after interference of ZNF32. *：P<0.05，and * *：P<0.01

图11 p53、p21和p27启动子区ZNF32结合序列 A：p53启动子区ZNF32结合序列；B：p21启动子区ZNF32结合序列；C：p27启动子区ZNF32结合序列

Fig. 11 Binding sequence of ZNF32 in p53，p21 and p27 promoter region A：The binding sequence of ZNF32 in p53 promoter region. B：The binding sequence of ZNF32 in p21 promoter region. C：The binding sequence of ZNF32 in p27 promoter region

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