Cloning and Expression Analysis of ZNF32 Gene in Goat

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

Abstract

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

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.

Figures/Tables 14

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

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

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

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

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

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

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

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

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

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

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

Fig. 9 Sequencing results of goat ZNF32 gene overexpression vector

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

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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