Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (12): 300-311.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0098
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SHENG Xue-qing(), ZHAO Nan, LIN Ya-qiu, CHEN Ding-shuang, WANG Rui-long, LI Ao, WANG Yong, LI Yan-yan()
Received:
2022-01-21
Online:
2022-12-26
Published:
2022-12-29
Contact:
LI Yan-yan
E-mail:1265393031@qq.com;liyanyan@swun.edu.cn
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.
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 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 |
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 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 |
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 |
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. 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. 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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