Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (4): 306-318.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1048
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ZHANG Qing-lan1,2(
), ZHANG Ya-ran2,3(), JU Zhi-hua2,3, WANG Xiu-ge2,3, XIAO Yao2,3, WANG Jin-peng2,3, WEI Xiao-chao2,3, GAO Ya-ping2,3, BAI Fu-heng4, WANG Hong-cheng1()
Received:2023-11-09
Online:2024-04-26
Published:2024-04-30
Contact:
ZHANG Ya-ran, WANG Hong-cheng
E-mail:1400304672@qq.com;zhang_ya_ran@126.com;wanghc@gznu.edu.cn
ZHANG Qing-lan, ZHANG Ya-ran, JU Zhi-hua, WANG Xiu-ge, XIAO Yao, WANG Jin-peng, WEI Xiao-chao, GAO Ya-ping, BAI Fu-heng, WANG Hong-cheng. Identification and Transcriptional Regulation Analysis of Core Promoter in Bovine TARDBP Gene[J]. Biotechnology Bulletin, 2024, 40(4): 306-318.
| 引物名称 Primer name | 引物序列 Primer sequence（5'-3'） | 产物大小 Product length/bp | 退火温度 Annealing temperature/℃ | 用途 Usage |
|---|---|---|---|---|
| TARDBP-F | ATGTCTGAATATATTCGGGTAACCG | 1 245 | 64.4 | CDS区扩增 |
| TARDBP-R | CTACATTCCCCAGCCAGAAGATTTA | |||
| TARDBP-GSP | CCGTGAAGCGAACGAAGCCAAACC | 496 | 68 | 5'RACE第一轮PCR |
| TARDBP -NGSP | AGATTCCCCCAGCCAGCATCGG | 230 | 5'RACE第二轮PCR | |
| pTARDBP-F | GCAAAGGGATAGAGGGAA | 1 954 | 55 | 启动子区扩增 |
| pTARDBP-R | CAGGGCGTCCTTAGTGAG | |||
| pGL3-P1 | CGG GGTACCGCAAAGGGATAGAGGGAA | 1 954 | 55 | 不同缺失片段扩增 |
| pGL3-P2 | CGG GGTACCGTTCAAACTACCGCACAATG | 1 498 | ||
| pGL3-P3 | CGG GGTACCTAGGGTGGCAAAGAGTTGG | 1 179 | ||
| pGL3-P4 | CGG GGTACCGAGAAGGAAATGGCAACCC | 854 | ||
| pGL3-P5 | CGG GGTACCAATGGCTTGGGCTCAACAG | 615 | ||
| pGL3-P6 | CGG GGTACCTATAGCCTTCAAGTTCACTCCC | 288 | ||
| pGL3-R1 | CCC AAGCTTCAGGGCGTCCTTAGTGAG | |||
| TARDBP-q-F | GTTTGGCTTCGTTCGCTTCA | 164 | 60 | RT-qPCR |
| TARDBP-q-R | CCTCTGTACAACGCCCAACA | |||
| β-actin-q-F | CATCGGCAATGAGCGGTTCC | 147 | ||
| β-actin-q-R | ACCGTGTTGGCGTAGAGGTC |
Table 1 Sequence information of bovine TARDBP gene primer
| 引物名称 Primer name | 引物序列 Primer sequence（5'-3'） | 产物大小 Product length/bp | 退火温度 Annealing temperature/℃ | 用途 Usage |
|---|---|---|---|---|
| TARDBP-F | ATGTCTGAATATATTCGGGTAACCG | 1 245 | 64.4 | CDS区扩增 |
| TARDBP-R | CTACATTCCCCAGCCAGAAGATTTA | |||
| TARDBP-GSP | CCGTGAAGCGAACGAAGCCAAACC | 496 | 68 | 5'RACE第一轮PCR |
| TARDBP -NGSP | AGATTCCCCCAGCCAGCATCGG | 230 | 5'RACE第二轮PCR | |
| pTARDBP-F | GCAAAGGGATAGAGGGAA | 1 954 | 55 | 启动子区扩增 |
| pTARDBP-R | CAGGGCGTCCTTAGTGAG | |||
| pGL3-P1 | CGG GGTACCGCAAAGGGATAGAGGGAA | 1 954 | 55 | 不同缺失片段扩增 |
| pGL3-P2 | CGG GGTACCGTTCAAACTACCGCACAATG | 1 498 | ||
| pGL3-P3 | CGG GGTACCTAGGGTGGCAAAGAGTTGG | 1 179 | ||
| pGL3-P4 | CGG GGTACCGAGAAGGAAATGGCAACCC | 854 | ||
| pGL3-P5 | CGG GGTACCAATGGCTTGGGCTCAACAG | 615 | ||
| pGL3-P6 | CGG GGTACCTATAGCCTTCAAGTTCACTCCC | 288 | ||
| pGL3-R1 | CCC AAGCTTCAGGGCGTCCTTAGTGAG | |||
| TARDBP-q-F | GTTTGGCTTCGTTCGCTTCA | 164 | 60 | RT-qPCR |
| TARDBP-q-R | CCTCTGTACAACGCCCAACA | |||
| β-actin-q-F | CATCGGCAATGAGCGGTTCC | 147 | ||
| β-actin-q-R | ACCGTGTTGGCGTAGAGGTC |
| 软件名Software name | 网址 Website | 用途 Purpose |
|---|---|---|
| NCBI | https://www.ncbi.nlm.nih.gov/ | 氨基酸序列 |
| https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi | 保守结构域分析 | |
| DNAMAN9.0软件 | 同源性比对 | |
| MEGA11.0软件 | 构建基因系统进化树 | |
| ExPASy | https://web.expasy.org/protscale/ | 理化性质分析 |
| TMHMM | https://services.healthtech.dtu.dk/service.php?TMHMM-2.0 | 蛋白跨膜结构域预测 |
| SignalP5.0 | https://services.healthtech.dtu.dk/service.php?Signal P-5.0 | 蛋白信号肽预测 |
| NetPhos-3.1 | http://www.cbs.dtu.dk/services/NetPhos/ | 蛋白磷酸化位点分析 |
| NPS@SOPMA | https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl | 蛋白质二级结构预测 |
| SWISS-MODEL | https://swissmodel.expasy.org/ | 蛋白质三级结构预测 |
| Meth Primer | http://www.urogene.org/methprimer/ | CpG岛预测 |
| AnimalTFDB4 | bioinfo.life.hust.edu.cn/AnimalTFDB4 | 转录因子结合位点预测 |
| TRANSFAC2.0 | https://genexplain.com/transfac-2-0/ |
Table 2 Software for bioinformatics analysis
| 软件名Software name | 网址 Website | 用途 Purpose |
|---|---|---|
| NCBI | https://www.ncbi.nlm.nih.gov/ | 氨基酸序列 |
| https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi | 保守结构域分析 | |
| DNAMAN9.0软件 | 同源性比对 | |
| MEGA11.0软件 | 构建基因系统进化树 | |
| ExPASy | https://web.expasy.org/protscale/ | 理化性质分析 |
| TMHMM | https://services.healthtech.dtu.dk/service.php?TMHMM-2.0 | 蛋白跨膜结构域预测 |
| SignalP5.0 | https://services.healthtech.dtu.dk/service.php?Signal P-5.0 | 蛋白信号肽预测 |
| NetPhos-3.1 | http://www.cbs.dtu.dk/services/NetPhos/ | 蛋白磷酸化位点分析 |
| NPS@SOPMA | https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl | 蛋白质二级结构预测 |
| SWISS-MODEL | https://swissmodel.expasy.org/ | 蛋白质三级结构预测 |
| Meth Primer | http://www.urogene.org/methprimer/ | CpG岛预测 |
| AnimalTFDB4 | bioinfo.life.hust.edu.cn/AnimalTFDB4 | 转录因子结合位点预测 |
| TRANSFAC2.0 | https://genexplain.com/transfac-2-0/ |
Fig. 1 PCR product of the bovine TARDBP gene CDS region M: DL5000 DNA marker. 1: CDS region of bovine TARDBP gene
| 物种 Species | 登录号 GenBank No. | 氨基酸序列相似性Similarity/% |
|---|---|---|
| 牛 Bos taurus | NP_001039950.2 | |
| 山羊 Capra hircus | XP_005690745.1 | 100 |
| 绵羊 Ovis aries | XP_027831445.1 | 100 |
| 犬 Canis lupus familiaris | XP_038516303.1 | 99.76 |
| 猪 Sus scrofa | XP_020950991.1 | 99.52 |
| 家猫 Felis catus | XP_003989534.1 | 99.03 |
| 人 Homo sapiens | NP_031401.1 | 97.58 |
| 苏门答腊猩猩Pongo abelii | NP_001127597.1 | 97.34 |
| 原鸡 Gallus gallus | NP_001026049.2 | 96.86 |
| 热带爪蟾 Xenopus tropicalis | NP_001231687.1 | 74.10 |
| 小鼠 Mus musculus | NP_001003898.1 | 67.39 |
| 大鼠 Rattus norvegicus | NP_001011979.1 | 66.67 |
Table 3 Amino acid sequence comparison of TARDBP between cattle and different species
| 物种 Species | 登录号 GenBank No. | 氨基酸序列相似性Similarity/% |
|---|---|---|
| 牛 Bos taurus | NP_001039950.2 | |
| 山羊 Capra hircus | XP_005690745.1 | 100 |
| 绵羊 Ovis aries | XP_027831445.1 | 100 |
| 犬 Canis lupus familiaris | XP_038516303.1 | 99.76 |
| 猪 Sus scrofa | XP_020950991.1 | 99.52 |
| 家猫 Felis catus | XP_003989534.1 | 99.03 |
| 人 Homo sapiens | NP_031401.1 | 97.58 |
| 苏门答腊猩猩Pongo abelii | NP_001127597.1 | 97.34 |
| 原鸡 Gallus gallus | NP_001026049.2 | 96.86 |
| 热带爪蟾 Xenopus tropicalis | NP_001231687.1 | 74.10 |
| 小鼠 Mus musculus | NP_001003898.1 | 67.39 |
| 大鼠 Rattus norvegicus | NP_001011979.1 | 66.67 |
Fig. 2 Phylogenetic tree of TARDBP gene The number on the branch is >50% of the self-explanatory value（1 000 repetitions）
| 氨基酸 Amino acid | 数目 Number | 比例Perc-entage/% | 氨基酸 Amino acid | 数目 Number | 比例Perc- entage/% |
|---|---|---|---|---|---|
| Ala（A） | 27 | 6.5% | Leu（L） | 20 | 4.8% |
| Arg（R） | 20 | 4.8% | Lys（K） | 20 | 4.8% |
| Asn（N） | 27 | 6.5% | Met（M） | 17 | 4.1% |
| Asp（D） | 22 | 5.3% | Phe（F） | 22 | 5.3% |
| Cys（C） | 7 | 1.7% | Pro（P） | 17 | 4.1% |
| Gln（Q） | 24 | 5.8% | Ser（S） | 41 | 9.9% |
| Glu（E） | 20 | 4.8% | Thr（T） | 15 | 3.6% |
| Gly（G） | 55 | 13.3% | Trp（W） | 6 | 1.4% |
| His（H） | 5 | 1.2% | Tyr（Y） | 6 | 1.9% |
| Ile（I） | 14 | 3.4% | Val（V） | 8 | 6.5% |
Table 4 Amino acid composition of bovine TARDBP protein
| 氨基酸 Amino acid | 数目 Number | 比例Perc-entage/% | 氨基酸 Amino acid | 数目 Number | 比例Perc- entage/% |
|---|---|---|---|---|---|
| Ala（A） | 27 | 6.5% | Leu（L） | 20 | 4.8% |
| Arg（R） | 20 | 4.8% | Lys（K） | 20 | 4.8% |
| Asn（N） | 27 | 6.5% | Met（M） | 17 | 4.1% |
| Asp（D） | 22 | 5.3% | Phe（F） | 22 | 5.3% |
| Cys（C） | 7 | 1.7% | Pro（P） | 17 | 4.1% |
| Gln（Q） | 24 | 5.8% | Ser（S） | 41 | 9.9% |
| Glu（E） | 20 | 4.8% | Thr（T） | 15 | 3.6% |
| Gly（G） | 55 | 13.3% | Trp（W） | 6 | 1.4% |
| His（H） | 5 | 1.2% | Tyr（Y） | 6 | 1.9% |
| Ile（I） | 14 | 3.4% | Val（V） | 8 | 6.5% |
Fig. 3 Analysis of TARDBP protein structure A: Prediction of bovine TARDBP protein conserved domain. B: Prediction of bovine TARDBP protein hydrophobicity. C: Prediction of bovine TARDBP protein transmembrane structure. D: Prediction of bovine TARDBP protein signal peptide. E: Prediction of secondary structure of bovine TARDBP protein. F: Tertiary structure prediction of bovine TARDBP protein. G: Prediction of phosphorylation site of bovine TARDBP protein
Fig. 4 Analysis of bovine TARDBP gene expression in different tissues The same capital letter indicates that the difference is not significant（P>0.05）, different capital letters indicate significant differences（P<0.01）
Fig. 5 5' RACE amplification electrophoresis of bovine TARDBP gene M: DL5000 DNA marker. 1: First round PCR products. 2: Second round PCR product
Fig. 6 Transcription start site of bovine TARDBP gene
Fig. 7 PCR products from the promoter region of bovine TARDBP gene M: DL5000 DNA marker. 1: The 5' flanking region（-1815/+139）of bovine TARDBP gene
Fig. 8 Prediction results of CpG island in the 5' regulatory region of bovine TARDBP gene A: The predict result by MethPrimer software. B: The specific location of CpG islands
Fig. 9 Prediction of potential transcription factor binding sites of bovine TARDBP gene promoter +1 indicates transcription initiation site; letters in in box indicates transcription factor binding sites. Overlapping regions of transcription factor binding sites are represented by different colored boxes
Fig. 10 Amplification and vector construction of different deletion fragments of bovine TARDBP gene promoter A: Electrophoretic amplification of bovine TARDBP gene promoter fragment by fragment deletion, M: DL5000 DNA marker, 1-6: add the missing fragments of bovine TARDBP gene promoter at double restriction sites. B: Electrophoresis of bovine TARDBP gene promoter double fluorescein reporter recombinant plasmid double enzyme digestion identification， M: DL5000 DNA marker, 1-6: recombinant plasmid
Fig. 11 Activity analysis of bovine TARDBP gene promoter in bovine mammary epithelial cells Different letters indicate significant differences（P<0.05）
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