Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (8): 118-128.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0177
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XING Li-nan(), ZHANG Yan-fang, GE Ming-ran, ZHAO Ling-min, CHEN Yan, HUO Xiu-wen()
Received:
2024-02-23
Online:
2024-08-26
Published:
2024-06-26
Contact:
HUO Xiu-wen
E-mail:1760349574@qq.com;huoxiuwen@imau.edu.cn
XING Li-nan, ZHANG Yan-fang, GE Ming-ran, ZHAO Ling-min, CHEN Yan, HUO Xiu-wen. Analysis of DoWRKY40 Gene Expression Characteristics and Screening of Interacting Proteins in Yam[J]. Biotechnology Bulletin, 2024, 40(8): 118-128.
引物名称Primer name | 引物序列 Primer sequences(5'-3') | 引物用途Primer usage |
---|---|---|
WRKY40-F | TTCTTCTTCTTCTTCTTCTTCT | 已知序列验证Sequence verification |
WRKY40-R | AACATTCAAAAGTCTCCCCCA | |
WRKY40-qF | AATGGAGCAATCATCACTTA | RT-qPCR |
WRKY40-qR | AACATCTCACTCAATCTTTC | |
18S-F | CCATAAACGATGCCGACCAG | 18S rRNA |
18S-R | AGCCTTGCGACCATACTCCC | |
WRKY40-SF | CGGGGTACCATGGAATCAATAACAATGGAGC | 亚细胞定位Subcellular localization |
WRKY40-SR | GCGGATCCTATTTGGAGAAAAACTAAGAA | |
WRKY40-Y-F | GCGGATCCATGGAATCAATAACAATGGAGC | 诱饵载体构建Decoy vector construction |
WRKY40-Y-R | CTGCAGATTTGGAGAAAAACTAAGAATTTTT | |
3'AD | GTGAACTTGCGGGGTTTTTCAG | 酵母质粒PCR Yeast plasmid PCR |
5'AD | CTATTCGATGATGAAGATACCCC |
Table 1 Primer sequences related to WRKY40
引物名称Primer name | 引物序列 Primer sequences(5'-3') | 引物用途Primer usage |
---|---|---|
WRKY40-F | TTCTTCTTCTTCTTCTTCTTCT | 已知序列验证Sequence verification |
WRKY40-R | AACATTCAAAAGTCTCCCCCA | |
WRKY40-qF | AATGGAGCAATCATCACTTA | RT-qPCR |
WRKY40-qR | AACATCTCACTCAATCTTTC | |
18S-F | CCATAAACGATGCCGACCAG | 18S rRNA |
18S-R | AGCCTTGCGACCATACTCCC | |
WRKY40-SF | CGGGGTACCATGGAATCAATAACAATGGAGC | 亚细胞定位Subcellular localization |
WRKY40-SR | GCGGATCCTATTTGGAGAAAAACTAAGAA | |
WRKY40-Y-F | GCGGATCCATGGAATCAATAACAATGGAGC | 诱饵载体构建Decoy vector construction |
WRKY40-Y-R | CTGCAGATTTGGAGAAAAACTAAGAATTTTT | |
3'AD | GTGAACTTGCGGGGTTTTTCAG | 酵母质粒PCR Yeast plasmid PCR |
5'AD | CTATTCGATGATGAAGATACCCC |
软件 Software | 用途 Usage |
---|---|
NCBI ORF Finder | CDS序列分析 CDS sequence analysis |
Prot Param | 蛋白理化性质分析Analysis in physicochemical properties of coding protein |
TMHMM Server 2.0 | 跨膜结构域预测 Prediction of protein transmembrane domain |
SOPMA | 蛋白结构预测 Prediction of protein structure |
DNAMAN | 氨基酸序列比对 Alignment of amino acid sequences |
CDD | 保守结构域分析 Analysis of conservative domain |
NCBI Blast | 同源性搜索及比对Homology search and alignment of amino acid sequence |
MEGA 5.0 | 系统进化树的构建 Construction of phylogenetic tree |
Table 2 Related software for DoWRKY40 bioinformatics analysis
软件 Software | 用途 Usage |
---|---|
NCBI ORF Finder | CDS序列分析 CDS sequence analysis |
Prot Param | 蛋白理化性质分析Analysis in physicochemical properties of coding protein |
TMHMM Server 2.0 | 跨膜结构域预测 Prediction of protein transmembrane domain |
SOPMA | 蛋白结构预测 Prediction of protein structure |
DNAMAN | 氨基酸序列比对 Alignment of amino acid sequences |
CDD | 保守结构域分析 Analysis of conservative domain |
NCBI Blast | 同源性搜索及比对Homology search and alignment of amino acid sequence |
MEGA 5.0 | 系统进化树的构建 Construction of phylogenetic tree |
Fig. 1 Extraction of RNA of yam and PCR of DoWRKY40 gene A: Photo of RNA electrophoresis detection; B: Verification of known DoWRKY40 sequences; M1: DL5000; M2: DL2000
Fig. 2 Coding sequence and deduced amino acid sequence of DoWRKY40 Red box: WRKY protein conserved sequence WRKYGQK; underline: C2H2 domain; *: termination codon
Fig. 7 Construction of cDNA library and identification A: Photo of RNA electrophoresis detection. B: Photo of ds cDNA electrophoresis detection. C: Photo of homogenization and defragmentation electrophoresis detection. Escherichia coli colony count (D) and PCR identification of 24 monoclonal insertion fragments (E); M1: DL5000 marker. 1-24: Selected 24 cloned colonies PCR
Fig. 8 Construction of bait vector and PCR identification of colony A: PCR of DoWRKY40 plasmid; 1-2: PCR amplification products; B: PCR of pGBKT7-DoWRKY40 colony; 1-6: PCR results of pGBKT7-DoWRKY40 positive clonal colony; M: DL5000 DNA marker
Fig. 10 Efficiency of cDNA libary screening (A), DoWRKY40 interaction protein screening (B) and plasmid PCR detection (C) B: 1-22: Numbering of screened positive clones; +: positive control; -: negative control; C: M1: DL5000 DNA marker; 1-6: PCR results of 1-6 plasmid
序号 No. | NCBI比对结果 NCBI blast results | E-value | 相似度 Similarity/% | 登录号 Accession number | 克隆数 Clone count |
---|---|---|---|---|---|
1 | 转录因子EGL1 Transcription factor EGL1 | 0.0 | 93 | XM-039282616.1 | 1 |
2 | DNA结合蛋白BIN4 DNA-binding protein BIN4 | 0.0 | 94 | XM-039264312.1 | 3 |
3 | 蛋白磷酸酶2C Protein phosphatase 2C | 0.0 | 86 | XM-039277539.1 | 1 |
4 | BAG家族分子伴侣调控因子7 BAG family molecular chaperone regulator 7-like | 0.0 | 95 | XM-039266610.1 | 1 |
Table 3 BLAST analysis of candidate proteins interacted with DoWRKY40
序号 No. | NCBI比对结果 NCBI blast results | E-value | 相似度 Similarity/% | 登录号 Accession number | 克隆数 Clone count |
---|---|---|---|---|---|
1 | 转录因子EGL1 Transcription factor EGL1 | 0.0 | 93 | XM-039282616.1 | 1 |
2 | DNA结合蛋白BIN4 DNA-binding protein BIN4 | 0.0 | 94 | XM-039264312.1 | 3 |
3 | 蛋白磷酸酶2C Protein phosphatase 2C | 0.0 | 86 | XM-039277539.1 | 1 |
4 | BAG家族分子伴侣调控因子7 BAG family molecular chaperone regulator 7-like | 0.0 | 95 | XM-039266610.1 | 1 |
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