Analysis of DoWRKY40 Gene Expression Characteristics and Screening of Interacting Proteins in Yam

doi:10.13560/j.cnki.biotech.bull.1985.2024-0177

Abstract

Abstract:

【Objective】WRKY, as a plant idiosyncratic transcription factor, is involved in plant growth and development. DoWRKY40 gene in yam（Dioscorea opposita Thunb.）was cloned and its expression pattern was analyzed. The protein interacting with DoWRKY40 was screened by yeast two-hybrid technique to explore the regulatory mechanism of WRKY40 in the process of yam expansion.【Method】DoWRKY40 gene was cloned from ‘Dahechangyu’ yam, and bioinformatics analysis, expression pattern analysis and subcellular localization were performed. Yeast libraries of yam tuber at different developmental stages were constructed and DoWRKY40 interacting proteins were screened.【Result】The length of open reading frame of DoWRKY40 was 927 bp; DoWRKY40 belonged to group II of the WRKY family and contained a typical WRKY transcription factor conserved domain, which was localized in the nucleus. It was closely related to Dioscorea cayenensis subsp. The expression of DoWRKY40 gene was the highest at 120 d after the growth and development of yam tuber, and it was expressed in the leaves, stems and tubers with tissue specificity. The library capacity of yam cDNA library was 1.484×10⁸; pGBKT7-WRKY40 decoy vector had no self-activation activity. Four kinds of proteins interacting with DoWRKY40 were screened from yam library by yeast two-hybridization, which were involved in plant growth and development, cell cycle regulation and cell expansion, signal transduction and environmental stress response, respectively.【Conclusion】The DoWRKY40 gene is cloned, which responds to the growth and development process of yam. The four interacting proteins are screened, suggesting that it plays a regulatory role in the expansion and signal transduction of yam cells.

Key words: Dioscorea opposita Thunb., WRKY40 gene, expression characteristics, subcellular localization, cDNA library, yeast two-hybrid, interaction protein screening

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.

Figures/Tables 13

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	已知序列验证Sequence verification
WRKY40-qF	AATGGAGCAATCATCACTTA	RT-qPCR
WRKY40-qR	AACATCTCACTCAATCTTTC	RT-qPCR
18S-F	CCATAAACGATGCCGACCAG	18S rRNA
18S-R	AGCCTTGCGACCATACTCCC	18S rRNA
WRKY40-SF	CGGGGTACCATGGAATCAATAACAATGGAGC	亚细胞定位Subcellular localization
WRKY40-SR	GCGGATCCTATTTGGAGAAAAACTAAGAA	亚细胞定位Subcellular localization
WRKY40-Y-F	GCGGATCCATGGAATCAATAACAATGGAGC	诱饵载体构建Decoy vector construction
WRKY40-Y-R	CTGCAGATTTGGAGAAAAACTAAGAATTTTT	诱饵载体构建Decoy vector construction
3'AD	GTGAACTTGCGGGGTTTTTCAG	酵母质粒PCR Yeast plasmid PCR
5'AD	CTATTCGATGATGAAGATACCCC	酵母质粒PCR Yeast plasmid PCR

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. 3 Prediction of secondary and tertiary structure and domain analysis of DoWRKY40 protein

Fig. 4 Amino acid sequence comparison（A）between WRKY40 and WRKY of other species and phylogenetic tree（B）

Fig. 5 Expression patterns of DoWRKY40 in different stages of yam tuber development（A）and different tissues of yam（B） ** P<0.01

Fig. 6 Subcellular localization of DoWRKY40

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. 9 Self-activation detection of the bait vector pGBKT7-DoWRKY40

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

Table 3 BLAST analysis of candidate proteins interacted with DoWRKY40

序号 No.	NCBI比对结果 NCBI blast results	相似度 Similarity/%	登录号 Accession number	克隆数 Clone count
1	转录因子EGL1 Transcription factor EGL1	93	XM_-039282616.1	1
2	DNA结合蛋白BIN4 DNA-binding protein BIN4	94	XM_-039264312.1	3
3	蛋白磷酸酶2C Protein phosphatase 2C	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	95	XM_-039266610.1	1

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