山药DoWRKY40基因表达特征分析及互作蛋白筛选

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

摘要/Abstract

摘要：

【目的】WRKY作为植物特异型转录因子，参与植物生长发育过程。克隆山药DoWRKY40基因，分析其表达模式，通过酵母双杂交技术筛选与DoWRKY40互作的蛋白，探究WRKY40在山药膨大过程中的调控机制。【方法】以‘大和长芋’山药为材料克隆DoWRKY40基因，并进行生物信息学分析、表达模式分析、亚细胞定位，构建山药块茎不同发育时期酵母文库，筛选与DoWRKY40互作蛋白。【结果】DoWRKY40的开放阅读框长为927 bp。DoWRKY40蛋白属于WRKY家族的第II组，含有一个典型的WRKY转录因子保守结构域，定位于细胞核。与几内亚薯蓣亲缘关系较近。DoWRKY40基因在山药块茎生长发育的120 d表达量最高，且在叶、茎和块茎中均有表达，具有组织特异性。山药cDNA文库的库容量为1.484×10⁸；pGBKT7-WRKY40诱饵载体无自激活活性。酵母双杂交法从山药文库中筛选到4类与DoWRKY40相互作用的蛋白，分别参与植物生长发育、细胞周期调节与细胞扩增、信号转导及环境胁迫应答等。【结论】克隆得到DoWRKY40基因，其响应山药的生长发育过程，筛选到的4类互作蛋白表明其在山药细胞膨大及信号转导中起调控作用。

关键词: 山药, WRKY40基因, 表达特征, 亚细胞定位, cDNA文库, 酵母双杂交, 互作蛋白筛选

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

邢丽南, 张艳芳, 葛明然, 赵令敏, 陈妍, 霍秀文. 山药DoWRKY40基因表达特征分析及互作蛋白筛选[J]. 生物技术通报, 2024, 40(8): 118-128.

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.

图/表 13

表1 WRKY40相关的引物序列

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

表2 DoWRKY40生物信息学分析相关软件

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

图1 山药RNA提取及DoWRKY40基因PCR A：RNA电泳检测图；B：DoWRKY40已知序列验证；M1：DL5000； M2：DL2000

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

图2 DoWRKY40的编码序列及推导的氨基酸序列红色框：WRKY蛋白保守序列WRKYGQK；下划线：C2H2结构域；*：终止密码子

Fig. 2 Coding sequence and deduced amino acid sequence of DoWRKY40 Red box: WRKY protein conserved sequence WRKYGQK; underline: C2H2 domain; *: termination codon

图3 DoWRKY40蛋白二级、三级结构预测及结构域分析

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

图4 WRKY40与其他物种WRKY氨基酸序列比对（A）及系统进化树（B）

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

图5 DoWRKY40在山药不同发育时期（A）和不同组织（B）中的表达模式

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

图6 DoWRKY40的亚细胞定位

Fig. 6 Subcellular localization of DoWRKY40

图7 cDNA文库构建与鉴定 A：RNA电泳检测图；B：ds cDNA合成电泳检测图；C：均一化与去小片段电泳检测图；D：大肠杆菌菌落计数；E：24个单克隆插入片段的PCR鉴定；M1：DL5000 marker；1-24：挑取的24个克隆菌落PCR

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

图8 诱饵载体构建与菌落PCR鉴定 A：DoWRKY40质粒PCR图；1-2：PCR扩增产物；B：pGBKT7-DoWRKY40菌落PCR图；1-6：pGBKT7-DoWRKY40阳性克隆菌落PCR；M：DL5000 DNA marker

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

图9 诱饵载体pGBKT7-DoWRKY40的自激活检测

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

图10 cDNA文库筛选效率（A）、DoWRKY40互作蛋白筛选（B）及质粒PCR检测（C） B：1-22：筛选的阳性克隆编号；+：阳性对照；-：阴性对照；C：M1：DL5000 DNA marker；1-6：6个质粒PCR

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

表3 DoWRKY40候选互作蛋白的BLAST分析结果

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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