玉米大斑病菌cDNA文库的构建及转录因子StMR1互作蛋白的筛选

doi:10.13560/j.cnki.biotech.bull.1985.2024-0121

摘要/Abstract

摘要：

【目的】 筛选玉米大斑病菌（Setosphaeria turcica）转录因子的互作蛋白，解析黑色素调控转录因子StMR1调控玉米大斑病菌致病性的分子机制。为解析玉米大斑病菌侵染过程中转录因子的调控网络，阐明病菌的致病机理提供参考。【方法】 收集玉米大斑病菌菌丝和孢子不同萌发阶段作为试验材料，采用Gateway方法构建玉米大斑病菌cDNA文库，使用同源重组的方法构建转录因子StMR1的诱饵载体，采用酵母双杂交技术筛选其互作蛋白并进行一对一验证。【结果】 构建的玉米大斑病菌文库插入的平均片段长度大于1 000 bp，初级文库及次级文库的库容量为1.2×10⁷和1.04×10⁷ CFU，重组率为100%，可以用于酵母双杂交筛选。成功构建可以用于筛库的诱饵载体pGBKT7-StMR1，经初筛与复筛得到3个互作蛋白，一对一验证短链脱氢酶、糖基转移酶、富含亮氨酸重复序列蛋白均与转录因子StMR1存在互作。【结论】 成功构建了丰富度高且质量好的玉米大斑病菌cDNA文库并筛选到了与转录因子StMR1互作的蛋白。

关键词: 玉米大斑病菌, cDNA文库, 转录因子, 酵母双杂交, 互作蛋白

Abstract:

【Objective】 To screen the interaction proteins of transcription factors of Setosphaeria turcica, and to analyze the molecular mechanism of melanin-regulated transcription factor StMR1 in regulating the pathogenicity of S. turcica. This study provides a reference for elucidating the regulatory network of transcription factors in the infection process of S. turcica and elucidating the pathogenic mechanism of the pathogen.【Method】 Different germination stages of hyphae and spores of S. turcica were collected as test materials, and the cDNA library of S. turcica was constructed by gateway method, The bait vector of transcription factor StMR1 was constructed by homologous recombination, and its interacting proteins were screened by yeast two-hybrid technology and verified one-to-one.【Result】 The average fragment length of the constructed library was greater than 1 000 bp, the library capacity of the primary library and the secondary library were 1.2×10⁷ and 1.04×10⁷ CFU, respectively, and the recombination rate was 100%, which could be used for yeast two-hybrid screening. The bait vector pGBKT7-StMR1 that could be used for screen library was successfully constructed, and three interacting proteins were obtained through primary screening and re-screening, and one-to-one verification of the interaction between short-chain dehydrogenase, glycosyltransferase and leucine-rich repeat protein and transcription factor StMR1 were verified.【Conclusion】 A high quality and abundant cDNA library was successfully constructed and the protein interacting with StMR1 was screened

Key words: Setosphaeria turcica, cDNA library, transcription factor, yeast two-hybrid, interaction protein

王秋月, 段鹏亮, 李海笑, 刘宁, 曹志艳, 董金皋. 玉米大斑病菌cDNA文库的构建及转录因子StMR1互作蛋白的筛选[J]. 生物技术通报, 2024, 40(6): 281-289.

WANG Qiu-yue, DUAN Peng-liang, LI Hai-xiao, LIU Ning, CAO Zhi-yan, DONG Jin-gao. Construction of cDNA Library of Setosphaeria turcica and Screening of Transcription Factor StMR1 Interacting Proteins[J]. Biotechnology Bulletin, 2024, 40(6): 281-289.

图/表 9

表1 试验所用引物

Table 1 Primers used in the experiment

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
pGBKT7-StMR1-F	TGGCCATGGAGGCCGAATTCCCGGGATGGTTTTCTGCACATACTGC
pGBKT7-StMR1-R	GGGGTTATGCTAGTTATGCGGCCGCTTAGCCGCGCGCAAGCTTTT
pGADT7-135640--F	CGACGTACCAGATTACGCTCATATGATGGCAGAGAAGAAGCAGCGC
pGADT7-135640-R	TGCAGCTCGAGCTCGATGGATCCTTATTGCTTCAGCGCGAGCA
pGADT7-169112-F	CGACGTACCAGATTACGCTCATATGATGCACCTCAACGACTTCCC
pGADT7-169112-R	TGCAGCTCGAGCTCGATGGATCCTCACGTCTGGATGGAATGGA
pGADT7-165582-F	TGGCCATGGAGGCCAGTGAATTCATGACATCACGACTCATCACC
pGADT7-165582-R	CTGCAGCTCGAGCTCGATGGATCCTTACCACTTCTCAACCG
M 13-F	GTAAAACGACGGCCAG
M 13-R	CAGGAAACAGCTATGAC
T7	TAATACGACTCACTATAGGGC
3-AD	AGATGGTGCACGATGCACAG

图1 文库材料选取 A-E：培养4、6、8、10和15 d；F：分生孢子；G：芽管；H：附着胞；I：侵入钉

Fig. 1 Library material selection A-E: Culture for 4, 6, 8, 10 and 15 d. F: Conidia. G: Germ tube. H: Appressorium.I: Penetration peg

图2 总RNA提取（A）及mRNA（B）和双链cDNA（C）电泳

Fig. 2 Total RNA extraction（A）and electrophoresis results of mRNA（B）and ds cDNA（C） (A) M: DL 5000 marker, 1-9: RNA；(B-C) M: DL 2000 marker, 1: mRNA,2: ds cDNA

图3 文库库容量及插入片段重组率鉴定 A：初级文库容量鉴定；B：1-24初级文库插入片段检测；C：核体系次级文库容量鉴定；D：1-24核体系次级文库插入片段检测；M：DL 2000 marker

Fig. 3 Identification of library capacity and insert recombination rate A: Identification of primary library capacity. B: 1-24 detection of primary library insert. C: Identification of the secondary library capacity of the nuclear system. D: 1-24 detection of nuclear system secondary library insert. M: DL 2000 marker

图4 诱饵载体pGBKT7-StMR1构建 A：目的片段扩增（1-3：目的片段；M：DL 5000 marker）；B：诱饵载体双酶切验证（1：双酶切；2：单酶切；M：DL 10000 marker）

Fig. 4 Construction of bait vector pGBKT7-StMR1 A: Target fragment amplification（1-3: target fragment; M: DL 5000 marker）. B: Verification of double digestion of bait vectors（1: digestion; 2: double single digestion; M: DL 10000 marker）

图5 诱饵载体pGBKT7-StMR1毒性及自激活活性检测

Fig. 5 Detection of toxicity and self-activating activity of bait vector pGBKT7-StMR1 A: SD/-Trp: pGBKT7-StMR1; B: SD/-Trp/X-α-gal: pGBKT7-StMR1; C: SD/-Trp/X-α-gal /AbA: pGBKT7-StMR1; D: SD/-Trp/Leu/X-α-gal/AbA: pGBKT7-53+pGADT7-T

图6 StMR1互作蛋白筛选 A：阳性克隆初筛；B：阳性克隆复筛；C：阳性克隆PCR验证

Fig. 6 StMR1 interacting protein screen A: Positive clone primary screening. B: Positive clone rescreen. C: PCR verification of positive cloning

表2 StMR1候选蛋白

Table 2 StMR1 candidate protein

基因号 Gene ID	基因登录号 GenBank No.	片段大小 Fragment size/bp	编码蛋白 Encoded protein	结构域 Pfam
SETTUDRAFT-165582	XM_008032228.1	1 396	短链脱氢/还原酶 Short chain dehydrogenase/reductase	短链脱氢酶\|烯酰基-（酰基载体蛋白）还原酶\|氪域
SETTUDRAFT-135640	XM_008027394.1	1 548	糖基转移酶 Glycosyltransferase	酵母中ALG2是一种1,3-甘露糖基转移酶
SETTUDRAFT-169112	XM_008027772.1	1 920	富含亮氨酸重复序列蛋白 Leucine-rich repeat	LRR_AMN1;富含亮氨酸的重复结构

图7 酵母双杂交一对一验证

Fig. 7 One-to-one verification of yeast two-hybrid

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