Construction of cDNA Library of Setosphaeria turcica and Screening of Transcription Factor StMR1 Interacting Proteins

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

Abstract

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

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.

Figures/Tables 9

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

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

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

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

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）

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

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

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

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

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