Cloning of Transcription Factor Gene StbZIP9 of Setosphaeria turcica, Expression Analysis and Screening of Its Downstream Target Genes

doi:10.7668/hbnxb.20193956

Abstract

Abstract:

Basic leucine zipper(bZIP)transcription factor protein is a kind of transcription factor with conservative structure and function in animals,plants and microorganisms.In order to clarify the function and mechanism of bZIP transcription factor in plant pathogenic fungi,and further determine its relationship with the growth,development and pathogenicity of the pathogen,the StbZIP9 gene was cloned from Setosphaeria turcica 01-23(GenBank No.XM _ 008032179.1).StbZIP9 is a member of the bZIP transcription factor family.The analysis of the gene structure and protein characteristics showed that the DNA sequence was 788 bp in length,with an open reading frame of 726 bp,encoding 241 amino acids.The encoded protein contained a highly conserved homologous domain BRLZ in fungi.The RNA-seq data of the gene during the growth and development of the pathogen and the process of infecting the host were analyzed.It was found that the expression level of StbZIP9 was 2 to 4 times higher than that in the appressorium and germ tube period compared with the mycelium period.After 24,72 h of infection of maize leaves,the gene expression increased from scratch and continued to increase,indicating that StbZIP9 was associated with appressorium development and germ tube formation and played an important role in the process of pathogen infecting host cells.Further,bioinformatics techniques were used to predict its binding conserved motifs and regulatory target genes.The binding motif was NNTWACGTNN,including the bZIP transcription factor recognition core sequence ACGT,and the downstream target genes of StbZIP9 were predicted according to the sequence.Combined with the expression pattern analysis using the RNA-seq data,four downstream target genes(protein IDs in the JGI database were :132893,163024,162798,40466)were obtained,and the functional annotation table was obtained.The functional annotation revealed its involvement in many biological processes, such as polymerization and transport of cell wall components, host infection, and spore dormancy. It will provide the basis for further elucidation of the molecular mechanism involved in the regulation of pathogen infection.

Key words: Setosphaeria turcica, Transcription factors, Gene cloning, Expression pattern analysis, Target gene screening

WANG Maocun, CAO Jiawei, ZHOU He, JIA Mingxuan, WEI Shuzhen, GONG Xiaodong, LIU Yuwei, GU Shouqin, DONG Jingao. Cloning of Transcription Factor Gene StbZIP9 of Setosphaeria turcica, Expression Analysis and Screening of Its Downstream Target Genes[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 158-165. doi: 10.7668/hbnxb.20193956.

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Figures/Tables 7

Fig.1 Amplification (A) and the structure mode of StbZIP9 (B)in S.turcica M.DL5000 DNA Marker;1.PCR result.

Fig.2 The advanced structure of StbZip9 protein of S.turcica A.Secondary structure, α-helix,extended strand, β-turn,and random coil are represented by the longest,second longest,second shortest,and shortest vertical lines,respectively;B.Three-level structure prediction,Rings Ⅰ,Ⅱ,Ⅲ.Three ring-like regions formed by curling;C.The protein conserved domain contains a BRLZ conserved domain.

Fig.3 StbZip9 protein BRLZ domain sequence alignment and protein phylogenetic analysis in S.turcica A.Comparison of the amino acid sequence of the BRLZ of StbZip9 with other fungi;.,:,★.The matching degree is from low to high;B.StbZip9 protein phylogenetic analysis and domain location species distribution.

Fig.4 StbZIP9 gene expression level during development and infection stages in S.turcica A.StbZIP9 gene expression level (FPKM) at different developmental periods;B. StbZIP9 gene expression level at different infection stages.

Fig.5 StbZip9 protein conservative motif prediction predicted by CISBP

Tab.1 Identification of the downstream target gene of the StbZip9 protein

蛋白ID Protein ID	正负链 Strand	分数 Score	阈值 P-value	基因注释 Gene ontology
1072501	-	14.032 3	8.30E-08	-
37808	+	13.887 1	2.20E-07	Ca²⁺结合肌动蛋白复合体
162798	-	13.741 9	4.14E-07	核糖体蛋白 L14p/L23e
1616769	+	13.741 9	4.14E-07	-
132893	+	13.741 9	4.14E-07	核酸结合酶,S1/P1核酸酶
28341	-	13.741 9	4.14E-07	-
40466	+	13.741 9	4.14E-07	己糖基转移酶活性
1042152	-	13.645 2	5.15E-07	-
163024	+	13.596 8	7.40E-07	囊外复合体亚基
80567	-	13.596 8	7.40E-07	DNA结合转录因子活性

Fig.6 Expression level of StbZIP9 downstream target gene in development and infection database

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