Cloning,Expression and Preliminary Bioinformatics Analysis of the Alkaline Tolerant Gene GhZAT12 in Gossypium hirsutum

doi:10.13560/j.cnki.biotech.bull.1985.2020-1516

Abstract

Abstract:

Some members of zinc finger protein（ZFP）transcription factor family play important roles in regulating plant growth,development,and response to abiotic stress. In this study,we aim to determine the role of GhZAT12,a C2H2-type transcription factor,in responding to adversity and stress. Bioinformatics analysis of the protein showed that the gene had the full length of 474 bp in coding region,encoding 157 amino acids with a predicted molecular mass of 17.074 kD. GhZAT12 protein did not contain a transmembrane domain. It was a non-secreted hydrophilic protein with 7 phosphorylation sites and no signal peptide. GhZAT12 contained two C2H2-type zinc finger domains,with a conserved L-box at the N-terminus,and a EAR-Motif at the C-terminus. Phylogenetic tree analysis demonstrated that GhZAT12 was closely related to the zinc finger protein in cocoa（XP_017982131.1）. The CDS sequence of GhZAT12 gene was cloned from cotton,and a subcellular localization vector was constructed. The GhZAT12-GFP fusion protein was observed for subcellular localization using the tobacco transient expression system. The results indicated that the GhZAT12 protein was localized in the nucleus. qRT-PCR analysis results showed that the relative expression of GhZAT12 in the root of Gossypium hirsutum was the highest,followed by the leaves and the lowest in the stem. At different times after alkaline stress,the expression of GhZAT12 gene generally increased first and then decreased,suggesting that GhZAT12 gene may play an important role in alkaline stress.

Key words: GhZAT12, Gossypium hirsutum, bioinformatics analysis, subcellular localization, qRT-PCR, alkaline stress

FAN Ya-peng, RUI Cun, ZHANG Yue-xin, CHEN Xiu-gui, LU Xu-ke, WANG Shuai, ZHANG Hong, XU Nan, WANG Jing, CHEN Chao, YE Wu-wei. Cloning,Expression and Preliminary Bioinformatics Analysis of the Alkaline Tolerant Gene GhZAT12 in Gossypium hirsutum[J]. Biotechnology Bulletin, 2021, 37(8): 121-130.

Figures/Tables 12

Table 1 Primer pairs used in the experiments

引物名称Primer name	引物序列Primers sequence（5'-3'）	描述Description
GhZAT12	F：ATGAAGAGAGGCAGAGATATTGATG	GhZAT12基因引物 Primer pairs for GhZAT12 gene
GhZAT12	R：CTATATAAAACAATGAACAACCGGA	GhZAT12基因引物 Primer pairs for GhZAT12 gene
GFP-GhZAT12	F：AGAACACGGGGGACTCTAGAATGAAGAGAGGCAGAGATATTG	GhZAT12亚细胞定位载体引物 Primer pairs for subcellular localization vector
GFP-GhZAT12	R：TAGTCAGGCGCGCCGGTACCTATAAAACAATGAACAACCGG
Actin（AY305733）	F：ATCCTCCGTCTTGACCTTG	内参基因引物 Primer pairs for internal reference gene
Actin（AY305733）	R：TGTCCGTCAGGCAACTCAT	内参基因引物 Primer pairs for internal reference gene
QGhZAT12	F：CTGCTCTGAATGACGGGTTG	GhZAT12荧光定量引物 Primer pairs for qRT-PCR of GhZAT12
QGhZAT12	R：CCGGAGTTGGTGTCACTTTC	GhZAT12荧光定量引物 Primer pairs for qRT-PCR of GhZAT12

Fig.1 Agarose gel electrophoretic analysis of PCR product of GhZAT12 M：DL2000 marker. 1-4：PCR amplification products of GhZAT12

Fig.2 Multiple sequence alignment of homologous amino acids in GhZAT12 protein XP_002314198.3 Populus trichocarpa, XP_002284111.1 Vitis vinifera, XP_017982131.1 Theobroma cacao, XP_008654280.1 Zea mays, NP_200790.1 Arabidopsis thaliana, NP_001237020.2 Glycine max, XP_021628881.1 Manihot esculenta, XP_002533110.1 Ricinus communis, XP_021887642.1 Carica papaya, XP_004147699.1 Cucumis sativus, XP_006345939.1 Solanum tuberosum, XP_012471399.1 Gossypium raimondii, XP_017618875.1 Gossypium arboretum, KAB2038530.1 Gossypium barbadense, XP_016740578.1 Gossypium hirsutum

Fig.3 Phylogenetic tree of ZAT12 homologous proteins in different species Parameters are set to be neighbor-joining,boostrap value=1 000. The numbers on the tree branches represent bootstrap confidence values as “Bootstrap” is 1000

Fig.4 Hydrophilic/hydrophobic analysis of GhZAT12 protein

Fig.5 Signal peptide analysis of GhZAT12 protein

Fig.6 Phosphorylation site analysis of GhZAT12 protein of G. hirsutum L.

Fig.7 Transmembrane helical projections of GhZAT12 protein of G. hirsutum L.

Fig.8 Secondary structure prediction of GhZAT12 protein

Fig.9 Tertiary structure prediction of GhZAT12 protein

Fig.10 Subcellular localization of GhZAT12 in tobacco leaves a,d：Confocal section of tobacco leave expressing GFP and GhZAT12-GFP,respectively. b,e：Bright field. c,f：Fluorescence and bright field merged image. Bar=50 μm

Fig.11 Relative expressions of GhZAT12 gene in different tissues and different times of alkaline stress in G. hirsutum L.

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