Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (10): 1877-1890.doi: 10.3864/j.issn.0578-1752.2022.10.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Drought Resistance Analysis of Transcription Factor GhMYB108 in Gossypium hirsutum

LIU RuiDa1,2(),GE ChangWei2,WANG MinXuan1,2,SHEN YanHui2,LI PengZhen2,CUI ZiQian2,LIU RuiHua2,SHEN Qian2,ZHANG SiPing2,LIU ShaoDong2,MA HuiJuan2,CHEN Jing2,ZHANG GuiYin1(),PANG ChaoYou2,3()   

  1. 1Hebei Agricultural University/Stare Key Laboratory of Cotton Biology (Hebei Base), Baoding 071001, Hebei
    2Institute of Cotton Research of Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology, Anyang 455000, Henan
    3Institute of Cash Crops, Xinjiang Academy of Agricultural Sciences, Urumqi 830000
  • Received:2021-12-22 Accepted:2022-01-28 Online:2022-05-16 Published:2022-06-02
  • Contact: GuiYin ZHANG,ChaoYou PANG E-mail:1165667680@qq.com;mhyzh@hebau.edu.cn;chypang@163.com

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Abstract:

【Objective】As one of the largest transcription factor families in plants, MYB genes play an important role in resisting stress. The MYB transcription factor GhMYB108 was cloned and analyzed to verify its role in drought stress response, which laid a foundation for further study on the molecular mechanism of GhMYB108 regulating drought tolerance in G. hirsutum.【Method】Through the analysis of unpublished drought transcriptome data, GhMYB108 was identified to drought response. The target gene was amplified from the root cDNA by polymerase chain reaction (PCR). Through bioinformatics analysis of gene structure characteristics, the sequence information and phylogenetic relationship of these genes were predicted. The obtained gene promoter sequences were analyzed by Plant Care website. The genes expression characteristics under different stress conditions were analyzed using Real time fluorescence quantitative PCR (qRT-PCR). The location of GhMYB108 protein was determined by subcellular localization. The transcriptional activity was tested in yeast cell; The GhMYB108 gene was silenced using Virus induced gene silencing (VIGS), and the gene silencing efficiency was detected by qRT-PCR. The phenotypic changes before and after drought treatment were observed and the survival rate was counted. The relevant physiological and biochemical indexes were measured by Solarbio Kit; The relationship between GhMYB108 and ABA was analyzed by spraying ABA and Fluridone on cotton leaves.【Result】GhMYB108 (Gh_A10G1563) was cloned from G. hirsutum, with 879 bp length and 292 amino acids. Its protein relative molecular weight and isoelectric point is 33.288 kD and 6.037, respectively. Multiple sequence alignment and conserved domain analysis showed that GhMYB108 contains two highly conserved MYB binding domains, which belongs to a typical R2R3 MYB transcription factor. Phylogenetic analysis of different species showed that GhMYB108 was highly homology with ATMYB108, ATMYB78 and ATMYB2, belonging to the same subfamily. Previous studies found that ATMYB108, ATMYB78 and ATMYB2 were related to drought and ABA signaling pathway. GhMYB108 located in the nucleus and had transcriptional activation activity. The expression level of GhMYB108 was the highest in roots and the lowest in stems, and was induced by abiotic stresses including natural drought, 18% PEG 6000 simulated drought, salt stress and low temperature. The GhMYB108 silenced plants showed a critical phenotype under natural drought conditions. Compared with the control, the silenced plants showed more serious wilting and decreased survival rate. Some physiological and biochemical indexes also changed significantly, such as accelerated leaf water loss rate, increased malondialdehyde content, decreased leaf relative water content and proline content, and decreased CAT and POD activities. Through DAB and NBT staining, the hydrogen peroxide (H2O2) and superoxide anion (O2-) were significantly accumulated in plants. By spraying the hormone ABA or Fluridone on cotton leaves, we found that GhMYB108 could be positively regulated by ABA signal.【Conclusion】GhMYB108 positively regulates cotton drought resistance and is positively regulated by ABA signal.

Key words: Gossypium hirsutum Linn, MYB transcription factor, abiotic stress, ABA

Fig. 1

Sequence analysis of GhMYB108 A: Sequence alignment of GhMYB108 with homologous Arabidopsis thaliana; B: Amino acid sequence alignment of GhMYB108 and MYB protein of other plants. R2, R3: Binding domain; C: Conserved domain analysis of GhMYB108 protein; D: Phylogenetic tree of GhMYB108 protein and MYB protein family of other plants; E: Phylogenetic tree of GhMYB108 protein and MYB protein family of Arabidopsis. Sequence display non full length"

Table 1

Anti stress and hormone related cis acting elements in the promoter sequence of GhMYB108"

位点名称 Site name 序列 Sequence 位点功能 Function of site
G-Box CACGTT 参与光反应的顺式调节元件Cis regulatory elements involved in photoreaction
ABRE ACGTG 参与脱落酸反应的顺式作用元件Cis acting elements involved in abscisic acid reaction
CGTCA-motif CGTCA 参与MeJA响应的顺式调控元件Cis regulatory elements involved in MeJA response
TC-rich repeats GTTTTCTTAC 参与防御和应激反应的顺式作用元件Cis acting elements involved in defense and stress response
TGA-element AACGAC 生长素响应顺式调控元件Auxin responsive Cis regulatory element

Fig. 2

Tissue expression analysis of GhMYB108 * and **: Significant differences and significant differences at 0.05 level and 0.01 level; ns: No significant difference. The same as below"

Fig. 3

Expression pattern of GhMYB108 in cotton A and E: Drought treatment; B and F: 18% PEG6000 treatment; C and G: 200 mmol·L-1 NaCl treatment; D and H: Low temperature treatment at 4℃. A-D: Root; E-H: Leaf. The drought treatment took the parallel time point as the control, and the other treatments took 0 h as the control"

Fig. 4

The subcellular localization of GhMYB108"

Fig. 5

Validation of transcriptional activation of GhMYB108 transcription factor in cotton 1: SD/-Trp/-Leu; 2: SD/-Trp/-His/-Ade/-Leu; 100, 10-1 and 10-2 indicate that the yeast solution is diluted 0, 10 and 100 times, respectively"

Fig. 6

GhMYB108 and its drought phenotype were silenced by VIGS technique A: Albino phenotype; B: The silencing efficiency of GhMYB108 was detected by qRT-PCR, 1-10: Strains injected with TRV2::GhMYB108; C: Survival rate; D: Phenotypes of VIGS and control plants before and after drought treatment and after rehydration"

Fig. 7

Physiological indexes and enzyme activity of VIGS and control plants were determined A: Water loss rate; B: Relative water content; C: Malondialdehyde content; D: Proline content; E: CAT activity; F: POD activity"

Fig. 8

DAB and NBT staining of VIGS and control plants before and after drought treatment A: DAB staining; B: NBT staining"

Fig. 9

Changes of GhMYB108 expression after ABA or Fluridone treatment A and B: Changes of GhMYB108 expression in roots and leaves after ABA treatment; C: Changes of GhMYB108 expression in roots and leaves after Fluridone treatment"

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