Functional Analysis of TaMYB1 Gene in Wheat Under Cadmium Stress

doi:10.13560/j.cnki.biotech.bull.1985.2023-0672

Abstract

Abstract:

【Objective】Members of the MYB family play key roles in diverse biological processes including regulation of plant growth and development, biotic and abiotic stress. However, there were few researches specifically focusing on the function of MYB transcription factors in the response to cadmium（Cd）stress in Triticum aestivum. Therefore, identifying Cd-responsive genes and illustrating their biological function may lay a foundation for wheat Cd-resistant or low-Cd breeding. 【Method】Firstly, the yeast cDNA library of wheat roots under Cd stress was constructed, and then the Cd-resistant gene was screened. Then, the expression patterns of Cd-resistant gene in different tissues under Cd stress were examined by quantitative real-time PCR（RT-qPCR）. The function of the gene was verified by virus-induced gene silencing, and physiological and biochemical indicators such as the Cd content, chlorophyll content, malondialdehyde（MDA）content, superoxide dismutase（SOD）and peroxidase（POD）activities of the control and gene-silenced plants were detected. Finally, the candidate Cd-resistant gene and its homologous genes in other plants were analyzed by evolutionary relationship, gene structure and expression profile in detail. 【Result】The results indicated that root development of wheat was mainly inhibited under Cd stress. Total 18 positive transformants were screened by cDNA library on a tolerance of 0.002 mol/L Cd, and five transformants encoded TaMYB1 protein, which grew well while the control yeast strains restrained in high Cd medium. Results of RT-qPCR indicated that TaMYB1 responded to Cd stress in the wheat. Compared to the control, TaMYB1 expression in TaMYB1-silencing wheat significantly decreased and the concentration of Cd in the roots and leaves as well MDA content were remarkably lower than that in the control plants, while the contents of chlorophyll and the activities of SOD and POD increased apparently in TaMYB1-silencing plants. Meanwhile, bioinformatics analysis found that TaMYB1 belonged to the 1R-MYB family with highly conserved MYB and CC domains, containing seven exons and six introns. TaMYB1 expressed the highest in the inflorescences of two nodes or internodes visible stage and maximum stem length reached stage, while the lowest in in pre-filling seeds and mid- and late-filling seeds. 【Conclusion】TaMYB1 plays a important role in wheat response to Cd stress.

Key words: Triticum aestivum, abiotic stress, cadmium, yeast, virus-induced gene silencing

ZHANG Yi, ZHANG Xin-ru, ZHANG Jin-ke, HU Li-zong, SHANGGUAN Xin-xin, ZHENG Xiao-hong, HU Juan-juan, ZHANG Cong-cong, MU Gui-qing, LI Cheng-wei. Functional Analysis of TaMYB1 Gene in Wheat Under Cadmium Stress[J]. Biotechnology Bulletin, 2024, 40(1): 194-206.

Figures/Tables 11

Table 1 Primer sequences

引物名称Primer name	引物序列Primer sequence（5'-3'）
TaMYB1-F	GTGAGTAAGGTTACCGAACTCCAAATGCAAATTGAG
TaMYB1-R	CGTGAGCTCGGTACCGGAGACGAGGCTAGCTCCGGC
TaMYB1-qPCR-F	AATTGAGGTCCAGAGACGACTG
TaMYB1-qPCR-R	GGTACTTCCCCTGGGCTTCG
TaActin-qPCR-F	CCAGGTATCGCTGACCGTAT
TaActin-qPCR-R	GCTGAGTGAGGCTAGGATGG

Fig. 1 Phenotype analysis of wheat plant under Cd stress A: Phenotype of wheat plants under Cd stress. B: Root length of wheat plants under Cd stress. C: Shoot length of wheat plants under Cd stress. D: Total biomass of wheat plants under Cd stress. ** indicates significant difference from control plants at P=0.01

Fig. 2 Quality detection of yeast two-hybrid library of wheat roots under Cd stress A: Total RNA of wheat roots under Cd stress. B: High-quality cDNA generated using SMART cDNA synthesis technology. C: Purified cDNA with column chromatography. D, E: PCR analysis of inserted clones from the yeast library

Fig. 3 Transformants with the medium containing different cadmium concentration A-C: Refer to the screening results of yeast two-hybrid library in the yeast medium containing 0.002, 0.003 and 0.004 mol/L CdCl2

Fig. 4 Growth curves of yeast colonies in the SD-Leu medium added with CdCl2 A: Growth curves of yeast transformants growing in SD-Leu medium. B: Growth curves of yeast transformants growing in 0.004 mol/L CdCl2 medium

Fig. 5 Expression analysis of TaMYB1 under Cd stress A: Root. B: Shoot. ** indicates significant difference from control plants at P=0.01, the same below

Fig. 6 Silencing of TaMYB1 enhanced wheat tolerance to cadmium A: Phenotypes of TaMYB1 gene-silenced and control plants under normal conditions and cadmium stress. B: Relative expression level of TaMYB1 gene in gene-silenced plants. C: Cadmium content of TaMYB1 gene-silenced and control plants after cadmium stress

Fig. 7 Physiological index detection of TaMYB1 gene-silenced and control plants under normal conditions and cadmium stress The data in A-D were all taken from wheat leaves

Fig. 8 Phylogenetic relationships of TaMYB1 and its homologous proteins in other species

Fig. 9 Sequence features and phylogenetic relationships of TaMYB1 and its homologous genes in wheat A: Phylogenetic tree of TaMYB1 and its homologous proteins was analyzed. B: Conserved motifs of TaMYB1 and its homologous protein. C: Structures of TaMYB1 and its homologous genes. D: The sequence logo of the conserved motif 1. E: The sequence logo of the conserved motif 2

Fig. 10 Tissue expression profile of TaMYB1 and its homologous genes in wheat

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