Expression and Functional Analysis of OsMATE34 in Rice under Mercury Stress

doi:10.13560/j.cnki.biotech.bull.1985.2025-0542

Abstract

Abstract:

Objective The multidrug and toxic compound extrusion transporter (MATE) plays a significant regulatory role in plant nutrient absorption, secondary metabolite transport, and detoxification of heavy metals and exogenous substances. Studying biological function of OsMATE34 in rice (Oryza sativa) may lay a foundation for a deeper understanding of the molecular mechanisms underlying MeHg accumulation in rice, and provide scientific reference for the breeding of low-MeHg rice varieties. Method Bioinformatics tools were used to analyze the physicochemical properties and phylogenetic tree of OsMATE34. Real-time quantitative PCR (RT-qPCR) was used to detect the relative expressions of OsMATE34 in different rice tissues under MeHg stress. The MeHg contents in different tissues of rice after MeHg exposure were measured, and the correlation between gene expressions and MeHg content was analyzed. A CRISPR/Cas9-mediated OsMATE34 knockout line was generated, and MeHg treatment was performed on seedlings. The MeHg contents in wild-type (WT) and osmate34 rice lines were measured to explore the effects of OsMATE34 on MeHg absorption and transport. Result The OsMATE34 gene was located on chromosome 8, encoding a protein of 489 amino acids with a relative molecular weight of 51.60 kD and a theoretical isoelectric point (pI) of 5.6. The protein was hydrophobic. RT-qPCR showed that MeHg stress inhibited the expression of OsMATE34 in roots, The expressions of OsMATE34 in the leaf sheath and leaves were significantly up-regulated with the increase of MeHg concentration, and the gene expression in stems was 20-fold higher than that of the control when treated with 80 nmol/L MeHg. The MeHg contents in rice tissues increased under MeHg stress, with the root accumulating significantly higher MeHg than the leaf sheath and leaf. There was a significant positive correlation between OsMATE34 gene expression and MeHg content in rice leaf sheath and leaves with the increase of MeHg concentration. Compared to the wild-type, the OsMATE34 knockout lines showed a significant reduction in MeHg content in the leaf sheath, leaf, and xylem, while there was no significant difference in the root. Conclusion The OsMATE34 gene is involved in the transport of MeHg from the root to the aboveground tissues. This study provides a foundation for further research on the molecular mechanisms of MeHg absorption and transport in rice.

Key words: MeHg, rice, OsMATE34, expression analysis

YANG Yue-qin, XING Ying, ZHONG Zi-he, TIAN Wei-jun, YANG Xue-qing, WANG Jian-xu. Expression and Functional Analysis of OsMATE34 in Rice under Mercury Stress[J]. Biotechnology Bulletin, 2026, 42(1): 86-94.

Figures/Tables 9

Table 1 Primer sequence for RT-qPCR

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
OsMATE34-qF	CATGAGACAAAGCGGCTGTG
OsMATE34-qR	TGAACATGGTGGTCACGGAG
OsActin-qF	GAGTATGATGAGTCGGGTCCAG
OsActin-qR	ACACCAACAATCCCAAACAGAG

Fig. 1 Analysis of hydrophilicity/hydrophobicity of OsMATE34 proteinRegions with values above 0 are OsMATE34 hydrophobic in character. Regions with values less than 0 are OsMATE34 hydrophilic in character

Fig. 2 Secondary and tertiary structure prediction of OsMATE34 proteinA: The secondary structure of OsMATE34, where blue indicates α-helix, purple indicates extended chain, yellow indicates irregular curl, and the abscissa indicates the position of each residue in the amino acid sequence of OsMATE34. B: The tertiary structure of OsMATE34, where dark blue indicates α-helix, light blue indicates irregular curl, and orange indicates extended chain

Fig. 3 Phylogenetic analysis of OsMATE34 homologous proteins

Fig. 4 Analysis of cis-acting elements of OsMATE34 proteinThe different colored squares indicate a portion of the growth hormone response element (blue), abscisic acid-induced cis-acting element (dark yellow), anaerobic-induced cis-acting element (purple), growth hormone-regulated cis-acting element (pink), methyl jasmonate-induced cis-acting element (green), cis-acting regulator involved in the regulation of zeinolysin metabolism (yellow), cis-acting related to the expression of meristematic tissue action regulatory element (light green), enhancer-like element involved in hypoxia-specific induction (gray), gibberellin-responsive element (light yellow), and MYBHv1 binding site (light pink)

Fig. 5 Expression analysis of OsMATE34 under MeHg stress

Fig. 6 Variation in MeHg concentration in rice tissuesDifferent lowercase letters indicate significant differences between treatments (P<0.05)

Fig. 7 Correlation analysis of OsMATE34 expression and MeHg concentration

Fig. 8 Growth conditions and MeHg contents of osmate34 and WT under hydroponic conditionsA: Mutated target site of OsMATE34 gene. B: Biomasses of osmate34 and WT in different tissues. C: Plant phenotypes of osmate34 and WT, scale =10 cm. D: MeHg contents of osmate34 and WT in different tissues. E: MeHg contents of osmate34 and WT in xylem sap. * indicates statistically significant difference in comparison to WT at P<0.05

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