Identification and expression analysis of the RBOH gene family of Isatis indigotica Fort. and the potential regulation mechanism of RBOH gene on H2O2 under salt stress.

Abstract

Key message: RBOH gene may regulate the resistance of Isatis indigotica Fort. to salt stress by mediating the production of H₂O₂. RBOH gene plays an important role in plant growth and development, abiotic and biotic stress response, and hormone signalling. However, studies on RBOH gene expression and molecular mechanism of Isatis indigotica Fort. under salt stress have not been reported. This study identified 10 genes of the I. indigotica RBOH gene family (IiRBOH) and divided them into five subfamilies (I-V). Genes within the same class show conserved structural features and similar amino acid sequences. Analysis of CRE suggested that IiRBOH genes might play roles in growth and development, metabolism, hormone regulation, and stress response. Two physiological indicators of I. indigotica treated with salt for different days were detected. It was found that the content of H₂O₂ in the I. indigotica tissue first increased, then decreased and increased again. The catalase activity also showed a trend of first increasing and then decreasing. The qRT-PCR results showed that these IiRBOH genes showed different expression patterns in response to salt stress, and some of these genes may be involved in the resistance of I. indigotica to salt stress. Through RT-PCR analysis and screening on the PlantCARE website, it was found that IiRBOHA and IiRBOHC not only possess W-box CRE but also exhibit high expression under salt stress. Y1H experiments were conducted with the WRKY genes predicted by phylogenetic analysis to regulate salt stress potentially, and it was discovered that IiWRKY6 and IiWRKY54 can directly activate the transcription of the IiRBOHA gene promoter. This study preliminarily explored the mechanism by which the RBOH gene in I. indigotica mediates H₂O₂ to resist salt stress, thus laying a foundation for further research on the biological functions of the RBOH gene in I. indigotica.