Cloning and function analysis of the high temperature inducible promoter pMsMBF 1c in alfalfa (Medicago sativa)

doi:10.11686/cyxb2018370

Abstract

Abstract: Plant thermotolerance is controlled by a complex and finely regulated network of transcription processes. High temperature inducible promoters can economically and efficiently activate or shut down the expression of key genes involved in regulating thermotolerance. Knowledge of these promoters is therefore important in plant functional genomics research and the molecular breeding industry. In the present study, a 1748 bp sequence upstream of MsMBF1c, which is a candidate gene involved in thermotolerance in alfalfa, was isolated using a digestion ligation PCR method, based on its coding sequence. Bioinformatics analysis showed that two conserved motifs representing the HSE and GATA binding sites involved in thermotolerance regulation existed in the MsMBF1c promoter region. There were 5 abscisic acid response cis-elements (ABRE, MYB2, MIC2, CBF and DPBF) and 2 MYB protein binding motifs in the MsMBF1c promoter region, which suggested MsMBF1c might regulate other abiotic stress response in addition to thermotolerance. A vector of pBI121-MsMBF1c::GUS was constructed and transformed into wild type Arabidopsis. The expression of GUS and AtMBF1c genes, was examined by qRT-PCR, and found to be up-regulated 5.4 and 4.8 fold, respectively, after heat stress. At the same time, histochemical staining analysis also showed the MsMBF1c promoter to be markedly induced by high temperature. To summarize, the MsMBF1c promoter was isolated and transformed into Arabidopsis, and was proved to be heat inducible by bioinformatics and gene expression data, and by histochemical staining analysis. This information provides technical and theoretical support for further investigation of heat regulation mechanisms and breeding of heat resistant alfalfa varieties.

Key words: high temperature, MBF1c, alfalfa, transgene, promoter

LI Xiao-dong, MO Ben-tian, MOU Qiong, LOU Fen, CHEN Wen-gui, CHEN Guang-ji, ZHANG Yu, HAN Yong-fen. Cloning and function analysis of the high temperature inducible promoter pMsMBF 1c in alfalfa (Medicago sativa)[J]. Acta Prataculturae Sinica, 2019, 28(1): 128-137.

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