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Two Promoters of Beta-Glucosidase Paralogs (ZmBGlu2 and ZmBGlu5) Highly Active in Tropical Young Maize Hybrid Seedlings

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Abstract

The beta-glucosidase (BGLU, EC 3.2.1.21) gene family in B73 maize contains 26 BGLU paralogs of unknown functional redundancy. Promoter sequences revealed that each paralog possesses a distinct set of cis elements and transcription factor binding sites that can modulate transcriptional activity in response to a variety of stimuli. Agrobacterium-mediated fast transient transformation is advantageous for studying large gene families in plants that have rapid recombinant protein expression. In this study, we applied an optimized method from tropical hybrids of maize seedlings in order to characterize some endogenous maize beta-glucosidase gene promoters to produce heterologous proteins. We evaluated germination kinetics and Agrobacterium susceptibility of NS9617, DK2038, and C/05 hybrids and one inbred (B-73) of which C/05 was the most susceptible hybrid to Agrobacterium infection. In order to investigate the promoter-driven gene expressions of BGLU2, BGLU5, BGLU7, and GLU12, 2 kb upstream nucleotides were cloned in pCAMBIA and pKGWFS7 vectors with β-glucoronidase (GUS) reporter (udiA) gene. GUS activity was quantified in root and leaf of C/05 maize seedlings, and two promoters (ZmBGlu2 and ZmBGlu5) were highly active in young maize seedlings comparable to the patented p35S CaMV viral promoter. We propose that the use of 9-day-old seedlings of C/05 for fast transformation assays is a rapid and cost-efficient alternative for protein characterization, functional evaluation of isogenes, and promoters. The maize cisgenic pZmBGlu2 and pZmBGlu5 promoters were expressed during germination (compatible with p35S CaMV), suggesting that they have potential heterologous protein expression and their use avoids intellectual property and biosafety conflicts.

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Acknowledgements

We are grateful to Consejo Nacional de Ciencia y Tecnológia in Mexico for CONACYT-Basic Science Grants 156563 and 151818 and to CINVESTAV Irapuato and CIBNOR staff Martha Reyes and Mayela Ornelas for technical support and Diana Dorantes for English editing.

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Authors and Affiliations

  1. Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Av. Instituto Politécnico Nacional 195, Col. Playa Palo de Santa Rita, 23096, La Paz, B.C.S., México

    Mario Rojas, Felipe Ascencio, Carlos Angulo & Gracia Gómez-Anduro

  2. Departamento de Ingeniería Genética, CINVESTAV, Unidad Irapuato, Km. 9.6 Libramiento Norte, 36821, Irapuato, México

    Axel Tiessen

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  1. Mario Rojas
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  2. Axel Tiessen
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  3. Felipe Ascencio
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  4. Carlos Angulo
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  5. Gracia Gómez-Anduro
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Correspondence to Gracia Gómez-Anduro.

Electronic supplementary material

Below is the link to the electronic supplementary material.

S1

Table showing the maximum values of gene expression of the β-glucosidases in Zea mays of Inbred line B73. (DOCX 17 kb)

S2

Schematic representation: Germination, cocultivation and GUS activity determination in days (DOCX 48 kb)

S3

Bioinformatics analysis of β-Glucosidases promoters. The sequences of the putative promoter region (2000 pb upstream start codon) of pZmBGlu2, 5, 7, 12 showing the transcription factor binding sites where one is the most distant position and 2000 is the nearest to the start codon. Each analysis was made with databases for maize from a database reported by (Chang et al. 2008). (PPTX 2542 kb)

S4

Histochemical GUS activity in cisgenic pZmBglu2 and pZmBglu5 and also 35S CaMV promoter. (DOCX 265 kb)

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Rojas, M., Tiessen, A., Ascencio, F. et al. Two Promoters of Beta-Glucosidase Paralogs (ZmBGlu2 and ZmBGlu5) Highly Active in Tropical Young Maize Hybrid Seedlings. Plant Mol Biol Rep 33, 1666–1674 (2015). https://doi.org/10.1007/s11105-015-0863-0

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