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Further Characterization of MUAS35SCP and FUAS35SCP Recombinant Promoters and Their Implication in Translational Research

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Abstract

Recombinant promoters are of high value in translational research. Earlier, we developed two recombinant promoters, namely MUAS35SCP and FUAS35SCP, and their transcriptional activities were found to be stronger than that of the most widely used CaMV35S promoter in dicot plants. Presently, we are reporting constitutive expression of both GUS and GFP reporters under the control of these promoters in several monocots, including rice, wheat, and pearl millet. We observed that these promoters could express the reporter genes constitutively, and their expression abilities were almost equal to that of the CaMV35S2 promoter. Plant-derived enriched PaDef (Persea americana var. drymifolia defensin) and NsDef2 (Nigella sativa L. defensin 2) antimicrobial peptides expressed under the control of these promoters arrest the growth of devastating phytopathogens like Pseudomonas syringae, Rhodococcus fascians, and Alternaria alternata. We observed that plant-derived NsDef2 and PaDef under control of these promoters showed approximately 80–90% inhibitory activity against Pseudomonas syringae. Hence, these promoters were constitutive and universal, as they can drive the expression of transgenes in both dicot and monocot plants. Alongside, these promoters could become a valuable tool for raising genetically modified plants with in-built resistance toward phytopathogens.

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Acknowledgements

This research work was supported by grants from core funds of Institute of Life Sciences, Department of Biotechnology, and Government of India. We are grateful to the Director, Institute of Life Sciences, for his utmost support in this study. We are also very thankful to Ms. Premaspada Glory Lima for her kind technical help and support. We are grateful for financial support from the SERB-DST, Government of India (Project Number- CRG/2021/001001).

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  1. Lini Sethi and Tsheten Sherpa have contributed equally to this work.

Authors and Affiliations

  1. Division of Plant and Microbial Biotechnology, Institute of Life Sciences, NALCO Square, Chandrasekharpur, Bhubaneswar, Odisha, 751023, India

    Lini Sethi, Tsheten Sherpa, Khushbu Kumari & Nrisingha Dey

  2. Regional Centre for Biotechnology, National Capital Region Biotech Science Cluster, Faridabad, Haryana (NCR Delhi), 121001, India

    Lini Sethi, Tsheten Sherpa & Khushbu Kumari

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  1. Lini Sethi
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ND planned and designed the experiments. LS, TS and KK performed the experiments. LS, TS and ND analyzed and organized the data. LS, TS, KK and ND wrote the manuscript.

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Correspondence to Nrisingha Dey.

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12033_2022_513_MOESM1_ESM.pdf

Fig S1 Gene integration analysis was performed for (a) FUAS35SCPGFP, MUAS35SCPGFP, by restriction digestion analysis with EcoRI/HindIII. (b, c) PCR amplification of Agrobacterium transformed clones of FUAS35SCPGFP, MUAS35SCPGFP, CaMV35SGFP, CaMV35S²GFP targeting respective promoters, gfp, nptII and rbcSE9 was done using appropriate primers (Table S1). M: 100bp DNA ladder (BioLit ProxiO 100bp DNA Ladder Plus #BLL005). Fig S2 Gene integration analysis was performed for (a) FUAS35SCPGUS, and MUAS35SCPGUS, by restriction digestion analysis with EcoRI/HindIII. (b, c) PCR amplification of Agrobacterium transformed clones of FUAS35SCPGUS, MUAS35SCPGUS, CaMV35SGUS, CaMV35S²GUS targeting respective promoters, gfp, nptII and rbcSE9 was done using appropriate primers (Table S1). M: 100bp DNA ladder. Fig S3 Gene integration analysis by PCR amplification of transformed Agrobacterium clones namely MUAS35SCPPaDef, FUAS35SCPPaDef, CaMV35SPaDef, CaMV35S2PaDef, MUAS35SCPNsDef2, FUAS35SCPNsDef2, CaMV35SNsDef2, and CaMV35S2NsDef2 was done by targeting (a) respective promoters (b) PaDef, NsDef2, nptII and rbcSE9, using appropriate primers (Table S1). M: 100bp DNA ladder. (c) Slot-western blot analysis of purified proteins (NsDef2 and PaDef) from agro infiltrated tobacco plants, along with vector control protein extract and buffer control is shown Supplementary file1 (PDF 731 kb)

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Sethi, L., Sherpa, T., Kumari, K. et al. Further Characterization of MUAS35SCP and FUAS35SCP Recombinant Promoters and Their Implication in Translational Research. Mol Biotechnol 64, 1356–1366 (2022). https://doi.org/10.1007/s12033-022-00513-0

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