Molecular cloning and functional characterization of the promoter of a novel Aspergillus flavus inducible gene (AhOMT1) from peanut

Peanut is an important oil and food legume crop grown in more than one hundred countries, but the yield and quality are often impaired by different pathogens and diseases, especially aflatoxins jeopardizing human health and causing global concerns. For better management of aflatoxin contamination, we report the cloning and characterization of a novel A. flavus inducible promoter of the O-methyltransferase gene (AhOMT1) from peanut. The AhOMT1 gene was identified as the highest inducible gene by A. flavus infection through genome-wide microarray analysis and verified by qRT-PCR analysis. AhOMT1 gene was studied in detail, and its promoter, fussed with the GUS gene, was introduced into Arabidopsis to generate homozygous transgenic lines. Expression of GUS gene was studied in transgenic plants under the infection of A. flavus. The analysis of AhOMT1 gene characterized by in silico assay, RNAseq, and qRT-PCR revealed minute expression in different organs and tissues with trace or no response to low temperature, drought, hormones, Ca2+, and bacterial stresses, but highly induced by A. flavus infection. It contains four exons encoding 297 aa predicted to transfer the methyl group of S-adenosyl-L-methionine (SAM). The promoter contains different cis-elements responsible for its expression characteristics. Functional characterization of AhOMT1P in transgenic Arabidopsis plants demonstrated highly inducible behavior only under A. flavus infection. The transgenic plants did not show GUS expression in any tissue(s) without inoculation of A. flavus spores. However, GUS activity increased significantly after inoculation of A. flavus and maintained a high level of expression after 48 hours of infection. These results provided a novel way for future management of peanut aflatoxins contamination through driving resistance genes in A. flavus inducible manner.

[1]  Ruoyu Zhang,et al.  Genome-wide identification and characterization of caffeoyl-coenzyme A O-methyltransferase genes related to the Fusarium head blight response in wheat , 2021, BMC genomics.

[2]  S. N. Nigam,et al.  Improved Genetic Map Identified Major QTLs for Drought Tolerance- and Iron Deficiency Tolerance-Related Traits in Groundnut , 2020, Genes.

[3]  R. Varshney,et al.  High-density SNP map facilitates fine mapping of QTLs and candidate genes discovery for Aspergillus flavus resistance in peanut (Arachis hypogaea) , 2020, Theoretical and Applied Genetics.

[4]  R. Varshney,et al.  Functional Biology and Molecular Mechanisms of Host-Pathogen Interactions for Aflatoxin Contamination in Groundnut (Arachis hypogaea L.) and Maize (Zea mays L.) , 2020, Frontiers in Microbiology.

[5]  J. Ncube,et al.  Current state of knowledge on groundnut aflatoxins and their management from a plant breeding perspective: Lessons for Africa , 2020 .

[6]  Sift Desk Journals Open Access Journals,et al.  Obliging Tactics to Mitigate the Intricate Problem of Aflatoxin Contamination in Peanut: A Review , 2019, SDRP Journal of Food Science & Technology.

[7]  V. Vadez,et al.  Isolation and functional characterization of three abiotic stress-inducible (Apx, Dhn and Hsc70) promoters from pearl millet (Pennisetum glaucum L.) , 2019, Molecular Biology Reports.

[8]  R. Varshney,et al.  Mitigating Aflatoxin Contamination in Groundnut through A Combination of Genetic Resistance and Post-Harvest Management Practices , 2019, Toxins.

[9]  Xingjun Wang,et al.  Integrated microRNA and transcriptome profiling reveals a miRNA-mediated regulatory network of embryo abortion under calcium deficiency in peanut (Arachis hypogaea L.) , 2019, BMC Genomics.

[10]  Xingtan Zhang,et al.  The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication , 2019, Nature Genetics.

[11]  Quanxi Sun,et al.  Genome-wide mining seed-specific candidate genes from peanut for promoter cloning , 2019, PloS one.

[12]  Dong-hong Min,et al.  Overexpression of TaCOMT Improves Melatonin Production and Enhances Drought Tolerance in Transgenic Arabidopsis , 2019, International journal of molecular sciences.

[13]  A. Gutierrez.  Hybrid Bt Cotton:A Stranglehold on Subsistence Farmers in India , 2018, Current Science.

[14]  R. Varshney,et al.  Employing Peanut Seed Coat Cell Wall Mediated Resistance Against Aspergillus flavus Infection and Aflatoxin Contamination , 2018 .

[15]  Scott Stewart,et al.  Effects of transgenic Bacillus thuringiensis cotton on insecticide use, heliothine counts, plant damage, and cotton yield: A meta-analysis, 1996-2015 , 2018, PloS one.

[16]  Chungui Lu,et al.  A wheat caffeic acid 3-O-methyltransferase TaCOMT-3D positively contributes to both resistance to sharp eyespot disease and stem mechanical strength , 2018, Scientific Reports.

[17]  D. Bhatnagar,et al.  Peanuts that keep aflatoxin at bay: a threshold that matters , 2017, Plant biotechnology journal.

[18]  P. Kaushal,et al.  Biotic stress inducible promoters in crop plants- a review , 2017 .

[19]  Rong-Long Pan,et al.  Overexpression of a novel peanut NBS‐LRR gene AhRRS5 enhances disease resistance to Ralstonia solanacearum in tobacco , 2016, Plant biotechnology journal.

[20]  R. Varshney,et al.  Identification of low Ca(2+) stress-induced embryo apoptosis response genes in Arachis hypogaea by SSH-associated library lift (SSHaLL). , 2016, Plant biotechnology journal.

[21]  D. Levin,et al.  Lignin biosynthesis in wheat (Triticum aestivum L.): its response to waterlogging and association with hormonal levels , 2016, BMC Plant Biology.

[22]  Yexiong Qian,et al.  Genome-wide identification and expression profiling of DNA methyltransferase gene family in maize , 2014, Plant Cell Reports.

[23]  C. A. Oliveira,et al.  Incidence of Aflatoxins in Oil Seeds and Possible Transfer to Oil: A Review , 2014, Food Engineering Reviews.

[24]  P. Bhatnagar-Mathur,et al.  Isolation and Functional Characterization of a Novel Seed-Specific Promoter Region from Peanut , 2013, Applied Biochemistry and Biotechnology.

[25]  Anna-Winona Struck,et al.  S‐Adenosyl‐Methionine‐Dependent Methyltransferases: Highly Versatile Enzymes in Biocatalysis, Biosynthesis and Other Biotechnological Applications , 2012, Chembiochem : a European journal of chemical biology.

[26]  D. Huson,et al.  Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks. , 2012, Systematic biology.

[27]  Tanya Z. Berardini,et al.  The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools , 2011, Nucleic Acids Res..

[28]  L. Thakur,et al.  Contaminants in milk and impact of heating: an assessment study. , 2012, Indian journal of public health.

[29]  Xiaowen Zhang,et al.  Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR , 2012, Molecular Genetics and Genomics.

[30]  J. Groopman,et al.  Aflatoxin: a 50-year odyssey of mechanistic and translational toxicology. , 2011, Toxicological sciences : an official journal of the Society of Toxicology.

[31]  Saori Amaike,et al.  Aspergillus flavus. , 2011, Annual review of phytopathology.

[32]  S. N. Nigam,et al.  Breeding Peanut for Resistance to Aflatoxin Contamination at ICRISAT , 2009 .

[33]  Michael A Grusak,et al.  Golden Rice is an effective source of vitamin A. , 2009, The American journal of clinical nutrition.

[34]  I. Carbone,et al.  Sexual reproduction in Aspergillus flavus , 2009, Mycologia.

[35]  R. Wu,et al.  Comparative functional analysis of three abiotic stress-inducible promoters in transgenic rice , 2009, Transgenic Research.

[36]  J. Richard.  DISCOVERY OF AFLATOXINS AND SIGNIFICANT HISTORICAL FEATURES , 2008 .

[37]  M. Thomashow,et al.  Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield. , 2007, Plant biotechnology journal.

[38]  Zhihao Wu,et al.  [The application of large-scale yeast two-hybrid to study protein--protein interaction]. , 2006, Yi chuan = Hereditas.

[39]  Masayuki Machida,et al.  Whole genome comparison of Aspergillus flavus and A. oryzae. , 2006, Medical mycology.

[40]  S. Roje.  S-Adenosyl-L-methionine: beyond the universal methyl group donor. , 2006, Phytochemistry.

[41]  A. Mushegian,et al.  Natural history of S-adenosylmethionine-binding proteins , 2005, BMC Structural Biology.

[42]  David M. Grant,et al.  The Legume Information System (LIS): an integrated information resource for comparative legume biology , 2004, Nucleic Acids Res..

[43]  A. J. Crowe,et al.  Sequence and Spacing of TATA Box Elements Are Critical for Accurate Initiation from the β-Phaseolin Promoter* , 2004, Journal of Biological Chemistry.

[44]  L. Josefsson,et al.  Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco , 1993, Plant Molecular Biology.

[45]  D. Luth,et al.  Activity of a maize ubiquitin promoter in transgenic rice , 1993, Plant Molecular Biology.

[46]  Roger N. Beachy,et al.  Tissue-specific and temporal regulation of a β-conglycinin gene: roles of the RY repeat and other cis-acting elements , 2004, Plant Molecular Biology.

[47]  D. Boulter,et al.  Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco , 2004, Molecular and General Genetics MGG.

[48]  Ron D. Appel,et al.  ExPASy: the proteomics server for in-depth protein knowledge and analysis , 2003, Nucleic Acids Res..

[49]  D. White,et al.  Comparison of Aspergillus ear rot and aflatoxin contamination in grain of high-oil and normal-oil corn hybrids. , 2003, Journal of food protection.

[50]  J. Noel,et al.  Characterization of phenylpropene O-methyltransferases from sweet basil: facile change of substrate specificity and convergent evolution within a plant O-methyltransferase family. , 2002, The Plant cell.

[51]  Stephane Rombauts,et al.  PlantCARE, a plant cis-acting regulatory element database , 1999, Nucleic Acids Res..

[52]  S. Clough,et al.  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[53]  Kenichi Higo,et al.  PLACE: a database of plant cis-acting regulatory DNA elements , 1998, Nucleic Acids Res..

[54]  D. Wicklow,et al.  Aflatoxin and cyclopiazonic acid production by a sclerotium-producing Aspergillus tamarii strain , 1996, Applied and environmental microbiology.

[55]  R. Wu,et al.  Isolation of an efficient actin promoter for use in rice transformation. , 1990, The Plant cell.

[56]  M. Bevan,et al.  GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants. , 1987, The EMBO journal.

[57]  Robert Kay,et al.  Duplication of CaMV 35S Promoter Sequences Creates a Strong Enhancer for Plant Genes , 1987, Science.

[58]  D. Ballance.  Sequences important for gene expression in filamentous fungi , 1986, Yeast.