Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects

Abouseadaa, Heba H.; Osman, Gamal; Ramadan, Ahmed M.; Hassanein, Sameh E.; Abdelsattar, Mohamed; Morsy, Yasser; Alameldin, Hussien F.; El-Ghareeb, Doaa K.; Nour-Eldin, Hanan A; Salem, Reda; Gad, Adel A; El-Khodary, Soheir; Shehata, Maher M.; Mahfouz, Hala; Eissa, Hala F.; Bahieldin, Ahmed

doi:10.1186/s12870-015-0570-x

Cited by 28 publications

(16 citation statements)

References 35 publications

(40 reference statements)

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“…These differences could be explained by the ability of some of the procedures in elimination of contaminating molecules. Sufficient purity does not guarantee successful amplification of a gene; there are also other factors such as concentration that also need consideration [21, 22]. High purity DNA was extracted from M 10 and M 321 using the three different extraction methods is shown in Figs.…”

Section: Resultsmentioning

confidence: 99%

Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize

2017

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BackgroundThe world’s top three cereals, based on their monetary value, are rice, wheat, and corn. In cereal crops, DNA extraction is difficult owing to rigid non-cellulose components in the cell wall of leaves and high starch and protein content in grains. The advanced techniques in molecular biology require pure and quick extraction of DNA. The majority of existing DNA extraction methods rely on long incubation and multiple precipitations or commercially available kits to produce contaminant-free high molecular weight DNA.ResultsIn this study, we compared three different methods used for the isolation of high-quality genomic DNA from the grains of cereal crop, Zea mays, with minor modifications. The DNA from the grains of two maize hybrids, M10 and M321, was extracted using extraction methods DNeasy Qiagen Plant Mini Kit, CTAB-method (with/without 1% PVP) and modified Mericon extraction. Genes coding for 45S ribosomal RNA are organized in tandem arrays of up to several thousand copies and contain codes for 18S, 5.8S and 26S rRNA units separated by internal transcribed spacers ITS1 and ITS2. While the rRNA units are evolutionary conserved, ITS regions show high level of interspecific divergence and have been used frequently in genetic diversity and phylogenetic studies. In this study, the genomic DNA was then amplified with PCR using primers specific for ITS gene. PCR products were then visualized on agarose gel.ConclusionThe modified Mericon extraction method was found to be the most efficient DNA extraction method, capable to provide high DNA yields with better quality, affordable cost and less time.

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Section: Resultsmentioning

confidence: 99%

Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize

2017

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“…The use of chemical fumigants including phosphine and methyl bromide is currently one of the most effective methods for controlling stored-product insects [3, 4]. However, frequent and widespread use of chemical fumigants has led to the development of resistance in stored-product insects [5]. Furthermore, due to the destruction of earth’s ozone layer, residue formation, and carcinogenicity, some chemical fumigants have been prohibited [6, 7].…”

Section: Introductionmentioning

confidence: 99%

Insecticidal Activity of Melaleuca alternifolia Essential Oil and RNA-Seq Analysis of Sitophilus zeamais Transcriptome in Response to Oil Fumigation

et al. 2016

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BackgroundThe cereal weevil, Sitophilus zeamais is one of the most destructive pests of stored cereals worldwide. Frequent use of fumigants for managing stored-product insects has led to the development of resistance in insects. Essential oils from aromatic plants including the tea oil plant, Melaleuca alternifolia may provide environmentally friendly alternatives to currently used pest control agents. However, little is known about molecular events involved in stored-product insects in response to plant essential oil fumigation.ResultsM. alternifolia essential oil was shown to possess the fumigant toxicity against S. zeamais. The constituent, terpinen-4-ol was the most effective compound for fumigant toxicity. M. alternifolia essential oil significantly inhibited the activity of three enzymes in S. zeamais, including two detoxifying enzymes, glutathione S-transferase (GST), and carboxylesterase (CarE), as well as a nerve conduction enzyme, acetylcholinesterase (AChE). Comparative transcriptome analysis of S. zeamais through RNA-Seq identified a total of 3,562 differentially expressed genes (DEGs), of which 2,836 and 726 were up-regulated and down-regulated in response to M. alternifolia essential oil fumigation, respectively. Based on gene ontology (GO) analysis, the majority of DEGs were involved in insecticide detoxification and mitochondrial function. Furthermore, an abundance of DEGs mapped into the metabolism pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database were associated with respiration and metabolism of xenobiotics, including cytochrome P450s, CarEs, GSTs, and ATP-binding cassette transporters (ABC transporters). Some DEGs mapped into the proteasome and phagosome pathway were found to be significantly enriched. These results led us to propose a model of insecticide action that M. alternifolia essential oil likely directly affects the hydrogen carrier to block the electron flow and interfere energy synthesis in mitochondrial respiratory chain.ConclusionThis is the first study to perform a comparative transcriptome analysis of S. zeamais in response to M. alternifolia essential oil fumigation. Our results provide new insights into the insecticidal mechanism of M. alternifolia essential oil fumigation against S. zeamais and eventually contribute to the management of this important agricultural pest.

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“…A nucleotide sequence obtained from such a mixed PCR product is consensus for several targets sharing the same priming sites in a single genome [9]. By contrast, DNA sequence data from an individual allele (called a haplotype) can provide considerable information to address various biological questions and to guide several practical applications [7,10]. Various methods have been investigated to obtain haplotype sequences from different genomes.…”

Section: Introductionmentioning

confidence: 99%

Molecular Characterization of two Maize Hybrids Based on Primer Bias

2018

Adv. Environ. Biol

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INTRODUCTIONThe taxonomic difficulty for several systematics, field ecologists, and evolutionary biologists is correctly determining an organism by a rapid, repeatable, and reliable method [1]. Advances in molecular genetics, sequencing technology, and bioinformatics have explored numerous molecular markers that can be easily sequenced and used for inferring phylogenetic relationships. One of these markers is the internal transcribed spacer (ITS), which is part of the non-coding nuclear ribosomal DNA cistron. It consists of the regions ITS1 and ITS2 that are separated by the gene 5.8S. The ITS region displays high rates of substitution that can manifest species and even subspecies divergences [2,3]. Therefore, the ITS sequence is one of the most frequently used DNA markers in plant phylogenetic and DNA barcoding analyses [4,5]. In addition, some genetic incidents exist that may mislead ITS-based phylogenetic investigations [6][7][8]. These incidents include the following: (a) existence of two alleles that may be heterozygous for each nuclear gene, (b) presence of several 18S-5.8S-26S repeats at one or more chromosomal locations, (c) some of these copies degenerate into pseudogenes, and (d) the near universality of most primers commonly used for the polymerase chain reaction (PCR) amplification of ITS sequences. Therefore, the direct sequencing of PCR-amplified ITS sequences would typically produce a mixture of allelic sequences with polymorphic nucleotide sites, which will appear as overlapping peaks. A nucleotide sequence obtained from such a mixed PCR product is consensus for several targets sharing the same priming sites in a single genome [9]. By contrast, DNA sequence data from an individual allele (called a haplotype) can provide considerable information to address various biological questions and to guide several practical applications [7,10]. Various methods have been investigated to obtain haplotype sequences from different genomes. A traditional method is to construct a random cloning library from the PCR products before sequencing of the target fragment [7]. Furthermore, it has been suggested that reducing the amount of genomic DNA and decreasing the number of PCR cycles [11]. In addition to using different PCR primer sets and reaction conditions result in allele bias [3,12,13]. Maize (Zea mays) is one of the most productive and widely grown crops worldwide. It has remarkable morphological and genetic diversity and is considered a foundational model for studying genetics and genomics. An improved de novo assembly and annotation of a maize reference genome have been recently conducted [14]. To investigate the wide diversity of maize genotypes, few studies on ITS sequencing [15,16] and microsatellites or simple sequence repeats [17,18] have been published. The phylogenetic relationships of Zea species inferred from ITS sequences were demonstrated to be highly concordant with Random Amplified Polymorphic DNA (RAPD) evidence [16,19]. The main objective of this study was to identify a molecular marker or a...

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Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects

Cited by 28 publications

References 35 publications

Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize

Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize

Insecticidal Activity of Melaleuca alternifolia Essential Oil and RNA-Seq Analysis of Sitophilus zeamais Transcriptome in Response to Oil Fumigation

Molecular Characterization of two Maize Hybrids Based on Primer Bias

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