1. A High-Resolution Melting Approach for Analyzing Allelic Expression Dynamics

Jiazheng Yuan, Muhammad Haroon, David Lightfoot, Yvan Pelletier, Qiang Liu and Xiu-Qing Li

Single nucleotide polymorphisms (SNPs) are single base pair mutations that provide new approaches to studies of allele transcript abundances. High-resolution DNA melting curve (HRM) analysis using a LightScanner (Hi-Res MeltingTM system with Idaho's LC Green) provides post- PCR detection of mutations and SNPs in genomic DNA. This study investigated whether the HRM analysis can distinguish alleles among potato (Solanum tuberosum) transcript abundances. Transcript properties of genes encoding seven carbohydrate metabolism enzymes/ proteins in various tissues and cold storage durations were studied. The HRM assay measured differential expression of alleles between different organs, between different storage treatments and stages of tubers from the same variety, and between different varieties with the same treatment. The RT-PCR amplicons were directly sequenced to assist the interpretation of HRM data. The cDNA HRM curves correlated well with the nucleotide polymorphisms of the cDNA sequences and the transcript abundance of alleles and therefore can serve as functional allele activity (FAA) markers. By combining the allelic specificity of HRM with simple PCR design, this technology can be applied to rapidly determine the most active allele of a gene among the cells analyzed.

2. Multigeneic QTL: The Laccase Encoded within the Soybean Rfs2/rhg1 Locus Inferred to Underlie Part of the Dual Resistance to Cyst Nematode and Sudden Death Syndrome

M.J. Iqbal, R. Ahsan, A.J. Afzal, A. Jamai, K. Meksem, H.A. El- Shemy and D.A. Lightfoot

Multigeneic QTL present significant problems to analysis. Resistance to soybean (Glycine max (L) Merr.) sudden death syndrome (SDS) caused by Fusarium virguliforme was partly underlain by QRfs2 that was clustered with, or pleiotropic to, the multigeneic rhg1 locus providing resistance to soybean cyst nematode (SCN; Heterodera glycines). A group of five genes were found between the two markers that delimited the Rfs2/rhg1 locus. One of the five genes was predicted to encode an unusual diphenol oxidase (laccase; EC 1.10.3.2). The aim of this study was to characterize this member of the soybean laccase gene-family and explore its involvement in SDS resistance. A genomic clone and a full length cDNA was isolated from resistant cultivar 'Forrest' that were different among susceptible cultivars 'Asgrow 3244' and 'Williams 82' at four residues R/H168, I/M271, R/H330, E/K470. Additional differences were found in six of the seven introns and the promoter region. Transcript abundance (TA) among genotypes that varied for resistance to SDS or SCN did not differ significantly. Therefore the protein activity was inferred to underlie resistance. Protein expressed in yeast pYES2/NTB had weak enzyme activity with common substrates but good activity with root phenolics. The Forrest isoform may underlie both QRfs2 and rhg1.

3. The Role of Green Fluorescent Protein (GFP) in Transgenic Plants to Reduce Gene Silencing Phenomena

Hany A. El-Shemy, Mutasim M. Khalafalla and Masao Ishimoto

The green fluorescent protein (GFP) of jellyfish (Aequorea victoria) has significant advantages over other reporter genes, because expression can be detected in living cells without any substrates. Recently, epigenetic phenomena are important to consider in plant biotechnology experiments for elucidate unknown mechanism. Therefore, soybean immature cotyledons were generated embryogenesis cells and engineered with two different gene constructs (pHV and pHVS) using gene gun method. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V31) as a target. However, sGFP(S65T) as a reporter gene was used only in pHVS as a reporter gene for study the relation between using sGFP(S65T) and gene silencing phenomena. Fluorescence microscopic was used for screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Protein analysis was used to detect gene expression overall seeds using SDS-PAGE. Percentage of gene down regulation was highly in pHV construct compared with pHVS. Thus, sGFP(S65T ) as a reporter gene in vector system may be play useful role for transgenic evaluation and avoid gene silencing in plants for the benefit of plant transformation system.

4. Antisense Phenotypes Reveal a Functional Expression of OsARF1 , an Auxin Response Factor, in Transgenic Rice

Kotb A. Attia, Amr F. Abdelkhalik, Megahed H. Ammar, Chun Wei, Jinshui Yang, David A. Lightfoot, Wagih M. El-Sayed and Hany A. El-Shemy

OsARF1 is the first full-length member of auxin response factor (ARF) gene family to be cloned from monocot plant. Using quantitative RT-PCR this study found that, the transcript abundance of OsARF1 was significantly higher in embryonic tissues than in vegetative tissues. To investigate the effect of OsARF1 on the phenotype of rice, a cDNA fragment of OsARF1 was inserted in inverse orientation to the 35S promoter in vector pBin438 to produce an antisense (AS) construction. The AS- OsARF1 construct was transferred into rice (Oryza sativa L. japonica) calli via Agrobacterium tumefaciens- mediated transformation. Molecular analysis of transgenic plants showed that the functional expression of OsARF1 was inhibited at mRNA level efficiently. The AS-OsARF1 plants showed extremely low growth, poor vigor, short curled leaves and tillered but were sterile. Therefore, the OsARF1 was shown to be essential for growth in vegetative organs and seed development.

5. An Account of Cloned Genes of Methyl-erythritol-4-phosphate Pathway of Isoprenoid Biosynthesis in Plants

Deepak Ganjewala, Shiv Kumar and Rajesh Luthra

Isoprenoids, also known as terpenoids, are biosynthesized by the condensation of the two C5 unit isopentenyl diphosphate (IPP) and isomer dimethylallyl diphosphate (DMAPP). Generally, plants use two separate pathways plastidial Methyl-erythritol-4-phosphate (MEP) and cytosolic acetate-mevalonate (MVA) pathways for formation of IPP. The genes, enzymes and intermediates of the MEP pathway have been unravelled in plants over the past few years. Interestingly, MEP pathway enzymes are encoded by nuclear genes but they function in plastids to produce precursors for isoprenes, monoterpenes, carotenoids, abscisic acid, gibberellins, and the side chain of chlorophylls, tocopherols, phylloquinones, and plastoquinone. In Arabidopsis thaliana, a complete set of genes of MEP pathway homologous to the E. coli MEP pathway genes have been identified. Although, these genes have been cloned and characterized from several other plants but overall information about them at one place is not available so far. Though, a range of reviews are available about their roles in isoprenoid biosynthesis and regulation. Therefore, we decided to compile the data on cloned and characterized genes of MEP pathway in plants. Also, we summarize the results of the previously published reports, particularly those which were based on incorporation of 13C-glucose or by application of specific inhibitors such as mevinolin and fosmidomycin to look into the MEP pathway in plants. In addition, we searched for the two key enzymes DXS and HMGR that could be assigned for the acetate-MVA and MEP pathway with the help of bioinformatics tools. Presence or absence of these enzymes can be correlated with respective isoprenoid biosynthetic pathways in plants.

6. Establishment of the Regeneration System for Vicia faba L.

Shimaa Bahgat, Omer A. Shabban, Osama El-Shihy, David. A. Lightfoot and Hany A. El- Shemy

A reliable regeneration system for faba bean has been difficult to establish and therefore, the genetic improvement of Vicia faba L. was delayed. The paper describes a method of somatic embryo induction in callus of V. faba. Two Egyptian faba bean cultivars 'Giza 2' and '24 Hyto' were used. Callus was induced from epicotyls and shoot tips cultured on MS or Gamborg medium supplemented with 3% sucrose and 0.025% (w/v) for each of ascorbic and citric acid, 0.8% agar and different concentrations of 10 mg/l BAP, 0.5 mg/l of each NAA and 2,4-dichlorophenoxyacetic acid (M1) and 1 mg/l BAP and 0.5mg/l NAA (M2) . The media with BAP, NAA and 2,4-D were optimal for embryogenic callus induction. Somatic embryos developed after transfer of the callus to 1/2 B5 medium with no plant growth regulators. There were various stages of somatic embryo development present including globular, heart-shaped, torpedo, and cotyledonary stages. Embryos developed into plantlets and plants were regenerated. RAPD analyses were performed to investigate the genetic stability of the regenerated plants obtained from different treatments and different explants. The cultivar Giza 2 exhibited more genetic stability than cultivar 24 Hyto. In conclusion, a regeneration system was established suitable for both gene transformation and the isolation of somaclonal mutants. The regeneration system will be used in order to improve the nutritional value of faba bean.

7. Cloning of a Novel Antifungal Promoter from Phaseolus vulgaris and the Determination of its Activity in Stably Transformed Nicotiana tabacum Plants

Eman A. Mahmoud, Solliman M. Mohei El-Din, Mourad A. M. Aboul-Soud, Ahmed M. Aboul-Enein, Ghanem A. Sobhy and Hany A. El-Shemy

To investigate the transcriptional regulation of gene expression, chimeric fusions, between the β-glucuronidase reporter gene (GUS) and the isolated promoter regions of the pvPDF gene (pvPDF-PRO: GUS), were constructed and introduced into Nicotiana tabacum. Analysis of transgenic pvPDF-PRO:GUS tobacco plants indicated that GUS activity was observed with all the promoter constructs with the strongest being in leaf followed by stem and roots. These results clearly demonstrate that pvPDF-PRO is a strong inducible and near-constitutive promoter and emphasize the great application potential for plant genetic engineering studies. Interestingly, a search for putative cis-acting elements in the pvPDF promoter architecture revealed the presence of some important transcription regulatory elements including: CAAT Box, TATA Box, CATA Box, and light regulatory elements (CCA1, GATA, GT-1). Taken together, these results further our understanding of the regulation of the pvPDF promoter activity.

8. The Interactions of the Largest Subunit of RNA Polymerase II with Other Cellular Proteins: a Bioinformatic Approach

Abhijit Shukla, Aparna Natarajan, Sukesh Bhaumik, Hany A. El-Shemy and David Lightfoot

The function of a protein is governed by its interaction with other proteins inside a cell. Therefore, it is important to identify the interacting partners of a particular protein to decipher its function. The protein interaction networks are generally determined by bioinformatic as well as experimental methodologies such as yeast two hybrid, mass spectrometry, immunoprecipitation, and fluorescence resonance energy transfer assays. Here, we have analyzed bioinformatically the interactions of Rpb1p (the largest subunit of RNA Polymerase II) with other proteins in yeast, using Cytoscape software and Biogrid/ Biomart database. We find that Rpb1p interacts with a large number of proteins involved in mRNA synthesis, processing, export, and other cellular processes. These results validate the application of such bioinformatic approach to determine the interactome for other cellular proteins.

9. Locus Interactions Underlie Seed Yield In Soybeans Resistant to Heterodera glycines

U.B. Karangula, M.A. Kassem, L. Gupta, H.A. El-Shemy and D.A. Lightfoot

In soybean (Glycine max L. Merr.) combining resistance to cyst nematode (SCN; Heterodera glycines I.) with high seed yieldremains problematic. Molecular markers linked to quantitative trait loci (QTL) have not provided a solution. Sets of markers describing a collection of favorable alleles (linkats) may assist plant breeders seeking to combine both traits. The objective of this analysis was to identify linkats in genomic regions underlying seed yield and root SCN resistance QTL. Used were groups of cultivars selected from a single recombinant inbred (RIL) population derived from 'Essex' by 'Forrest' (ExF). The yield was measured at four locations. SCN resistance was determined in greenhouse assays. The mean seed yield was used to define 3 groups (each n = 30), high, medium and low. SCN resistance formed 2 groups (SCN resistant (n = 21) and SCN susceptible (n = 69)). Microsatellite markers (213) alleles were compared with seed yield and root SCN (Hetrodera glycines) resistance using mean analysis. The number, size and position of potential linkats were determined. Loci, genomic regions and linkats associated with seed yield were identified on linkage group (LG) K and with root resistance to SCN e on LG E, G, and D1b+W. A method to identify co-localized genomic regions is presented.

10. Different Responses of Two Genes Associated with Disease Resistance Loci in Maize (Zea mays L.) to 3-allyloxy-1,2-benzothiazole 1,1-dioxide

Jiazheng Yuan, Jennifer Tedman, Liakat Ali, Jie Liu, Jeff Taylor, David Lightfoot, Michiaki Iwata and K. Peter Pauls

Probenazole (3-allyloxy-1,2-benzothiazole 1,1-dioxide, PBZ) is a bactericide and fungicide that acts by inducing plant defense systems. It has been shown to induce the expression of NBS-LRR genes like RPR1 (rice probenazole-response gene) in rice (Oryza sativa L.) and systemic acquired resistance (SAR)-like disease resistance. Two maize (Zea mays L.) genes Zmnbslrr1 (a NBS-LRR gene, cloned from a disease resistance analog PIC11 based) and Zmgc1, (a putative guanylyl cyclase- like gene) have both been associated with quantitative resistance loci (QTL) for resistance to Fusarium graminearum. PIC11 was associated with Fusarium stalk rot and ZmGC1 showed resistance to Gibberella ear rot caused by F. graminearum. The objectives of the current study here were to characterize the Zmnbslrr1 gene and to determine whether it and Zmgc1 respond to the inducer PBZ. The transcript abundance of Zmnbslrr1 expression was significantly reduced in corn seedlings of the Gibberella ear rot resistant genotype CO387 48h after PBZ treatment. In contrast, the transcript abundance of the maize Zmgc1 gene increased more than 10-fold 8h after the treatment. Therefore, the two genes do not appear to be coordinately regulated by PBZ.