Articles
9(3) 2016 issue
9(3) 2016 issue
May 2016 issue
Southern Cross Publishing©2016
Australia
Australia
Molecular modeling and docking studies of phytoalexin(s) with pathogenic protein(s) as molecular targets for designing the derivatives with anti-fungal action on Alternaria spp. of Brassica
Rajesh Kumar Pathak1, 2, Gohar Taj*1, Dinesh Pandey1, Virendra Kumar Kasana3, Mamta Baunthiyal2 and Anil Kumar1
1Department of Molecular Biology & Genetic Engineering, College of Basic Sciences, Humanities, G. B. Pant University of Agriculture, Technology, Pantnagar-263145, Uttarakhand, India
2Department of Biotechnology, G. B. Pant Engineering College, Pauri Garhwal-246194, Uttarakhand, India
3Department of Chemistry, College of Basic Sciences & Humanities, G. B. Pant University of Agriculture & Technology, Pantnagar-263145, Uttarakhand, India
Abstract
The present study used molecular modeling and docking based approaches to test some proteins viz, ABC transporter, Amr1, Beta-tubulin, Cutinase, Fusicoccadiene synthase and Glutathione transferase of Alternaria brassicicola as possible molecular target of phytoalexins during pathogenesis or defense response. Molecular Operating Environment (MOE) was used to predict 3D structures of above proteins which were subsequently docked with phytoalexins which included Camalexin, Brassilexin, Rutalexin and Spirobrassinin by Molegro Virtual Docker. The results of molecular docking of Spirobrassinin with the above targets showed greater affinity as revealed from binding energy in the range of -73.09 to -94.46 Kcal/mol. Accordingly five derivatives of Spirobrassinin were further designed and docked against each target proteins, so as to detect phytoalexin(s) having the antifungal potential. The molecular modeling and docking experiments identified two derivatives of Spirobrassinins, with binding energy in the range of -77.50 to -85.88 Kcal/mol, respectively, which could be used for protection of Brassica plants against infection by Alternaria spp including Alternaria brassicicola and Alternaria brassicae, main pathogen of Alternaria blight in rapeseed mustard. Further studies and downstream validation would give way to use the above phytoalexin(s) as a substitute for hazardous fungicides to control plant diseases.
Pages 172-182 | Full Text PDF | DOI: 10.21475/poj.16.09.03.p7654
.
The role of sucrose metabolizing proteins in hyperosmotic stress tolerance in sweet sorghum (Sorghum bicolor L. Moench)
Anathi Njokweni1, 2, Omodele Ibraheem1, 2, 3, Bongani Ndimba*1, 2
1National Proteomics Service Unit, Agricultural Research Council, Nietvoorbij-Infruitec Campus, Stellenbosch, 7600, South Africa
2Proteomics Research Group, Department of Biotechnology, University of the Western Cape, Robert Sobukwe Road, Bellville, 7535, South Africa
3Department of Biochemistry, Federal University Oye-Ekiti, Ekiti State, Nigeria
Abstract
Sugar accumulation is a common metabolic response under drought stress conditions. However, studies on the underlying molecular mechanisms of sugar accumulation under stress remain restricted. This study explores the role of sucrose metabolizing proteins in conferring tolerance to drought-induced hyperosmotic stress, and ultimately osmotic adjustment in sorghum. The effect of stress on sugar content, enzyme activity and gene expression was investigated. Sweet sorghum varieties (ICSV213 and ICSB338) differing in levels of drought tolerance were subjected to a 10-day water deficit period. Brix plant sap analysis indicated elevated total soluble sugar levels under water stress conditions in both varieties with ICSV213 demonstrating a higher brix content than ICSB338. HPLC analysis gave a decrease in sucrose levels and an increase in glucose and fructose concentrations in both varieties with ICSV213 demonstrating higher hexose levels. Enzyme activity levels of invertase, sucrose phosphate synthase and sucrose synthase were found to increase under stress in both varieties with ICSV213 invertase displaying the highest activity when compared to other sucrose metabolizing enzymes. Transcriptional expression of invertase and sucrose phosphate synthase (SPS) genes was significantly up regulated in ICSV213 under stressed conditions, whereas sucrose synthase (Susy) levels remained low in both varieties. Data obtained for sorghum variety ICSV213 points towards a relationship between hyperosmotic stress tolerance and the accumulation of solutes and sucrose metabolism proteins. Consequently variety ICSV213 may therefore be an excellent target for future development of hyperosmotic stress tolerant sweet sorghum.
Pages 183-190 | Full Text PDF | DOI: 10.21475/poj.16.09.03.p7724
.
Microsatellites mining in date palm (Phoenix dactylifera L.) and their cross transferability across Arecaceae family
Kalathil Palliyarakkal Manju1, Ramaswamy Manimekalai*2, Sudalaimuthu Asari Naganeeswaran1, Vadivel Arunachalam3, Anitha Karun1
1Biotechnology Section, Central Plantation Crops Research Institute (ICAR), Kasaragod, Kerala, India
2Biotechnology Section, Sugarcane Breeding Institute, Coimbatore, Tamil nadu, India
3ICAR Research Complex for Goa, Goa, India
Abstract
Phoenix dactylifera L. (date palm) is the first publicly available nuclear genome sequence under Arecaceae family and indeed under the entire order Arecales. These genomic sequences were mined microsatellites which could be used for marker assisted selection for important traits. We have developed an integrated, configurable and time effective microsatellite mining and annotation pipeline for this purpose. Microsatellite survey of date palm whole genome shotgun sequences using the developed pipeline detected a total of 166,760 perfect repeats with an average of one SSR per 2.2kb. Putative functions of these SSR-containing sequences within the proximity of genic regions were predicted. Primers targeting the functionally important SSR regions were designed and a set of eight SSR primers synthesized and were validated in vitro across five members of the Arecaceae family (coconut, arecanut, oilpalm, date palm and palmyrah palm). We have found that 28.6% of the SSRs are common among the members of arecaceae family. Those SSRs could be used in molecular marker analysis of less studied palm family members like arecanut and palmyrah palm.
Pages 191-197 | Full Text PDF | Supplementary Data xls | DOI: 10.21475/poj.16.09.03.p7793
.
Genome-wide transcriptome profiling of Gossypium spp. roots during early growth after infection with Rotylenchulus reniformis
Yonathan Tilahun*
Center for Molecular Biology, Alabama A&M University, Normal AL, 35762, USA
Abstract
Plant-nematode interactions have been studied extensively. The identification of genes expressed in root organs subsequent to nematode infection has been studied to a lower extent. Fewer still, potential resistance genes have been identified in cotton towards Rotylenchulus reniformis the reniform nematode (RN). Currently, there have been three cotton genomes completely sequenced. Gossypium, or cotton, genomes sequenced include: Gossypium hirsutum cv TM1, Gossypium arboreum, and Gossypium raimondii. This study imparts knowledge of differentially expressed genes (DEGs) after individual domesticated and wild cotton plants have been infected with approximately 50,000 juvenile RNs. After extraction of Total RNA from infected roots and subsequent next generation 454 pyrosequencing methods were employed, FastQC quality control measures were completed prior to Trimomatic-0.32 trimming from 33,788 reads, resulting in a significant decrease in the number of input reads (3454 input reads or DEGs). Putative descriptions were made for 634 input reads or DEGs, many of which were repeated multiple times as predicted proteins/enzymes or partials. The definition of 52 DEGs were made and enriched through the assignment of gene ontology (GO) terms that highlight categories of host plant genes for potential targets in future downstream transformation of cotton plants, especially those involved in strengthening systemic acquired resistance (SAR) or the hypersensitive response (HR) that occurs most often in plants when in defense against pathogen attack. This study reveals known protein/enzymes (52), unknown protein/enzymes (582) and unassigned DEGs (2820).
Pages 198-204 | Full Text PDF | Supplementary Data PDF | DOI: 10.21475/poj.16.09.03.p7816
.
Isolation and molecular characterization of a novel Na+/H+ antiporter gene, AlNHX2, from Aeluropus littoralis and comparison of AlNHX1 and AlNHX2
Zahra Sami and Abbas Alemzadeh*
Department of Crop Production and Plant Breeding, College of Agriculture, Shiraz University, Shiraz, Iran
Abstract
Halophytes have high tolerance against salinity and it is expected that these plants have special proteins that allow them to thrive under salinity conditions. Hence, to understand molecular aspects of vacuolar Na+/H+ antiporter, which has a possible role in salt tolerance in halophytic plants, a novel Na+/H+ antiporter gene, AlNHX2, was isolated and characterized by rapid amplification of cDNA ends (RACE) technique. The results revealed that AlNHX2 is expressed in leaves, stems and roots and its expression in leaves is 1.767 and 1.269 times higher than stems and roots, respectively. This gene has an ORF with 1617 bp in length, a 3'-UTR region with 245 pb, and a 5'-UTR region with 187 bp which encodes a 538 amino acid protein shared a high homology with those putative vacuolar Na+/H+ antiporters of higher plants. Putative phosphorylation sites within AlNHX1 and AlNHX2 were determined using the prediction software, and the binding of 14-3-3 protein to specific domains within AlNHX1 and AlNHX2 was predicted. The results also revealed this binding is induced by a protein kinase-mediated phosphorylation of a specific Thr or Ser residue in these domains. The results also revealed that the activities of AlNHX1 and AlNHX2 are regulated by PKC, p38MAPK and GSK3. In addition, the findings reported here show the interaction of CaM protein with C-terminal of AlNHX2. Autoinhibitory domain at C-terminal region of AlNHX2 that can suppress the protein activity in under normal growth conditions was also found.
Pages 205-212 | Full Text PDF | DOI: 10.21475/poj.16.09.03.pne35
.
Characterization of a pumpkin mRNA encoding a Cyclin-Dependent Protein Kinase (CDK) potentially involved in phloem development
Paul Starsky Herrera-Pola, Beatriz, Xoconostle-Cαzares, Roberto Toscano-Morales, Roberto Ruiz-Medrano*
Department of Biotechnology and Bioengineering CINVESTAV-IPN, Ave. IPN 2508, Zacatenco, 07360 Mιxico DF, Mιxico
Abstract
The phloem is involved in the delivery of nutrients but also of long-range signals that regulate diverse processes. Several different RNAs have been found in the phloem translocation stream, which could have a role in signaling. In a previous work, we have found several RNAs in pumpkin phloem sap exudates in response to viral infection, among them a transcript encoding a potential cyclin-dependent protein kinase (CDK). In this study, we report the further characterization of the aforementioned CDK mRNA. The complete sequence of this mRNA, which we termed CmCDKP (for Cucurbita maxima CDK from Phloem), was obtained (GenBank no. AIQ82912.1). The phylogenetic analysis of the virtual translation of this sequence showed that CmCDKP is closely related to those involved in transcriptional regulation via RNA polymerase II CTD phosphorylation, or splicing control. The mRNA accumulated to highest levels in pollen tissue. Interestingly, the mRNA localized to the companion cell-sieve element complex in the phloem, developing phloem, and also in isolated cells in the shoot apical meristem, suggesting a role in early phloem development.
Pages 213-219 | Full Text PDF | DOI: 10.21475/poj.16.09.03.pne114
Rajesh Kumar Pathak1, 2, Gohar Taj*1, Dinesh Pandey1, Virendra Kumar Kasana3, Mamta Baunthiyal2 and Anil Kumar1
1Department of Molecular Biology & Genetic Engineering, College of Basic Sciences, Humanities, G. B. Pant University of Agriculture, Technology, Pantnagar-263145, Uttarakhand, India
2Department of Biotechnology, G. B. Pant Engineering College, Pauri Garhwal-246194, Uttarakhand, India
3Department of Chemistry, College of Basic Sciences & Humanities, G. B. Pant University of Agriculture & Technology, Pantnagar-263145, Uttarakhand, India
Abstract
The present study used molecular modeling and docking based approaches to test some proteins viz, ABC transporter, Amr1, Beta-tubulin, Cutinase, Fusicoccadiene synthase and Glutathione transferase of Alternaria brassicicola as possible molecular target of phytoalexins during pathogenesis or defense response. Molecular Operating Environment (MOE) was used to predict 3D structures of above proteins which were subsequently docked with phytoalexins which included Camalexin, Brassilexin, Rutalexin and Spirobrassinin by Molegro Virtual Docker. The results of molecular docking of Spirobrassinin with the above targets showed greater affinity as revealed from binding energy in the range of -73.09 to -94.46 Kcal/mol. Accordingly five derivatives of Spirobrassinin were further designed and docked against each target proteins, so as to detect phytoalexin(s) having the antifungal potential. The molecular modeling and docking experiments identified two derivatives of Spirobrassinins, with binding energy in the range of -77.50 to -85.88 Kcal/mol, respectively, which could be used for protection of Brassica plants against infection by Alternaria spp including Alternaria brassicicola and Alternaria brassicae, main pathogen of Alternaria blight in rapeseed mustard. Further studies and downstream validation would give way to use the above phytoalexin(s) as a substitute for hazardous fungicides to control plant diseases.
Pages 172-182 | Full Text PDF | DOI: 10.21475/poj.16.09.03.p7654
.
The role of sucrose metabolizing proteins in hyperosmotic stress tolerance in sweet sorghum (Sorghum bicolor L. Moench)
Anathi Njokweni1, 2, Omodele Ibraheem1, 2, 3, Bongani Ndimba*1, 2
1National Proteomics Service Unit, Agricultural Research Council, Nietvoorbij-Infruitec Campus, Stellenbosch, 7600, South Africa
2Proteomics Research Group, Department of Biotechnology, University of the Western Cape, Robert Sobukwe Road, Bellville, 7535, South Africa
3Department of Biochemistry, Federal University Oye-Ekiti, Ekiti State, Nigeria
Abstract
Sugar accumulation is a common metabolic response under drought stress conditions. However, studies on the underlying molecular mechanisms of sugar accumulation under stress remain restricted. This study explores the role of sucrose metabolizing proteins in conferring tolerance to drought-induced hyperosmotic stress, and ultimately osmotic adjustment in sorghum. The effect of stress on sugar content, enzyme activity and gene expression was investigated. Sweet sorghum varieties (ICSV213 and ICSB338) differing in levels of drought tolerance were subjected to a 10-day water deficit period. Brix plant sap analysis indicated elevated total soluble sugar levels under water stress conditions in both varieties with ICSV213 demonstrating a higher brix content than ICSB338. HPLC analysis gave a decrease in sucrose levels and an increase in glucose and fructose concentrations in both varieties with ICSV213 demonstrating higher hexose levels. Enzyme activity levels of invertase, sucrose phosphate synthase and sucrose synthase were found to increase under stress in both varieties with ICSV213 invertase displaying the highest activity when compared to other sucrose metabolizing enzymes. Transcriptional expression of invertase and sucrose phosphate synthase (SPS) genes was significantly up regulated in ICSV213 under stressed conditions, whereas sucrose synthase (Susy) levels remained low in both varieties. Data obtained for sorghum variety ICSV213 points towards a relationship between hyperosmotic stress tolerance and the accumulation of solutes and sucrose metabolism proteins. Consequently variety ICSV213 may therefore be an excellent target for future development of hyperosmotic stress tolerant sweet sorghum.
Pages 183-190 | Full Text PDF | DOI: 10.21475/poj.16.09.03.p7724
.
Microsatellites mining in date palm (Phoenix dactylifera L.) and their cross transferability across Arecaceae family
Kalathil Palliyarakkal Manju1, Ramaswamy Manimekalai*2, Sudalaimuthu Asari Naganeeswaran1, Vadivel Arunachalam3, Anitha Karun1
1Biotechnology Section, Central Plantation Crops Research Institute (ICAR), Kasaragod, Kerala, India
2Biotechnology Section, Sugarcane Breeding Institute, Coimbatore, Tamil nadu, India
3ICAR Research Complex for Goa, Goa, India
Abstract
Phoenix dactylifera L. (date palm) is the first publicly available nuclear genome sequence under Arecaceae family and indeed under the entire order Arecales. These genomic sequences were mined microsatellites which could be used for marker assisted selection for important traits. We have developed an integrated, configurable and time effective microsatellite mining and annotation pipeline for this purpose. Microsatellite survey of date palm whole genome shotgun sequences using the developed pipeline detected a total of 166,760 perfect repeats with an average of one SSR per 2.2kb. Putative functions of these SSR-containing sequences within the proximity of genic regions were predicted. Primers targeting the functionally important SSR regions were designed and a set of eight SSR primers synthesized and were validated in vitro across five members of the Arecaceae family (coconut, arecanut, oilpalm, date palm and palmyrah palm). We have found that 28.6% of the SSRs are common among the members of arecaceae family. Those SSRs could be used in molecular marker analysis of less studied palm family members like arecanut and palmyrah palm.
Pages 191-197 | Full Text PDF | Supplementary Data xls | DOI: 10.21475/poj.16.09.03.p7793
.
Genome-wide transcriptome profiling of Gossypium spp. roots during early growth after infection with Rotylenchulus reniformis
Yonathan Tilahun*
Center for Molecular Biology, Alabama A&M University, Normal AL, 35762, USA
Abstract
Plant-nematode interactions have been studied extensively. The identification of genes expressed in root organs subsequent to nematode infection has been studied to a lower extent. Fewer still, potential resistance genes have been identified in cotton towards Rotylenchulus reniformis the reniform nematode (RN). Currently, there have been three cotton genomes completely sequenced. Gossypium, or cotton, genomes sequenced include: Gossypium hirsutum cv TM1, Gossypium arboreum, and Gossypium raimondii. This study imparts knowledge of differentially expressed genes (DEGs) after individual domesticated and wild cotton plants have been infected with approximately 50,000 juvenile RNs. After extraction of Total RNA from infected roots and subsequent next generation 454 pyrosequencing methods were employed, FastQC quality control measures were completed prior to Trimomatic-0.32 trimming from 33,788 reads, resulting in a significant decrease in the number of input reads (3454 input reads or DEGs). Putative descriptions were made for 634 input reads or DEGs, many of which were repeated multiple times as predicted proteins/enzymes or partials. The definition of 52 DEGs were made and enriched through the assignment of gene ontology (GO) terms that highlight categories of host plant genes for potential targets in future downstream transformation of cotton plants, especially those involved in strengthening systemic acquired resistance (SAR) or the hypersensitive response (HR) that occurs most often in plants when in defense against pathogen attack. This study reveals known protein/enzymes (52), unknown protein/enzymes (582) and unassigned DEGs (2820).
Pages 198-204 | Full Text PDF | Supplementary Data PDF | DOI: 10.21475/poj.16.09.03.p7816
.
Isolation and molecular characterization of a novel Na+/H+ antiporter gene, AlNHX2, from Aeluropus littoralis and comparison of AlNHX1 and AlNHX2
Zahra Sami and Abbas Alemzadeh*
Department of Crop Production and Plant Breeding, College of Agriculture, Shiraz University, Shiraz, Iran
Abstract
Halophytes have high tolerance against salinity and it is expected that these plants have special proteins that allow them to thrive under salinity conditions. Hence, to understand molecular aspects of vacuolar Na+/H+ antiporter, which has a possible role in salt tolerance in halophytic plants, a novel Na+/H+ antiporter gene, AlNHX2, was isolated and characterized by rapid amplification of cDNA ends (RACE) technique. The results revealed that AlNHX2 is expressed in leaves, stems and roots and its expression in leaves is 1.767 and 1.269 times higher than stems and roots, respectively. This gene has an ORF with 1617 bp in length, a 3'-UTR region with 245 pb, and a 5'-UTR region with 187 bp which encodes a 538 amino acid protein shared a high homology with those putative vacuolar Na+/H+ antiporters of higher plants. Putative phosphorylation sites within AlNHX1 and AlNHX2 were determined using the prediction software, and the binding of 14-3-3 protein to specific domains within AlNHX1 and AlNHX2 was predicted. The results also revealed this binding is induced by a protein kinase-mediated phosphorylation of a specific Thr or Ser residue in these domains. The results also revealed that the activities of AlNHX1 and AlNHX2 are regulated by PKC, p38MAPK and GSK3. In addition, the findings reported here show the interaction of CaM protein with C-terminal of AlNHX2. Autoinhibitory domain at C-terminal region of AlNHX2 that can suppress the protein activity in under normal growth conditions was also found.
Pages 205-212 | Full Text PDF | DOI: 10.21475/poj.16.09.03.pne35
.
Characterization of a pumpkin mRNA encoding a Cyclin-Dependent Protein Kinase (CDK) potentially involved in phloem development
Paul Starsky Herrera-Pola, Beatriz, Xoconostle-Cαzares, Roberto Toscano-Morales, Roberto Ruiz-Medrano*
Department of Biotechnology and Bioengineering CINVESTAV-IPN, Ave. IPN 2508, Zacatenco, 07360 Mιxico DF, Mιxico
Abstract
The phloem is involved in the delivery of nutrients but also of long-range signals that regulate diverse processes. Several different RNAs have been found in the phloem translocation stream, which could have a role in signaling. In a previous work, we have found several RNAs in pumpkin phloem sap exudates in response to viral infection, among them a transcript encoding a potential cyclin-dependent protein kinase (CDK). In this study, we report the further characterization of the aforementioned CDK mRNA. The complete sequence of this mRNA, which we termed CmCDKP (for Cucurbita maxima CDK from Phloem), was obtained (GenBank no. AIQ82912.1). The phylogenetic analysis of the virtual translation of this sequence showed that CmCDKP is closely related to those involved in transcriptional regulation via RNA polymerase II CTD phosphorylation, or splicing control. The mRNA accumulated to highest levels in pollen tissue. Interestingly, the mRNA localized to the companion cell-sieve element complex in the phloem, developing phloem, and also in isolated cells in the shoot apical meristem, suggesting a role in early phloem development.
Pages 213-219 | Full Text PDF | DOI: 10.21475/poj.16.09.03.pne114