In silico analysis and transient expression of wound-inducible promoter MPI in tomato (Lycopersicon esculentum Mill. cv. CH)
Zahra Hajiahmadi, Reza Shirzadian-Khorramabad*, Mahmood Kazemzad, Mohammad Mahdi Sohani
Plant Biotechnology Department, Faculty of agricultural sciences, University of Guilan, Rasht, P.O.Box 4199613776, Iran
Department of Energy, Materials and Energy Research Center, P.O. Box 14155-477, Tehran, Iran
Abstract
Various genetic promoters are applied to generate transgenic plants. Constitutive promoters such as CaMV35S are mostly being used; however, the constitutive expression of a gene could be harmful and lead to many changes in plant metabolism, growth and development. Subsequently, using inducible promoters in plant transformation has become more useful. In this project, efficiency of the wound-inducible promoter of Maize Proteinase Inhibitor (MPI) in tomato Lycopersicon esculentum Mill. cv. CH, as a new genetic background, was investigated. Therefore, the MPI was amplified following insertion into pTZ57R/T, sequencing and subsequently ligation into binary vector pBI121. Recombinant plasmids were transferred into Agrobacterium tumefaciens AGL1 strain. The results of in silico sequence analysis of MPI specified methyl jasmonate (MeJA) and abscisic acid (ABA) as responsive motifs, which might play a role in wound signaling pathway. Therefore, tomato leaves were transformed by PBI:MPI:GUS using agroinfilteration approach following GUS expression analysis by applying histochemical GUS and qRT-PCR assays. Our results demonstrated that induction of the MPI promoter only occurred in the wounded leaves and not detected in the control leaves. GUS transcripts in the wounded leaves containing MPI promoter were 4.1 folds higher than those containing CaMV35S, and GUS histochemical assays also revealed that the MPI give higher color intensity (2.64 folds) compared to the CaMV35S. Thus, the MPI could be considered as an acceptable working and inducible promoter to generate dicot transgenic plants harboring insecticidal genes such as different types of cry genes, which just expressed when plants are being attacked by pests.
Pages 118-126 | Read More| doi: 10.21475/poj.10.03.17.pne411……………………………………………………………………………………………………………………………………………………..
Defatted coagulant seeds of Moringa oleifera and Moringa peregrena mediate alleviation of cadmium toxicity in wheat (Triticum aestivum L.) plant
Raifa A. Hassanein, Amal F. Abdelkader*, Heba M. Faramawy
Department of Biological Sciences, College of Science, King Faisal University, P.O.380, El-Ahassa 31982, Saudi Arabia
Department of Botany, Faculty of Science, Ain Shams University, 11355, Cairo, Egypt
Abstract
Contaminated soil with toxic heavy metals, particularly cadmium, is an environmental originated challenge from various industrial activities such as effluents and mining and affecting crop growth. Cadmium removal from the soil is a problem, therefore we introduced our approach for reinforcing cadmium tolerance in wheat plant by applying powder (1g/kg soil) of fat-free seeds 'defatted seed or seed-cake' from Moringa oleifera and Moringa peregrena trees before wheat exposure to three levels of soil contamination with cadmium in soil solution (0.5, 1.0 and 1.5 mM) for 35 days. Our data showed that accumulation of Cd in wheat was concentration-dependent and was 4-folds higher in roots than in the shoots. In shoots and roots, cadmium accumulation was 3- and 2-folds higher at 1.5 mM Cd, respectively. Shoot length and dry mass have reduced by 30 and 58%, respectively. In addition, the photosynthetic pigments have decreased by 50% and the protein profile was altered in leaf. Moringa defatted seeds powder had conferred protection against cadmium toxicity in wheat. Upon defatted seeds pre-treatments, Cd accumulation has diminished by 3 and 2-folds in shoots and roots, respectively and wheat growth and physiological parameters have improved spectacularly. Protein polymorphism and SDS-gel electrophoresis confirmed the appearance of eight newly expressed protein bands in response to Moringa pre-treatments in absence of Cd. Eleven new bands were also expressed under the combined effect of cadmium and Moringa together in wheat leaf and weighed: 261.38, 217.77, 177.38, 135.4, 114.0, 97.0, 74.14, 51.6, 38.2, 24.0 and 13.5 KDa. We believed that the new bands have participated with wheat tolerance. In conclusion, we suggested two strategies: first, Moringa seeds have coagulated cadmium in the soil preventing its absorption and accumulation in wheat plants. Second, wheat newly expressed protein has assisted cadmium detoxification via binding it to some peptides, and/or through sequestrating cadmium in the vacuole as metallothioneins and/or polychelatins.
Pages 127-133 |Read More| Supplementary Data|doi: 10.21475/poj.10.03.17.pne423……………………………………………………………………………………………………………………………………………………..
Isolation, expression, and characterization of the serine protease inhibitor gene (600Hbpi) from Hevea brasiliensis leaves, RRIM600 cultivar
Dutsadee Chinnapun*, Sarawoot Palipoch
School of Medicine, Walailak University, Nakhon Si Thammarat 80161, Thailand
Abstract
First-strand cDNA encoding a serine protease inhibitor was synthesized from RNA extracted from Hevea brasiliensis leaves, RRIM600 cultivar. A full-length cDNA of RRIM600 H. brasiliensis protease inhibitor (600Hbpi) (GenBank accession no. KJ471471) was obtained from reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The primers for 600Hbpi were created from alignments of H. brasiliensis RRIM600 latex protease inhibitor (Hb-PI) (GenBank accession no. EU295479) and H. brasiliensis protease inhibitor protein 1 (PI1) (GenBank accession no. AY221985). 600HbPI encodes a 70 amino acid protein and is a member of the potato inhibitor I (PI-I) family of serine protease inhibitors. Multiple sequence alignment of homologous PI-I family proteins revealed one motif WPEL of 600HbPI conserved across the PI-I family. The coding region for the active site of 600HbPI was predicted as Met46-Glu47. 600Hbpi was cloned into the pFLAG-ATS vector. Recombinant 600HbPI was expressed as 11 kDa proteins in Escherichia coli strain BL21. Protease inhibition analysis showed that recombinant 600HbPI is more effective at inhibiting subtilisin A than chymotrypsin but did not inhibit trypsin protease. These results indicate that the recombinant 600HbPI encoded a functional protease inhibitor that specifically targets the chymotrypsin and subtilisin classes of serine proteases.
Pages 134-138 |Read More| doi: 10.21475/poj.10.03.17.pne450……………………………………………………………………………………………………………………………………………………..
Data mining approaches highlighted transcription factors that play role in thermo-priming
Fereshteh Izadi*, Hamid Najafi Zarrini, Ghaffar Kiani, Nadali Babaeian Jelodar
Department of Plant Breeding and Biotechnology, School of Molecular Genetics and Genetics Engineering, College of Crop sciences, Sari Agricultural Sciences and Natural Resources University (SANRU), Mazandaran, Farah Abad Road, Iran. Postal code: 4818168984
Abstract
Abiotic stresses including heat are major threats to crop plants especially considering the global warming facts. A pre-exposure to heat stress can prime plants and enable them to encounter a severe dose of stress which is lethal to unprimed plants. In this study, in order to identifying potential key elements involved in priming responses to heat stress we analyzed a microarray series in Arabidopsis thaliana. To this end we conducted differential expression analysis, clustering, annotation and network construction by using the publically available tools. In agreement with experimentally validated results we noticed that different genes were differentially expressed between primed and unprimed plants from which genes encoding retro-elements and proteins involved in chromatin remodeling were noteworthy. The topology analysis of constructed network highlighted the role of TFs including zinc finger and DREB in heat acclimation after priming phase. A total of 33 transcription factors were differentially expressed in primed versus unprimed plants five of which were detected to be hub and bottleneck nodes in genes network that may play a role in heat stress priming and memory additionally as potential targets to discover new insights to improve stress resistance in crop plants.
Pages 139-145 |Read More| Supplementary Data| Supplementary Excel| doi: 10.21475/poj.10.03.17.pne468……………………………………………………………………………………………………………………………………………………..
RAPD marker assisted evaluation of chloroplast DNA variation in twelve hosta taxa
Hasan Mehraj*, Subarna Sharma, Kouhei Ohnishi, and Kazuhiko Shimasaki
The United Graduate School of Agricultural Sciences, Ehime University, Ehime 790-8556, Japan
Laboratory of Vegetable and Floricultural Science, Faculty of Agriculture and Marine Science, Kochi University, B200 Monobe, Nankoku, Kochi 783-8502, Japan
Research Institute of Molecular genetics, Kochi University, B200 Monobe, Nankoku, Kochi 783-8502, Japan
Abstract
Hostas are major ornamental plants for perennial shady nurseries. They have striking foliage with attractive flowers, and their leaves are sometimes used as vegetables in Japan and Korea. The objective of this study was to estimate the genetic identity and genetic distance of twelve Hosta species and to build a UPGMA cluster for them using Random Amplified Polymorphic DNA (RAPD). Rhizomes of hostas were collected from Shikoku Island, Japan. Newly emerged unfurled hosta leaves were disrupted with Nuclei Lysis solution and then purified the genomic cpDNA. The cpDNA was dehydrated as a final point for extraction. We amplified the cpDNA of twelve species by means of 20 RAPD 10mer markers (Kit A: OPA-01 to OPA-20). The amplified DNA was run in 0.8% agarose gel using 1000 kb DNA ladder. Out of 20 RAPD markers, the genomic cpDNA was successfully amplified for fourteen. Fourteen markers generated a total of 102 fragments. We found maximum genetic distance (0.655) and minimum genetic identity (0.520) between H. sieboldiana - H. longissima species and between H. sieboldii - H. longissima. Estimated minimum genetic distance (0.071) and maximum genetic identity (0.931) were found between H. kikutii var. polyneuron - H. longipes var. caduca. The UPGMA dendrogram revealed the relationship among the 12 hosta species. The results confirmed our hypothesis of large genetic variability among the species, which distinguishes the genotypes clearly. The RAPD 10mer markers were found useful in application to the evaluation of the genetic variability and diversity within species.
Pages 146-152 |Read More| doi: 10.21475/poj.10.03.17.pne506……………………………………………………………………………………………………………………………………………………..
Advances in detection of stress tolerance in plants through metabolomics approaches
Naeem Khan*, Shahid Ali, Muhammad Adnan Shahid, Ardashir Kharabian-Masouleh
Department of Plant Sciences, Quaid-I-Azam University Islamabad, Pakistan
Plant Epigenetics and Development, Northeast Forestry University, Harbin, China
Horticultural Sciences Department, IFAS, University of Florida, USA
Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, Australia
Abstract
Heat and drought stresses are presently the principal risk on world’s food quantity, limiting yield. Both of these two stresses affect plants metabolism, physiological and morphological processes, which ultimately reduces the productivity. The plant cell develops different stress induced self-defence mechanisms to reduce the effect of stresses. These defence mechanisms are developed by modifying gene expression pattern, which results in qualitative and quantitative deviations in proteins synthesis, leading to the modulation of certain metabolic and defensive pathways. New metabolic profiling technologies offer a great opportunity for biologist to understand defence mechanism of plants under stress conditions. Metabolomics technologies presently enabled the using of different multi-variate analyses, generated from various hyphenated and chromatographic discovery systems, such as gas or liquid chromatography together with mass spectrometry, or nuclear magnetic resonance (NMR) based methods. Investigation and mining of metabolomics data can be done through a blend of different statistical methods, such as independent component analysis and analysis of variance. Metabolomics in combination with gene expression, protein interaction and other different regulatory pathways can be useful to diverse organisms with trivial alterations. In recent time, this technology has been used to investigate drought tolerance in plant crops to find particular stress related patterns in metabolic expression. These studies identified the vital roles of primary and secondary metabolites associated with abiotic stress tolerance.
Pages 153-163 |Read More| doi: 10.21475/poj.10.03.17.pne600……………………………………………………………………………………………………………………………………………………..
Identification of putative metabolic biomarker underlying cooked rice elongation
Nnaemeka Emmanuel Okpala, Lixin Duan, Guoan Shen, Guiquan Zhang, Xiaoquan Qi*
Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
Guangdong Key Laboratory of Plant Molecular Breeding, College of Agriculture, South China Agricultural University, Guangzhou 510642, China
University of Chinese Academy of Sciences, Beijing, China
Abstract
Rice (Oryza sativa L.) is a staple food for over half of the world’s population. However, rice grain quality is one of the major problems facing rice breeders across the world. Cooked rice elongation, cooked rice expansion, and water absorption have been identified as some of the parameters used in gauging rice grain quality. Biomarkers such as proteins or metabolites can be used to differentiate among a pattern of variations among various sampled (e.g., various locations within a plant, various germplasm accessions); consequently, they present a type of internal validation for a given biological state. In the present study, we investigated the putative metabolite biomarkers associated with the variation of cooked rice elongation for Hua Jing Xian 74 (receptor), Basmati 370 (donor), and five hybrid lines resulting from a cross of these parent lines. We also investigated their cooked rice expansion and water absorption properties. After carrying out cooked rice elongation studies, metabolomics studies, correlation analyses, V-plot analyses, and thorough searches in public metabolite databases (Metlin, Massbank and KEGG), and in-house secondary metabolite database, we identified a metabolite with molecular weight of 280.25 and retention time of 6.4 min as a putative biomarker associated with cooked rice elongation in the varieties investigated. We also discovered that changes in cooked rice elongation and changes in cooked rice expansion follow a similar pattern; however, it appears that cooked rice elongation and cooked rice expansion do not affect water absorption in these rice lines. Our findings may facilitate the improvement of the cooked rice elongation of hybrids resulting from the crosses of Basmati 370 and Hua Jing Xian 74. Our results also offer interesting insight into cooked rice elongation, cooked rice expansion, and water absorption.
Pages 164-168 |Read More| doi: 10.21475/poj.10.03.17.pne670RSS