MODERN GENETICS JOURNAL (MGJ)
Year:2015 | Volume:9 | Issue:4 (39)|Papers Count:17

Plants produce variety of secondary metabolites which are economically important as drugs, flavor and fragrances, dye and pigments, pesticides, and etc. Several problems such as level of production, heterogeneous quality and raw material supply affect production of secondary metabolites in plants. The growing demands in today’s market place for renewable natural products lead scientist to consider in vitro plants material cultures as an alternative method for production of secondary phytochemicals. Plant cell culture and tissue culture under sterile condition have been used to produce and extract plants secondary metabolites and multiplication. However, most of trails fail to produce desired products and new strategies to improve production of secondary metabolites are needed. Elicitors including biotic and abiotic compounds, trigger formation of secondary metabolites. Phytohormones such as methyl jasmonate and gibberellic acid and polysaccharides such as chitosan have been used as biotic elicitors. Helium-neon soft laser and ultrasonic are examples of abiotic elicitors. Lack of enough knowledge about biosynthetic pathways and mechanisms involve in plant metabolite production is one of the main problems in production of secondary metabolites. Where the productivity of the desired metabolites is limited by the lack of particular precursors, biotransformation using an exogenous supply of biosynthetic precursors, genetic manipulation and metabolic engineering may improve the accumulation of compounds. Amino acids are commonly used as secondary metabolite precursors. Any foreign gene of interest can be transferred to hairy root clones in order to produce genetically modified hairy roots and improve the production of secondary metabolites. Cell immobilization in calcium alginate along with suitable bioreactor system can enhance the production of secondary metabolites in vitro. The present review is covering biotechnological methods and useful applications to optimize and scale up production of plant secondary metabolite.

Lipases represent the most widely used class of enzymes in biotechnological applications and organic chemistry so that as the third industrial enzyme are significant and they are object of many studies. Bacterial lipases are members of the structural superfamily of a/b hydrolases that catalyze the hydrolysis and synthesis of a variety of acylglycerols at the interface of lipid and water. Lipases from thermophiles often show their extreme stability at elevated temperatures and in organic solvent. So, they play important roles in structural investigations and industrial applications. The structural comparison of a/b hydrolases and lipases represented that the insertion of x3 domain has not, until now, been seen within the a/b hydrolase canonical fold. In order to study the effect of the X3 domain of Bacillus thermocatenulatus lipase, deletion of X3 domain is applied by using SOE-PCR and the recombinant btl2 gene is cloned in pPTZ57R/T and pPicZaB as cloning and expression vectors, respectively and after linearization of recombinant pPicZaB, integrated further into the genome of Pichia pastoris GS115 by using electroporation. The recombinant Pichia pastoris was cultivated on YPD medium and the expression of extracellular BTL2 lipase was induced by adding methanol to the cultivation. The enzyme expression assay using p-nitrophenyl palmitate (pNPP) and SDS-PAGE analysis showed production and secretion of lipase to medium. The secreted lipase to medium was purified with one step ion exchange chromatography and enzyme confirmed with western bloting. Investigations showed that the mutated BTL2 lipase expressed and secreted. These results represented that Pichia pastoris could be an interested system for expression of many recombinant proteins in industry.

Denaturing gradient gel electrophoresis (DGGE) is a molecular fingerprinting method capable of detecting differences among double-stranded DNA fragments according to melting temperature. In present study, genetic diversity of exon-7 of Na+/K+ ATPase among several Artemia populations were studied. The study was conducted with six different Artemia populations as A. urmiana, Parthenogenetic Artemia Maharlu Lake, Parthenogenetic Artemia Urmia Lake, Parthenogenetic Artemia Incheh Lake from Iran, and A. franciscana and A. sinica using RFLP, DGGE and sequencing methods. After DNA extraction, 280 bp fragment of exon-7 of this gene was amplified using genus specific primer. Due to unsuccessful digestion by 8 different restriction digestion enzymes in RFLP analysis, the exon was analyzed by DGGE method. According to recent electrophoresis several new pattern and polymorphisms were detected by DGGE, Which were not identified by RFLP. Authenticity of the found polymorphism was checked by the fragment sequencing. The calculated genetic distance among the Artemia populations, based on the nucleotide diversity, proposed this theory that probably exon-7 is an important part of Na+/K+ ATPase gene in Artemia genus which play an important role in Artemia speciation.

A-thalassemia is a set of hereditary anemia, which occurs frequently not only in the countries of the Mediterranean seacoast but also in the tropics and subtropics of the world including South East Asia and southern Iran. The a-thalassemia can be generally categorized as: silent a-thalassemia carrier, a- thalassemia carrier, hemoglobin H disease, and a-thalassemia major (hydrops fetalis). Peripheral blood samples were obtained by venopuncture from ten healthy volunteers and ten silent a-thalassemia carrier patients. Blood samples were divided into two parts with one exposed to 3 Gy gamma rays generated from a 60Co source and the other without any irradiation. The blood samples were either immediately used for the comet assay or left at 37oC CO2 incubator for 48 h in RPMI-1640 medium. Chromosome analysis was carried out exclusively on first division metaphases. For all samples, 70 metaphases were observed. Obtained data were statistically analyzed using SPSS (version 15) software. Results showed that the frequency of spontaneous aberration was similar in all the study groups. In normal individuals, in all the chromosomes except chromosome 1, a random distribution of break points proportional to their lengths based on their DNA content was observed. In silent a-thalassemia carrier individuals, in all the chromosomes, a random distribution of break points proportional to their lengths based on their DNA content was observed. Results showed that the frequency of apoptosis in leukocytes of freshly drawn blood was similar in two study groups. Incubation of non-irradiated samples in culture medium and CO2 incubator at 37oC for 48 h greatly increased the frequency of apoptosis in all samples. However the frequency of apoptosis in silent a-thalassemia carrier was significantly higher than control. Irradiated samples kept for 48 hours in incubator prior to processing for comet assay, showed higher frequency of apoptosis compared to non-irradiated samples. However the frequency of apoptosis in silent a-thalassemia carrier and control were similar in two study groups.

Barley is one of the important cereals around the world which is used as food and feed. It is belonged to Gramineae and includes cultivated and wild types. Genetic diversity losses resulted to sever vulnerability of cultivated crops against environmental stresses, pests and diseases which cause yield reduction. In order to study genetic diversity, 64 barley lines were evaluated by AFLP molecular markers. The lines were cultivated in Gorgan Agricultural research station under a complete block design arrangement with three replications. The goal of this study was molecular evaluation and determination of genetic diversity between studied barley lines and using the resulted information in parent selection for crosses and production of suitable populations in subsequent generations. Eight AFLP primer combinations produced 749 bands which 93 band were polymorphic. The maximum and minimum polymorphic bands was produced by E-ACA/M-CTA (16 bands) and E-ACA/M-CTC (7 bands) primer combinations, respectively. E-ACA/M-CAA primer combination showed the highest PIC value, thus it can be used for determination between much closed barley genotypes. Cluster analysis of molecular traits based on Jaccard similarity coefficient, divided studied lines into 7 clusters. Principle coordinate analysis confirmed the cluster analysis results. The highest genetic distance or lowest genetic similarity was between line 41 and Sahra (land race). Therefore these two genotypes would be good source in breeding programs to cross and production of population with high genetic diversity.

This study was conducted to assess the low molecular weight glutenin subunits allelic variation among 97 lines of bread and durum wheat landraces using 3 primer pairs for loci (Glu-A3, Glu-B3 and Glu-D3). Results showed significant variation among durum and bread wheat landraces. In general, five amplified band segments by primer Glu3-D.2, inclusive four alleles a, b, c and d among landraces were identified with the frequency of 0.013, 0.853, 0.026 and 0.054, respectively, and allele b was the most frequent. Four amplified band patterns by primer Glu3-3.1, inclusive 6 alleles a, b, c, d, e and f were identified with the frequency of 0.474, 0.474, 0.525, 0742, 0.742 and 0.164, respectively and allele d and e were the most frequent. Four amplified band patterns by primer Glu3-3.2 inclusive 10 alleles a, b, c, d, e, f, g, h, i and j were identified with the frequency of 0.010, 0.536, 0.010, 0.010, 0.010, 0.020, 0.659, 0.546, 0.010 and 0.010, respectively, and allele g was the most frequent. Using Nei’s genetic variation index (H), allelic variation for primers Glu3-D2, Glu3-3.1 and Glu3-3.2 was 0.314, 0.829 and 0.709, respectively. Identified variations in this study may be valuable as allelic diversity in bread and durum wheat breeding programs to improve the quality of their end products.

The vascular endothelial growth factor signaling pathway through VEGFR receptors is well known as one of the crucial regulators of angiogenesis. Deficiencies in angiogenesis regulation leading to neovascularization which is an essential process in solid tumor growth and metastasis. Inhibition of VEGFR2 tyrosine kinase receptor has been developed as a systemic treatment strategy for cancer. Sorafenib is one of the most important anti-angiogenic tyrosine kinase inhibitors, with high binding capacities to angiogenic kinases primarily VEGFR-2 were recently approved for treatment of patients with advanced cancer. Two major problems have been noticed during the clinical development of angiogenesis inhibitors including significant clinical toxicities and drug resistance. To avoid such side effects, the natural compounds (phytochemicals) as VEGFR2 inhibitor in comparison with sorafenib are the best substitutes. Several offline tools, online tools and databases were used to obtain 3D structure of some phytochemicals, Sorafenib and VEGFR2. Then, Docking studies of natural compounds and Sorafenib with combination of VEGFR2 was down using Molegro Virtual Docker version 6. The study revealed Lutein, Homoharringtonine and Curcumin as potential drug compounds showing better binding efficiency and lower energy with VEGFR2 as target. Lutein, Homoharringtonine and curcumin require -172.5 Kcal/mol,-170 Kcal/mol and -165.3 Kcal/mol respectively and they were suggested as potential candidate phytochemicals for laboratory assessment.

OIlseed crops such as soybeans (Glycin max L.) have significant contribution to human energy. Knowledge of genetic diversity based on various markers, is very important for the development of breeding programs, especially in the selection of parents. Cold stress is one of the environmental factors that is caused oxidative stress and physiological, morphological and biochemical changes in cells and plants. Soybean is a thermophile plant and sensitive to low temperatures. In this study, cold tolerance of 16 soybean genotypes were evaluated using some physiological characteristics and their association with ISSR molecular marker analysis were investigated. From 12 primers which showed polymorphic bands, up to 94 bands were scored, out of which 77 (81.91%) were polymorphic. Cluster analysis of genotypes for physiological traits under cold stress level had approximate similarity with dendrogram of molecular data, and Mantel's test confirmed this relative similarity. Based on regression analysis, some informative markers associated with physiological traits studied in cold conditions were identified. The study found that molecular and morphological markers together are the perfect tool for the identification of superior genotypes.

Supplementary experiments especially for estimation of general and special combining abilities are necessary for introducing and supply of new silkworm hybrids. These genetic parameters investigated at four Japanese pure lines including Xihang1, Xihang2, Xihang3 and 101433 and three Chinese pure lines including Koming1, Koming2 and Y. Twenty hybrids obtained using Chinese and Japanese pure lines crossing. Effects of general combining abilities at Japanese pure lines (JGCA) were significant for total traits except pupae vitality percentage, and double cocoon percentage. Also, effects of general combining abilities at Chinese pure lines (CGCA) were significant for total traits except produced cocoon number, low cocoon percentage, and cocoon shell percentage. Effects of special combining abilities (SCA) were significant for alive larvae number, alive pupae number, produced cocoon number, cocoon shell weight, and cocoon weight. Cocoon weight and cocoon shell weight traits had high additive genetic variance and also they are under heterotic and non-additive genetics effects. Therefore, crossbreeding is the effective method for improvement of these economical characters in studied pure lines.

Sodium azide (NaN3) is a mutagenic agent that causes point mutations in the genome. Induced mutagenesis is a useful method for the improvement of a character(s) in the plants. Artemisia vulgaris plants which called Berenjasf in Iran is contained useful secondary metabolites. The production of mutant colonies of hairy root cultures of this plant could be a suitable strategy for increasing secondary metabolites. In this study, hairy roots were induced in in vitro condition using strain A4 of Agrobacterium rhizogenes. Mutagenesis using NaN3 at 5 concentrations (1-5 mM), was applied. The culture medium without sodium azide was used as the control. To monitor mutagenesis, PCR products obtained from ISSR and RAPD markers were analyzed on agarose gel. Results were analyzed by the NTYSIS software. Results showed that four out of five concentrations of sodium azide caused mutagenesis. Only 1mM sodium azide did not show mutagenesis in the samples. The lowest similarity coefficient compared to the control was observed 21%, by RAPD and 33% by ISSR, both of which were obtained by 2 mM sodium azide concentration.

The low level expression of transgenes and their silence through successive generations are the most important factors limiting the production of recombinant proteins in plants. Choosing an appropriate plant tissue for expression, and improving of the gene cassette could increase the expression of the transgene transferred to the plant. To address this problem, in this study, the increase of expression level of GUS gene in tobacco seed was considered by designing and preparation of seed specific construct and transferring it into the tobacco explents. The signal sequence in upstream and KDEL in downstream of the GUS gene were integrated as signals for protein retention in endoplasmic reticulum. The matrix attachment region (MAR) sequence was placed downstream of the GUS gene to prevent silencing. The whole fragment was expressed under the control of Napin, the seed specific promoter. This construct was transferred to A.tumefaciens LBA4404 and then used to transform tobacco leaf explants. Analysis of regenerated plants by using PCR and specific primers of nptII and GUS indicated the successful transfer of these genes to plantlets. The RT-PCR reaction results showed that nptII is transcribed in both tissues while GUS is only transcribed in the seed tissue. This finding is expected when considering that Nos promoter (which controls the nptII transcription) is a constitutive and Napin is a seed specific promoter. Expression of GUS in seeds of selected plants was investigated using SDS-PAGE and histochemical assay. The nearly 60 kDa band in the transgenic plant sample and also the results of histochemical assay indicate the expression of GUS gene in transgenic seeds of tobacco.

Despite superior medicinal properties, endangered and high value for industries of Birch, there are not any studies on its genetic diversity and structure. The main aims of this study were evaluated of genetic diversity, differentiation and genetic bottleneck of Betula pendula in Iran. The data collected in five natural populations and 13 microsatellite markers were used in this study. The results were shown that the genetic diversity indices including number of alleles, effective number of alleles, heterozygosity and gene flow were different for each primer pair. Highest FST was 0.4 for L7-3, so it can be introduced as an informative marker for identification and classification of B. pendula populations. The results of the analysis of molecular variance (AMOVA) were shown that the genetic differences among populations were 34% that confirm the clear genetic variation within populations. The population Sangdeh have the highest percentage of polymorphic alleles, number of alleles, effective number of alleles, Shannon Index, private alleles and expected heterozygosity. The Marmishoo and Shahrastanak populations were shown heterozygosity excess. It seems to be occurring a genetic bottleneck in Marmishoo and Shahrastanak populations. It is necessary to study on the "Conservation Biology Techniques" in those populations.

Leptin gene could be the candidate genes for growth traits because of its role in food intake regulation, energy expenditure and blood pressure. In order to investigate leptin gene polymorphism blood samples were collected from 120 Kurdi sheep located at the Breeding Center of north Khorasan Kurdi sheep. DNA was extracted by salting out method and PCR-SSCP was used to amplify a fragment of 275 bp from third exon and to genotype all animal for this gene. The results showed that this region is polymorphic; three patterns AA, AB and BC were observed with the frequencies 0.3, 0.275 and 0.425 resepectivily. The results revealed that there was no significant association between the genotypes and growth triats.

Characterization of genetic diversity has long been a major goal in tobacco breeding programs. In this research, the genetic diversity of tobacco germplasm including 90 oriental and semi oriental tobacco genotypes was investigated using simple sequence repeat markers. Herein, 26 microsatellite primer pairs out of 278 microsatellite primer pairs were able to clarify existence polymorphism in individuals DNA. Results of fingerprinting of genotypes using 26 microsatellite primer pairs revealed 67 alleles. The mean number of alleles per locus (na) and the effective allele number (ne) were 2.481 and 2.159 respectively. The expected heterozygosity ranged from 0.326 to 0.662 with an average of 0.512. Based on Jaccard's similarity coefficient, similarity values among genotypes were varied among 0.04 to 0.78 which imply the existence of genetic diversity in studied germplasm. Classification of tobacco germplasm via UPGMA clustering algorithm distinguished it into 4 separate groups. Results of cluster analysis manifested that the resultant classification did not accompany with the geographical distribution of genotypes.