Iranian Journal of Biotechnology
Volume:6 Issue: 2, Spring 2008

This paper reviews recent strategies used for increasing specific yield and productivity in high cell density cultures. High cell density cultures offer an efficient means for the economical production of recombinant proteins. However, there are still some challenges associated with high cell density cultivation (HCDC) techniques. A variety of strategies in several aspects including host design consideration, tuning recombinant protein expression, medium composition, growth methodologies, and even control and analysis of the process have been successfully employed by biotechnologists to increase yield in high cell density cultures. Although most researches have focused on Escherichia coli, other microorganisms have the potential to be grown at high density and need further investigation. In recent years, information on physiological changes of hosts during different phases of cultivation derived from functional genomics, transcriptomics and proteomics is being used to overcome the obstacles encountered in high cell density cultivation and hence increase productivity. Keywords: High cell density culture; Recombinant protein; Expression system.

The present study describes approaches for the identification of individual beech trees using maternal tissues from their seeds or fruits. Four microsatellite markers were used for genetic analysis of seedlots from Fagus orientalis Lipsky, a highly out-crossing tree species. Seeds from 11 single-tree harvests belonging to one population, (7 seeds from each), as well as non-paranchymatic maternal tissues of the seeds (single woody pericarps) were genotyped. No prior information about the genotypes of the mother trees was available for the seedlot samples. Two methods for detection of mother tree genotypes were adopted; (1) analysis of woody pericarps and their applicability for a direct molecular identification of the mother trees; and (2) analysis of maternal half-sib families and usefulness contaminations and inference of the seed parents from the offspring. A comparison of the multi-locus genotypes from pericarps with those of the inferred from the offspring revealed absolute identity. Analysis of the half-sib families revealed that the maternal genotypes can be inferred from offspring genotypes due to codominant Mendelian inheritance of the microsatellites. Differences among the seeds from the same mother tree suggested that paternal genotypes and gene flow have more influence on differentiation. The results of this research show that microsatellite analysis is a suitable means to monitor the number of trees included in commercial seedlot samples and detect seed contaminations.

Glutathione S-transferases (GSTs) are a major family of detoxification enzymes which possess a wide range of substrate specificities. Interest in insect GSTs has primarily focused on their role in insecticide resistance. In this study, following World Health Organization (WHO) routine susceptibility test, DNA was extracted from specimens of Anopheles stephensi collected from the Kazeroon district in the Fars province as control area and Saravan, Chabahar, Nikshahr districts in Sistan and Baluchistan province representing major malarious areas under insecticide treatment, in Iran. The (Glutathion S-transferase Epsilon class 2) GSTe2 gene including exon I and II and the full sequence of intron I, belonging to An. stephensi specimens were then amplified. The size of the resulting amplicons from the control area and the insecticide treated areas were 492 and 489 bp, respectively. These fragments were purified and then sequenced from both ends. The comparison of total amplified fragments among Kazeroon and Nikshahr and/or other populations of the Sistan and Baluchistan province (Saravan and Chabahar) showed 98% and 97% similarities, including 9-11 nucleotide substitutions, none of which had led to any amino acid change, within these populations. Comparison of the nucleotide sequence of GSTe2 in An. stephensi populations with that of the major world malaria vector, Anopheles gambiae revealed 86% homology, while amino acid similarity between the two species was approximately 90%. However, the main difference between the two susceptible and resistance groups in An. stephensi populations is related to their intron sequence with a distance of 8-9%, while this distance among resistance populations from the Sistan and Baluchistan province varied by approximately 0-4%. The results obtained from this study serve as a first report and baseline data regarding the structure of GSTe2 gene, including exon I, exon II and intron I in susceptible and resistance field specimens of An. stephensi. However, the integration of these data into the malaria control program still remains a challenge in Iran and neighboring countries, especially Afghanistan and Pakistan.

The genetic diversity among 56 almond (Prunus dulcis) genotypes was analysed using 35 microsatellite markers and 14 morphological traits. Analysis of morphological traits revealed a wide range of variation among the studied genotypes. Out of 35 simple sequence repeats (SSRs) markers, 25 were polymorphic, producing 215 alleles that varied from 2 to 16 with an average of 8.76 alleles per locus. Regression analyses revealed a positive correlation between the CPPCT03 locus and kernel yield, kernel percentage, grain weight, leaf length and tree altitude. The results of analysis of molecular variance (AMOVA) indicated that approximately 4.5% of genetic variance was observed between the collection sites. Based on SSR data, cluster analyses showed that the studied almond genotypes were classified into five main groups. The results of the present study showed that microsatellite markers could be successfully used to assay genetic diversity among Iranian almond landraces/cultivars and to identify informative markers for improving traits in breeding programs.

Schizophrenia is a severe neuropsychiatric disorder with symptoms such as hallucination, delusion and mental disorder. It is a complex disorder, in which genetic components play a crucial role in its pathogenesis. Among candidate genes for schizophrenia, Neuregulin 1 (NRG1) gene is the most important gene, association of which with the illness has been confirmed in several studies. Single nuclotide polymorphisms (SNPs) located 5 upstream of NRG1 have shown significant association with schizophrenia in several populations. Here, we describe a designed simple Multiplex Tetra-Primer Amplification Refractory Mutation System - polymerase chain reaction (PCR) for genotyping single SNP (SNP8NRG221533) in the human NRG1 gene. No restriction site was found for distinguishing T and C alleles of this SNP. The developed method proved to be simple, rapid and cost effective. This technique was used to compare SNP8NRG221533 in 95 schizophernics and 95 healthy controls. Our data demonstrate that there is a significant difference between allelic and genotypic frequencies of the two groups. These preliminary results confirm the association of the NRG1 gene with schizophrenia in an Iranian population.

Silymarin production by hairy root culture of Milk thistle (Silybum marianum L. Gaertn) was investigated using Agrobacterium rhizogenes AR15834. Hairy roots were induced by injection or inoculation of explants with A. rhizogenes. One month old hairy roots were dissected from the explants and grown in Murashing and Skoog (MS) liquid medium. Polymerase Chain Reaction (PCR) using the B gene and the B-glucoronidase (GUS) assays were used for identification of the transformed hairy roots. Flavonolignan levels in the hairy roots were determined by high-performance liquid chromatography (HPLC). Five different components were isolated; taxifolin, silychristin, silydianin, silybin and isosilybin, with the following quantities, 0.009, 0.041, 0.042, 0.007 and 0.011 mg g-1 dry weihht, respectively. Silybin was the major flavonolignan. Produced by hairy roots culture may serve as a useful system for producing silymarin or studying its biosynthetic pathways.

Resistance gene analog polymorphism (RGAP) markers linked to Fusarium head blight resistance (FHB) and co-localize with Qfhs.ndsu-3BS were identified using F3 plants and F3:5 lines derived from a Wangshuibai (resistant) / Seri82 (susceptible) cross. The mapping populations were genotyped using 50 degenerate primers designed based on the known R genes. Out of the 50 designed primer combinations, eight showed polymorphism and produced 16 RGAP markers. Out of the 16 RGAP markers, two were integrated into the major QTL for FHB resistance, Qfhs.ndsu-3BS. Composite interval mapping (CIM) analysis detected two QTLs in a genomic region that were coincident with Qfhs.ndsu-3BS, thus explaining up to 12.5% of the phenotypic variations. The nucleotide sequence analysis of the positive subjected RGAP markers showed that known R-genes, namely Pto and Pto-like genes, may be considered as FHB candidate resistance genes underlying Qfhs.ndsu-3BS and may be used in future studies.