Iranian Journal of Biotechnology
Volume:6 Issue: 4, Autumn 2008

Tomato yellow leaf curl virus (TYLCV) is a major tomato virus in tropical and subtropical regions. In this study, 134 accessions of Solanum lycopersicum and six accessions of Solanum peruvianum were assessed for resistance to an Iranian isolate of TYLCV. Plants were inoculated using whiteflies (Bemisia tabaci) and the reaction of plants was evaluated based on either disease symptoms or viral DNA amplification. All accessions of S. lycopersicum had demonstrated various degrees of disease symptoms. However, all six accessions of S. peruvianum were resistant and remained symptomless. Phenotypic evaluation was confirmed by amplification of a 670bp TYLCV DNA fragment in all tested accessions of S. lycopersicum. Based on both phenotypic and molecular evaluations, no accession provided complete resistance to TYLCV, whereas nine accessions were assessed as tolerant. The high level of resistance noted in whitefly inoculated accessions of S. peruvianum was not observed in graft inoculated plants of these accessions. The TYLCV DNA fragment was detected five weeks post-inoculation when plants were inoculated by grafting. These results suggested that accessions of S. peruvianum may be merely resistant to vector inoculation of TYLCV.

Haploid microspore-derived embryos (MDEs) of wheat were obtained by in vitro androgenesis. These embryos were employed to evaluate the transient expression of GUS gene (uidA) following particle bombardment. Using the Bio-Rad PDS-1000/He system, the physical parameters including rupture disk pressure (900, 1100 and 1350 psi); microprojectile travel distance (6 and 9 cm); gold particles size (0.6 mm, 1 mm and 1.6 mm), DNA and microcarrier concentrations (0.5 mg of DNA with 150 g of gold particles or 1.0 g of DNA with 300 mg of gold particles/bombardment) and bombardment numbers (1x (single) and 2x (double)) were assessed. The effect of high osmoticum in the bombardment medium (0.3 M mannitol and 0.4 M maltose) and the age of embryos were also evaluated. Optimal expression in MDEs was obtained using the following conditions of double bombardment at 1350 psi, 9 cm target distance, a 1 mm gold particle size, 1.0 mg of DNA with 300 mg of gold particles/bombardment, and osmotic pretreatment of 4-6 weeks old embryos using 0.4 M maltose for 6 h before and 16 h after bombardment. The optimized transformation protocol presented in this study is expected to improve devalopment of commercial transgenic wheat lines expressing desirable agronomic traits.

Bacillus sp. GUS1, isolated from soil of the citrous garden in Iran was screened on gelatin agar medium for their ability to produce alkaline protease. The zymogram analysis of partial purification of the enzymes showed the presence of at least three proteases. The enzymes were active in a wide pH range (6.0-12.0) and stable in the alkaline range (8.0-12.0). The optimum temperature for the proteases was 70 °C. The irreversible thermoinactivation of the proteases showed that the enzymes retained 100% of its original activity after 60 min of incubation at 70 °C and, therefore, the proteases are highly stable at 70 ºC. Enzymes were mostly inhibited by PMSF, while the enzyme activity was retained to about 90% and 80% in the presence of 2-mercaptoethanol and iodoacetate, respectively. These data show that most of these proteases seem to be serine alkaline protease. Addition of SDS also marginally influenced the protease activity. Enzyme activity was remained more than 90% and 80% in the presence of 1 and 10 mM EDTA respectively. On the other hand, adding Ca2+ did not influence the enzyme activity. Our results suggested that the enzymes are not metalloproteases and are Ca2+-independent.

Enteroviruses are the causative agents of a number of diseases in humans. Group B coxsackieviruses are believed to be the most common viral agents responsible for human heart disease. Genomic data of enteroviruses has allowed developing new molecular approaches such as Nucleic Acid Sequence Based Amplification (NASBA) for detection of such viruses. In this study, coxsackievirus B3 (CVB3) was detected in virus-infected cell culture and specimens of artificially infected mice with specific primers using Reverse Transcription - Polymerase Chain Reaction (RT-PCR) and NASBA techniques. According to the results, both techniques could be used for the detection of viruses in cell culture and artificially infected animals. NASBA reaction was simpler to perform than RT-PCR. The only variable factor that had to be optimized with NASBA is KCl concentration. The optimal concentration of KCl was determined as 90 mM. Serial dilutions of 1 mg of total RNA showed that both RT-PCR and NASBA could detect the virus at 10-5 dilution. Analyses of heart and spleen samples from infected animals were positive for presence of Coxsackievirus B3 with both RT-PCR and NASBA. In conclusion, NASBA offers some advantages over RT-PCR and is a suitable alternative technique for the sensitive detection of CVB3 in contaminated samples.

Human granulocyte colony stimulating factor (hG-CSF) induces proliferation and differentiation of granulocyte progenitor cells. This glycoprotein is currently being used for treatment of neutropenia, in patients who have undergone bone marrow transplantation. So far, different researchers have tried to enhance hG-CSF biological activity and stability. In this study, Polymerase Chain Reaction (PCR) based site-directed mutagenesis was performed on hG-CSF cDNA. The final amplified DNA fragment was cloned into the pBluescript sk(-) plasmid and after verification of the desired mutations by sequencing, it was subcloned into the pET-21a(+) vector and expressed in Escherichia coli BL21. The mutant G-CSF product was analyzed by SDS-PAGE and Western-blot analyses. The results show that the recombinant mutant G-CSF has been cloned and expressed successfully in prokaryotic system. This research aimed to produce a new recombinant hG-CSF expected to show enhanced biological characteristics in contrast to those of the native hG-CSF. The analysis of its function and biological characteristics remain to be examined.

In this study, the full-length M2 gene of the avian influenza virus (H9N2) was isolated, analyzed and studied in detail. Total RNA was extracted and cDNA of the M2 mRNA was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) using random hexamer oligoes; specific primers were used for amplification of the M2 open reading frame (ORF) region. PCR was able to amplify the desirable fragment (294-bp) of the spliced M2 gene. The nucleotide sequence homology between the Iranian isolate and other H9 and H5 subtypes of influenza A from different hosts and geographical areas deposited in GenBank ranged from 92 to 98% and the amino acid sequence homology ranged from 97 to 100%.