Optimizing expression of antiviral cyanovirin-N homology gene using response surface methodology and protein structure prediction.

Cyanovirin-N (CVN) is well known as an anti-HIV protein. The efficient production of low cost microbicides for preventing the HIV-infection  has lately become a requirement worldwide. The aim of the present study was to optimize the expression of antiviral Cyanovirin-N homology gene found in the indigenous strain of Nostoc ellipsospourum LZN using Response Surface Methodology (RSM) and Protein Structure Analysis. Optimization of three induction factors (IPTG concentration (0.1, 0.55 and 1mM), temperature for bacterial growth (20, 28.5 and 37°C) and induction time (4, 10 and 16h) was done using RSM and Box-Behnken Design. Total RNA extraction was performed and mRNA levels were quantified in each experimental design by one-step SYBR qPCR. Protein structure was predicted using I-TASSER server. The full-length sequence of LZN-CVN gene is 306 bp in length, due mostly to five mutations. RSM analysis showed that the optimum condition to obtain maximum fold change was a concentration of 0.6mM IPTG, temperature set to 29°C and a 12h long induction time. The extracted protein from periplasmic fraction (8 kDa) was verified via SDS-PAGE. The high percentage of LZN-CVN similarity was demonstrated with PDB (Protein Data Bank) accession code of 2rp3A (CVN domain B mutant) and the ligand binding sites were related to N42, V43, D44, G45, S52, N53 and E56 residues. Different expression systems could assist in the development of anti-HIV proteins in a large scale. The LZN-CVN protein was successfully expressed in the E.coli system. RSM could be applied to a series of mathematical and statistical methods for modeling and analysis of responses which are influenced by various variables of interest.

DNA isolation protocol for the medicinal plant lemon balm (Melissa officinalis, Lamiaceae).

Lemon balm (Melissa officinalis) is a medicinal plant that is widely used as a sedative or calmant, spasmolytic and antibacterial agent and sleep aid. This has led to a high demand for lemon balm products, resulting in the extinction of this species in some of its natural habitats. Molecular techniques have increasingly been used in plant diversity conservation and isolation of PCR amplifiable genomic DNA is an important pre-requisite. Lemon balm contains high levels of polyphenols and polysaccharides, which pose a major challenge for the isolation of high-quality DNA. We compared different genomic DNA extraction protocols, including traditional phenol-chloroform DNA extraction protocols and two commercial kits for DNA purification for their ability to produce good-quality DNA from fresh leaves of five lemon balm genotypes. Quality and quantity of the DNA samples were determined using 0.8% agarose gel electrophoresis and a spectrophotometer. The DNA purity was further confirmed by PCR amplification using barley retrotransposon LTR base primers. The spectral quality of DNA as measured by the A(260)/A(280) ratio ranged from 1.46 to 2.37. The Fermentase genomic DNA purification kit and the CTAB extraction protocol using PVP and ammonium acetate to overcome the high levels of polyphenols and polysaccharides yielded high-quality DNA with a mean A(260)/A(280) ratio of 1.87. The quantity of DNA and its PCR purity were similar with all the protocols, but considering the time and cost required for extraction of DNA from a large number of samples, the CTAB protocol using PVP and ammonium acetate is suitable for lemon balm.

Analysis of the spatio-temporal distribution of Eurygaster integriceps (Hemiptera: Scutelleridae) by using spatial analysis by distance indices and geostatistics.

Eurygaster integriceps Puton (Hemiptera: Scutelleridae) is the most serious insect pest of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) in Iran. In this study, spatio-temporal distribution of this pest was determined in wheat by using spatial analysis by distance indices (SADIE) and geostatistics. Global positioning and geographic information systems were used for spatial sampling and mapping the distribution of this insect. The study was conducted for three growing seasons in Gharamalek, an agricultural region to the west of Tabriz, Iran. Weekly sampling began when E. integriceps adults migrated to wheat fields from overwintering sites and ended when the new generation adults appeared at the end of season. The adults were sampled using 1- by 1-m quadrat and distance-walk methods. A sweep net was used for sampling the nymphs, and five 180° sweeps were considered as the sampling unit. The results of spatial analyses by using geostatistics and SADIE indicated that E. integriceps adults were clumped after migration to fields and had significant spatial dependency. The second- and third-instar nymphs showed aggregated spatial structure in the middle of growing season. At the end of the season, population distribution changed toward random or regular patterns; and fourth and fifth instars had weaker spatial structure compared with younger nymphs. In Iran, management measures for E. integriceps in wheat fields are mainly applied against overwintering adults, as well as second and third instars. Because of the aggregated distribution of these life stages, site-specific spraying of chemicals is feasible in managing E. integriceps.

Chloroplast microsatellites markers to assess genetic diversity in wild and cultivated grapevines of Iran.

To assess the haplotype diversity and genetic relationship between them, A set of 69 Iranian cultivated accessions, six European cultivars and an accession of Vitis labrusca along with 63 wild grapevine individuals were studied using chloroplast microsatellite markers. Results showed that among analyzed cpssr loci only ccmp 3 and ccmp10 were polymorphic within cultivars and only ccmp3 was polymorphic in wild grape individuals. The size variants of both loci combine in a total of 4 different haplotypes. All the 4 haplotype are displayed in the cultivars while only 2 are presented in wild grapes. Sultani or keshmeshi Bidane cultivar has the haplotype III that there is not this haplotype among the wild grapes of studied regions. Concerning to existence of both haplotypes I and II in the number of Iranian cultivated and wild grapes, it is possible to consider that the wild grapes are ancestor of some of our native cultivars.

Evaluation of Tolypothrix germplasm for phycobiliprotein content.

Twenty Tolypothrix strains, including 15 strains of T. tenuis, three strains of T. ceylonica and one strain each of T. nodosa and T. bouteillei, were evaluated for their phycobiliprotein content and composition. Significant differences among the Tolypothrix strains were found at both inter- and intra-specific levels in the production of phycobiliprotein constituents--phycocyanin (PC), allophycocyanin (APC) and phycoerythrin (PE). Four specific parameters, viz. PC or PE content, total phycobiliprotein and total protein content, and percentage of phycobiliproteins, in a mixture of total proteins were used to select four T. tenuis and one T. ceylonica strain as useful for phycobiliproteins production.