فصلنامه ژنتیک نوین
سال نهم شماره 2 (پیاپی 37، تابستان 1393)

Atend of 2010 year, about 34 million people living with HIV. With current therapy AIDS is still not curable. Griffithsin (GRFT) is a potent anti-HIV protein that binds to HIV and inhibits cell to cell transmission of virus. In this study, first GRFT gene sequence was optimized. Then pBIgR-KD and pBIgR-ZR vector containing GRFT gene plus N termination of Zera SP ZERA and endoplasmic reticulum retention signal peptide (SEKDEL) was constructed. To obtaining transformants, young leaf explants of Nicotiana tobacco cv. Samsun were transformed with agrobacterium LBA4404 strain containing pBIgR-KD and and pBIgR-ZR. Formed shoots on MS medium containing 1 mg/l BA and 0/1 mg/l NAA and 100 mg/l kanamycin were transferred to root inducing medium (½ MS hormone free) containing 50 mg/l Kn. PCR using GRFT and 35S primers and sothern blot analysis showed succefull integration of GRFT transgene in tobacco genome. RT-PCR and real time PCR showed expression of GRFT gene at transcriptome level with high differences of expression transgenic lines. A band with corresponding size (27 KD for dimer GRFT protein) was detected based on SDS-PAGE analysis. Western blot analysis with anti-GRFT antibody confirmed production of GRFT protein as monomer (13 KD) and dimer (27 KD). Based on ELISA data, targeting to protein bodies using Zera signal peptide resulted in higher amount of recombinant GRFT in comparison to ER targeting via KDEL signal peptide. Based on our results high level of recombinant protein expression is achievable by optimizing of gene codon, selection of suitable host plant and application of suitable promoter and signal peptides.

There is growing concerns of over utilization of fish stocks in the world so that marine biodiversity is under extreme pressure by commercial fishing activities. As a solution, molecular genetic studies are being applied to determine genetic characteristics and also to select desirable genes or gene combinations in commercially important species. Silver pomfret (Pampus argenteus) is one of the most important marine food fish species in Persian Gulf and Oman Sea. In this study, genetic variability and population/s structure of Silver Pomfret were studied by genotyping 144 individuals from 5 localities (Sistan and Blauchestan, Hormozgan, Busher and Khuezstan provinces) using 11 microsatellite loci. Results of Fst (0.040) and Rst (0.129) showed low genetic differentiation among regions examined. Analysis of molecular variance (AMOVA) revealed 4% of genetic variation between groups and high genetic diversity observed within groups. This study suggests allelic richness and high genetic diversity of species across Persian Gulf and Oman Sea. Therefore, using microsatellite loci can provide scientific data of genetic resources of this commercial important species in the area.

Red flesh apple is one of the Iranian apple genotypes containing high level of anthocyanin in the cortex and flesh of fruit which causes red color in fruits. In order to assess genetic diversity of these native red flesh genotypes, 20 genotypes from red and white flesh fruits were selected and evaluated using 11 SSR primer pairs among which seven pairs showed polymorphism and a total of 56 alleles were generated. The number of alleles per locus varied from 3 to 11 with an average of 8 and also the average number of effective alleles was 5.74 per SSR locus. Furthermore, the averages of expected and observed heterozygosity were 0.6 and 0.77, respectively. The cluster analysis based on Dice similarity coefficient and UPGMA method distinguished genotypes into 7 groups. According to the dendrogram, no significant relation between classification and geographical origin was observed therefore those genotypes originating from the same area classified into different groups. As MYB10 gene is responsible for color of red flesh apple, we used the information about polymorphism in the promoter of the gene from all the studied genotypes. Considering self-incompatibility of apple, it seems that crossing between heterozygote red flesh genotypes with MYB10 and white fleshes have resulted in generation of the red and white flesh apples that are closely related.

Vinca (Catharanthus roseus) is a model medicinal plant. Roots of this plant is a place for accumulation of both alkaloids ajmalicine and serpentine. These materials are used for treatment of cardiovascular diseases, such high blood pressure. This plant is also very important, for production of alkaloids, vinblastine and vincristine, which are used in cancer treatment. Ethylene treatment significantly increased production metabolites of this plant. The objective of this research was investigation of ethylene effect on expression of some genes and detection of responsible genes for increase these metabolite. Effect of ethylene on the expression of T16H, G10H, DAT and AVLBS genes, at time course of 6, 12, 24, 48 and 72 hours, by Real time RT-PCR was investigated. Expression of T16H, DAT and AVLBS genes were up-regulated at 6 h after treatment with ethylene and down-regulated at 12 hours, then followed steady pattern. Also, the expression of G10H was increased at 72 h, and did not follow the pattern of the other three genes. According to results of the G10H gene is important gene to respond to ethylene.

This research was conducted to study the resistance inheritance of common bean to BCMNV virus. The Cross used in the experiment was between cultivars Goli (the resistant parent) and Derakhshan (the sensitive parent). Six generation seed obtain from Khomain bean national station and as randomized complete block design with four replications, planting and inoculate in plastic pot. Evaluation of plants performed with Elisa test and IC-RT-PCR 21 day after planting, and by observation at 45 day after planting. Resistance inheritance study with use of generation mean analysis, indicate that additive and dominant component have important role in control of infection rate. And simple model of additive – dominant can explain for genetic changes. Also determined that, for all traits under study, traits reduce genes are dominant and also this traits controlling genes with according to symptom and largeness are at different place. Means of broad-sense and narrow-sense heritability account 74 and 54 percent respectively. Thus selection for highest resistance can be effective. Accounting of effective factors number (genes) indicate that a great effect gene have role to genetic resistance control (probably I gene) and seem one or some small effect gene assistant to I gene for resistance induction.

In the present study 22 fish belonging to the species Capoeta trutta, C. damascina, C. aculeatae and C. buhsei from Sirvan (Palangan), Armand, Alvand, Sorkhakan – Balarood, Marbor, Chenarkhoshkeh, Beheshtabad, Dopolan and Ab-Vanak Rivers in Zagros region were analyzed for their COI barcods. In total 13 haplotypes were retrieved. The mean intraspecific barcod sequence divergence was 0.13 % based on K2P base substitution model. The results showed that the genus Capoeta is monophyletic and compared to the other species analyzed here, are of closer affinity to the species Barbus barbus. The maximum pairwise divergence was revealed between C. trutta and C. aculeatae (7.3%) and the least divergence level was for C. buhsei and C. damascina (2.7%). According to the measured divergence coefficients, the oldest and latest divergence times for the studied species are 9.6-14.38 and 3.55-5.19 MY (Million years).

Association analysis allows precise and fast methods for positioning of genes controlling quantitative traits. For this purpose, present study was conducted to assess the association of 27 primer pairs and 16 morphological traits recorded on 36 accessions belonged to 7 species of genus Brassica by stepwise regression using SAS software. Twenty-seven SSR primers amplified 130 alleles which 127 were polymorphic bands, with an average of 4. alleles per locus. Polymorphic information content (PIC) ranged from 0.16 to 0.49, with an average of 0.77. Primers O113-D02a and Ra2-E12 had the highest PIC. The result of stepwise regression analysis showed significant relationship among 27 SSR markers and at the least one of the morphological traits. Therefore, it is possible to use these markers along with morphological data in canola breeding programs for identification of suitable parents especially in segregation populations. A significant amount of morphological variation was explained by markers BRMS-008 and BRMS-024 indicating that genes associated with these traits are possibly located in chromosomal loci close together. The most variation of oil content (0.99) was accounted by BRMS-001, BRMS-005, BRMS-007, BRMS-008, BRMS-029, BRMS-031, BRMS-040, Ni4-D09, Na14-G06, Na12-F03, Na12-D04, O113-D02a, Ra2-E12 and Ni2-F02 markers. In this study, all studied loci were uniformly distributed around traits. Therefore, coding genes of agronomcal traits could identify by sequencing of loci with highest R2. Also markers with highest association to traits can be used for saturating linkage maps.

Anethum graveolens is one of medcinal plant that use in traditional and modern medicine and some of populations cultivate all of Iran. There is alittle information about cytogenetic status of cultivated populations. Thus, this study was carried out in order to evaluate cytogenetic variation of 15 populations of dill (Anethum graveolens) in a completly randomaized design with three replications using aceto iron hematocylin staining. Measured cytological traits were include number of chromosomes, length of longest and shoetest chromosomes, ratio of longest to shoetest chromosome, ratio of long arm to short arm of chromosomes, total of form, and symmetric index of chromosome. The results indicated that all populations had the basic chromosome number of eleven (x=11 and 2n=22). According to symmetry classes of Stebbins, one population from Gorgan and one from Bahbahan were placed in symmetric class of 1A, one population from Poland was placed in symmetric class of 2B and the rest of populations were placed in symmetric class of 2A. Investigation of asymmetry index showed that populations of Friedan, Poland and Tiran had the lowest symmetry while populations of Gorgan, Behbahan and Shiraz had the highest asymmetry. Cluster analysis classified populations into four groups. This grouping may be used to determine the evolutionary relationships and classification position of populations and for selecting the appropriate populations in future crossing with the purpose of creating diversity.

Among proteins involved in gene silencing and creation of small RNA, argonaute proteins play an important role that has been shown to possess endonucleolytic activity. In the present study, we aim to use bioinformatics tools for analyzing putative argonaute genes in forest strawberry. A BLAST analysis was performed against the available strawberry genomic sequence draft to identify argonaute sequences using previously reported argonaute sequences of Arabidopsis. Sequence alignment, 3D structure, phylogenetic tree and conserved domains were generated for strawberry argonautes. Results showed that 13 putative argonaute genes are spread on different chromosomes of forest strawberry. There were three classes of argonaute proteins similar to that of Arabidopsis, and each varies in length from 845 to 1956 AA, with an average of 1076 AA and 120 kDa. Forest strawberry argonaute proteins have PAZ, PIWI and MID conserved domains. argonaute 6 contained a new domain GT1-Sucrose-synthase which has not been reported for plants and animals previously. Our results showed that forest strawberry have three classes of argonautes and a new argonaute that needs more studies.

In order to mapping QTLs of 9 phenological and morphological traits of "Seri M82×Babax" drived recombinant inbred line population of wheat, an experiment was conducted in two alpha lattice with two replications under normal and salt stress conditions in 2012. Days to heading, days to anthesis, plant height (cm), spike number, grain number in spike, biological yield, thousand grain weight, harvest index and grain yield were measured. QTL analysis was conducted by composite interval mapping method separately for each trait in each condition and mean of two conditions. Based on combined analysis of variance, the main effect of genotype was high significant for all studied traits and transgressive segregation in both directions (positive and negative) was observed. Twenty seven QTLs were found for the studied traits. Phenotypic variation that were explained by these QTLs, varied from 5-16%. The highest and lowest phenotypic variations were related to days to anthesis and thousand grain weight. Highest and loest LOD scores were obtained for the QTLs of harvest index in mean of two conditions and thousand grain weight in salt condition. Most of mapped QTLs were unstuble. Therefore, use of MAS in this population for studied traits seen to be unefficient.

The aim of this study Investigating the genetic variation in the collection germplasm Sistan grapevine native and non-native cultivars using REMAP marker. To study the genetic diversity of grapevine native and non native cultivars of Zahak research station in Sistan, 33 grape varieties (Vitis vinifera L.) were evaluated by by 7 primer combination REMAP combination ISSR primers and retrotransposons based primer from of Ty1-copia and Ty3-gypsy families. The observed polymorphism for markers MS1 / Gret1 Ra, MS5 / Gret1Rb in both native and non-native varieties were 80 and 75%, respectively. The results revealed that the Gret1 retrotransposon has more transcription and translocation trough the genome. Cluster analysis based on Jaccard's similarity and UPGMA algorithm showed that the population is divided into two groups at (0.44) similarity; one group with twelve sub-groups and one group with one varieties. The results of principal component analysis extracted six components that explained 50.44 % of the total variation among the studied population.

Fructans as the main wheat stem reserves are accumulated during salt stress and their remobilization into grains can be significant to reduce severe yield loss. To study the effect of salinity on wheat fructan metabolism at molecular level, expression of the key genes including sucrose-sucrose fructosyl transferase (1-SST) and sucrose- fructan fructosyl transfarease (6-SFT) involved in fructan biosynthesis, fructosyl exohydrolase (1-FEH) and vacuolar invertase (IVR) involved in fructan and sucrose degradation, respectively, and sucrose transporter (SUT1) was analyzed in Bam as a salt-tolerant and Ghods as a salt-sensitive wheat cultivars. Control and salt treatments were applied by irrigation water with EC of 0.5 and 12 dSm-1, respectively from seedling stage in the greenhouse with three replicates using completely randomized design. Gene expression analysis was carried out at anthesis on leaf and stem by real Time PCR and stem fructan measurement was done at five points with 7-day intervals during seed filling period. The results showed the up-regulation of stem 1-SST and 6-SFT and the increase of fructan content in Bam and down-regulation of these genes and decrease of fructan content in Ghods under salinity. In leaf tissues of both cultivars, 1-SST, 1-FEH and SUT1 up-regulated under salt stress. Based on the obtained results, the most remarkable difference of fructan metabolism involved gene expression under salt stress between Bam and Ghods at anthesis was related to stem 6-SFT gene. The results showed that the up-regulation of 6-SFT, the higher fructan production and having greater capacity of stem reserves in Bam correlates with its higher grain yield under salt stress.

In the recent years, symptoms of viral diseases such as mosaic, stunt and yield reduction has been observed at corn fields. Maize dwarf mosaic virus (MDMV), in the north, and Iranian Johnsongarss mosaic virus (IJMV), in the most regions of Iran, are important viruses in corn fields. For detection of these viruses from Golestan province corn fields, 350 plant samples from the fields were selected and then samples survived from DNA-ELISA with specific antiserums of two viruses and RNA production is accomplished. Extracted cDNA were replicated by specific primers at PCR test. Results of ELISA test showed that corn fields are infected to two viruses simultaneously in the some regions. That some of the corn samples response positive. Also infection of MDMV observed in samples of zea and Johnsongrass. All of studied regions are infected to IJMV. Positive responses in PCR test with used primers supported the results of ELISA test and existence two section of reproduction fore per virus in electrophoresis showed simultaneously infection samples for two viruses. Also results of PCR product electrophoresis from symptoms infected to Millet-pearl show only section of MDMV. For result of this study the most zea, johnsongrass with mosaic, yellow and stunt infected to one or both of IJMV and MDMV.

Bacillus cereus is a Gram-positive aerobic or facultative anaerobic, spore-forming bacterium that is widely distributed environmentally. The organism causes emetic and diarrheal food poisoning syndromes. This study was aimed at determining the of enterotoxigenic hblA gene carried by B. cereus strains isolated from Schizothorax zarudnyi samples in Iran using specific primers. PCR is a rapid and prompt method for detection of presence of enterotoxigenic B. cereus in food, to ensure that food products are safe for consumption. We provided 35 Schizothorax zarudnyi from Chah nime in sistan. Selective plating on polymixin-pyruvate-egg yolk-mannitol-bromocresol purple agar (PEMPA) was used for isolation of B. cereus from Schizothorax zarudnyi. Enterotoxin producing ability of all 13 isolates obtained from the samples was judged by reverse passive latex agglutination (RPLA) test and the presence of hblA gene was screened by PCR. B. cereus and enterotoxigenic B. cereus were found to be in 37.2 and 25.8 per cent of fish samples, respectively. All the diarrhoeal enterotoxin producing isolates showed the presence of hblA gene, but hblA gene was not present in any of the non-enterotoxigenic isolates tested in this study. This indicated that the data of hblA gene specific PCR is a promising result in differentiating of enterotoxigenic B. cereus from non- enterotoxigenic ones. PCR is a good alternative for a RPLA and PCR is able to distinguish Enterotoxigenic B. cereus isolates from non Enterotoxigenic B. cereus in shorter time and with less cost.