فصلنامه ژنتیک نوین
سال هشتم شماره 4 (پیاپی 35، زمستان 1392)

Interferon gamma (IFN-g) is one of the most important proteins used in diagnostic and therapeutic especially in virus disease therapy and regulation of immune system actions. There are several ways for production of interferon gamma protein as an active ingredient of many medicines and one of these ways is using plants and molecular farming. In latest years chloroplast transformation due to having many advantages such as ability to accumulate large amounts of foreign protein in chloroplast, being free of human pathogens and expression of several genes simultaneously has been considered. The purpose of this research was to check feasibility of interferon gamma production using chloroplast transformation in a dicistronic operon. Interferon gamma gene was cloned in pKCZ vector. Then gold particles coated with this vector were bombarded on tobacco leaves by gene gun. The pieces of leaves were transferred to shoot regeneration medium containing spectinomycin. Thereafter the regenerated shoots were transferred to MS medium for rooting. Presence of interferon gamma gene and it’s expression in regenerated plants was confirmed by PCR and RT-PCR. Additionally gene expression in protein level was confirmed by SDS-PAGE and dot blot. Finally four regeneration rounds were performed to obtain homoplasmy.

Garra rufa, belongs to the Cyprinidae family; this fish is one of the important ecological species. In this study, six microsatellite locations were used to investigate the levels of genetic variation of Garra rufa in the Fahlyan and Sarab Bahram rivers in Fars province. In this research 56 samples used (28 samples per each river). The mean number of allele’s level at the population was 11.41 which are more than the reported values for freshwater fishes. The expected and observed heterozygosity were 0.497 and 0.847, respectively. Almost all of loci showed deviation from Hardy-Weinberg equilibrium. It seems that there is a low separation between two populations of this species in the studied areas due to high level of gene flow (Nm=17.212) between two regions and low Fst (0.02). According to these analyses, it seems that, Garra rufa has desirable genetic diversity in investigated regions.

Plant pathogens secret their effector proteins into host cell in order to suppress plant defense system. Knowing precise molecular aspect of this invasion helps for the development of novel desease control strategies. Using yeast secretion system, a wheat leaf rust cDNA library was cloned upstream of invertase coding ORF in an expression vector and sent to the mutant yeast which is deficient for invertase production. CDNA cloneds containg signal peptid were able to secret recombinant invertase outside of the host cell and provided glucose for their growth. Using this sytem, a novel gene here we named ptGSP1, was isolated, and its expression was confirmed in the germinated and haustorial stages of the rust life sycle. Also, fast evolution of PtGSP1 coding region was deduced from comparing sequences of this gene in the geographical starins. Its secertion nature, size of the deduced protein, fast evolution of the ORF and its expression at huastorial intimate stage, all emphesis on its possible role during the pathogenesis.

Abiotic stresses such as drought, cold and salinity have negative effects on yield and growth plant, so tolerance to them has been main target of plant breeders. DREB transcription factors are member of AP2/ERF family. Analysis of promoter region of genes which responsive to drought and low temperature stresses and ABA-independent, led to the discovery of 9pb region with TACCGACAT sequence that named DER. Proteins which are encoded by DREB genes have attached to this sequence and regulate gene expression. WDREB2 or TaDREB gene expression is induced by drought, cold, abscisic acid and salinity stresses. This gene was transferred to wheat using Agrobacterium-mediated gene transformation method. Molecular analysis with PCR technique confirms that 20 plants out of 40 plants are transgenic. Also gene expression analyses with Real-Time PCR Technique have showed that the most part of transgenic plants have increased expression between 2 to 19 times, more than non-transgenic plants. This increasing of expression which demonstrate the efficiency of 35S promoter in monocot plants, can lead to the raising of expression in downstream genes and enhancement of tolerance to environmental abiotic stresses.

In this research, in order to detect polymorphism in the exon 4 and 3' untranslated region (3' UTR) loci, 90 blood samples were collected randomly from Makoei sheep. DNA was extracted from blood samples and was amplified a fragment of 273 bp in size from TNFα gene exon 4 and 3' UTR. The Single-Strand Conformation Polymorphism (SSCP) method were used for genotyping. Electrophoresis of PCR products were done on acrylamide gel that observed the polymorphic in this region. Based on SSCP patterns, three genotypes EE, OE and RE obtained with frequencies of 0.4667, 0.3556 and 0.1777, respectively. The genotypes in this locus deviated from HWE. The effect of observed genotypes on growth traits was investigated, and TNFα genotypes were found to be associated with birth weight and average daily gain from birth to weaning. Comparison of means indicated that EE genotypes had higher birth weight and average daily gain from birth to weaning (4.32 and 0.224 kg/day, respectively) than the other genotypes. No significant associations of TNFα genotypes with other traits were detected.

In foundation of appropriate breeding program, awareness of gene action for controlling quantitative traits is high important. In this study, a complete diallel cross design was used to estimate of gene effects on controlling some traits, genotype × year interaction, variance component, general and specific combining ability and heterosis in cantaloupe group. Five traits, including fruit maturity, yield, fruit weight, number of fruits and flesh thickness evaluated over two years. Analysis of variance showed the genetic difference among genotypes. General and specific combining ability, reciprocal, maternal and non-maternal effects were significant for all the traits. Fruit maturity had the highest specific heritability, while yield had the lowest specific combining ability (0.73 and 0.08 respectivley). Resultd also show that additive gene effects were the most important with respect to fruit maturity, fruit weight and flesh thickness. Dastjerdi had the highest general combining ability for fruit maturity and fruit weight, while for the number of fruits, Samsori had the highest general combining ability. Non-additive gene effects mainly controlled number of fruits and yield. In this study, the crosses including Rishbaba × Savei and Tiltorogh × Savei had the positive and significant specific combining anility for yield. Therefore, these crosses could be considered for production of high yielding cultivars. Results also indicated that for fruit number and yield traits, hybrid production in order to benefit more than additive gene effects is of higher priority. But for the genetic improvement of earliness and flesh thickness selection is more efficient. Furthermore, the selection based on progeny testing can be used to modify the weighted cantaloupe fruit.

Grain hardness has an important role for discriminating marketability and commercial classes in world trade in hexaploid wheat (Triticum aestivum L.). Hardness (Ha) locus controlled by a single locus comprised of the Puroindoline a, Puroindoline b and Grain Softness Protein-1 (Gsp-1) genes. A large deletion in the puroindoline a (Pina) gene or single nucleotide polymorphisms (SNPs) in the puroindoline b (Pinb) gene results in hard grain texture. When both puroindolines are wild-type, grain texture is soft. When either of the puroindoline alleles is absent or alter by mutation, then the result is hard texture. In this study, some new Iranian commercial cultivars were investigated for their kernel hardness and puroindoline alleles using STS-PCR and SNPs genotyping. The results indicated that approximately half of the cultivars had Pina-D1b or Pinb-D1b allele and genotype the other half of the cultivars not identified and to identify other cultivars should be used to their detector primers. In this study, Gonbad, Sirvan, Oroom, Bahar, Aflak and Moqan3 cultivars had Pina-D1b allele and zaree cultivar showed Pinb-D1b allele. Pina-D1b alleles showed the most frequency among our cultivars. This study showed that cultivars with hardness genotype have accordance with Inframatic machine (NIR) results. Correlation coefficient (r = 0.74) between hardness index (HI%) and water absorption (WA%) by using Inframatic machine (NIR) was significant (α<0.0001). Our knowledge about the genetic basis of kernel hardness could provide useful information in breeding programs of bread wheat.

Psoralen is one of the important secondary metabolites in Psoralea corylifolia. The important aspect of medicinal value of psoralen can be indicated to anticancer activity against leukemia, lung and breast cancers. In this research due to the pharmaceutical importance of psoralen, pathway of psoralen synthesis was studied. For the first time we isolate and characterized the gene coding psoralen key enzyme from the leaves of the Psoralea corylifolia by the different molecular techniques. Result of molecular analysis revealed that the length of this gene was 1237 bp. Also, the sequence analysis of nucleotides by using available bioinformatics data in NCBI found that psoralen synthase gene has 93% similarity with this gene in Ammi magus.

This study was conducted to determine polymorphism Intron 4 of Ghrelin gene in West Azerbaijan native chickens using PCR-SSCP. Blood samples were randomly taken from 100 birds. DNA was extracted from the blood samples using Aljanabi and Martinez method. 1% agarose gel was used for determining its quality. Intron 4 of Ghrelin gene encompassing 458 bp was amplified with PCR and PCR products analyezed using SSCP method. The PCR products were electrophoresed in 8% polyacryl amide gel and stained with silver nitrate. Based on SSCP analysis of PCR product with Polyacryl amide three genotypes AA, AB and BB were identified respectively. Shanon index, Nei index and observed heterozygosity were 0.58, 0.39 and 0.42, respectively. Results revealed that the Ghrelin gene had high degree of polymorphism and the relationship between polymorphism of Ghrelin gene and native chickens economic traits could be assessed in chickens breeding programs.

Genetic diversity is the basis of the plants breeding that is created from natural evolution and It is an important components of sustainable systems Assessment of genetic diversity in plants is very important for breeding programs and conservation of genetic resources. Studying diversity is a crucial step in plant breeding and it could be achieved by cytogenetic studies and karyotype analysis. In order to study the cytogenetic variation, of 19 garlic ecotypes were collected Iranian land races. After preparation of samples in most cases, five replicates of praper cells selected and the length of the long and short arms of chromosomes and total length of chromosomes were measured using Karyotype Analysis software (ver. 1.5). Other karyotypic parameters were calculated in Excel software. The results showed that the basic number of chromosomes in the Bojnourd ecotype is x =7 (2n =2x =14) and in the rest are x =8 (2n =2x =16). Data were analyzed using JMP8 software in a completely randomized unbalanced design that revealed a significant difference (P ≤ 0.01) between the short arm, long arm and the total length of chromosome. Tukey's method was performed to compare the means. cluster analysis was performed applying Ward method that divided the ecotypes in two symmetric and asymmetric groups. Minimum Euclidean distance observed between Semnan and Tabas ecotypes, the smallest chromosome belonged to Yazd ecotype and the largest chromosomes belonged to Aliabad ecotype. Also the most symmetrical karyotype was Yazd and the most asymmetrical karyotype was Khaf ecotype.

Spirlin (Alburnoides bipunctatus) is a stream species that has a good abundance in southern basin of Caspian sea, but it is supposed to be extincted in many inland waters of Europe. There hasn’t been any study about genetic diversity of this species yet. In this study, 84 fish were collected from Tilabad, Shirabad and Kaboodwal streams in Golestan Province (28 samples from Tilabad, 28 samples from Shirabad and 28 samples from Kaboodwal) to investigate their population structure. DNA was extracted by phenol-chloroform method and screened for 6 microsatellite loci. The average of expected heterozygosity and observed heterozygosity were 0.893 and 0.817, respectively. The analysis of molecular variance showed high genetic diversity (98%) within investigated populations. The Fst value was 0.024 which indicates a low genetic differentiation between the Tilabad, Shirabad and Kaboodwal populations. In examining deviations from Hardy-Weinberg equilibrium, two loci in Tilabad population and two loci in Kaboodwal population were balanced and other loci had a significant diviation from Hardy-Weinberg equilibrium (P ≤ 0.05). According to UPGMA cluster analysis based on genetic distance, populations of Tilabad and Shirabad rivers haven’t been separated, but possibly Kaboodwal river population has been separated from two other rivers’ populations.

An F5 population of 150 lines derived from a cross between sepidroud (Indica) and Gharib (Indica) cultivars were evaluated in a drought stress and normal conditions in a field to detect QTLs of drought tolerance index and its associated traits. Based on the genetic linkage map containing 131 polymorphic SSR and 52 AFLP markers which covered 14.1063 cM of the rice genome with an average distance of 5.81 cM between markers, 32 main effect QTLs (M-QTLs) and 21 epistatic QTLs (E-QTLs) were identified for grain yield and grain weight in normal and stress conditions and eight drought tolerance indices by QTL IciMapping software using inclusive composite interval mapping (ICIM) analysis. For grain yield one M-QTL and one E-QTL and two M-QTLs and two E-QTLs were detected in normal and stress conditions, respectively. For grain weight also was identified five M-QTLs and one E-QTL in normal condition and four M-QTLs in stress condition. For each mean productivity index (MP), geometric mean productivity (GMP), harmonic mean (HM) and stress tolerance index (STI) three QTLs on chromosomes 1, 7 and 11, for each stress susceptibility index (SSI), Yield stability index (YSI) and Yield index (YI) two QTLs on chromosomes 1 and 7 and for tolerance index (TOL) two QTLs on chromosomes 6 and 7 were detected. The phenotypic variation controlled by each M-QTL and E-QTL ranged from 5.93 to 21.12% and 3.98 to 18.86%, respectively. The total phenotypic variation of M-QTLs for grain yield and grain weight in normal and drought conditions and indices MP, GMP, HM, TOL, SSI, STI, YI and YSI was 7.43%, 18.6%, 49.71%, 31.09%, 16.27%, 16.01%, 16.2%, 23.84%, 19.26%, 19.11%, 18.6% and 19.26%, respectively. The main effect of genomic regions associated with grain yield under normal and drought stress conditions, and drought tolerance indices can be used in breeding programs to produce high-yielding cultivars and yield stability.

Among the several species belong to the genus Papaver, P. bracteatum with abundant thebain and P. somniferum with a variety of alkaloids have special pharmacological importance. In the current investigation, properties and symmetrical karyotypes of the two species were studied. Results revealed that both species are diploid but the basic chromosome number varied between two species. The basic chromosome number in P. bracteatum was X =7, while it was X=11 in P. somniferum. P. bracteatum and P. somniferum were placed on 3A and 3B class based on Stebbin’s asymmetry categories, respectively. The results of the current study demonstrated that P. somniferum is evolutionary advanced comparing with P. bracteatum.