نشریه مهندسی ژنتیک و ایمنی زیستی
سال ششم شماره 1 (پیاپی 11، بهار و تابستان 1396)

After nitrogen, phosphorus (P) is the most essential element in plants. With recent advances in molecular biology, an opportunity for manipulation of plants to increase P uptake is provided. Construction of multipurpose vectors to enhance the efficiency of phosphorus uptake conferring improved plant yield as well as conferring herbicide tolerance will be valuable in plant genetic engineering of economically important crops. In this study the PSTOL1 gene from the wild rice Kasalas variety was isolated and cloned next to the glyphosate herbicide tolerance gene, each under independent promoters and terminators. Uptake efficiency of P and PSTOL1 function were confirmed by measuring phosphorus uptake in the bacteria culture medium. It was expected that the PSTOL1 gene would improve the plant's root structure and be effective in developing drought-tolerant plants. As PSTOL1 was cloned behind the CaMV 35S promoter and this promoter is recognizable by bacterial transcription factors, analysis of gene function could be performed by measurement of P uptake from the bacterial culture medium. The results showed that, compared to the control, bacteria containing PSTOL1 absorbed two times more phosphorus from the culture medium. Thus, the function and expression of this gene was confirmed. It is expected that Agrobacterium containing the recombinant plasmid (pUEs-PSTOL1) constructed in this study can be used in the genetic engineering of different crops and lead to increase yield, improved root structure, and drought tolerance by increasing efficiency of phosphorus uptake as well as providing resistance to glyphosate herbicides. The resulting recombinant plasmid is without a selectable marker for antibiotic resistance which would be a biosafety advantage.

Sclerotinia stem rot disease caused by Sclerotinia sclerotiorum is one of the most important diseases of sunflower. The use of resistant genotypes is potentially an economically useful method for its control. In this study, the expression level of the genes encoding phenylalanine ammonia-lyase 2 (PAL2) and thaumatin-like protein (TLP) were measured in the LC106-C and RHA265 genotypes of sunflower inoculated with SSU53 isolate of S. sclerotiorum by real time PCR technique. The results revealed that transcript levels of both the PAL2 and TLP genes were significantly affected by genotype, time after inoculation and genotype-time of inoculation interactions. In particular there were significant differences between the expression of PAL in two genotypes 6 and 48 hours after infection. Concerning the expression of the TLP gene, the maximum difference between two genotypes was observed 48 hours after infection. The high expression level of studied genes in a resistant line compared to a susceptible one confirms the involvement of these genes in partial resistance of sunflower to Sclerotinia stem rot disease. It also confirms the resistance of LC106-C to the disease at the molecular level. The findings of this study can be useful in sunflower breeding programs for producing cultivars resistant to Sclerotinia stem rot disease.

Milk production traits and its components are quantitative and polygenic traits affected by many genes. This study was conducted to determine the polymorphism in alpha-lactalbumin and β-lactoglobulin genes using PCR-SSCP method in East Azarbaijan native buffalo. Milk samples were collected from 150 buffaloes of East Azarbaijan native province and then DNA samples were extracted using the pronase method. After DNA extraction specific primers were used for amplification of a 392 bp fragment of exon 1 and intron 2 of the alpha-lactalbumin gene and a 452 bp fragment of exon 2 of the β-lactoglobulin gene. To identify polymorphisms, the PCR products were electrophoresed on polyacrylamide gel (SSCP method) and stained with silver nitrate. Three different banding patterns were observed in samples 1, 2 and 3 for the alpha-lactalbumin gene (with 87.5%, 10.71% and 1.78% frequency) and five different band patterns were observed for the for the β-lactoglobulin gene, with 9.80%, 58.82%, 19.60, 9.80% and 1.96% frequency. The results indicate that the different patterns may be due to polymorphism in these fragments.

Licorice (Glycyrrhiza glabra L.) is one of the most important medicinal plants and contains bioactive compounds such as triterpene saponins (glycyrrhizin) and phytosterols. Squalene synthase (EC 2.5.1.21) is a membrane- bound enzyme that converts two farnesyl diphosphate molecules into squalene, a key precursor for sterol and triterpene biosynthesis. In this study, the coding sequence of squalene synthase 1 in Iranian native licorice was cloned in pTZ57R/T and the characterization of its polypeptide was predicted by using bioinformatic tools. The cDNA of GgSQS1 is 1242bp and encodes a 413 amino acid polypeptide. Bioinformatic analysis revealed that the deduced GgSQS1 polypeptide had high similarity with squalene synthase1 of members of the glycyrrhiza genus. Subcellular analysis showed that the activity of this polypeptide is in the endoplasmic reticulum. The molecular weight of this polypeptide is 47.3 kDa with a pI value of 8.18. Two transmembrane domains and two protected regions were detected in the amino acid sequence. The three-dimensional protein structure was predicted using I-TASSER software. In the predicted structure, transmembrane helixes, hydrophobic amino acids, the conserved sequence and amino acids on the protein surface were identified using Chimera software. Studies of Squalene synthases1 gene expression showed that the highest and lowest expression occur in the root and green organs of the plant, respectively.

Growth, development and productivity of plants are greatly affected by various abiotic stresses. Physiological and molecular studies have shown that naturally occurring plant polyamines (PAs) are involved in conferring abiotic stress tolerance in plants. Polyamine oxidases (PAOs) are FAD-dependent enzymes associated with polyamine catabolism in peroxisomes, the apoplast and the cytoplasm. In plants, increasing evidence supports the idea that PAO genes play essential roles in abiotic and biotic stresses responses. In this study, bioinformatic analysis identified eight putative PAO genes (VvPAO1–VvPAO8) in grape (Vitis vinifera) using the released 12×assemble grape genomic sequences. Phylogenetic analysis indicates that these VvPAOs can be classified into three subgroups as found in Arabidopsis and rice and also reveal that grape PAO proteins are more closely related to Arabidopsis than to those in rice. The VvPAO genes contained zero to nine introns and were distributed across 6 out of the 19 chromosomes in grape. Promoter analysis showed the presence of several cis-regulatory elements related to stress and hormone responses in regulatory regions, indicating their probable role in stress response. Microarray-based expression analysis of VvPAO orthologs in Arabidopsis under abiotic stresses showed that transcript levels of PAO genes were up-regulated significantly by such treatments, indicating their vital roles during stress adaptation. The results obtained provide basic information for future research on the functions of PAO genes in grape.

Excess salinity is one of the most important problems of agricultural production. Rapeseed is a superior oilseed due to the high quality of oil, large amounts of polyunsaturated fatty acids and oil yield. Rapeseed is classified as a semi-salt-tolerant plant although yields decrease more than in many other crops when salinity is higher than the threshold. Therefore breeding for increasing salt tolerance would be of interest. Changes of protein expression were investigated in a Safi7 tolerant genotype in order to identify the molecular mechanisms of salinity tolerance in rapeseed. NaCl concentrations of 0 (control), 150 and 300 mM caused a significant increase of Na content in leaves and a decrease of shoot dry weight, shoot height, leaf K content and the leaf Kﳖ ratio. 110 repeatably-appearing protein spots were identified on two-dimensional electrophoresis (2-DE) gels and among them, 37 spots showed significant expression changes based on changes in the induction factor (IF). Among them, 5 spots showed significant statistically changes at the 95% confidence level, with 1 spot being up-regulated and the other four spots down-regulated. Identification of the spots was performed by LC-MS/MS mass spectrometry analysis. The identified proteins play a key role in energy production and photosynthesis. Our results indicated that these proteins can also play a role in rapeseeds tolerance to salt stresses.

The use of an appropriate reference gene for assessment of DNA and genome quality and analysis of transgenic crops is very important. In this research, we have evaluated the SadI gene as a potential reference gene in cotton. First, DNA was extracted, and then a PCR reaction was done using appropriative primers designed for Vector NTI. The PCR products were 623 and 544 bp fragments. The fragments were cloned in pTZ57R/T plasmid. Recombinant plasmids were identified by PCR colony hybridization with appropriative primers and M13 primers and then were sequenced. The sequencing results showed that these two fragments are related to two different copies of the SadI gene that in one of copies a 79 bp deletion had occurred in the second interon. This research is the first report from sequencing of a reference gene showing the presence of two different copies of the SadI gene, and affirming that it is a suitable reference gene for different studies.

Heavy metal over-dosage is a predominant concern in soil pollution worldwide due to high stability of these elements as well as their their health side-effects on many organisms including humans. Experiments were conducted to study the effects of lead on growth characteristics (germination rate, plant fresh and dry weight, leaf number, leaf length and width) and some physiological traits (leaf and root lead concentration, relative water content and malondialdehyde, hydrogen peroxide, protein, bio-concentration factor and chlorophyll content) of Lepidium sativum as CRD in three replications. Different lead concentrations (0, 1, 2, 3, 4 and 5 mgL-1) were included in MS medium upon which seeds were cultured. The results revealed that lead concentrations from 3-5 mgL-1, led to significant increases in malondialdehyde and hydrogen peroxide contents. The highest lead concentration was recorded at 5 mgL-1of lead in leaves and roots. The highest amount of Bio-Concentration Factor was recorded at 1 and 3 mgL-1 Pb. The greatest amount of chlorophull a, leaf number and protein content was found in control plants and plants subjected to 1 mgL-1 lead treatment. For chlorophyll b, the lowest content was recorded in 5 mgL-1 lead. Pb concentration up to 2 mgL-1 had no significant effects on germination rate and the length and width of leaves, but any Pb increment from 3 mgL-1 upward, significantly affected the above-mentioned traits. Our studies make it evident that growing cress in Pb-polluted soil should be avoided if the plant is to be used as food. However, if the idea is to take advantage of the hyper-accumulation capacity of a plant in a soil decontamination program, cress would be an excellent candidate.

Drought stress is one of the most important factors reducing wheat quality and quantity. In the past years research on stress, especially drought stress, has focused on plant resistance genes and their mechanism of action. This research has revealed that a majority of the genes that have important roles in biotic and abiotic stress resistence encode transcription factors. In this survey, the level of expression of the TaMYB73 gene, a gene whose product is involved in transcription in several wheat cultivars (cultivars Chamran2, Afghani calk, Sistan, Arg and Ofogh) was studied under five draught conditions (5, 10, 15, 20 and 25 drought percent) by real-time PCR. Catalase and glycol peroxidase enzyme levels were also measured in drought conditions. The results show that TaMYB73 gene expression levels and catalase and glycol peroxidase enzyme activity in the Arg cultivar was higher than in the other cultivars. Arg may thus be considered as resistant cultivar.

Salinity is one of the important abiotic stresses which affecting growth and performance of plants. A Plant’s response to salinity depends on the genotype, salt intensity and duration of the stress. Plant sodium transporter activity is the most important salt tolerance mechanism in plants. In the present study, the expression pattern of the Na transporter gene was investigated in roots of three barley genotypes (Sahara3771, an Iranian advanced line (A line) as a salt-tolerant cultivar and Clipper as salt-susceptible) by quantitative real-time-PCR. The plants were exposed to 0, 100 and 200 mM NaCl at the seedling stage and root samples were harvested 24 hours, 3 days and 3 weeks after salt treatment. Also, root length, wet and dry weight were measured. The results indicated that root fresh and dry weight decreased with increasing salt concentration and duration of treatment. Analysis of variance revealed significant differences of Na transporter gene expression among the genotypes with respect to salinity levels and sampling stages. Salinity x genotype, salinity x sampling stage, genotypes x sampling stage and salinity x genotype x sampling stage were all significant. Increased expression of the gene was associated with salt tolerance in the genotypes. In response to 100 and 200 mM NaCl, the mRNA level of Na transporter gene decreased in Clipper. In Sahara3771 and the A-Linen gene expression decreased and increased respectively in response to 100 and 200 mM NaCl. Under 200 mM NaCl treatment, gene expression in A-Line increased more strongly than in Sahara3771. The results suggest that in these genotypes, salinity tolerance is related to greater ability to sequester Na into sub-cellular compartments.

Considering the importance of the herb borage and its growing use in traditional medicine and the pharmaceutical industry, the waste resulting from the indiscriminate exploitation of wild plants, the need to cultivate this plant is widely seen as commercially necessary. To overcome the negative effects of salinity, the use of calcium supplements to improve the growth environment is an essential requirement. In order to investigate this issue we preformed a factorial experiment in a completely randomized design with three replications. Factors included salinity levels (0, 3, 6, 9, 12ds / m-1) and calcium at two levels 0 and 5 mM, respectively. The seeds were grown in Gldanbh for hydroponics. One week after planting treatments were applied. One month after treatment the leaves were sampled. The results showed that with increasing salinity levels, the element sodium and the amount of biomass and the guidance and catalase activity decreased. The high salinity levels and the presence of high levels of calcium chloride total chlorophyll and Falyt peroxidase and polyphenol oxidase were increased but there was decreased proline. Results of Khasl 2D electrophoresis showed that the expression of some proteins in high levels of salinity and chloride increased calcium levels and also different proteins expressed this protein was likely to create resistance and important loss of salt.

basal stem rot is an important fungal disease of sunflower. It reduces growth and crop yield worldwide. The aim of this study is identification of resistant lines and confirmation of the accuracy of QTL analysis done for resistance to this disease in previous research in order for their use in sunflower breeding programs. In this study the expression of transcription factors (AP2 Domain, HD-Zip and MYB Family) responsible for resistance to disease was studied in susceptible (RHA265) and resistant (LC106-C) sunflower lines challenged with Sclerotinia fungal isolate (SSU53) using Real Time PCR. The results showed that the expression of the studied transcription factors is different in resistant and susceptible sunflower lines. In particular, expression of the AP2 domain and MYB Family increased significantly in resistant line LC106-C as compared to a susceptible one (RHA265). The results suggest a positive role of AP2 domain and MYB Family in resistance mechanisms of sunflower plant in response to Sclerotinia fungal disease. According to these results, the line LC106-C can be used as a resistant line for resistance to Sclerotinia disease. In addition, the highest expression level of genes in the resistant line compared to susceptible one confirmed the phenotypic data used in QTL analysis in our previous research. The findings of this study should be useful in sunflower breeding programs for producing cultivars resistant to disease.

Phospholipase D (PLD) and its product phosphatidic acid play important roles in cellular processes in plants including growth and developmentand stress responses. PLD genes constitute an important gene family in higher plants. The PLD family has been identified in Arabidopsis, rice, cotten and grape. In this study, 16 PLD genes were identified in the Medicago truncatula L. on the basis of protein domains, evolutionary relationship, gene architecture and sequence identity. They were grouped into six clads; α (4 genes), β (2 genes), γ (2 genes), δ (3 genes), ε (2 genes), ζ (2 gene) and φ (1 gene). The gene expression pattern of MtPLD4, MtPLD9 in the α subgroup and MtPLD13, MtPLD15 in the β/γ subgroup were investigated in four-week-old seedlings of Medicago truncatula under salt stress (23 ds/m NaCl) using qRT-PCR. MtPLD4 gene expression compared to that in control plants was increased 48 hr post-treatment. MtPLD13 gene expression decreased 48 hr post-treatment. Expression of the MtPLD9 and MtPLD15 genes increased in the early hours and then decreased compared to control plants. The results showed that the α subgroup MtPLD genes in particular are valuable candidates for further functional genomics analysis.

Recombinant or genetically modified microorganisms (GMMs) have high potential to provide valuable services to the agriculture sector. For example, it is possible to produce GM microbial biopesticides and biofertilizers with higher efficiencies, high quality animal feed supplements (enzyme sources and probiotics), functional and enriched foods, and also bioremediation of polluted agricultural environments. Regarding the high potential and proven advantages of these kinds of microorganisms, reduction of the concerns about their risks will open the way for their commercialization in the near future. During risk assessment of GMMs, some characteristics, including the level of durability, gene flow and possible horizontal gene flow to other microbial flora in the environment, possible effects on non-target organisms - especially useful microorganisms in the rhizosphere – and, finally, human health and food safety concerns, must be evaluated. Although, numerous studies have been dedicated to the environmental risk assessment of GM plants, the knowledge about gained is unfortunately still insufficient, causing their large scale production and commercialization to be very slow. This paper reviews possible benefits of GMMs and also their possible environmental effects related to divers agricultural environments with emphasis on GM PGPRs (biofertilizers) and GM microbial agents used as biopesticides for control of plant pests and diseases. Finally, it will be argued that the benefits and environmental effects of GMMs should be compared with the negative effects of chemical fertilizers and pesticides which are widely used in agriculture sector, and also with the potential effects of wild type microorganisms.

Potato, Solanum tuberosom, an annual plant of the Solanaceae family is one of the important crop plants and food. This review is based on genetic engineering researches which has been conducted on this plant untill now. A variety the of transgenic potato plants which have been created to improve its agronomic characteristics, adaptation to the environment, resistance to abiotic stresses, resistance to fungal, bacterial and viral pathogens, resistance to pests, production of recombinant proteins and edible vaccines are described.