مجله زیست فناوری گیاهان زراعی
پیاپی 5 (پاییز و زمستان 1392)

E. coli O157: H7 is an important zoonotic pathogen that causing severe gastrointestinal disease such as hemorrhagic diarrhea and hemolytic uremic syndrome. The first step of bacterial pathogenicity is، attaching to the host cell. EspA is a protein molecule and forms as filamentous structure that is transiently expressed on the outer membrane surface and interacts with the host cell during the early stage adhesion and forms bacterial biofilm، this filamentous structure make it a strong immunogenic. Tir is bacterial protein that translocated to host cell after expression in the bacteria by type III secretion system (T3SS) and integrated in to host cell membrane. Intimin can attach to the host cell by conducting to Tir. The purpose of this study is constructing bivalent gen which contains virulence factor mention before and transferring in to target plant in order to production edible vaccine against E. coli O157: H7. After bioinformatics investigation the two bivalent gen construction (espA-Tir،) were attached by peptide linker، and the gens were constructed. After codon optimization based on tobacco; construction were cloned in expression vector which contains CaMV35s promoter. After transformation and regeneration، the expressions of transgenes were showed in analysis.

Mentha species is a genus of family Lamiaecae، and is well known for its great medicinal and economic values. in order to evaluate the genetic variation of 15 genotypes belonging to three species M. longifolia، M. pulegium and M. Sp.، used were from 10 ISSR primer. The average percent of polymorphism in all accessions was 94. 70. The primers of IS11 & IS9 having the best primers parameters were introduced in this study. Low coefficient of similarity based on Jaccard (0. 17 - 0. 56) have showed a high genetic diversity of Studied genotypes. Cluster analysis based on Jaccard coefficient and UPGMA method classified the genotypes in tree major groups and the PCoA analysis confirmed the results of clustering. Analysis of molecular variance (AMOVA) among and within species showed more intra-specific variation (98. 00%) in comparison with inter-species variation (2. 00%). Evaluation of genetic diversity within species with an average of Nei’s gen diversity analysis and Shannon’s information index، showed that diversity within species of M. longifolia (H=0. 165، I =0. 460) was o more than other populations while genetic diversity within species of M. sp. (H=0. 102، I=0. 194) was less than other species. The results showed that the overall addition to geographic distance، gene flow between populations in the wild populations، ecological conditions is determinating genetic distance and genetic diversity. Also because hybridization between species during evolution of mint،is similarity between species than within species similarities.

Artemisia species due to have different compounds of terpenoids are considered the most important medical plants. Artemisinin، a sesquiterpene with antimalarial and anticancer properties، and tritenpenes، squalene and β-amyrin، are important medicinal compounds which are produced by Artemisia species. Since farnesyl diphosphate is the precursor of all tri- and sesquiterpenes، expression of farnesyl diphosphate synthase (FDS)، squalene synthase (SQS) and β-amyrin synthase in three developmental stages are studied in seven Artemisia species native of Iran by real-time PCR. Furthermore، artemisinin content was determined by HPLC. Our results showed A. annua has maximum artemisinin content in budding stage and A. diffusa and A. spicigeria have minimum artemisinin content in vegetative stage. In this manner expression of FDS has no difference between the species and although its effective role in biosynthesis of artemisinin، it is not useful to manipulate for increase of artemisinin. Also lower expression of SQS means we will have higher artemisinin but the revers is not true. Also A. scoparia in flowering stage is the best source to access of squalene and β-amyrin.

Trichoderma spp. is considered as promising biological control agents. Antagonistic Trichoderma species control numerous of plant pathogenic fungi. The highest value of degrading enzymes production (chitinases and glucanases) between known micro- organisms belongs to this genus. Hence that chitinases have effective role in myco-parasitism mechanism of plant pathogenic fungi، therefore superior bio-control potential may then be found in isolates which produce a high capacity of these enzymes. Application of genetic engineering and direct mutation in fungal genome in order to increase T. harzianum antagonistic potential via enhancement in chitinase activity has some advantages. In this study، we use 250 Gry gamma irradiation dose to induce mutation in T. harzianum. Then، chitinase activity of 20 mutants have been evaluated and 15 mutants showed higher enzyme activity than wild type isolate. Thus، induced mutation by gamma irradiation in order to improve antagonistic capacity of native iranian Trichoderma spp. to obtain effective and useful bio- control agents is advisable.

Drought stress is a major constraint for barley production and yield stability in rainfed ecosystems. An advanced backcross breeding strategy was used to identify quantitative trait loci (QTLs) associated with yield and yield components in a BC3 population derived from an interspecific cross between six-rowed spring barley (H. vulgare) ‘Azhul’ and wild barley (H. spontaneum) line ‘Spontaneum I’، under drought stress. The linkage map constructed by 170 SSR molecular markers covered a total length of about 1008. 7 cM. For ten agronomical characteristics، 27 QTLs were determined. The phenotypic variation explained by individual QTLs ranged from 5. 7% to 34. 8% and the LOD scores ranged between 3 and 12. 4. A total of 12 new QTLs were identified، where at ten QTLs the exotic introgression caused an improved trait performance، under drought stress. Four QTLs contributed by ‘Spontaneum I’ on chromosomes 1H، 2H، 3H and 7H were found to significantly increase chlorophyll content، days to maturity، flag leaf length and number of tillers per spike، respectively. In conclusion، the results of this study showed that Hordeum spontaneum، the wild progenitor of barley، is a potential source of useful genetic variation for barley breeding programs.

microRNAs are small non-coding RNAs approximately 21-22 nucleotides long and have been identified as negative regulators of gene expression in the post-transcriptional level in plants and animals. Temperature is one of the most important climate change factors and higher temperatures affect agriculture and crop production adversely. In this study، the expression pattern of miR398 and its target gene NtGT5b were measured in root and leaf tissues of sunflower (Helianthus annuus) in various heat stress conditions (0h، 1. 5h، 3h، 6h) at 42 °C by qRT-PCR. The results showed different pattern of miRNA expression in both root and leaf tissues during mild، moderate and severe heat stress، respectively. miR398 was highly up regulated and its target gene down regulated in leaf tissue that showed thermo tolerance of this tissue in term of heat stress. There is a different miRNA expression patterns in leaf and root tissues under various stress conditions which is an indication of induced signal transmission after heat stress. Investigating miR398 network shows that this miRNA through presences of important genes and transcription factors such as AFO and INO has the ability to change the pattern of gene expression of many processes such as growth، development and response to stress.

Black zira is a perennial herb with highly valuable impact in the pharmaceutical industries. To overcome the existing limitations of the field، achieving efficient regeneration methods provides the possibility of genetic manipulation and improvement of traits in this plant. Our objective in this research work is to develop an efficient regeneration protocol of bleack zira being used in genetic improvement programs. After optimizing the conditions for sterilization and seed germination، sterile cotyledonary seedlings were obtained on MS basal medium. We investigated efficient callus induction، rooting and shoot regeneration methods on Murashige and Skoog’s medium containing different concentrations of growth regulators including BAP، NAA and IAA in a factorial experiment based on completely randomized design with 3 replications. A significant increase in callus، rooting and regeneration from rootlet explants was observed. We noted that BAP and NAA were more capable for callus initiation and long-persistent development. It is shown that the most frequency and dry weight of callus was obtained on medium MS supplemented with 0. 5 or 1mg/l BAP and NAA. Often frequency of rooting was influenced by NAA growth regulator. Also BAP and IAA in single stage regeneration in this study were found to be effective so that the addition of IAA to the culture medium caused reduction in callus development and showed direct shoot regeneration from cotyledon node، hypocotyl and rootlet explants.

Charcoal Rot is one of the most important fungal diseases in soybean particularly in northern region of Iran. This disease caused some damages to yield quality and quantity (up to 20%) especially in dried and worm seasons. Today there is not many researches in regards to enzymatic and non-enzymatic study in response to disease. In this study super oxide and hydrogen peroxide radicals have measured، also، super oxide dismutase، and catalase enzymes، as well as ascorbic acid changes، alphatochopheroll، carotenoid and TBARM (cellular oxidative levels) in soybean genotypes during growth stages (flowering، packing pod and grain filling) at two environmental conditions (control and disease). The result showed superoxide and hydrogen peroxide radicals content have increasing during growth stages، as the most amount accorded in final stage of sampling (grain filling). Katul cultivar and DPX × For a hybrid had more tolerance to charcoal rot disease due to antioxidant defense systems.

Hulled wheats are among the earliest domesticated Triticeae. They possess many important agronomic traits such as tolerance to biotic and abiotic stresses، higher grain protein quality and quantity، and higher micronutrients concentration. These characteristics has made hulled wheat a useful germplasm resource and highly valuable in wheat breeding programs. Hulled wheats are found in different regions of Iran but their genetic potential is not exploited owing to lack of genomic information. In this research، two hulled wheat accessions (Zarne and Jonghan) collected from farmers field in two distantly located villages in the Chehar-Mahal Bakhtiari province with other 28 diploid، tetraploid and hexaploid wheat genotypes were studied using seventeen Simple Sequence Repeats (SSR) primers. The SSR markers used were related to the A and B genomes of wheat and generated between 2 to 11 alleles per locus with the average polymorphism information content (PIC) of 0. 53 per locus. The high value of PIC showed that a high level of polymorphism was present for accessions classification. The dendrogram derived from SSR data clustered two accessions of hulled wheat (Zarne and Jonghan) with two T. dicoccoides (wild emmer) in a single group، while T. dicoccum (domesticated emmer) appeared in a distantly related class. The three T. durum (free-threshing) were placed in a separate cluster، distant from T. dicoccum. These results showed that Zarne and Jonghan genotypes have closer affinity to T. dicoccoides than T. dicoccum genotypes.

Bread wheat (Triticumaestivum) is one of the most important world food crops that are exposed to many pathogen. During the previous expression-profiling experiments، in addition to major resistant genes to disease in wheat، some defense-related genes such as S-Like RNase gene have been identified. Here to study expression pattern of this gene in several fungal wheat diseases، some bioinformatics and laboratory studied were performed. In bioinformatics studies، several microarray libraries infected with Fusarium، Spike blight and Stripe rust were considered. In laboratory experiments Septoria tritici bloth was studied. So the level of expression was measured at 8 time interval، from 0h to 6 days after inoculation by Mycosphaerella graminicola in Wangshuibai as a resistant wheat cultivar and Falat as a susceptible wheat cultivar by semi quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The results show that the maximum expression of this gene، depending on types of disease and resistant cultivars، is obtained up to 24 hours after the inoculation. Thus، according to this results it can be concluded that this gene plays an important role in resistance to diseases and، along with the main gene، increase and maintain resistance to many fungal diseases in wheat. Also this gene was confirmed by Nucleotide Blast.

Drought stress is one of the most important abiotic stresses that deteriorates rice agriculture of Iran. One of the best ways to establish drought stress tolerance in plants is miRNA mediated post transcriptional gene regulation. MiRNAs are small 19-24 nt regulatory RNAs and play important role in regulating plant gene expression in biotic and abiotic stress. In this study، we selected five miRNAs for promoter analysis and evaluation of differential expression of them under drought stress in roots. Three of them including miR162، miR169 and miR172 are conserved in many plants and the others including miR1425 and miR1880 are rice specific miRNAs. In addition، upstream screening of MIRNA genes showed that upstream region of some MIRNA genes like MIR172 are enriched with important regulatory elements like DRE and ABRE. Quantitative Realtime-PCR was used in this study for analyzing differential expression of evaluated miRNAs. Studying the differential expression of miRNAs in roots under drought condition showed that miR169 was up-regulated but conversely، miR172 was down-regulated. The rest of miRNAs in our study did not show significant differential expression under drought stress. It can be concluded that NF-YA and AP2 as the most important target genes for miR169 and miR172 respectively can play critical roles in response to drought stress.

In order to study of genetic diversity of barley genotypes، 14 pair’s primers of SSR were used in 20 genotypes. After genomic DNA extraction and PCR reaction، Primers produce 266 polymorph bands and mean of polymorph band per primer was 19. The highest value of polymorphic information content (PIC) belonged to Hvm60 and Hvm20 primers (0. 88 and 0. 73، respectively) and the lowest value of PIC was belonged to Bmac0032 primer (0. 32) and mean of PIC for all primers were 0. 6. Jacard similarity coefficient was calculated and ranged from 0. 3 to 0. 96 among genotypes. The highest genetic similarity (0. 96) belonged to genotypes no. 6 and 5 and the lowest (0. 3) was belonged to genotypes no. 13 and 10. Clustering dendrogram based on UPGMA method classified genotypes to 5 main groups. Results of PCOA classification were similar to results of cluster analysis. Evaluation of genotypes by SSR molecular marker could discriminate two row and six row genotypes and also hulled and hulless genotypes. In other hand the similar parents in pedigree of some genotypes (e. g. between 3 and 12 and between 14 and 18) had an important effect on this classification. Results of this study revealed that this molecular marker has a valuable potential for evaluation and discrimination of barley genotypes.

MicroRNAs (miRNAs) play important roles in numerous processes in plants including development، tissue proliferation، differentiation، hormone signaling and responses to biotic and abiotic stresses. SPL، the plant-specific transcription factors، are regulated by miRNAs and play important roles in several processes including tissue development، response to biotic and abiotic stress and induction of several other transcription factors and membrane proteins. In this study we selected miRNAs that regulate SPL transcription factors expression in rice. Later، the differential expression of these miRNAs are evaluated using qRT-PCR and Stem-loop primers. Results of shoot differential expression under drought stress showed that miR529 was down-regulated but conversely، miR535 was up-regulated. However، significant differential expression of miR156 was not observed in our study. Likewise، root differential expression under drought condition showed up-regulation of miR535، but miR529 and miR156 did not show any significant differential expression. Although all of these miRNAs are involved in regulating the expression of the same genes، but their diverse differential expressions highlight the complexity of gene-regulatory networks in various environmental conditions. Based on results of this study، it can be suggested that compared to miR535، miR156 and miR529 play more important roles in regulating the development and flowering process via controlling SPL transcription factors whereas، miR535، miR529 and relatively lesser miR156 are responsible for SPL transcription factor regulation under stress.

Peroxidase and Catalyse are two important enzymes involved in reaction of many crop species to salt tolerance. Assessment of activity of these enzymes in the seedlings of barley during salt stress and using quantitative data for mapping of QTLs involved in salt tolerance has been addressed in this study. Parents of four barley mapping populations were exposed to the different concentrations (0 mM، 100mM and 200 mM) of salt (NaCl) and the level of activity of Catalase and Proxidase were quantified in the seedlings stage. There was a high variation in enzymatic activities of parents of OWB population (Dom and Rec) in 200mM of NaCl. In order to map QTLs involved in salt tolerance in OWB، 94 doubled haploid lines were exposed to 0 mM and 200mM of NaCl and the activity of Catalase and Peroxidase were quantified. Mapping of QTLs was performed using MAPQTL5 software. The results showed that the position of QTLs depends on both concentration of salt and on the type of enzyme. For Peroxidase activity there was no QTL mapped despite a high variation between individuals of mapping population while for Catalase in the concentration of 200 mM of NaCl، two QTLS in Chromomes 3 (3H) and 4 (4H) were mapped. Two other minor QTLs with LOD values slightly lower than the threshold were mapped in chromosomes 5 (1H) and 7 (5H). The results showed that quantification of the level of activity of Catalase can be used as quantitative data for mapping of QTLs involved in salt tolerance in barley

The chromosomal arm 1RS of rye (Secale cereale L.) is a valuable resource for improving the properties of wheat (Triticum aestivum L.) which has been translocated with the short arm of a wheat group-1 chromosome (1AL. 1RS، 1BL. 1RS، 1DL. 1RS). This arm carries rust resistance genes; Lr26 for leaf rust (Puccinia triticina)، Sr31 stem rust (P. graminis) and Yr9 for stripe rust (P. striiformis). These genes were located 6. 6 cM from the Sec-1. This close linkage has been used as a marker for identification of rust resistance genes. In this study، the presence of Sec-1 was examined in 66 Iranian cultivars and 70 abroad regional accessions، using rye-specific primer “O-SEC”. The rust resistance gene by the presence of Sec-1 was verified in 15 (23%) Iranian cultivars and 2 (3%) abroad accessions. Moreover، because of the rye-specific primer can distinguish between 1AL. 1RS and 1BL. 1RS translocations، the presence of these genes were identified in 14 (21%) cultivars and 2 (3%) abroad accessions in chromosome 1B. Also among all cultivars and accessions، the presence of the resistance genes was verified in chromosome 1A of «Sholeh» cultivar (1. 5%). On the whole، the presence of rust resistance genes in Iranian wheat cultivars appeared to be better than abroad wheat accessions. This is because many Iranian wheat materials are commercial cultivars and in their pedigree have cultivars carrying rust resistance genes. The results of this study can be used for the production of new wheat cultivars in breeding programs.