مجله بیوتکنولوژی کشاورزی
سال هفتم شماره 1 (پیاپی 17، بهار 1394)

Some important strategies to increase recombinant protein yield in plants include expression of protein in suitable tissue, improvement of protein stability and accumulation by targeting into subcellular organs using specific targeting signals. In this perspective, seed-based platforms are attractive because they allow recombinant proteins to stably accumulate at a relatively high concentration in a compact biomass, which is beneficial for extraction and downstream processing. On the other hand, the ER contain chaperons and isomerase for folding of nascent proteins, which can promote correct protein folding leading to recombinant protein stability and accumulation. In this research for human Gamma interferon expression in tobacco seeds and targeting into ER, we used seed specific promoter (Napin), Kozak sequence at 5' end and KDEL sequence in 3' end of the IFNγ gene. The fragment was cloned into pGEM®-T Easy vector and after confirmation by PCR, restriction enzyme analysis and sequencing, was subcloned into a plant binary vector (pBI121). This construct cassette was transferred to A.tumefaciens LBA4404 and then used to transform tobacco explants. The transformed plants were screened on antibiotic-contained media. The presence of the transgenes was confirmed in the transformants by PCR and expected 432bp (IFNγ) and 795bp (nptII) sequences were amplified. Digestion result indicated that the mentioned construct completely and correctly transferred into tobacco genome. Transcription of IFNγ gene was confirmed by RT-PCR. Analysis of transgenic plants by SDS-PAGE represented that IFNγ protein is being expressed in seeds. Our results indicate that plant seeds have potential for production of recombinant proteins as ‘natural bioreactors’.

Detoxification of deoxynivalenol (DON) is one of the molecular mechanisms of resistance to Fusarium Head Blight desease in wheat, which causes by Fusarium graminearum. Mycotoxin producing Fusarium spices have some enzymes to reduce toxic effects. They can change trichothecenes to less toxic compounds by using trichothecene 3-O-acetyletransferase (Tri101) enzyme. This enzyme converts trichothecene to less toxic compounds by replaceing OH group on C3 with acetyl group. It had shown that a yeast acetyltransferase, AYT1, can convert DON to 3A DON and reduce it toxicity. In this study synthetic AYT1 gene was used for tobacco transformation and transiently expressed in wheat plants. The results showed that the synthetic AYT1 expresses in tobacco as well as wheat. Acetyltransferase activity analyses by thin layer chromatography confirmed that this enzyme can convert DON to 3-A DON that leads to reduce poisonous effect of mycotoxin.

Oilseed crops such as canola have important role to produce oil and energy needed for human. Information about genetic variability based on different markers, particularly molecular markers has played a key role in designing breeding programs. To study the genetic diversity in 24 genotypes of rapeseed, the 10 microsatellite primers were used according to previous studies. The results showed that the average polymorphic information content for assessing primers was 0.55 and the mean observed and expected heterozygosity for all primers, were 0.35 and 0.41 respectively. NA12-E09 locus had the highest rate (1.0893) of Shannon diversity index, which represents diversity among the population, whereas loci RA2-A11 and OL10-G06 had the lowest Shannon index. Cluster analysis based on molecular data using Jaccard's similarity coefficient and UPGMA method, grouped rapeseed varieties into the third major group. Accordingly, CR3133 variety of Canada origin which is located in a separate group showed lower similarity compared with other varieties. In general, rapeseed genotypes showed intra-species diversity based on microsatellite markers could be used in plant breeding programs.

After about one hundred year after its identification, Foot and Mouth Disease (FMD) is the first viral disease in animals that impose a major risk to the world's livestock industry. The main method to identify animals vaccinated from animals infected with FMD is using of non-structural 3ABC protein as antigen in the ELISA kit. Hence, 3ABC gene of FMDV serotype O was isolated with PCR using specific primers containing BamHI and HinDIII restriction sites. The isolated fragment was then inserted in pTZ57R/T vector for cloning and nucleotide characterization. In order to produce recombinant antigens, 3ABC was inserted in pET21a (+) vector and then transform to E. coli BL21 (DE3). Recombinant protein production was induced with 1.5 mM IPTG. Production of recombinant proteins confirmed with SDS electrophoresis and Western blotting. The molecular weight of recombinant protein was determined approximately 50 kDa. The results showed that the produced protein can be used as an antigen in the ELISA kit for animals.

Iran is known as one of the most important pear genetic resources because of being near to the pear diversity centers and having more than 10 species of genus Pyrus. On the other hand, Iran with acreage of 17,000 hectares, is one of the pear production centers. Despite its unique geographical location, there is no sufficient genetic information among pear germplasm in Iran and it is not clear what genetic relationship is between Iranian local genotypes and imported genotypes. In this study, Inter Simple Sequence Repeat (ISSR) markers was used for clustering of 10 Iranian Pear genotypes and positioning of their genetic variation among 13 imported genotypes. Fifteen ISSR primers were screened that yielded a total of 257 amplified products, of which 213 bands (81.6%) were polymorphic. Dendrogram of pear cultivars with ISSR banding patterns was drawn with NTSYS V. Pc-2.02e software using Dice coefficient and UPGMA (Unweighted Pair-Group Method Arithmetic Average), method. The 23 genotypes of pear were clustered in to three groups. The first group includes Conference, Mohammad Ali, Sardroudi, Bartlett, Red Bartlett, Harrow Sweet, Pass Crassane, Doyenneducomice, Anjou, Palestine,Lies Bonne, Duchesse and Daregazi. Kolikhaj, Sebri, Khuj labtorsh, Dom kaj, Khuj Katesar, Khuj Mordab Anzali and Shah Miveh belong to the second group. The third group includes SK10 and SK13. Cultivars were classified according to the geographical origin and the figures belonging to P. communis and P. pyrifolia, were completely separated. Iranian commercial cultivars including: Dom kaj, Shah miveh, Sebri with different Khuj accessions were placed in a group, that seems that thay have originated from different Khuj. While the three cultivars: Mohammad Ali, Sard roudi and Daregazi revealed closer to the European figures and possibility they have different origin than different Khuj And have a common ancestor with European figures. The results of this study indicate that ISSR markers have higher level of discrimination in evaluating genetic diversity and fingerprinting of pear genotypes.

Thermophilic microorganisms are the main sources of valuable thermostable hydrolytic enzymes hence they have very importance in different industries. In comparison with other amylolytic enzymes, application of alpha amylase in starch processing industries, fermentation and carbohydrate products is a great importance and they could also be substituted with chemical hydrolysis of starch in industries. Determination of optimal conditions for thermostable α-amylase production by native strain Bacillus licheniformis was the aim of this study. Bacillus licheniformis strain has been isolated from Qinarje Hotspring (Ardebil Prov.) recently which have been identified and registered as the name of Bacillus licheniformis-AZ2 in bacterial collection of agricultural biotechnology department of Bu-Ali Sina University. In this article, possibility of the amylolytic activity of this microorganism has been evaluated using Gram’s iodine staining method. Results showed that maximum growth rate and also amylase production in basal medium conditions which was inoculated with 1% (V/V) inoculums achieved at 40˚C and pH=9, 84 hours after inoculation. Amylase assay also showed that the optimum enzyme activity was achieved at 80˚C and pH=7, so this enzyme has been considered to be thermostable. Our results showed that Bacillus licheniformis-AZ2 strain produced thermostable α-amylase with characteristics suitable for application in starch processing and other food industries.

The objective of the present study was to design and optimize an economic fermentation process for mass production of a previously isolated native lepidopteran active Bacillus thuringiensis strain (YD5). Firstly, optimum pH and temperature of the strain were studied at Erlenmeyer flask level (incubator). The results showed that the optimum pH and temperature were 7 and 30° C, respectively. To optimize economic medium culture for mass production of the strain, different kinds of available agricultural and food industry wastes were used. This included molasses (sucrose), milk permeate (lactose) and starch liquor (polysaccharide) as carbon source, corn steep liquor as nitrogen source, and sea minerals as mineral sources. The experiments at Erlenmeyer flask level showed that the maximum growth and spore/crystal production by the strain was observed when medium containing 3% hydrolyzed starch, 3% corn steep liquor and 0.003 % sea minerals were used. In the next experiments the optimized economic medium and growth conditions of the strain were evaluated in a batch bioreactor. Also, the effects of primary inoculation concentration, aeration and pH on growth rate, and spore/crystal production were evaluated. The results showed that the maximum growth and spore/crystal production was achieved when 2% preculture concentration and 90% oxygen saturation were used. As result of the designed bioprocess in the present study, the spore/crystal and δ-toxin production were achieved to 5.5 × 109 CFU/ml and 740 mg/l, respectively.

Black root rot of chickpea caused Fusarium solani f. sp pisi is one of the most important fungual diseases in Iran, specially in Razavi and Northern Khorasan provinces. Because of scientific lack about genetic diversity populations of this fungi, the aim of study was determination of genetic diversity and pathogenesity of isolates using RAPD and AFLP markers. Sampling was carried out from 50 fields of major growing-chickpea in Razavi and Northern Khorasan provinces. Pathogenecity test conducted with chickpea seedling using root dip methods and locating infected wheat seeds around tap roots confirmed 28 isolates as Fusarium solani. Host range study of F. solani isolates with inoculation eight plant species including (Lentis, Bean, Soybean, Pea, Chickpea, Tomato, Melon and Watermelon) showed that all the pathogenic isolates caused root rot only in chickpea and pea. In RAPD- PCR, from 12 primers, only 7 primer showed polymorphism well. Based on this marker, 28 isolates Fsp placed in different genotype groups, without considering geographical regions. In AFLP technique, 4 primer combinations (EcoRI/MseI) produced 330 scorable bands of which 110 bands were polymorphic (34%). Also the pair-wise genetic distance was from 0.06 to 0.78. The dendrogram constructed using UPGMA method, distinguished 4 main groups among 20 isolates of Fsp that was confirmed by multi-dimensional scaling. AFLP technique could separate the isolates with different levels of pathogenecity.

Tissue culture is a useful and applicable method in micropropagation and plants mass production in a short period of time irrespective of growing and planting season. Meanwhile it is a new and effective method to breed plants through gene transformation. Despite these advantages, a useful tissue culture system for effective regeneration is required for many plants such as White top (Cardaria draba). White top contains a lot of sulforaphan for which it is considered a medicine plant which in turn highly requires a suitable protocol for its effective regeneration. In this study, the samples gathered in Kerman Township were used and the ability of the plant for callus induction and shoot regeneration was studied in vitro in two separate factorial experiments in randomized complete block design, BAP: in 7 levels and NAA in 4 levels. The first experiments were carried on cotyledon explants and the second one on hypocotyl explants with 3 replications in the years 2010 and 2011. Considering the statistical analysis (p≤0.05), 3 mg/L BAP plus 0.5 mg/L NAA treatment and 3 mg/L BAP plus 1 mg/L NAA hormone treatment had the callus induction and the most suitable number of shoots for cotyledon and hypocotyl explants, respectively. The best root generation was for cotyledon and hypocotyl explants using only 1 mg/L NAA treatment and 0.5 mg/L BAP plus 1 mg/L NAA treatment, respectively. All the samples were placed on MS liquid containing 2 mg/L IBA and then they were elongated in the full strength medium without growth regulators for 2 weeks. After that, they were kept in the pots containing sterilized peat moss under greenhouse conditions for 4 weeks which resulted in 90% success in the plants regeneration.

Sugarcane is one of the most important industrial plants. There are many ways to improve the agronomic traits of this plant, such as genetic engineering and gene transfer methods. These modern techniques depend on optimizing the regeneration of plants from callus tissue. In this study, we attempted to optimize all different stages of tissue culture for regenerating the plants from callus tissue derived from terminal roll lives of two commercial sugarcane cultivars, CP48-10 and CP69-1062. Two types of cutting were applied, ring cutting and half-cylinder cutting from roll leaves. Among these cutting types, ring cutting from the roll leaves gives better results. Using solid MS medium with 3 mg per liter 2,4-D could attain the maximum callus production. To produce shoots from callus, the hormone combination of 1 mg per liter BAP and 2 mg per liter Kinetin showed the best response. Seedlings produced in MS medium containing 5 mg per liter IBA, 1 mg per liter Kinetin and 3 mg per liter NAA accompanied by 3 g per liter activated charcoal rendered the most root production. In the treatments with activated charcoal, the regenerated seedlings had superiority for the traits, days to root emergence, root length and number of roots in plants.

Fungal diseases are the major factors of sugar beet yield losses worldwide. Expression of pathogenesis-related proteins such as chitinases is considered as one of the plant defense responses against pathogens. Chitinases are cell wall degrading enzymes which have been shown to have high antifungal activity against a wide range of phytopathogenic fungi. In this study, two diploid sugar beet genotypes, SBSI-02 and SBSI-04, were used for transformation through Agrobacterium tumefaciens strain LBA4404 harboring the pBI-BCH plasmid containing the chi gene under the control of the CaMV35S promoter and the nptII selectable marker gene. Leaf blade with attached shoot bases were used as explant substratum for transformation. Green shoots were successfully screened using different concentration of kanamycin. PCR screening with chi-specific primer showed the presence of the transgene in more than 50% of regenerated kanamycin-resistant plants. Dot blot analysis confirmed the integration of at least one copy of the chi gene into the genome of putative transgenic plants. Western blot analysis revealed chitinase protein accumulation in transgenic plants. The content of chitinase protein varied among the five T0 transgenic plants. Bioassay analysis using detached leaves and at the whole plant level revealed increased resistance of T0 transgenic sugar beet lines against the fungal pathogen Alternaria alternata compared to non-transformed control plants.

Single-strand specific (sss) nucleases (S1/P1 family) are widely used in molecular genetics studies. The most widely used sss-nucleases are those plant endonucleases with the ability of specific cleavage of mismatch loops in heteroduplex DNA molecules. Because of the valuable activity of these endonucleases, enzymatic cleavage of heteroduplex DNA molecules is one of the most common techniques of point mutation detection in genomes. In this study, gene sequences encoding single-strand specific endonucleases were isolated from parsley (Petroselinum crispum L.) using heterologous primers in PCR reactions. The isolated gene sequences, after cloning and molecular identification, were designated as pars I and pars II. Physicochemical, structural and phylogenetic analysis using software tools, verified PARS I and PARS II as two members of S1/P1 family with a substantial homology to heteroduplex DNA specific endonucleases. However, it is essential to produce pure enzyme preparations as native or recombinant forms to determine mechanisms of activity and substrate preferences of these nucleases on heteroduplex DNA substrates.