مجله بیوتکنولوژی کشاورزی
سال یازدهم شماره 1 (پیاپی 34، بهار 1398)

 It is reasonable to investigate the signaling pathways in diseases such as mastitis in dairy cattle, which are more likely to be affected by epigenetics.   In this research, 5 microscopic RNA molecules with the accession numbers bta-mir-223, bta-mir 21-5p, bta-mir-181, bta-mir-146a and bta-mir-16a in the sources were identified and extracted from the literatures. Using mirtarbase and targetcan databases, respectively, the confirmed and predicted target genes were extracted for each of the top microRNAs, respectively. Subsequently, the expression of the predicted and confirmed target genes for each of the top microRNAs in the mammary gland tissue was investigated at the NCBI database. In addition, using the DAVID software, cellular signaling pathways were identified in KEGG.   Based on the results, bta-mir-146a microRNA through effects on of 7 proteins, and microRNA bta-mir-223 through effects on 11 proteins and micro RNA bta-mir21-5p through effects on 9 proteins, involved in various cellular signaling pathways, sought to be involved in mastitis in dairy cattle. The results also showed that two bata-mir-181 and bta-mir-16a played an important role in crucial pathways such as the GnRH signaling pathway, Estrogen signaling pathway, Progesterone-mediated oocyte maturation Oxytocin Signaling Pathway and MAPK Signaling Pathway.   This study shows that the microRNAs can be used as molecular markers to dissect molecular etiology of mastitis in dairy cattle.

 So far, various methods have been used to investigate the structure of the population using the markers available in the whole genome (single-nucleotide polymorphism (SNP)), each of which has Weakness and strength. In the present study, an unsupervised network clustering (SPC), a data-mining method, was used to survey the population structure of the Sarabi and Najdi cows. The study population 424 cattle consisted of 213sarabi cattle and 211 Najdi cattle, sequenced with Illumina Bead Chip 40 K v 2 for single nucleotide markers. SORTING POINTS INTO NEIGHBORHOOD(SPIN) was used to analyze population structure. After editing data, 27859 autosomal markers were analyzed. Clustering results based on the similarities and differences between nucleotides led to the classification of two base populations and nine clusters. Comparison the number of samples and other existing methods for population layering, the use of the SPIN method with high computational efficiency and the needn`t for prior assumptions makes it possible to analyze the structure of populations.

 The aim of the present study was to compare the effects of two different levels of Zataria multiflora with Avilamycin antibiotic and probiotic based on Bacillus subtilis on humoral and cellular immunity and expression of MUC2 gene in broiler chickens of Arian strain.   This study was conducted in a completely randomized design with five experimental groups with 4 replicates and 25 observations in each replicate. Experimental groups included basal diet and basal diet containing 100 mg Bacillus subtilis, 150 mg avilamycin, 200 mg and 400 mg Zataria multiflora essence, respectively. In order to evaluate the humoral and cellular immunity, the antibody produced against the sheep's red blood cell, white blood cell differentialcounts and the volume percentage of red blood cells were determined. Expression of MUC2 gene from the intestinal tissues was investigated by Real time PCR.   The results showed that response to red blood cells of sheep, immunoglobulin G and M was not affected by experimental groups(p>0.05). The number of white blood cells and the ratio of heterophil to lymphocyte were not affected by the experimental groups (p>0.05). The expression of MUC2 gene in the chickens that consumed the basal diet included Zataria multiflora was significantly increased compared with the control group.   The results showed that the use of Zataria multiflora essence could be having moderating effects on the immune system. It, by increasing the expression of MUC2 gene, could be having improving effect on the digestive system performance of broiler chickens.

 Determination of genetic variation in plant material is an initial step for identification, preservation and maintenance of heritable reserves as well as the basis for genetic research and breeding programs. The aim of this research was to study the genetic diversity and categorization of cumin ecotypes collected from North, Razavi and South Khorasan provinces based on RAPD and ISSR markers. Ultimately, the aim was to answer the following questions: Is there a genetic diversity between cumin populations and is there a correlation between the genetic diversity and geographic diversity of cumin ecotypes?   In this research, 20 different cumin ecotypes were cultivated for DNA extraction in the College of Agriculture of university of Birjand. The quantity and quality of the extracted DNA were evaluated using Nano drop and 1% agarose gel electrophoresis. For genetic variation, nine primers of RAPD and 30 ISSR primers were used.   The results of the analysis of molecular variance (AMOVA) showed that there were variations between and within populations. Based on the RAPD and ISSR markers, 10 and 9 percent of the variation were between populations and 90 and 91 percent were within populations, respectively. Based on the RAPD and ISSR markers the percentage of polymorphism 62% and 70%, the mean of polymorphic content was 0.26 and 0.3, the mean of Shannon's information index was 1.37 and 1.9, and the mean of the information index 2.1 and 2.76 were obtained, respectively. The results PCoA showed that for the RAPD marker, the first two components were accounted 40.4% and for ISSR marker, however, the first four components were accounted for 49.6% of the variation. The cluster analysis based on the similarity matrix grouped the ecotypes in 6 and 7 clusters, respectively.          According to the obtained information, such as the polymorphism, the large amplitude between the similarity coefficients (jacquard) and the grouping of ecotypes in distinct clusters, there is a genetic diversity between the studied cumin ecotypes. The other hand, in most cases, there was the correlation between the genetic variation in the collected ecotypes and geographic location.

 Agrobacterium rhizogenes results in the formation of hairy root phenotype in plants. The hairy roots have high growth rate and genetic stability and able to produce plant metabolites. In this study, the optimum parameters for induction and growth conditions of radish hairy roots has been investigated.   The effect of explant type (leaves, stems and cultures), co-culture medium (MS, ½MS, B5 and ½B5) and A. rhizogenes strains (A4, ATCC15834 and LBA9402) were evaluated based on factorial experiments in a CRD design with three replications. Transformation percentage was calculated based on the number of appearing hairy roots and was considered as an index for determining the optimum conditions. Transgenic nature of hairy roots was confirmed by PCR and specific primers for rolB and virD genes. The dry and fresh biomass and the amount of phenolic and flavonoid compounds of hairy roots were measured.   ATCC15834 had the highest ability to induce hairy roots in radish under different conditions. The ½B5 medium was the most suitable co-culture medium and the cotyledon explant was the best plant material for induction of radish hair roots. The growth rate among hairy root clones was significantly different based on dry and fresh biomass and the highest amount of phenolic and flavonoid compounds in hairy root cultures was 2.9 and 3.6 times more than normal plant, respectively.   The optimum conditions for induction of hairy roots must be determined in each plant and the hairy root cultures of radish have a high capability to produce secondary metabolites.

 The objective of this study was to assess the genetic diversity of some commercial apple cultivars in Khorasan Razavi province using 40 RAPD primers. DNA was collected from fresh leaves of apple samples studied and extracted based on dellaporta method. In order to investigate polymorphism, the PCR analysis was performed for each primer and the marker indices were calculated. The genetic similarity and distance were evaluated using Jaccard coefficient by UPGMA method and PcoA analysis by NTSYS-PC ver 2.2, respectively. The genetic structure analysis was performed using STRUCTURE software ver 2.3.   The percentage of polymorphic bands was different from 37.5% for OPJ13 marker to 100% for OPA7 markers. The highest and lowest PIC parameter was related to OPA4 (0.48) and OPM6 (0.2), respectively. With respect to cluster analysis, the samples were grouped into two main clusters and five subgroups. The highest similarity was between Golab Esfahan-Golomkani, Margenduft-Low Red Roem Beauty and Alimori Khorasan Dalago, while the lowest similarity was between Golab Esfahan-Fuji Rossa and Golab Esfahan-Low Red Roem Beauty cultivars. The classification based on PcoA analysis was different with UPGMA. In addition, the obtained result of Bayesian analysis showed lack of admixture of the apple cultivars studied.   According to the results of RAPD analysis, this marker can be used for recognition of polymorphic regions, assessment of genetic distance and management of apple genotypes and cultivars.

 In dairy cattle, the increase in milk yield has been accompanied by a more negative energy balance during early lactation and a decrease in fertility. As the hormone leptin is involved in regulation of nutritional status and reproductive function, this hormone is an interesting protein to investigate during the periparturient period in dairy cattle when many changes take place both in energy metabolism and reproductive physiology. The aim of this research was to study leptin gene expression in adipose tissue of Holstein dairy cattle.   Tissue sampling from subcutaneous adipose tissue of 20 Holstein cattle on the 20th day of lactation, with the same gestational age (second pregnancy) and mean body weight of 680 ± 80 kg was performed and RNA was extracted. Extracted RNA were immediately stored at -80°C.The Quality and quantity of RNA were evaluated and cDNA was synthesized and Real Time PCR was performed. PCR Products were electrophoresed on 2% agarose gel and were evaluated level of gene expression in the studied tissue.   Results showed that the leptin gene was expressed in the subcutaneous adipose tissue. These results may show that leptin plays a particular role in fat metabolism.   During the periparturient period the cow mobilizes her fat reserves (i.e. adipose tissue) and it appears that all her energy is going to the production of milk, and that other processes like reproduction and immunity, get a lower priority. Because fertility, just as milk production, is also an economically important trait, fertility should be considered as part of a dairy cattle breeding program. Generally, further studies are needed to clarify role of leptin in the physiology of fat metabolism and other materials. This would help us to better understand the mechanisms for the known effect of nutritional factors and body fatness on various functions.

 High level expression of recombinant protein in Escherichia coli often results in aggregation of the expressed protein molecules into inclusion bodies. Cysteines in the protein contribute to this process. Intermolecular and intramolecular disulfide bonds formation in Grapevine fanleaf virus (GFLV)-coat protein (CP), a cysteine-rich protein, and lead to aggregation when the recombinant protein was overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins.   In this paper, SDS/MPD method used as a unique approach to refold the structure some protein in the presence of the denaturing agent, Sodium Dodecyl Sulfate (SDS). This was made possible by addition of the amphipathic solvent 2,4-Methyl-2-PentaneDiol (MPD), used as protecting but also as refolding agent for these proteins. For this purpose, the inclusion bodies containing insoluble proteins of GFLV CP were solubilized by denaturing with SDS, then the soluble proteins were purified by the size exclusion chromatography, and finally, the purified proteins were refolded using SDS/MPD method.   Transmission electron microscopy images confirmed the reassembly GFLV VLPs using SDS/MPD method.   MPD modulate the denaturing properties of SDS, therefore, for the first time, a simple and effective method to refolded GFLV VLP from the SDS-denatured state.