JOURNAL OF AGRICULTURAL BIOTECHNOLOGY
Year:2021 | Volume:12 | Issue:4|Papers Count:10

Objective Today, global increasing in corn prices has led to the replacement of wheat instead of corn in poultry diets. The NSPs in the cell wall prevents the uptake of wheat nutrients by poultry. Using xylanase is one of the methods to remove NSPs. Xylanases can digest xylan. The aim of current investigation was to produce xylanase (xynA) gene as a supplement of poultry diets in E. coli. Materials and methods In present study, the thermostable xylanase (xynA) gene in a bacterial secretory expression system along with: Shine-Dalgarno sequence, Usp45 signal peptide and T7 promoter was cloned in plasmid pET-22b (+) from E. coli strain BL21 (DE3) using The heat shock method. IPTG were used at different times to induce the expression of recombinant xylanase gene. In addition, catalytic indicators were done according to the DNS method. Results PCR colony confirmed the presence of 743 bp recombinant xylanase gene sequence on 1% agarose gel. SDS-PAGE showed protein with 27 kDa molecular weight. Also, the activity of recombinant protein was 189. 47 (unit/ml) using DNS method at optimum temperature of 65 ° C and pH=6. As well as, michaelis-menten curve determined Km and Vmax 4. 1 (mg/ml) and 87 (unit/ml), respectively. Conclusions Xylanase are the most important complementary enzymes in poultry diets that are added to wheat-based diets. Results showed that produced recombinant xylanase is thermostable enzyme with acceptable activity and concentration. Furthermore, because of the secretory expression system, it is produced at a lower cost.

Objective Nowadays, plant components with anti-cancer effects and multi-mechanisms are desired by scientists. Studies have shown which to control of multi-factors diseases like cancer, should emphasize on anti-cancer multi-mechanisms components. One of the most common cancers in today's societies is glioblastoma. Therefore, in this research, the effect of hydro alcoholic extract of saffron corm as an anti-tumor drug on the survival of glioblastoma cells was investigated. Materials and methods In this study, extract of corm saffron (Crocus sativus L. ) was prepared by softening method. The U87 cell line (human primary glioblastoma cell line) was prepared from a pasteurized cell bank, multiple times passage, and the cells reached the required number. Cytotoxic drugs and extract of saffron curry were performed simultaneously and simultaneously based on MTT colorimetric assay with certain concentrations of drug and extract in 24. 48, and 72 hours. The apoptotic changes of. 120 and 240 mg ml-1 of extract and control were measured by using flow cytometry and propidiodideid. In addition, ROS was measured for each of the concentrations of extract and control by fluorimetry and staining with DCF-DA. Results Results of MTT test shown that the highest cytotoxic effect was related to 400 μ g ml-1 hydro alcoholic corm extract with 90 % toxicity and treatment 200 μ g ml-1 with about 60 % toxicity stayed on second position. Temzolamide drug 125 μ M had the highest toxicity of effect 55 %. The highest amounts of apoptotic cell death 87 and 95 % were related to 240 μ g ml-1 hydro alcoholic corm extract in 24 and 48 hours after treat respectively based on flow cytometry data. ROS test at 6 hours after treat shown that the highest amount of adsorption were related to 240 and 120 μ g ml-1 hydro alcoholic corm extracts respectively. Conclusion The results 24, 48, and 72 hours treatment of saffron corn extract and temzolamide showed their anti-tumor effects on glayobastoma cancer cells, and the results were dose-dependent and time-dependent. The results of the cytotoxic, apoptotic and oxidative stress determined that saffron corm extract have anti-tumor effects on U87 cancer cell line significantly (P <0. 0001) level and can increase oxidative stress on them.

Objective About half of the human genome is covered by repetitive sequences. These sequences have a large share in the other mammalian genomes, therefore studying this part of the genome can provide researchers valuable information on evolution. The aim of this study was to sequencing and assembly the whole genome of Iranian Bactrian camels to identify transposable elements and their distribution in the genome of this species. In addition, the results of Iranian Bactrian camels were compared with non-Iranian Bactrian camels and dromedary camels. Materials and methods In this study, the whole genome of six Iranian Bactrian camels was sequenced to transposable elements identification. Iranian Bactrian camel whole genome sequenced using Illumina HiSeq 2000 system in paired-end. FastQC and Trimmomatic software were used to quality control and quality filtering of raw sequencing reads, respectively. CLC Genomics Workbench (CLC Bio, Aarhus, Denmark) was used to de novo assembly of trimmed reads. Also, we used the RepeatMasker program to search for transposable elements using a homology-based method. Results Results of the assembling of sequenced genomes showed that the genome size in these samples ranged from 1. 9 to 1. 97 Gb. In the present study, the percentage of transposable elements for six Iranian Bactrian camels was 29. 89% on average of the whole genome. The percentage of LINE sequences for the Iranian Bactrian camel was 17. 58% on average. So, these sequences were considered as the largest group of transposable elements in the Bactrian camel in this study. SINE elements showed a lower number in comparison with LINEs. So that, only 3. 45% of the total Bactria camel genome length was dedicated to the SINEs. In accordance with the results of Iranian dromedary camels, no Alu element was identified in the genome of Iranian Bactrian camels. Conclusion Shortage of genomic and biological information about camels is one of the inhibiting factors in advancing the breeding goals and programs. Although this study is not enough alone, it can be a step towards starting the production of genomic data for camels. Continuing this kind of study and integrating biological and genomic information will provide the ground for the start of modern breeding in Iranian camels.

Objective Immunotoxins are one of the most promising ways in therapeutic fields, specially cancer therapy which have a unique toxin-antibody structure, and kill the cancer cell by passing through the cell membrane and entering the target cell. The aim of this study was to engineer and design bovine pancreatic enzyme (RNase A) for to escape from ribonuclease inhibitor (RI) as a toxin segment in designing of immunotoxins used in therapeutic fields. Materials and Methods After bioinformatics investigation using PyMol software, engineering and substitution of target amino acid in wild gene coding RNase A sequence (K91A) were performed. The wild type and recombinant proteins in E. coli (BL21(D3) strain) were expressed and refolded, and then approved using SDS-PAGE. The purification of produced proteins was conducted using an immobilized-metal affinity chromatography for His-tagged proteins. Results The results showed that the protein concentrations were 3 and 2. 2 g/L for wild RNase A and recombinant enzyme. The hydrolysis activity measurement of the produced enzymes, in presence of different concentrations of RI, indicated that recombinant variant had lower sensitivity and higher catalytic activity in comparison with the wild enzyme. The results obtained from enzyme activity of recombinant enzyme compared to the wild enzyme, in presence or absence of RI, demonstrated that the interaction effect between RNase A and RI can be disrupted by manipulation of amino acids involved in this interaction. Conclusion In respect to the current results, it was concluded that RNase A enzyme can be used as a promising drug in engineering and production of immunotoxins.

Objective Lodging is the permanent horizontal growth of stem and has been considered as a limiting factor in cereal production. Identification of traits that affect grain yield is very important in breeding programs. Furthermore, genetic diversity is essential for breeding programs and increasing selection efficiency. This research was set up to study genetic diversity, evaluation of biomechanical traits, assess lodging stem and detect quantitative traits loci (QTLs) in a RIL population of bread wheat. Materials and methods A large population including 225 bread wheat lines was assessed in a randomized complete block design with two replications in research field of Shahid Bahonar University of Kerman during 2017-2018 growing season. To define biomechanical properties of stems, a universal testing machine equipped with 3-point bend and shear test probes was used. Results Results of ANOVA showed significant difference between wheat genotypes for all traits. Modulus of elasticity of stem had the highest response to selection (21. 65) in comparison with other traits. Moreover, this trait had high genetic variation. Based on these results, modulus of elasticity of each individual plant stem can be used as an effective factor in selection programs. Plant height had the highest narrow sense heritability (0. 59) among evaluated traits. Cross section area had strong and significant correlation with moment of inertia (r=0. 818**). In the molecular assay of this research, linkage map and QTL mapping of traits performed by inclusive composite interval mapping (ICIM) method. Linkage map was constructed based on diversity array technology (DArTs) and SSR markers. Conclusions A total of 12 QTLs have been identified which had LOD higher than 2. 5. These QTLs were controlling 6 biomechanical traits including: specific shear energy, maximum shear energy, moment of inertia, flexural stiffness, maximum specific bending strength and modulus of elasticity. The information of identified QTLs could be used in wheat breeding programs using marker assisted selection.

Objective Camelina (Camelina sativa L. Crantz) is a fast-growing oil crop belonging to the Brassicaceae family that can tolerate drought, salinity, cold, and many diseases and pests. Camelina seed has precious oil and protein with a number of potential attributes or benefits in both the human food and animal feed industry. Camelina also is being deemed as promising species to produce biodiesel and jet fuel in margin lands of the globe. Monolignols, as the precursor of lignin, are the common compounds in both lignification and soluble chemicals that have important roles in both normal development of healthy plants and defense-related responses in infected plant subjects. The regulatory mechanisms underlying the biosynthesis of these multifaceted secondary metabolites are poorly understood. Material and Methods Our current study presents the mode of gene expression and analyzes data to investigate the role of monolignol biosynthesis genes in the normal development growth of Camelina. We considered the transcript level of those genes that were covered 12 different tissues in major developmental stages during the life cycle of the Camelina. Using the R programming environment, we could have visualized the pattern of gene expressions with transcript per million (TPM) data in the heatmap. Results The results revealed the similarities as well as differences in gene expression patterns in both regulatory and functional gene groups among different tissues. Moreover, tissue-specific genes in different developmental stages were recognized. Conclusions The scrutiny in the literature related on biotic stress experiments in Camelina and also other species determined considerable differences in transcript levels and gene regulation patterns for the genes especially for members of the gene group encoding cinnamoyl-CoA reductase (CCR). Association of the latter genes CsCCR4 and CsCCR2 in particular involved in monolignol biosynthesis with the resistance of Camelina to pathogens contributes to providing a preliminary view to contemplate the future research options in various Camelina breeding programs.

Objective The present study was designed to identify the physiological and molecular responses of drought stress induced metabolism in plants. Since the studied plants include resistant and susceptible wheat crop, it is important to know the drought tolerance mechanisms in these cultivars in order to improve the resistance and tolerance of drought stress to the climate in Iran. Materials and Methods For this purpose, a factorial experiment based on completely randomized design with three replications was conducted in Mohaghegh Ardebil University in 2018-2019. The main factors included drought stress (35, 60 and 85% of field capacity (control)) and sub factor included three wheat cultivars (Pishgam, Pishtaz and Baharan). Drought stress in the three-leaf stage was applied for 10 days and then the seedlings were sampled to check the total protein content of the solution, the sugar content, proline content and the expression of proline-5-D gene expression-5-carboxyxyl synthase (P5CS) of the leaf tissue sample. Results The results showed that with increasing severity of drought stress, the amount of proline, soluble sugar and total protein increased. The highest and lowest levels of total protein, respectively, belonged to the leading cultivar under conditions of strict control and stress. The highest amount of soluble sugar (75. 76 and 91. 66 mg / g, respectively, wet weight of leaves) in spring wheat and severe drought stress and the lowest (57. 59 and 48. 7 mg / g, respectively). Examination of P5SC gene expression showed that the number of transcripts of this gene increases under stress and this shows that this gene plays an important role in responding to stress in plants. Baharan showed higher drought resistance than other cultivars by increasing P5CS gene expression and significant proline production and accumulation under drought stress. Conclusions In general, it is inferred that drought-induced expression key gene involved in the biosynthesis of proline (P5CS) cause increased levels of proline leaves and also with the accumulation of soluble sugar probably causes more stress tolerance in cultivars.

Objective Animal genetic resources are playing a vital role in ensuring food security and maintaining genetic diversity. Iran has a variety of livestock genetic resources and So far there are about 8. 44 million registered cows. The number of native cattle is 2044000 head, which accounts for about 27% of the animal population. These breeds are adapted to different climatic conditions of Iran and their genetic diversity is due to their domestication process over the centuries. Native breeds have unique features such as the disease resistance gene and the presence of the A2 variant in the milk of some native cows has been confirmed. According to reports, there has been a 27% decrease in the native breed from 2003 to 1977. Crossbreed populations have increased over the past few decades, and they have grown by about 22 percent. Therefore, the purpose of this article is to review the situation of native cattle and conservation strategies in the country. Preservation and conservation of native cow breeds In order to conserve of native breed, activities have done include the establish of native cattle stations, the implementation of composite breed projects to conserve blood percent of native cattle, native cattle genomic projects, cooperation with the private sector to purify native populations, the allocation of subsidies, save of genetic material at the Gene Banks of the Animal Breeding Center and the National Center for Genetic Resources and these actions have not been successful. Results The animal genetic conservation programs in developing countries are confounded by different challenges and the efforts of conservation of animal genetic resources are minimal. So the future strategy for native breeds should be a combination of breed improvement and genetic conservation. Conclusions Developing a medium-term and long-term strategy for breeding, considering the potential of each breed in different regions separately, and policy-making and presenting strategic plans is a suitable solution. The following factors are needed to economize the native livestock such as establishment of regional gene banks in accordance with the principles of conservation, cooperation animal husbandry, governmental and non-governmental organizations, appropriate coordination and cooperation between different organizations, research institutes, Academic and animal science associations and breeding companies are required.

Objective Estimates show that in the world about 30 million goats and in Iran about five million Cashmere goat are bred. One of the most important of these breeds is the Raini Cashmere goat. Steroid hormones perform their physiological functions by regulating the transcription of target genes. Receptors for these hormones play a role in osteoporosis, inflammation and cancer through transcription networks. Estrogen receptor (ER) is a transcription factor that mediates the function of estrogen in many physiological and pathological processes. There are two estrogen receptors, ESR1 and ESR2, which are located in the reproductive organs. Reproduction rate, especially fertility, is one of the most important economic traits in animal production and is regulated by genetic and environmental factors. Therefore, the aim of this study was to investigate the expression profile of ESR2 gene in different tissues of fluffy goats using Real Time PCR. Materials and methods Sampling of testis, ovarian, uterine, breast, brain, kidney and heart tissues was performed from a male and a female goat (3 replicates from each tissue). Total RNA was extracted from the tissues and the quality and quantity of the extracted RNA were evaluated. The cDNA was then synthesized and Real Time PCR was performed. The GAPDH housekeeping gene was used as a control. Melting curves were examined and data obtained from Real Time PCR were analyzed. Results The results of Real Time PCR curves and observation of electrophoresis of PCR products on 2% agarose gel showed that ESR2 gene was expressed in testis, ovary, uterine, breast, brain, kidney, and heart tissues. The highest level of expression was observed in the ovary and then the brain and uterus and the lowest level of expression was related to kidney, heart and testis tissues. Conclusions According to the results of the present research and the results of other studies, it can be concluded that ESR2 plays an important role in female fertility, because in the present study it was found that ESR2 is much more expressed in ovarian tissue than other tissues. Therefore, ESR2 is likely to be essential for female fertility, and the results of this study provide the basis for future research to describe the role of the ESR2 gene as a candidate gene for better fertility and normal physiology in domestic animals, especially goats.

Objective HSP90 is a member of heat shock proteins (HSPs) family which produces in response to environmental stress factors and has different roles in cells. In this study, the differential expression of HSP90 gene during the day and its relationship with ambient temperature and foraging rate of worker bees were investigated. Material and methods The worker bees’ commuting in front of hive and ambient temperature in five different times of day (at 7: 00, 9: 30, 12: 00, 14: 30 and 17: 00) were recorded for three days. The relative transcription level of HSP90 gene was measured in those times using real-time PCR method. The effects of day and different times in a day were subjected to the model as random and fixed effects, respectively, for analyzing worker bees’ commuting and ambient temperature. The regression between these traits was also run. Results Results showed that time of day has significant effect on ambient temperature, worker bees’ commuting and relative expression of HSP90 (P < 0. 01). The highest and lowest ambient temperature were recorded at 12: 00 pm and 7: 00 am, respectively. The highest and least worker bees’ commuting were obtained at 17: 00 and 12: 00 pm, respectively. In addition, the highest and lowest HSP90 gene transcription level were recorded at 62: 00 pm and 7: 00 am o’ clock, respectively. The regression analysis revealed that worker bees’ commuting and relative expression of HSP90 gene were increased with increasing of ambient temperature; the commuting of bees was reduced in temperature above 39 ̊ C. Furthermore, the worker bees’ commuting was increased with increasing of HSP90 expression to three-fold, which was decreased with more relative expression level. Conclusion In general, the trend of worker bees’ commuting, ambient temperature, and relative expression of HSP90 gene were clear during the day. When the ambient temperature was increased, the relative transcription level of HSP90 gene was increased in response to heat stress, while the number of commuter bees was decreased. The management of heat shock can be recommended to improve nectar gathering by commuter bees.