مجله بیوتکنولوژی کشاورزی
سال دوازدهم شماره 1 (پیاپی 38، بهار 1399)

 Cadmium is a highly toxic and widespread soil pollutant threatening human and animal. Plants effectively help to eliminate environmental pollution by up taking of heavy metals and tolerate cadmium stress through a variety of mechanisms, but biochemical pathways and genes involved in the response of plants to cadmium stress have not been fully and comprehensively identified.  

Following proteomic studies on the Arabidopsis thaliana mutant in which AT2G37050 (receptor like kinase gene) was knocked-out, it was identified that 150 proteins that were present in the wild (control) plant have been disappeared in the mutant plant. In current study, biological function of AT2G37050 gene/GO terms has been investigated by GeneMANIA and agriGO algorithms. GO term is a controlled vocabulary system describing biological entities in three aspects (biological process, molecular function, and cellular component) in different organisms.  

Bioinformatics studies resulted from the GeneMANIA algorithm showed that the AT2G37050 gene is involved in the biological process of response to cadmium ion. The agriGO algorithm was then used to study GO terms at three levels of biological process, molecular function and cellular component, and role of the AT2G37050 gene and biological process of response to cadmium ion was reconfirmed. In addition, significant GO terms (FDR <0.05) such as "extracellular region", "plasmodesmata", "vacuole membrane" and "chloroplast" are associated with the mechanisms involved in plant tolerance to cadmium stress. This is another supporting evidence, which shows association of AT2G37050 gene and "response to cadmium ion". 

In addition to suggesting a new effective gene in response to cadmium stress, the result of current study can be considered in order to construction of transgenic plants, which are able to purify soil from cadmium contamination.

Plants that have a symbiotic relationship with arbuscular mycorrhizal fungi (AMF) compared with non-mycorrhizal plants have a higher ability to resist against various biotic and abiotic environmental stresses such as drought, salinity, pests, and plant diseases. This study aim is studying different species of AMFs in Kerman province. The identification of these microorganisms is to provide the basis for further studies on the use of these microorganisms in germination, growth, and proliferation of medicinal plants.

To molecular and morphological identification of arbuscular mycorrhizal fungi, sampling was done from the rhizosphere of medicinal plants in some areas of Kerman province. Arbuscular mycorrhizal fungi spores were isolated from the sampled soils by the wet sieve method. The grouping was done based on spore microscopic slides and morphological characteristics such as spore color, shape, surface ornamentation, size, wall structure and spore characteristics in water. DNA was extracted from single spores. A Part of ribosomal DNA was amplified by nested PCR using SSUmaf and LSUmAr primers in the first step, SSUmCf, and LSUmBr in the second step. After horizontal agarose gel electrophoresis and see the related band the PCR product was sent for sequencing. Then sequencing results were analyzed using BLAST, Gene Runner and Clustal Omega Softwares, and the phylogenetic tree was plotted using MEGA 7 software.

According to morphological and molecular studies, Viscospora viscosa, Septoglomus deserticola, Funneliformis caledonium, Funneliformis mosseae, and Diversispora spurca were identified. Finally, the existence and manner of species identity were investigated in the phylogeny tree.

Since the morphological features used in the identification and classification of mycorrhizal fungi are limited, molecular methods can be used for safer classification, and in this way, Recognize differences of Similar species morphologically and similarity of different species in appearance.

 Lentil (Lens culinaris) is the third most important grain legume in the world after chickpea (Cicer arietinum L.) and pea (Pisum sativum L.).  Therefore, the aim of this research was (1) to determine the genetic diversity and structure of molecular variation of genotypes and (2) to identify relationships among genotype for conservation, management, and utilization of these genotypes in crop breeding programs.  

Total genomic DNA was extracted from leaves of each genotype following a CTAB protocol.  After DNA extraction, DNA samples were amplified using polymerase chain reaction (PCR).  Evaluation of the genetic diversity of 90 lentil genotypes was performed using 30 SSR markers.

 A total of 145 polymorphic alleles were produced with an average of 4.06 alleles per locus. The highest number of alleles produced belonged to SSR 80 primer with eight alleles and the lowest number of alleles belonged to SSR96 primer with three alleles.  Polymorphic information content (PIC) for the markers ranged between 0.35 to 0.83 with an average of 0.63.  Maximum and minimum PIC have belonged for SSR80 and SSR28, respectively.  Shannon index value was variable from 1.94 (for the SSR80) to 0.829 (for the SSR48). The expected heterozygosity was observed in genotypes ranged from 0.845 (SSR80) to 0.422 (SSR48). The mean of this index obtained with 0.643.  Clustering analysis was performed using Neihbour-Joining algorithm and population structure analysis was performed using Bayesian method.  The best number of sub-populations was identified as three.  Genotypes were identified within the different sub-populations that most of genotypes in sub-populations were not separated based on their geographic origins.

Results of this study revealed that SSR marker has highly potential for evaluation. This marker could separate all of genotypes very well.

In this study for the first time, different strains of Agrobacterium rhizogenes, co-cultivation times, and concentrations of acetosyringon in leaf explants of Echium khuzistanicum were examined to produce hairy roots.

The leaves of 21-day-old plant, different concentrations of acetosyringone (0,100,200 µM) and 3 strains of Agrobacterium (A4, ATCC15834 and 11325) were used for hairy root induction. In each explant, surficial wounds were created by a syringe contaminated with the bacterium and put for 10 minutes in bacterial suspension. After infection, the explants were transferred in the ½ MS solid medium supplemented with ascorbic acid (100 mg/L) to a light free growth chamber with 25 ° C temperature for 48 or 72 hours. PCR technique was used to confirm transformation by gene amplification with the rolBprimers. HPLC analysis was conducted on a high performance liquid chromatography system for shikonin measurement.  

The results of this study showed that, the first hairy roots emerged from wounded places after 14-21 days. The presence of A. rhizogenes T-DNA in the hairy root genome was confirmed by PCR using specific primers for rolB gene. Strain ATCC15834 had the highest rate of hairy root induction at 100 and 200 µM concentrations of acetosyringone indicating that the phenolic compound, acetosyringone, was effective in increasing hairy root induction. Generally, High concentrations of acetosyringone and more co-cultivation time together resulted in a significant increase in the hairy root induction in three strains of Agrobacterium rhizogenesis. Line 1 of hairy root produced 254.4±0.56 µg/g FW of shikonin.  

For the first time the results of this research showed that transformation and hairy root induction in E.khuzistanicum is possible by Agrobacterium rhizogenes and it can be used in the gene transfer and hairy root culture in order to shikonin production.

 Heat shock protein 70 (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The purpose of the study was to identify the polymorphisms of heat shock protein 70 gene of Khuzestan native chickens using SSCP technique. 

For this purpose, blood samples were collected from 130 native chicken from Shosh, Ramhormoz, Andimeshk cities and Jihad native chicken breeding station (Bavi city). After DNA extraction, the target area (892 base pair) was amplified using polymerase chain reaction (PCR) method and sequenced.    

PCR-SSCP analysis of these regions indicated the presence of polymorphisms in the beginning of the coding region and the sequencing of the PCR products confirmed and identified two polymorphic sites in this region: a transition A → G in position +259 (A259G) and a transversion C→G in position +277 (C277G). The results showed that Hsp70 gene is polymorphic in the studied area. Moreover, using the chi-square (X2) showed that the locus allele frequencies is not in Hardy-Weinberg equilibrium. Furthermore, the observed heterozygosity and the effective number of alleles demonstrated high diversity in the population. Moreover, Haplotype H3 has the highest frequency in the population. It occurs A259G mutants in this haplotype.   

Thus, these valuable gene pools should be conserved to be used in the future if needed. The selection at Jihad native chicken breeding station (Bavi city) has led to increased inbreeding and, as a result, the abundance of haplotype H3 in the population.

 Drought stress is a major problem that plagues world's arable lands and poses major limitations to plant growth and productivity. Lavandula angustifolia L. is a medicinal and aromatic plant that has great value for its essential oil. Due to the relatively resistant nature of this plant to water deficit, it can be a good substitute for plants with high water demand. To identification of some of genes involved in response to drought stress, we carried out transcriptome analysis under normal and drought condition using RNA-Seq.  

Illumina HiSeq. 2000 was applied for sequencing of flower and leaf mRNAs under control and stress conditions. Also the activity of some antioxidant enzymes  and level of Malondialdehyde and Dityrosine were measured.

Among a total of 264126 transcripts, 1083 DEGs in flower and 150 DEGs in leaf were identified in response to the drought stress. Gene Ontology Enrichment of drought responsive DEGs including catalytic activity, response to stimulus and binding were identified. KEGG analysis showed different pathways associated with stress such as biosynthesis of secondary metabolites, glutathione and proline metabolism and plant hormone signal transduction. Also some unigenes related to antioxidant enzymes were highlighted in response to drought.  

 The transcriptome data generated here is the first report of RNA-Seq for Lavandula angustifolia L. under drought stress. Biochemical analysis results in this study were consistent with the RNA-seq results. Our findings offer insights into the molecular networks of Lavandula angustifolia L.  in response to drought stress.

Drought is the most common environmental stress, which approximately limits production in 25 percent of the world's land. Drought stress causes changes in plant morphology, physiology and profile of genes expressions. One of the effects of oxidative stress due to drought stress is damage to telomeric DNA. The predominant mechanism of telomere conservation in most eukaryotes is dependent on telomerase activity, which prevents shortening of the chromosome end. In the present study the expression of telomerase gene has been investigated in two sunflower tolerant and susceptible genotypes using real time PCR technique. 

The inbred lines; ENSAT254 and LC1064C were planted in a completely randomized design with three replications in the greenhouse. The plants were kept at 100% crop capacity up to 8 leaf stage in terms of hydration. After this stage, a number of pots were kept at the same capacity, but some others were kept at 80%, 60% and 40% crop capacity. Leaf samples were taken at two times; 7 and 21 days after stress application. RNA was extracted from leaf samples using RNX-plus TM extraction solution (Sinaclone Co., Iran) and real time PCR was conducted using specific telomerase gene primers. Analysis of variance and mean comparison were performed using SAS 9.4 software. Charts were drawn with Excel 2016.

The results of the mean comparisons showed that the expression of the genes encoding the telomerase enzyme in the sensitive and resistant genotypes of sunflower is different, so that the expression of the gene encoding telomerase enzyme in the resistant genotype (ENSAT254) increased significantly compared to the sensitive one (LC1064C).   

Since telomerase plays an essential role in maintaining genome integrity and stability of cell cycle, therefore, the higher expression of telomerase gene in tolerant genotype (ENSAT254) compared to susceptible one (LC1064C) probably suggests that this gene is involved in resistance of sunflower to drought stress.

 Orchids seeds are so small, lacking nutrients and barely germinate in natural condition. For germinating in natural conditions, should associatie with fungi, especially mycorrhizal fungi. Drawing on the tissue culture technique and meeting all necessary growth and development conditions, the limitations can be overcome.  

After being surfaced cleaned, the capsules containing seeds were placed in 1% Teepol solution as a disinfectant and wetting agent for 20 minutes to completely remove pathogens; then were washed and placed under laminar air flow, disinfected with HgCl2 0.2 for 10 minutes, Bavistin and Streptomycin each one 0.03% for 5 minutes in a entirely sterile environment. All the culture media were simultaneously ready and the seeds were uniformly inoculated in a totally sterilized environment and incubated.   

The percentage and germination rate were affected by seed age and plant growth regulators. The formation of spherule and chlorophyll synthesis were highest in the third stage of seed culture. Based on the type of treatment, the formation, development and differentiation of protocorm were observed between 43-76 days after culture, which was statistically significant at 1% compared to control.  

Considering the data analysis results, the success rate from the germination stage to the differentiation of protocorm in orchid seeds is mostly different based on seed age in each spices and cultivars, the type of plant growth regulators used, The ratio and composition of the two types of auxin and the ratio of auxin to cytokinin and vice versa are different. Therefore, it is necessary that new complementary experiments be done for each spices and cultivars based on the difference in the time taken from pollination to embryo ripening.

 Evaluation and conservation of native chickens as future genomic resources are essential. In this study, genomic diversity of four Marandi breeds was investigated using whole genome sequencing technique.  

 Blood samples were taken from four chickens in East Azerbaijan province, Iran. Whole genome sequencing (paired end sequencing) was done by Illumina Company) Hiseq 2500). Data quality control was performed by FastQC program. Whole genome sequencing data were aligned with genome reference (Gallus_gallus-5.0/galGal5) using MEM algorithmimplemented in burrows wheeler aligner program (BWA). Single nucleotide polymorphisms (SNPs) and small insertions and deletions (INDELs) were identified by the GATK program. Annotation of SNPs and Indels was done using SnpEff program. Genetic diversity of 4 chicken genomes was calculated with VCFtools.   

The short sequences were compared with the reference genome of over 99% and with the mean depth of 7X coverage. In this study, 8.7 million SNPs and 9.1 Indels were identified with the most counts of them in the intron and intergenic regions. The mean of observed and expected heterozygosity percentages for SNPs in four chicken genomes were 0.33 and 0.35, respectively.  

Results from annotation showed that percentage of the silent SNPs (74.64%) is higher than that the nonsynomous SNPs (missense and nonsense) in Marandi chicken genome. The results obtained from this research can be useful for Marandichicken breeding and conservation programs.

The estrogen receptor (ER) is a transcription factor that mediates the function of the hormone estrogen in many physiological and pathological processes. The expression of the estrogen receptor (ESR1), which encodes the alpha estrogen receptor (ER-α), is tissue specific. For example, the expression of ESR1 in the endometrium and mammary gland is very high and low in the placenta and skin. The gene is located on human chromosome 6 and is also known as Era, ESRA, ESTRR, and NR3A1. Several studies have shown that genetic variants in this gene are associated with breast cancer sensitivity. The aim of this study was to investigate ESR1gene expression in different tissues of Raini Cashmere goat using Real Time PCR.  

One male and one female goats were selected for tissue sampling. Samples were taken from tissues of the heart, kidneys, brain, ovaries, uterus, breast and testicles (3 repetitions of each tissue) during slaughter at the slaughterhouse. RNA was extracted and cDNA was synthesized. Real Time PCR was performed using SYBR Green method to study relative gene expression. GAPDH gene was used as housekeeping gene. Pfaffl method was used to analyze achieved data. 

  The study of ESR1 gene expression in the tissues of the heart, kidney, brain, ovary, uterus, mammary gland and testis of goat using Real Time PCR showed that this gene is expressed in all studied tissues. The highest levels of expression were observed in the kidney, ovary, uterus and testis tissues, and the lowest levels of expression were observed in the brain and heart tissues.  

Based on the results of the present study and the results of other researchers, it can be concluded that estrogen receptors play an important role in spermatogenesis and male fertility, because in the present study it was found that ESR1 is much more expressed in reproductive tissues than in non-reproductive tissues. Therefore, estrogen and its receptors are likely to be essential for male fertility, and the results of this study provide a basis for future research to describe the role of ESR1 gene as a candidate gene for better fertility and natural physiology in domestic animals, especially goats.