Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 15 num. 2 lang. es <![CDATA[SciELO Logo]]> <![CDATA[<b>Genetic differentiation between Cinta Senese and commercial pig breeds using microsatellite</b>]]> Background: Cinta Senese (CS) is an autochthonous Tuscan breed, which risked extinction since the ‘60s. Results: Monitoring the genetic variability of the actual population by use of DNA molecular markers is essential to address a correct breeding policy, finalized to obtain the race preservation and its fitness in the future. 17 SSRs autosomal markers and 1 associated to the X chromosome were used to genotype 86 individuals belonging to the CS and 12 belonging to two main white races Landrace (L), Large White (LW) and crosses between LW and L and L and CS widespread in Tuscany and used in the recent past to obtain hybrids with the CS. Conclusions: A dendrogram of similarity measures the relative genetic distance between individuals in the population. Data show that CS pigs have a distinct genotype from L, LW, LW x L and L x CS. <![CDATA[<b>Microsatellite markers in candidate genes for wood properties and its application in functional diversity assessment in <i>Eucalyptus globulus</i></b>]]> Background: Functional genetic markers have important implications for genetic analysis by providing direct estimation of functional diversity. Although high throughput sequencing techniques for functional diversity analysis are being developed nowadays, the use of already well established variable markers present in candidate genes is still an interesting alternative for mapping purposes and functional diversity studies. SSR markers are routinely used in most plant and animal breeding programs for many species including Eucalyptus. SSR markers derived from candidate genes (SSR-CG) can be used effectively in co-segregation studies and marker-assisted diversity management. Results: In the present study, eight new non reported SSRs were identified in seven candidate genes for wood properties in Eucalyptus globulus: cinnamoyl CoA reductase (CCR), homocysteine S-methyltransferase (HMT), shikimate kinase (SK), xyloglucan endotransglycosylase 2 (XTH2), cellulose synthase 3 (CesA3), glutathione S-transferase (GST) and the transcription factor LIM1. Microsatellites were located in promoters, introns and exons, being most of them CT dinucleotide repeats. Genetic diversity of these eight CG-derived SSR-markers was explored in 54 unrelated genotypes. Except for XTH2, high levels of polymorphism were detected: 93 alleles (mean of 13.1 sd 1.6 alleles per locus), a mean effective number of alleles (Ne) of 5.4 (sd 1.6), polymorphic information content values (PIC) from 0.617 to 0.855 and probability of Identity (PI) ranging from 0.030 to 0.151. Conclusions: This is the first report on the identification, characterization and diversity analysis of microsatellite markers located inside wood quality candidate genes (CG) from Eucalyptus globulus. This set of markers is then appropriate for characterizing genetic variation, with potential usefulness for quantitative trait loci (QTL) mapping in different eucalypts genetic pedigrees and other applications such as fingerprinting and marker assisted diversity management. <![CDATA[<b>Intein-mediated expression of cecropin in <i>Escherichia coli</i></b>]]> Different strategies have been used to overcome the difficulties to produce antimicrobial peptides. Here we used Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT-System, New England Biolabs) for the expression of the antimicrobial peptide cecropin to reduce its sensitivity to intracellular proteases and use its inducible self-cleaving capability to remove the carrier. Cecropin was cloned into suitable expression vector pTYB11, and expression induced by IPTG in Escherichia coli ER2566. The use of 22ºC induction allowed the expression of cecropin with its intein carrier in soluble form. Cell extracts were purified by chitin affinity chromatography and intein-mediated splicing of the target protein was achieved by thiol addition, obtaining a final yield of 2.5 mg cecropin/l. Cecropin cleaved from the intein had its proper biologically active form, showing a micromolar antimicrobial activity against Vibrio ordalii, Vibrio alginolyticus and Escherichia coli. <![CDATA[<b>Development of cDNA-derived SSR markers and their efficiency in diversity assessment of <i>Cymbidium</i> accessions</b>]]> Cymbidium spp. are popular flowering plants. Assessment of the genetic diversity in cultivated Cymbidium facilitates conservation of germplasm and subsequent cultivar improvement. Thus, it is important to develop more efficient polymorphic DNA markers. Although more motifs (403) were identified and more primers (206) were designed in the genomic library compared to the cDNA library, a larger number of successful primers were obtained from the cDNA library (59.9%) than from genomic DNA library (51.1%). However, higher PIC and gene diversity were identified in genomic SSRs. The average allele number per locus was also higher in genomic SSRs (7.3) than EST-SSRs (5.2), among the 24 evaluated Cymbidium accessions. AT/TA was comparatively high in EST-SSRs, while this motif was not as common in genomic SSRs. The CTT/AAG/TCT/AGA/TTC/GAA and TGC/GCA/GCT/AGC/CTG/CAG motifs were the most abundant tri-nucleotide sequences in EST-SSRs, while GTT/AAC/TGT/ACA/TTG/CAA was the most frequent in genomic SSRs. The number of repeats ranged from 3 to 12 in EST-SSRs. Currently, 52 novel polymorphic SSR markers have been evaluated, which will be useful for germplasm assessments, core set construction, evaluation of genetic diversity, and marker assisted selection (MAS) based Cymbidium breeding. <![CDATA[<b>Improved isolation of good-quality total RNA from the optic stalk of Mud crab, <i>Scylla paramamosain</i></b>]]> An improved and efficient protocol was developed based on the TaKaRa RNAiso Plus Kit (Code: D9108A) for isolating good-quality total RNA from the optic stalk of mud crab, Scylla paramamosain. The protocol was based on the Trizol method with modifications. The carapace overlapping the optic stalk was retained with RNA in regular protocol. In order to remove the abundant deposition correlative with the carapace which makes the isolation of RNA particularly difficult, 5M potassium acetate solution (pH = 6.0) was added before the precipitation of RNA, and the temperature of RNA deposition was also decreased to -70ºC to ensure the stabilization of RNA. Good-quality total RNA from the optic stalk of S. paramamosain could be easily isolated with this modified protocol and three conventional methods were also employed to confirm the quality of RNA. This improved method would be helpful in facilitating molecular research of crabs involving RNA from the optic stalk. <![CDATA[<b>Genetic diversity and population structure analysis of strawberry (<i>Fragaria </i>x<i> ananassa</i> Duch.) using SSR markers</b>]]> In total, 18 simple sequence repeat (SSR) markers were used to analyze the genetic diversity and population structure of 59 accessions of cultivated strawberry (Fragaria x ananassa Duch.) from Korea, Germany, United States, United Kingdom, and Japan. In total, 101 alleles were detected with an average of 5.6 per locus and 21 specific alleles were identified. Notably, one genotype (Blonoli from Germany) possessed a maximum of 10 different unique alleles specific to each genotype. The gene diversity varied from 0.027 (EMPaEKO1B) to 0.791 (CFACT110), with an average value of 0.509. PIC values ranged from 0.026 to 0.762 (average 0.454). A model-based structure analysis revealed the presence of two populations. The accessions that were clearly assigned to a single population in which > 70% of their inferred ancestry was derived from one of the model-based populations. However, two accessions (3.4%) in the sample were categorized as having admixed ancestry. Here, we report detailed information on commercially grown strawberry accessions from five different origins using SSR markers. These results couldbe used for broadening the genetic base of commercially grown varieties. <![CDATA[<b>Association between AA-NAT gene polymorphism and reproductive performance in sheep</b>]]> Arylalkylamine N-acetyltransferase (AA-NAT) is critical enzyme in Melatonin (MLT) biosynthesis for MLT regulating the animal seasonal breeding. In this study, DNA sequencing methods were applied to detect the polymorphisms of the AA-NAT gene in 179 Chinese sheep belonging to two non-seasonal reproduction breeds and two seasonal reproduction breeds. One mutation at exon 3 (NM_001009461:c.486A &gt; G) was firstly described at the sheep AA-NAT locus. Hence, we described the SmaI PCR-RFLP method for detecting EX3 486A &gt; G mutation, frequencies of the AA-NAT-G allele varied from 0.871 to 0.908 in two non-seasonal reproduction breeds and 0.517 to 0.578 in two seasonal reproduction breeds. The associations of SmaI polymorphism with estrus traits was analyzed in non-seasonal reproduction breeds sheep and seasonal reproduction breeds sheep, the significant statistical results were found between them, the GG genotype frequencies was higher in non-seasonal reproduction breeds (p < 0.001), while, the GA genotype frequencies was higher in seasonal reproduction breeds (p < 0.05). Hence, the EX3 486A &gt; G mutation could facilitate association analysis and serve as a genetic marker for Chinese sheep breeding and genetics. <![CDATA[<b>Construction of an engineered alpha 1-antitrypsin with inhibitory activity based on theoretical studies</b>]]> Background: The elastase inhibitor α-1-antitrypsin (AAT), is a member of the serpin superfamily of protease inhibitors. AAT has a characteristic secondary structure of three-β-sheets, nine-α-helices and a reactive central loop (RCL). This protein inhibits target proteases by forming a stable complex in which the cleaved RCL is inserted into β-sheet-A of the serpin, leading to a conformational change in the AAT protein. Spontaneous polymerization and instability of AAT are challenges with regard to producing drugs against AAT-deficient diseases. Therefore, the purpose of many investigations currently is to produce drugs with lower degrees of polymerization and higher stabilities. In order to investigate the effect of the N-terminal segment (residues 1-43) on AAT structure, molecular dynamic (MD) simulation was used to study structural properties including Root-mean-square deviation (RMSD), internal motions, intramolecular non-bonded interactions and the total accessible surface area (ASA) of native and reduced AAT. These properties were compared in native and truncated AAT. Results: Theoretical studies showed no noticeable differences in the dynamic and structural properties of the two structures. These findings provided the basis for the experimental phase of the study in which sequences from the two AAT constructs were inserted into the expression vector pGAPZ and transformed into Pichia pastoris. Results showed no differences in the activities and polymerization of the two AAT constructs. Conclusions: As small-scale medicines are preferred by lung drug delivery systems, in this study AAT was designed and constructed by decreasing the number of amino acids at the N-terminal region. <![CDATA[<b>Expression of RNA polymerase IV and V in <i>Oryza sativa</i></b>]]> RNA polymerase IV and V are principal players in the RdDM pathway, where their current study has shown interaction of several factors that control DNA silencing of intergenic regions and siRNA production. DNA silencing is an important process during cell differentiation, nuclear structure and viral control. However, RNA pol IV and V are yet to be study in model monocot systems like Oryza sativa that can provide further information on genetic silence mechanism in plats. We show the expression pattern of these polymerases in tissues extracts of Oryza sativa. Detectable amounts of these polymerases are found in specific adult plant tissues and particularly expressed during somatic embryogenesis but not during early stages of normal embryo development. The use of synthetic auxin leads to an induction of both RNA pol IV and V in scutellum tissue where nuclear localization may be required for genome reorganization and gene silencing. <![CDATA[<b>Sheep 7SK promoter for short hairpin RNA expression</b>]]> Background: Gene silencing mediated by small interfering RNA (siRNA) has become a powerful biological tool for the regulation of gene expression. For the synthesis of siRNA by vector-based expression systems, several mammalian small nuclear RNA (snRNA) promoters have been cloned and shown different transcriptional efficiencies. Results: In this study, we identified a sheep 7SK snRNA (s7SK) promoter based on the highly conserved polymerase III promoter elements. Promoter activity was measured by promoter-driven shRNA expression to suppress expression of an exogenous reporter gene and endogenous sheep gene. Conclusions: The knock down assay demonstrated that the s7SK induced more stronger inhibition effect than human U6 and H1 promoters. The use of this native sheep 7SK promoter for shRNA expressionis an important component for development of RNAi-based gene therapy and production of transgenic animals in sheep species.