Cloning Genes Research Articles

Differential interferon system gene expression profiles in susceptible and resistant gynogenetic clones of gibel carp challenged with herpesvirus CaHV

Interferon (IFN) system plays a vital role in the first line of defense against viruses. In this study, we first identified multiple transcripts of 15 IFN system genes, including PRRs (TLR2, TLR3, RIG-I, and LGP2), PRR-mediated IFN signal pathway (MyD88, MITA, and MAVS), IFN regulatory factors (IRF1, IRF3, IRF7, and IRF9), IFNs (IFNφ1 and IFNφ3), and ISGs (Mx and viperin), and one transcript of TLR9 in de novo transcriptome assembly data of gibel carp head-kidney. Multiple nucleotide alignments and phylogenetic analysis of common region showed that the transcripts of every of the 15 IFN system genes were classified into two homologs with distinctly divergent sequences, indicating that hexaploid gibel carp may be an allopolyploid. During Carassius auratus herpesvirus (CaHV) infection, gibel carp resistant clone H significantly suppressed CaHV replication with markedly less viral loads than those in highly susceptible clone A+ and moderately resistant clone F. Then, qPCR analyses were performed to reveal their differential and dynamic expression changes during CaHV infection in head kidney, spleen and liver among three gibel carp gynogenetic clones. Through qPCR and hierarchical clustering analysis, 8 genes, such as RIG-Is, LGP2s, IRF1-B, IRF3s, IRF7s, IRF9-B, Mxs, and viperins, were identified as candidate resistant-related genes. They remarkably increased their expression in immune tissues of three clones after CaHV infection. Significantly, the up-regulation folds of these genes in clone A+, F and H were related to their resistance ability to CaHV, progressively increasing from susceptible clone to resistant clone at 1 dpi. The positive correlation to the resistance ability suggested that resistant clone H immediately triggered stronger IFN response. IFNφ3 showed a different dynamic change and was sharply induced in moderately resistant clone F at 3 dpi. The other 5 IFN system genes (TLR2, TLR3, TLR9, MyD88, and MITA) maintained a low expression level after CaHV challenge. Interestingly, the A or B copies/homologs of almost these IFN system genes exhibited differential transcript abundance in immune tissue after CaHV challenge, suggesting A or B homologs might occur dominant or biased expression of homeologs during gibel carp evolution. These data provide candidate resistant-related genes for disease-resistance breeding of gibel carp.

Cloning and transformation human glucocerebrosidase gene in Agrobacterium tumefaciens LBA4404 strain

Cloning and transformation human glucocerebrosidase gene in Agrobacterium tumefaciens LBA4404 strain

Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions.

Some mammalian reference genes, which are widely used to normalize the qRT-PCR, could not be used for this purpose due to its high expression variation. The normalization with false reference genes leads to misinterpretation of results. The silversides (Odontesthes spp.) has been used as models for evolutionary, osmoregulatory and environmental pollution studies but, up to now, there are no studies about reference genes in any Odontesthes species. Furthermore, many studies on silversides have used reference genes without previous validations. Thus, present study aimed to was to clone and sequence potential reference genes, thereby identifying the best ones in Odontesthes humensis considering different tissues, ages and conditions. For this purpose, animals belonging to three ages (adults, juveniles, and immature) were exposed to control, Roundup®, and seawater treatments for 24 h. Blood samples were subjected to flow-cytometry and other collected tissues to RNA extraction; cDNA synthesis; molecular cloning; DNA sequencing; and qRT-PCR. The candidate genes tested included 18s, actb, ef1a, eif3g, gapdh, h3a, atp1a, and tuba. Gene expression results were analyzed using five algorithms that ranked the candidate genes. The flow-cytometry data showed that the environmental challenges could trigger a systemic response in the treated fish. Even during this systemic physiological disorder, the consensus analysis of gene expression revealed h3a to be the most stable gene expression when only the treatments were considered. On the other hand, tuba was the least stable gene in the control and gapdh was the least stable in both Roundup® and seawater groups. In conclusion, the consensus analyses of different tissues, ages, and treatments groups revealed that h3a is the most stable gene whereas gapdh and tuba are the least stable genes, even being considered two constitutive genes.

Effects of Microcystis aeruginosa on the expression of nuclear receptor genes in Daphnia similoides sinensis

Nuclear receptor (NR) genes form a conserved superfamily, which is involved in organism metabolism, reproduction, development, homeostasis, and resource allocation. Microcystis aeruginosa can inhibit the growth and reproduction of Daphnia. However, whether M. aeruginosa can affect the expression of Daphnia NR genes is unknown. In total, 18 NRs were identified in this study based on previous Daphnia similoides sinensis transcriptome data. In treatments containing M. aeruginosa, the gene expression of the NR1 subfamily (E75a, E75b, HR3, HR96, NHR-1, HR97a, HR97g, and NHR97) and the NR2 subfamily (RXR, TLL, PNR, and SVP) were down-regulated 59% and 79%, respectively. In treatments containing M. aeruginosa, although the expression of 78% of the genes showed a similar trend in clones 1 and 2, the expression of 42% of the genes in clone 3 showed the opposite trend compared to clones 1 and 2, suggesting that the adaptability and molecular mechanism differ in individuals with different Microcystis tolerance genotypes.

Development of a Molecular Marker for Fruiting Body Pattern in Auricularia auricula-judae

The fruiting body pattern is an important agronomic trait of the edible fungus Auricularia auricula-judae, and an important breeding target. There are two types of fruiting body pattern: the cluster type and the chrysanthemum type. We identified the fruiting body pattern of 26 test strains, and then constructed two different near-isogenic pools. Then, we developed sequence characterized amplified region (SCAR) molecular markers associated with the fruiting body pattern based on sequence-related amplified polymorphism (SRAP) markers. Ten different bands (189–522 bp) were amplified using 153 pairs of SRAP primers. The SCAR marker “SCL-18” consisted of a single 522-bp band amplified from the cluster-type strains, but not the chrysanthemum strains. This SCAR marker was closely associated with the cluster-type fruiting body trait of A. auricula-judae. These results lay the foundation for further research to locate and clone genes controlling the fruiting body pattern of A. auricula-judae.

Increased pregnancy losses following serial somatic cell nuclear transfer in goats.

Serial cloning by somatic cell nuclear transfer (SCNT) is a critical tool for the expansion of precious transgenic lines or resetting the lifespan of primary transgenic cells for multiple genetic modifications. We successfully produced second-generation cloned goats using donor neonatal fibroblasts from first-generation clones. However, our attempts to produce any third-generation clones failed. SCNT efficiency decreased progressively with the clonal generations. The rate of pregnancy loss was significantly greater in recloning groups (P<0.05). While no pregnancy loss was observed during the first round of SCNT, 14 out of 21 pregnancies aborted in the second round of SCNT and all pregnancies aborted in the third round of SCNT. In this retrospective study, we also investigated the expression of 21 developmentally important genes in muscle tissue of cloned (G1) and recloned (G2) offspring. The expression of most of these genes in live clones was found to be largely comparable to naturally reproduced control goats, but fibroblast growth factor 10 (FGF10), methyl CpG binding protein 2 (MECP2) and growth factor receptor bound protein 10 (GRB10) were differentially expressed (P<0.05) in G2 goats compared with G1 and controls. To study the effects of serial cloning on DNA methylation, the methylation pattern of differentially methylated regions in imprinted genes H19 and insulin like growth factor 2 receptor (IGF2R) were also analysed. Aberrant H19 DNA methylation patterns were detected in G1 and G2 clones.

Identification of metolachlor mineralizing bacteria in aerobic and anaerobic soils using DNA-stable isotope probing.

The influence of soil environmental factors such as aeration on the ecology of microorganisms involved in the mineralization and degradation of the popular soil-applied pre-emergent herbicide, metolachlor is unknown. To address this knowledge gap, we utilized DNA-based stable isotope probing (SIP) where soil microcosms were incubated aerobically or anaerobically and received herbicide treatments with unlabeled metolachlor or 13C-metolachlor. Mineralization of metolachlor was confirmed as noted from the evolution of 14CO2 from 14C-metolachlor-treated microcosms and clearly demonstrated the efficient utilization of the herbicide as a carbon source. Terminal restriction fragment length polymorphisms (T-RFLP) bacterial community profiling performed on soil DNA extracts indicated that fragment 307bp from aerobic soil and 212bp from anaerobic soil were detected only in the herbicide-treated (both unlabeled metolachlor and 13C-metolachlor) soils when compared to the untreated control microcosms. T-RFLP profiles from the ultracentrifugation fractions illustrated that these individual fragments experienced an increase in relative abundance at a higher buoyant density (BD) in the labeled fractions when compared to the unlabeled herbicide amendment fractions. The shift in BD of individual T-RFLP fragments in the density-resolved fractions suggested the incorporation of 13C from labeled herbicide into the bacterial DNA and enabled the identification of organisms responsible for metolachlor uptake from the soil. Subsequent cloning and 16S rRNA gene sequencing of the 13C-enriched fractions implicated the role of organisms closely related to Bacillus spp. in aerobic mineralization and members of Acidobacteria phylum in anaerobic mineralization of metolachlor in soil.

Mapping and screening of the tomato Stemphylium lycopersici resistance gene, Sm, based on bulked segregant analysis in combination with genome resequencing

BackgroundTomato gray leaf spot disease caused by Stemphylium lycopersici (S. lycopersici) is considered one of the major diseases of cultivated tomatoes. The only S. lycopersici resistance gene, Sm, was derived from the wild tomato species S. pimpinellifolium. Sm has been identified as an effective source of gray leaf spot resistance in tomatoes and has been mapped to tomato chromosome 11. In this study, the first bulked segregant analysis (BSA) combined with genome resequencing for the mapping and screening of the Sm candidate gene was performed.ResultsBased on the resequencing results, we identified 50,968 Diff-markers, most of which were distributed on chromosome 11. A total of 37 genes were located in the interval of 0.26-Mb. The gene loci of resistant and susceptible lines were sequenced successfully using PCR products. The relative expression levels of candidate genes in resistant and susceptible lines were confirmed via qRT-PCR, Solyc11g011870.1.1 and Solyc11g011880.1.1 were identified through qRT-PCR. A marker, D5, which was cosegregated with the resistant locus, was identified according to the mutation of the Solyc11g011880.1.1 trait in the resistant line.ConclusionsThe Sm gene was mapped to the short arm of chromosome 11. The candidate genes Solyc11g011870.1.1 and Solyc11g011880.1.1 displayed expression patterns related to the resistance response. This study will be valuable for Sm cloning and Sm gene breeding in tomato.

Cloning and Sequence Analysis of Gene Encoding Flavonol Synthase from Trung Du Teas Growing in Thai Nguyen

In this study, we conducted the cloning and sequencing gene encoding FLS from the two green and purple Trung Du tea cultivars. The length of FLS gene is 996 bp, encodes 331 amino acid. Results of FLS gene analysis showed that green and purple Trung Du cultivars has 13 nucleotide variants total as compare with FLS sequence published on Genbank. Nucleotide sequence differences lead to change amino acid sequence of key functional motives in FLS like motif characterizes the 2OG-Fe (II) oxygenase superfamily, the PxxxIRxxx-EQP motif at the N-terminal (18→29) determines the FLS activity, the CPQ/RPxLAL motif) is the binding site of 2-oxoglutarate. How amino acid position changes affect FLS activity need further research. FLS gene isolates are sources for further research to aim elucidating the function of this enzyme.&#x0D;

Non-muscle myosin IIB (Myh10) is required for epicardial function and coronary vessel formation during mammalian development.

The coronary vasculature is an essential vessel network providing the blood supply to the heart. Disruptions in coronary blood flow contribute to cardiac disease, a major cause of premature death worldwide. The generation of treatments for cardiovascular disease will be aided by a deeper understanding of the developmental processes that underpin coronary vessel formation. From an ENU mutagenesis screen, we have isolated a mouse mutant displaying embryonic hydrocephalus and cardiac defects (EHC). Positional cloning and candidate gene analysis revealed that the EHC phenotype results from a point mutation in a splice donor site of the Myh10 gene, which encodes NMHC IIB. Complementation testing confirmed that the Myh10 mutation causes the EHC phenotype. Characterisation of the EHC cardiac defects revealed abnormalities in myocardial development, consistent with observations from previously generated NMHC IIB null mouse lines. Analysis of the EHC mutant hearts also identified defects in the formation of the coronary vasculature. We attribute the coronary vessel abnormalities to defective epicardial cell function, as the EHC epicardium displays an abnormal cell morphology, reduced capacity to undergo epithelial-mesenchymal transition (EMT), and impaired migration of epicardial-derived cells (EPDCs) into the myocardium. Our studies on the EHC mutant demonstrate a requirement for NMHC IIB in epicardial function and coronary vessel formation, highlighting the importance of this protein in cardiac development and ultimately, embryonic survival.

High incidence of multidrug-resistant fecal E. coli producing ESBLs and carried ST131 in Jordanian adults

Background: Escherichia coli is part of the human intestine normal flora, although it has the potential of causing variety of invasive and diarrheal diseases. It is also a frequent cause of community- and hospital-acquired urinary tract infections. Intestinal E. coli has the potential to develop rapidly multidrug resistant (MDR) and to emerge as extended-spectrum β-lactamases (ESBLs)-producer. &#x0D; Methods: Over the period of July through November, 2015; 287 stool samples were collected from Jordanian adults who visited the students’ clinic of the University of Jordan. Fecal samples were collected and cultured for isolation of E. coli. The isolates were investigated for antimicrobial susceptibility, and molecular method of polymerase chain reaction (PCR) was performed for the detection genes of ST131 clone, blaCTX-M group I, blaCTX-M-15, blaNDM-1, blaVIM, blaIMP, blaOXA-48, blaKPC and fluoroquinolones resistance (gyrA and parC).&#x0D; Results: A total of 105/287 E. coli isolates (36.6%) were found to be multi-drug resistant (MDR) to at least 3 classes of antibiotics, of these 45.1% were ESBL-producers. A total of 51 representative MDR isolates indicated the following; 49% were found positive for ST131 clone, 58.8% were resistant for ciprofloxacin, and 41.2% were positive for CTX-M group I and CTX-M-15, respectively. All these MDR isolates were also positive for mutated both gyrA and ParC genes, and only 6 / 51 isolates (11.8%) were positive for each blaNDM-1 and blaKPC. One out of 51 MDR isolates (2%) was positive for blaVIM, and none of these isolates was positive for blaIMP nor blaOXA-48 genes.&#x0D; Conclusion: This study indicated that a relatively high rates of commensal fecal E. coli isolates from Jordanian adults were MDR, ESBLs-producer and belonging to ST131 clone. Also, high rates of CTX-M-15 and fluoroquinolones resistance were found among MDR E. coli isolates. &#x0D;

De novo transcriptome of the cosmopolitan dinoflagellate Amphidinium carterae to identify enzymes with biotechnological potential

Dinoflagellates are phytoplanktonic organisms found in both freshwater and marine habitats. They are often studied because related to harmful algal blooms but they are also known to produce bioactive compounds for the treatment of human pathologies. The aim of this study was to sequence the full transcriptome of the dinoflagellate Amphidinium carterae in both nitrogen-starved and -replete culturing conditions (1) to evaluate the response to nitrogen starvation at the transcriptional level, (2) to look for possible polyketide synthases (PKSs) in the studied clone (genes that may be involved in the synthesis of bioactive compounds), (3) if present, to evaluate if nutrient starvation can influence PKS expression, (4) to look for other possible enzymes of biotechnological interest and (5) to test strain cytotoxicity on human cell lines. Results showed an increase in nitrogen metabolism and stress response in nitrogen-starved cells and confirmed the presence of a type I β-ketosynthase. In addition, L-asparaginase (used for the treatment of Leukemia and for acrylamide reduction in food industries) and cellulase (useful for biofuel production and other industrial applications) have been identified for the first time in this species, giving new insights into possible biotechnological applications of dinoflagellates.

Identification of major quantitative trait loci for root diameter in synthetic hexaploid wheat under phosphorus-deficient conditions.

Synthetic hexaploid wheat (SHW) possesses numerous genes for resistance to stress, including phosphorus (P) deficiency. Root diameter (RDM) plays an important role in P-deficiency tolerance, but information related to SHW is still limited. Thus, the objective of this study was to investigate the genetic architecture of RDM in SHW under P-deficient conditions. To this end, we measured the RDM of 138F9 recombinant inbred lines derived from an F2 population of a synthetic hexaploid wheat line (SHW-L1) and a common wheat line (Chuanmai32) under two P conditions, P sufficiency (PS) and P deficiency (PD), and mapped quantitative trait loci (QTL) for RDM using an enriched high-density genetic map, containing 120,370 single nucleotide polymorphisms, 733 diversity arrays technology markers, and 119 simple sequence repeats. We identified seven RDM QTL for P-deficiency tolerance that individually explained 11-14.7% of the phenotypic variation. Five putative candidate genes involved in root composition, energy supply, and defense response were predicted. Overall, our results provided essential information for cloning genes related to P-deficiency tolerance in common wheat that might help in breeding P-deficiency-tolerant wheat cultivars.

The Evolution of gene regulation research in Lactococcus lactis.

Lactococcus lactis is a major microbe. This lactic acid bacterium (LAB) is used worldwide in the production of safe, healthy, tasteful and nutritious milk fermentation products. Its huge industrial importance has led to an explosion of research on the organism, particularly since the early 1970s. The upsurge in the research on L. lactis coincided not accidentally with the advent of recombinant DNA technology in these years. The development of methods to take out and re-introduce DNA in L. lactis, to clone genes and to mutate the chromosome in a targeted way, to control (over)expression of proteins and, ultimately, the availability of the nucleotide sequence of its genome and the use of that information in transcriptomics and proteomics research have enabled to peek deep into the functioning of the organism. Among many other things, this has provided an unprecedented view of the major gene regulatory pathways involved in nitrogen and carbon metabolism and their overlap, and has led to the blossoming of the field of L. lactis systems biology. All of these advances have made L. lactis the paradigm of the LAB. This review will deal with the exciting path along which the research on the genetics of and gene regulation in L. lactis has trodden.

Molecular cloning, expression, and characterization of four novel thermo-alkaliphilic enzymes retrieved from a metagenomic library

BackgroundEnzyme discovery is a promising approach to aid in the deconstruction of recalcitrant plant biomass in an industrial process. Novel enzymes can be readily discovered by applying metagenomics on whole microbiomes. Our goal was to select, examine, and characterize eight novel glycoside hydrolases that were previously detected in metagenomic libraries, to serve biotechnological applications with high performance.ResultsHere, eight glycosyl hydrolase family candidate genes were selected from metagenomes of wheat straw-degrading microbial consortia using molecular cloning and subsequent gene expression studies in Escherichia coli. Four of the eight enzymes had significant activities on either pNP-β-d-galactopyranoside, pNP-β-d-xylopyranoside, pNP-α-l-arabinopyranoside or pNP-α-d-glucopyranoside. These proteins, denoted as proteins 1, 2, 5 and 6, were his-tag purified and their nature and activities further characterized using molecular and activity screens with the pNP-labeled substrates. Proteins 1 and 2 showed high homologies with (1) a β-galactosidase (74%) and (2) a β-xylosidase (84%), whereas the remaining two (5 and 6) were homologous with proteins reported as a diguanylate cyclase and an aquaporin, respectively. The β-galactosidase- and β-xylosidase-like proteins 1 and 2 were confirmed as being responsible for previously found thermo-alkaliphilic glycosidase activities of extracts of E. coli carrying the respective source fosmids. Remarkably, the β-xylosidase-like protein 2 showed activities with both pNP-Xyl and pNP-Ara in the temperature range 40–50 °C and pH range 8.0–10.0. Moreover, proteins 5 and 6 showed thermotolerant α-glucosidase activity at pH 10.0. In silico structure prediction of protein 5 revealed the presence of a potential “GGDEF” catalytic site, encoding α-glucosidase activity, whereas that of protein 6 showed a “GDSL” site, encoding a ‘new family’ α-glucosidase activity.ConclusionUsing a rational screening approach, we identified and characterized four thermo-alkaliphilic glycosyl hydrolases that have the potential to serve as constituents of enzyme cocktails that produce sugars from lignocellulosic plant remains.

Construction and identification of lentiviral vector mediated GATA-3 gene RNA interference in mouse

Objective:The aim of this study was to construct and identify a lentiviral vector for RNA interference of mouse GATA-3.Method:The coding region of the GATA-3 gene sequences were analyzed for a single-stranded oligo design and synthesis.PCR was performed for splicing reaction of synthetic oligo,which was synthesized into the lentiviral vector and transformed into the competent cell DH5α.An expression vector shRNA-GATA-3 was constructed after sequencing and identifying the gene sequence of the recombinant clones.Accoding to the target gene sequence ,four pairs are as following: siRNA-685 group(LV3-GATA-3-Mus-685),siRNA-1152 group (LV3-GATA-3-Mus-1152),siRNA-1615 group(LV3-GATA-3-Mus-1615) and control group ,which were designed and syntheized and builded into the lentiviral carrier.The shRNA vectors were cotransfected with expression vectors into the 293T cell line,and the optimal interference sequence was screened by QPCR. The 293T cells were cotranfected with the constructed lentiviral interference vectors and packing plasmids,the viruses were packed,the virus stock was collected and concentrated by ultracentrifugation,and the titer of the viruses was determined. Result:Gene sequence proved that the recombinant clone gene sequence was correct,and the lentiviral vector was successfully constructed. The relative expression of GATA-3 mRNA in siRNA-1615 group(0.004) was significantly reduced after RNA interference comparing with that in the control group(0.022),siRNA-1152 group(0.009) and siRNA-685 group(0.009). The screened LV3-GATA-3-Mus-1615 ,as the most effective interference sequence,was constructed into the lentiviral vector.The titer of the concentrated virus of LV3-GATA-3-Mus-1615 was 5×108 TU/ml in the viral supernate of 293T cells collected at 48 hours after co-transfection. Conclusion:Alentiviral RNAi expression vector targeting the GATA-3 gene (LV3-GATA-3-Mus-1615) was successfully constrcted and identified.

Extraction and purification of recombinant intact human Parathyroid Hormone (hPTH) from bacterial cell

Objective: The intact human Parathyroid hormone (hPTH) is one of the biopharmaceutical drug produced by biotechnology, this hormone can be provided in a good amount using simple batch culture, and therefore the main purpose of this study was the production of purified bioactive intact hPTH.Methods: A cloning BL21(DE3) strain (which already cloned in our Lab. by synthetic human Parathyroid hormone (shPTH) gene) was grown in LB medium and the cloning gene expression was induced by addition of IPTG to the medium. The recombinant fused protein was purified from the bacterial cell lysate by affinity chromatography and underwent proteolysis by Enterokinase enzyme to obtain the target hPTH, and it's further purified by DEAE and SP Sepharose ion exchange chromatography. RP-HPLC analysis and biological activity on the MCF-7 breast cancer cells were done for both the standard and produced hPTH. Furthermore, the haemolysis ability of the latter was tested on the human RBC.Results: 225ng/ml of hPTH was produced after SP chromatography which detected by specific PTH ELISA kit, standard and produced hPTH showed the same peaks in the same retention time when analyzed by HPLC. The produced hPTH haemolysis assay showed the ability of the produced hPTH to partially haemolyze the RBC in a concentration above 200µg/ml. The bioactivity assay for the produced and standard rhPTH demonstrated that both compounds had a biological activity on the MCF-7 cells with IC50 of about 84.4 and 75.7 µg/ml for standard and produced hPTH respectively, and no significant difference was detected between them.Conclusions: Via suitable purification processes, the biologically active hPTH with structure similar to the standard ones as detected by RP-HPLC and bioactivity assay, can be obtained by using already cloned isolate with shPTH gene for hormone production. The hormone showed haemolysis effect on the RBC similar to the normal secreted hPTH.

Forward Genetics by Sequencing EMS Variation-Induced Inbred Lines

In order to leverage novel sequencing techniques for cloning genes in eukaryotic organisms with complex genomes, the false positive rate of variant discovery must be controlled for by experimental design and informatics. We sequenced five lines from three pedigrees of ethyl methanesulfonate (EMS)-mutagenized Sorghum bicolor, including a pedigree segregating a recessive dwarf mutant. Comparing the sequences of the lines, we were able to identify and eliminate error-prone positions. One genomic region contained EMS mutant alleles in dwarfs that were homozygous reference sequences in wild-type siblings and heterozygous in segregating families. This region contained a single nonsynonymous change that cosegregated with dwarfism in a validation population and caused a premature stop codon in the Sorghum ortholog encoding the gibberellic acid (GA) biosynthetic enzyme ent-kaurene oxidase. Application of exogenous GA rescued the mutant phenotype. Our method for mapping did not require outcrossing and introduced no segregation variance. This enables work when line crossing is complicated by life history, permitting gene discovery outside of genetic models. This inverts the historical approach of first using recombination to define a locus and then sequencing genes. Our formally identical approach first sequences all the genes and then seeks cosegregation with the trait. Mutagenized lines lacking obvious phenotypic alterations are available for an extension of this approach: mapping with a known marker set in a line that is phenotypically identical to starting material for EMS mutant generation.

Expression, Docking, and Molecular Dynamics of Endo-β-1,4-xylanase I Gene of Trichoderma virens in Pichia stipitis.

It is essential that major carbohydrate polymers in the lignocellulosic biomass are converted into fermentable sugars for the economical production of energy. Xylan, the major component of hemicelluloses, is the second most naturally abundant carbohydrate polymer comprising 20–40% of the total biomass. Endoxylanase (EXN) hydrolyzes xylan into mixtures of xylooligosaccharides. The objective of this study was to genetically modify Pichia stipitis, a pentose sugar fermenting yeast species, to hydrolyze xylan into xylooligosaccharides via cloning and heterologous extracellular expression of EXNI gene from locally isolated Trichoderma virens species. Pichia stipitis was engineered to carry the EXNI gene of T. virens using pGAPZα expression vector. The open reading frame encodes 191 amino acids and SDS-PAGE analysis revealed a 24 kDA recombinant protein. The EXNI activity expressed by recombinant P. stipitis clone under standard conditions using 1% beechwood xylan was 31.7 U/ml. Molecular docking and molecular dynamics simulations were performed to investigate EXNI-xylan interactions. Free EXNI and xylan bound EXNI exhibited similar stabilities and structural behavior in aqueous medium. Furthermore, this in silico work opens avenues for the development of newer generation EXN proteins that can perform better and have enhanced catalytic activity.

Genome-wide association study of pre-harvest sprouting resistance in Chinese wheat founder parents.

Pre-harvest sprouting (PHS) is a major abiotic factor affecting grain weight and quality, and is caused by an early break in seed dormancy. Association mapping (AM) is used to detect correlations between phenotypes and genotypes based on linkage disequilibrium (LD) in wheat breeding programs. We evaluated seed dormancy in 80 Chinese wheat founder parents in five environments and performed a genome-wide association study using 6,057 markers, including 93 simple sequence repeat (SSR), 1,472 diversity array technology (DArT), and 4,492 single nucleotide polymorphism (SNP) markers. The general linear model (GLM) and the mixed linear model (MLM) were used in this study, and two significant markers (tPt-7980 and wPt-6457) were identified. Both markers were located on Chromosome 1B, with wPt-6457 having been identified in a previously reported chromosomal position. The significantly associated loci contain essential information for cloning genes related to resistance to PHS and can be used in wheat breeding programs.