Advancements in Life Sciences (E-Journal, University of the Punjab)
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Plant genome editing using engineered nucleases and success of CRISPR/Cas9 system
Get PDFDevelopment of new plant breeding techniques have facilitated easy manipulation of plants at genetic level. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein9 (Cas9) system is a valuable addition in programmable nucleases. The CRISPR/Cas9 system uses an RNA component to recognize a target DNA sequences and it has shown promising results with respect to simultaneous editing of multigenic plant traits. In this review, components of CRISPR/Cas9, their construction and its methods of delivery to plant cells are analyzed. Variation in nucleotide sequence of the protospacer adjacent motif, codon optimization and progress in web-based bioinformatic tools, will make CRISPR/Cas9 systems more efficient for plants. Development and optimization of protocols to efficiently target all plant species is still under development. Along with this, methods to inspect induced mutation and efficiency of the system have also been reviewed. Auxiliary improvements and understanding are still required to expand the CRISPR/Cas9 systems to target complex genome architectures and epigenetic elements
Confirmation of root-knot nematode resistant gene Rmi1 using SSR markers
Get PDFBackground: The Root Knot Nematode (RKN) is a serious economic threat to various cultivated crops worldwide. It is a devastating pest of soybean and responsible to cause severe yield loss in Pakistan. The cultivation of resistant soybean varieties against this pest is the sustainable strategy to manage the heavy loss and increase yield. There is an utmost need to identify RKN resistant varieties of soybean against cultivated in Pakistan. The presented study is an attempt to identify and confirm the presence of resistant gene Rmi1 in soybean. Method: Molecular studies have been done using Simple Sequence Repeat (SSR) marker system to identify resistant soybean varieties against Root Knot Nematode (RKN) using fifteen (15) indigenous cultivars and four (4) US cultivars. DNA was isolated, purified, quantified and then used to employ various SSR markers. The amplified product is observed using gel documentation system after electrophoresis. Results: Diagnostic SSR markers Satt-358 and Satt-492 have shown the presence of Rmi1 gene in all resistance carrying genotypes. Satt-358 amplified the fragment of 200 bp and Satt-492 generated 232 bp bands in all resistant genotypes. This study confirmed the Rmi gene locus (G248A-1) in all internationally confirmed resistant including six (6) native varieties.Conclusion: These investigations have identified six (6) resistant cultivars revealing the effective and informative sources that can be utilized in breeding programs for the selection of RKN resistance soybean genotypes in Pakistan.
Re-engineering of RuBisCO for the purpose of producing algal biofuels may be a lost cause
Get PDFProponents of algal biofuels hope that photosynthesis could be improved, which would make them economically feasible. One of the means to do that would be by engineering and making the slow enzyme RuBisCO more efficient. In this commentary article I discuss the possibility it could be a lost cause, because the proper place of the enzyme in the evolutionary history is still misunderstood. The interest in algal biofuels was only moderate prior to 2006-2007 [1], after which the publications of research articles on this subject bloomed. One of the articles that facilitated the ignition of the scientific debate was written by Chisti in 2007 [2] and as of 29 August 2016, this article has been cited totally 6276 times, including self-citations (Google Scholar Data). Regardless of the hype, the prospects of biofuel production from microalgal biomass are unrealistic in the near future. Research teams in our department have published two articles on the subject– in 2012 [3], concerning biodiesel, and in 2015 [4], concerning other types of algal fuels: bioethanol, biohydrogen, biogas, hydrocarbons, and direct burning of algal biomass. In both manuscripts the conclusions are clear: mass production of algal biomass and its subsequent treatment to extract valuable components remain difficult and expensive and as such, algae should be best utilized as food and not as fuel.Now I’d like to add more to the discussion of algal biofuels by focusing on only one of the significant obstacles. If we would really like to produce biofuels in a fast and efficient way, a primary goal would be to fixate CO2 as quickly into organic compounds as possible. This leads straight to the point. The enzyme RuBisCO, responsible for the first major step of the carbon fixation, is very slow, being only able to fix 3 CO2 molecules per second. Not only that - the problem with RuBisCO is also tied to its oxygenase ability. Petkov et al [3] have already pointed out that if the algae are not supplied with additional CO2, after the depletion of the naturally available CO2 the enzyme starts using O2 as a substrate due to its oxygenase ability. As a result photorespiration levels are higher, and the quantity of the biomass is low. Regrettably, supplying algae with additional CO2 adds to the cost. Chatterjee & Basu [5] discuss the possibilities of reengineering and improving RuBisCO, as they describe it as “the world's most abundant protein is perhaps the most incompetent enzyme”. The authors claim that familiar tools of the Green Revolution are providing diminishing returns and according to them RuBisCO is a tempting target.There is certain research dedicated to re-engineer the RuBisCO and changing CO2/O2 specificity factor. Unfortunately, results from experiments with cyanobacteria are so far met with mixed success. Durall and Lindbald [6] have written a very detailed review article about engineering approaches in cyanobacteria for increased carbon fixation. Concerning RuBisCO substitutions of certain amino acids may lead to an increase of the specificity factor, but simultaneously to a decrease of CO2 fixation rate. Some experiments lead to a decrease of the specificity factor and there are also some who report both increase of the specificity factor and the CO2 fixation rate. But overall, these researchers say that “it has not been possible to significantly improve the RuBisCO specificity for CO2, the affinity to CO2 and/or the carbon fixation rate”, “the enzyme may already be optimized through evolution”, and “it still remains unclear if a significant improvement of the carboxylation by RuBisCO can be achieved”. Could the enzyme be indeed already optimized? This is a very interesting question that needs to be discussed in-depth. Evolutionary, RuBisCO is described as a relic of a bygone age, it has evolved under conditions different from these of today and it hasn’t changed significantly since then [7]. This could easily make some researchers think that RuBisCO is probably maladapted and non-efficient in its function. But could it be that it is simply misunderstood? This question was asked by Morell et al [8] in 1992, more than two decades ago, but since scientists continue suggesting RuBisCO could be improved, probably it’s still relevant today. Researchers may hope that engineering methods would significantly improve RuBisCO’s performance, however, if it hasn’t already happened before by natural means, is this hope justified? Here it must be reminded that during the later stages of the Earth history, internal CO2 concentration mechanisms appeared, for example, the C4 mechanism in higher plants [9]. It seems that evolutionary forces pushed neither for drastic modifications of the structure of RuBisCO, nor for an alternative catalytic system. Instead, they pushed for supplementary mechanisms that concentrate CO2 and saturate RuBisCO’s catalytic capacity.ConclusionI suggest the engineering efforts should focus on improving these, rather than touching RuBisCO. But so far the goal to create fast growing algae which are independent of additionally supplied CO2 remains unmet and I still think that the future of obtaining biofuels from algal biomass remains bleak
Functional characterization of fifteen hundred transcripts from Ziarat juniper (Juniperus excelsa M.Bieb)
Get PDFBackground: Ziarat juniper (Juniperus excelsa M.Bieb) is an evergreen and dominant species of Balochistan juniper forests. This forest is providing many benefits to regional ecosystems and surrounding populations. No functional genomics study is reported for this important juniper plant. This research is aimed to characterize the Ziarat juniper functional genome based on the analyses of 1500 transcripts. Methods: Total RNA from shoot of Juniperus excelsa was extracted and subjected for transcriptome sequencing using Illumina HiSeq 2000 with the service from Macrogen, Inc., South Korea. The Illumina sequenced data was subjected to bioinformatics analysis. Quality assessment and data filtration was performed for the removal of low-quality reads, ambiguous reads and adaptor sequences. The high-quality clean reads data was deposited in the Sequence Read Archive (SRA) at NCBI, and used for downstream processes. Fifteen hundred transcripts were randomly chosen and used for functional characterization.Results: As a result of homology search 80.3% transcripts showed significant similarities and were placed in significant similarities category, 19.3% transcripts showed low similarities and assigned to the ‘‘unclassified’’ category while 0.4% transcripts are defined as no hits. The functional characterization results showed that most (18%) of the transcripts are involved in metabolism, followed by 11.7% in transcription and 11.5% as structural protein. 8.8% transcripts are engaged in stress response, whereas the transcripts involved in growth and development constituted 6.7%. Transcripts involved in signal transduction represented 5.6%, while 3.5% facilitating transport and 34.1% are involved in hypothetical functions.Conclusion: The functional annotation data produced in this study will be very useful for future functional genome analysis of Juniperus excelsa.
DNA Barcoding: Amplification and sequence analysis of rbcl and matK genome regions in three divergent plant species
Get PDFBackground: DNA barcoding is a novel method of species identification based on nucleotide diversity of conserved sequences. The establishment and refining of plant DNA barcoding systems is more challenging due to high genetic diversity among different species. Therefore, targeting the conserved nuclear transcribed regions would be more reliable for plant scientists to reveal genetic diversity, species discrimination and phylogeny.Methods: In this study, we amplified and sequenced the chloroplast DNA regions (matk+rbcl) of Solanum nigrum, Euphorbia helioscopia and Dalbergia sissoo to study the functional annotation, homology modeling and sequence analysis to allow a more efficient utilization of these sequences among different plant species. These three species represent three families; Solanaceae, Euphorbiaceae and Fabaceae respectively. Biological sequence homology and divergence of amplified sequences was studied using Basic Local Alignment Tool (BLAST).Results: Both primers (matk+rbcl) showed good amplification in three species. The sequenced regions reveled conserved genome information for future identification of different medicinal plants belonging to these species. The amplified conserved barcodes revealed different levels of biological homology after sequence analysis. The results clearly showed that the use of these conserved DNA sequences as barcode primers would be an accurate way for species identification and discrimination.Conclusion: The amplification and sequencing of conserved genome regions identified a novel sequence of matK in native species of Solanum nigrum. The findings of the study would be applicable in medicinal industry to establish DNA based identification of different medicinal plant species to monitor adulteration
Detection of Endothelin 2 (Et-2) as a novel biomarker of renal dysfunction in patients of liver Cirrhosis with Ascites
Get PDFBackground: Hepatorenal dysfunction in liver cirrhosis is a condition in which there is progressive kidney failure. It is a serious complication that can lead to death. Endothelin is an endothelial cell-derived peptide which is involved in renal dysfunctioning by generating oxidative stress in individuals suffering from liver cirrhosis. It can be used as important biomarker of hepatorenal dysfunction. The present study was aimed to detect endothelin-2 in the blood of patients of liver cirrhosis with ascites as important biomarker correlated to the generation of oxidative stress and renal dysfunctioning. Methods: 50 blood and ascitic fluid samples were collected from the patients of liver cirrhosis with ascites as case group and 25 from normal individuals as control group. Endothelin-2 was detected by PCR in serum and ascetic fluid of patients and its quantification was done by Real-time PCR. ET-2 was quantified in serum samples but was not quantifiable in ascetic fluid might be due to its very low concentration. Blood urea, creatinine and uric acid with GFR were estimated as renal dysfunction parameters. Anti-oxidative enzymes glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MAD) as biomarkers of oxidative stress were estimated in order to calculate oxidative stress. The obtained data was evaluated statistically by using t-test and Pearson correlation.Results: Positive correlation was found between individual parameters of oxidative stress and renal dysfunction. Overall positive and highly significant (P<0.05) correlation of CAT, SOD, MDA, GSH levels with each other was observed, a significant (P<0.05) and positive correlation of oxidative stress parameters were found with urea, creatinine and uric acid with Glomerular filtration rate (GFR).Conclusions: It was concluded that the production of ET-2 under oxidative stress generates renal dysfunction in patients of liver cirrhosis with ascites
Association patterns of volatile metabolites in urinary excretions among Type-2 Non-Insulin dependent diabetes patients
Get PDFBackground: Patterns of volatile metabolites in urine are important to detect abnormalities associated with diabetes. Present study was conducted to find out the excretion patterns of endogenously produced alcohols in urine for type 2 (Non-Insulin Dependent) diabetes mellitus. A cross sectional analytical study was conducted with duration extended from Jan to Mar 2015.Methods: The current study included 40 patients with chronic type 2 diabetes mellitus. In total, 10 sex and age matched subjects with no history of any disease were considered as controls. Blood sugar was estimated by autoanalyzer using standard kit of Merck following manufacturer`s instructions. Urine sugar was quantitatively detected by biuret reagent using titration technique. Urinary alcohol was identified and estimated by gas chromatography. Urinary ketone bodies were estimated by urinary strip.Results: It was observed that level of fasting blood sugar was significantly increased (P<0.001) in patients as compared to their controls. The blood sugar and urinary alcohol in patients were 3.0% and 6.0% respectively. Urinary ketone bodies were found to be 2+. On the other hand urine sugar, alcohol and ketone bodies were not detected in the negative control subjects.Conclusions: It is concluded that urinary alcohol is endogenously produced in patients with type 2 diabetes due to uncontrolled hyperglycemia. However further work is needed to find out the ratio of urinary and blood alcohol which may confirm the present findings
Screening for drought tolerance: comparison of maize hybrids under water deficit condition
Get PDFBackground: Maize is an important cereal crop, grown throughout the globe for human food and livestock feed, but biotic and abiotic factors had shown adverse effects on biomass and grain yield. Changing climatic conditions have imposed drought (water scarcity) as a major problem to combat with yield losses and biomass in maize and other cereals. Methods: The prescribed study was conducted to evaluate F1 hybrids at seedling subject to 45% moisture level. The data was recorded and subjected to multivariate analysis to find the significant variation attributed by various traits under stress conditions for efficient root/shoot development.Results: Significant differences were found in F1 hybrids for all studied traits. Higher heritability was found for root length, shoot length and fresh shoot weight, while fresh shoot weight, dry shoot weight and dry root weight showed higher genetic advance. Significant correlation was found for dry root weight to fresh shoot length and fresh root length, fresh root weight to fresh root/shoot weight ratio and fresh shoot weight. The hybrids Sh-139×B-316, Raka-poshi×B-316, B-327×B-316, Sh-139×EV-340, EV-1097Q×EV-347, EV-1097Q×EV-340, EV-1097Q×Pop/209 and B-327×EV-340 showed higher and positive heterosis and heterobeltiosis for most of the studied traits. To assess the overall variation on dependent structure, we used multivariate analysis, an important tool in breeding program, for efficient selection.Conclusions: EV-1097Q×Pop/209 and Sh-139×EV-340 showed significant results for root and shoot development under various water stress regimes at seedling stage, thus further studies should be carried out to find out the known and un-known loci regarding root and shoot development traits in high yielding maize cultivars under arid/semi-arid regions
Qualitative Phytochemical Analysis and Microbial Inhibitory Activities of Pacific Rain Tree (Samanea saman (Jacq.) Merr.) Pods
Get PDFBackground: Crop diseases and human health are always at stake and the emerging problem on the use of synthetic anti-pathogens and medicine is one of the most difficult to combat. The first step towards determining such capabilities among plants is to determine their phytochemicals.Methods: Eight preliminary phytochemical tests was done on Samanea saman which includes, test for alkaloids saponins, flavonoids, tannins, glycosides, steroids, terpenoids and resins. Powdered pods were subjected to ethanol and aqueous extraction. Extracts were also tested for its antifungal and anti-microbial properties against Fusarium oxysporum, E. coli and S. aureus,respectively.Results: Out of the eight phytochemical tests done, seven (7) were found to be present both on the ethanol and aqueous extracts namely, alkaloids, saponins, tannins, glycosides, steroids, terpenoids and resins. However, flavonoids is absent. The statistical results exhibited that there is a significant difference on the inhibitory effects against in-vitro bioassay of Fusarium oxysporumwhich is known to cause crop wilts and the two bacterial pathogens E. coli and S. aureus.Conclusions: The presence of such phytochemicals in Samanea saman pods revealed that it can be a basis of new, natural and non-synthetic treatments. This finding suggests that its pods can be used as antibacterial and antifungal source
Genetic diversity and evolutionary analysis of Citrus Tristeza Virus p20 gene in Pakistan: insights into the spread and epidemiology
Get PDFBackground: Citrus tristeza virus (CTV) is a widespread disease and the most destruction causing agent of citrus. Pakistan is ranked amongst the top ten citrus producing countries around the globe and it contributes about 2% to its foreign exchange earnings. Based on this assumption it is very important to monitor and determine the evolutionary forces and the phylogeography of Pakistani CTV population. Methods: A total of 49 sequences of p20 gene from Pakistan were phylogenetically compared with CTV sequences worldwide. These sequences were analyzed for their genetic diversity and evolution using a Bayesian Probability approach and predicted secondary structure.Results: Phylogenetic analysis using Bayesian probability inference and predicted secondary structures diversity of CTV indicated that Pakistani isolates were not diverse from global isolates. Lineage analysis showed that CTV was introduced in Pakistan in three individual events from various parts of the world. After that CTV dispersed in Pakistan via vector transmission or by use of infected propagating material by local farmers.Conclusions: Our study confirmed multiple introductions of CTV in Pakistan and also confirmed the dissemination of CTV within Pakistan. This study also shows that the mutations are present in the predicted secondary structure of the p20 protein, however, it is not known if it affects the pathogenicity of the virus