Abiotic stresses affect plant productivity by modulationg various physiological and biochemical processes. Studies were performed to evaluate the influence of seed priming on the performance of barley varieties under late sown and abiotic stress conditions. For this purpose, a series of experiments was conducted in field and green house of University of Agriculture, Faisalabad, and glass house of Texas A&M University, USA. In first pot experiment, seeds of two barley varieties (viz. Haider-93 and Frontier-87) primed with water (hydropriming), CaCl2 solution (osmopriming) and Enterobacter sp. strain FD17 culture (biopriming) were sown in pots. After seedling establishment, drought levels (viz. 80, 60 and 40% water holding capacity) were imposed. In second pot experiment, same varieties and seed priming treatments were followed except after seedling establishment salinity levels (viz. 50, 100 and 150 mM NaCl) were imposed. Third experiment was carried out in hydroponics. Seedlings were raised in sand filled polythene bags by using same varieties and seed priming treatments. After stand establishment seedlings were transplanted in hydroponics then, osmotic (-0.8 MPa using PEG) and ionic (-0.8 MPa using NaCl) stresses were imposed. In fourth experiment, same procedure was followed as in the third experiment except cadmium (Cd) toxicity stress levels (viz. 0, 8 and 12 mg L-1 water) were imposed. In fifth experiment, seeds of USA cultivar Solum were primed with water (hydropriming) and CaCl2 (osmopriming), and sown in pots. At reproductive stage two levels of heat stress viz. control (25/18°C day/night) and heat stress (35/25°C day/night) were applied. In all pot and hydroponics experiments dry seed was taken as control. The pot and hydroponics experiments were carried out using completely randomized design (CRD) with factorial arrangement having four replications, except fifth experiment in which six replications were used. In sixth experiment, same varieties and seed priming treatments, as in first pot experiment, were followed and sown in field at November 30 and December 30. The experiment was conducted by using randomized complete block design (RCBD) with split-split plot arrangement having four replications. In first and second experiments, drought and salinity decreased plant growth, yield and chlorophyll contents, and perturbed the water and nutrient relations; while, increased accumulation of osmolytes and lipid peroxidation in both barley varieties, as compared to control. Moreover, salinity increased the sodium (Na) accumulation while decreased potassium (K) accumulation. However, seed priming improved plant growth, yield, tissue water status, cell membrane stability, chlorophyll contents and accumulation of phenolics, total soluble proteins, free proline and glycine betaine contents while decreased the malondialdehyde (MDA) content in both varieties under stressed conditions, as compared to unprimed control. The gretest improvement in yield under drought was caused by biopriming; whereas, under moderate and severe salt stress by biopriming and osmopriming, respectively. Moreover, biopriming improved the grain zinc (Zn), manganese (Mn) and boron (B) contents. In third and fourth experiments, osmotic, salt as well as Cd stress decreased the seedling growth and dry biomass in both varieties while increased the osmolytes and lipid peroxidation, as compared to control. Moreover, NaCl salt stress and Cd stress increased Na and Cd contents in barley, respectively. However, seed priming enhanced seedling growth, fresh and dry biomass, chlorophyll contents, phenolics, total soluble proteins, free proline and glycine betaine contents while decreased MDA, Na and Cd contents under stressed conditions, as compared to unprimed control. Under osmotic and Cd stress biopriming was most effective, while, under salt stress osmopriming was superior in improving barley performance. In fifth experiment, terminal heat stress hampered the plant growth, yield, leaf gas exchange and chlorophyll photochemistry while increased the phenolics and lipid peroxidation, as compared to control. However, seed priming improved the photosynthesis, stomatal conductance, carboxylation use efficiency (CUE), quantum yield of photosystem II (QY), electron transport rate (ETR), chlorophyll contents, phenolics and cell membrane stability while decreased MDA content under terminal heat stress, as compared to unprimed control, and osmopriming was superior in this regard. In sixth experiment, late sowing caused a reduction in emergence, growth, grain yield, dry matter accumulation, grain filling duration, chlorophyll contents, and grain crude protein and starch contents in both barley varieties, as compared to optimum sowing time. However, seed priming improved emergence, plant height, crop growth rate (CGR), total dry matter accumulation (TDM), leaf area index (LAI), grain filling rate, yield and related traits, and grain crude protein and starch contents under both optimum and late sowing, as compared to unprimed control. The greatest improvement was caused by osmopriming followed by biopriming. The economic analysis showed that late sowing decreased economic returns as well as benefit cost ratio (BCR) which was improved by seed priming treatments. Among all, biopriming caused maximum improvement in BCR and marginal rate of return (MRR). In all pot and field experiments, variety Haider-93 performed better than Fronteir-87. In conclusion, abiotic stresses and late sowing decreased the plant growth and yield by negatively affecting plant physiological processes. However, performance of barley varieties was effectively improved by seed priming treatments under stressed conditions by improving the water relations, nutrient relations, osmolytes accumulation, photosynthesis, chlorophyll contents and decreasing the lipid peroxidation under stressed conditions.
پاکستانی معاشرے میں حدود وقصاص قوانین سے واقفیت اور ان پر عمل درآمد کے حوالے سے عوامی شعور کی کمی ہے، جس کی وجہ سے لوگ ان قوانین کے نفاذ کے لیے جدوجہد نہیں کرتے ہیں اور نہ گرم جوشی کا مظاہرہ کرتے ہیں۔ صحیح معنوں میں لوگ شریعت کی روح سے واقفیت رکھتے ہی نہیں۔ وہ اسلام کو صرف عبادات اور اخلاقیات کا مجموعہ سمجھتے ہیں ۔ اسلام تو حقوق اللہ اور حقوق العباد دونوں کا مجموعہ ہے ۔ عبادت سے تو صرف جنت ملتی ہے اور خدمت سے خدا ملتا ہے ۔ اس فلسفہ سے آگاہی ہر شخص کو نہیں۔ لہذا اس امر کی ضرورت ہے کہ عوام الناس میں اسلامی معاشرے کی اخلاقی اقدار کے تحفظ ، انسانیت کی خدمت کا جذبہ بیدار کیا جائے ، تاکہ اسلامی قوانین کے نفاذ میں کوئی رکاوٹ پیدا نہ ہو۔
Hazrat Muhammad (SAW) is the last Apostle to human beings. He was gifted with a divine Deen having complete code of life. Every field of life has been discussed in the Holy Quran and Sunnah of the Prophet r. As an Apostle, head of the state and army commander, He guided the mankind and provided an excellent example in all the perspectives of life. As a commander of the Islamic forces, the Holy Prophet r fought twenty seven Ghazwat after migration to Madina. In Zeqaida 6 AH, during the pact of Hudaibia a complete turn was taken by Muslims. After this event, the Muslim army role changed to offensive rather than defensive. Immediately, after the pact, the Holy Prophet r attacked on Khyber in Muharram 7th AH, while the whole Hijaz region was captured during the Ghazwa Fath-i-Makkah. In this article, the strategy and tactics employed by the Holy Prophet r during Hudaibia truce have been discussed. These tactics are useful and beneficial in modern era warfare also. As an ideal for all the Ummah, lessons should be extracted by the commanders to defend their motherland and ideological boundries.
Thecurrent study involves cloning, sequence analysis, expression, secretion, purification and different factors influencing the secretion of ovine growth hormone (oGH) gene isolated from local ovine breed (Lohi). On the basis of conserved sequences, two forward and one reverse primers were designed for the amplification ofoGH gene. The forward primers contained NdeI, NcoI restriction sites whereas the reverse primer contained a BamHI site at their 5’ end. Total RNA was isolated from pituitary gland of Lohi by using Guanidium-thiocyanate-chloroform extraction method. cDNA was synthesized by RT-PCR using gene specific primers. Moreover,genomic DNA was isolated from the blood sample of Lohi and was amplified by using four sets of primers designed on the basis of conserved sequence of the ovine growth hormone (oGH) gene. These were ligated into pTZ57R/T by the dT. dA tailing technique and used to transform into E. coli DH5α. The sequences of the DNA obtained from multiple colonies were compared with already published ovine GH gene sequence using multiple sequence alignment software “Clustal W”. The sequence analysisrevealed only one amino acid change when compared to previously reported OaST (Ovis aries somatotropin) or oGH gene sequences of Indian and Australian breeds. It showed 99% homologies with bubaline, bovine and 100 percent homology with caprine GH genes of the local breeds. The sequence of the GH ofLohi was submitted to "Data bank of Japan" which bears an accession number AB244790. In the present study, wereport secretion of recombinant oGH into the periplasmic space and inner membrane of E. coli under the influence of variant signal sequences. For periplasmic translocation the recombinant proteins were expressed under the influence of pelB leader sequence of pET 22b vector. The effect of different factors i.e., glycerol in the medium, use of E .coli strain BL21 DE3 and pLys S ,chemical chaperon (ZnCl2) and IPTG concentration were studied to enhance the expression while osmotic shock conditions were also optimized and studied the effect of glycerolandZnCl2 concentration on the release of oGH by using freeze thaw method. Best result of 22% expressed roGH on 12% SDS-PAGE was observed at 20mM (final concentration) IPTG after 4 hrs of fermentation at 370C in LB modified medium with 50µM ZnCl2 in BL21DE3 E. coli strain. The optimized freeze thaw method including 25% glycerol with 50µM ZnCl2 enhanced the relase of oGH upto 24% in the periplasmic space of E. coli. The oGH thus found was further purified by FPLC and authenticated by Western blot analysis. Although the recovery of oGH was enhanced but still there was a need to enhance the production of accurate size (22 kDa) growth hormone which was bit higher (25 kDa) by using pelB leader sequence. For this purpose different signal peptides i.e.,DsbA,STII and natural oGH signal peptide were utilized. Amongst the signal sequences the DsbA signal sequence was found to exhibit the best expression, size and secretion of oGH into the inner membrane of E. coli.We further studied the expression of oGH and targeting to the inner membrane using signal sequence (DsbA) in E.coli cell. Factors such as temperature, IPTG induction, expression conditions were studied and showed diverse optical density with different media compositions. The optimum expression level of oGH in terrific broth medium was at 25ºC on induction with 20μM IPTG in early logarithmic phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of oGH to the inner membrane of E. coliwith DsbA signal sequence at the N terminus of roGH. The protein was easily solublized by 40% acetonitrile with ~90% purity and was identified by Western blot and analysis on MALDI/TOF confirmed a size of 21059Da. Relatively high soluble protein yield of 65.3gm/L of oGH was obtained. The biological function of oGH was confirmed by HeLa cell line proliferation.It was observed that DsbA signal sequence on the basis of its hydrophobicity gave best results of 22kDa protein in membrane bounded form as compared to pelB and reference native signal sequence of oGH which resulted in 25kDa oGHsecreted mainly into cytoplasm. Despite of cost effective single step purification we encountered a problem with low yield. We developed a novel strategy for the high yield of functional recombinant ovine growth hormone (roGH) directed to the inner membrane of E. coli.In order to enhance the yield of soluble fraction, bacterial cells were grown under osmotic stress (4% NaCl in terrific broth medium) and effect of compatible solutes (sorbitol, glycine betine, glycylglycine and mannitol) were studied on the soluble expression of roGH. Other factors; temperature, induction time, induction by IPTG and lactose were also studied. It was observed that fermentation of roGH construct with DsbAss was best achieved with 0.6M mannitol, 50μM ZnCl2, 50mM glycylglycine at the time of induction with 50μM IPTG in the early logarithmic phase at OD600 ~3.10 in TB medium at 25ºC in shaking flask culture at 150rpm. These optimized conditions resulted in very high expression ~32% of soluble roGH which was recovered by ultra centrifugation (density centrifugation) from the inner membrane of E. coli.The unbelievably high yield, 443mg/L was obtained as compared from previos yield. The roGH was confirmed by Western blot analysis . Furthermore the effect of amino acid substitution in the tripartite structure of DsbA signal sequence (DsbAss) on co-translation of recombinant oGH inE. coli was studied. Six amongst the eight constructs were designed on the basis of increasing hydrophobicity in H domain of DsbA signal sequence to make it more efficient for the translocation of oGH through SRP (signal recognition particle)mechanism. For this purpose all the alanines in the hydrophobic domain of DsbA signal sequence were replaced by Isoleucine one by one, while lysine in the N terminal and serine in the C-terminal regions were substituted by arginine and cysteine respectively. The substitution of arginine in the N-terminalresulted in very low expression and secretion while cysteine substitution in the C region totally impaired the expression and secretion of the recombinant protein. it was observed that not only the hydrophobicity but the position of amino acid in the hydrophobic core also effects thecleavage of signal sequence from recombinant product. The substitution of alanine with the isoleucine residue in H domain of DsbA signal sequence resulted in; (a) at position 11 with respect to signal peptidase site in the H domain impaired the correct processing of oGH protein while (b) isoleucine at position 9 resulted in correctly processed recombinant oGH protein in the inner membrane.The results showed that the replacement of alanine amino acid at position 11 with reference to signal peptidase site in the hydrophobic core of the DsbA ss interferes with the binding of DsbA ss hydrophobic region to Ffh protein of SRP. This resulted in weak or no binding of Ffh with DsbA ss and consequentlyoGH protein was localised in the cytoplasmic fraction rather than membrane. Thus, the gene mutation from alanine residue to isoleucine specifically at position 11 with respect to signal peptidase site changed the whole mechanism of protein translocation through DsbA ss. It was hypothesized that alanine at position number 11 with respect to the signal peptidase site is crucial for SRP routing of recombinant proteins .