Genetic Variability Of Sahiwal And Cholistani Cattle Breeds Of Pakistan Usin Mitochondrial D-Loop Sequences
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Publisher: 2011 Dissertation note: Pakistan is rich in cattle genetic resources. The phenotypic and genetic diversity of animal breeds in Pakistan is very vast. Efforts to manage and utilize these genetic resources efficiently are lacking due to lack of both awareness and weakness of Government institutions. The genetic data of dairy cattle breeds (Sahiwal and Cholistani) is not yet been studied for their genetic identification, conservation and to find the genetic diversity among them and it needs to be established. For this study the blood samples( 25 samples from each breed) were collected from their home tracts and livestock farms. Unrelated animals with typical phenotypic features known for Sahiwal and Cholistani cattle breeds were selected from their breeding areas and Government livestock farms. Blood samples from true representative individuals of Sahiwal breed were collected from Research Centre for the Conservation of Sahiwal Cattle (RCCSC), Jahangirabad, Khanewal, Semen Production Unit (SPU) Qadirabad and Barani Livestock Production & Research Institute (BLPRI), Kherimurat District Attock. Cholistani cattle samples were collected from Govt. Livestock Farm, Jugaitpeer, Bahawalpur. Sampling from siblings was avoided to minimize inbreed samples as it results in depleting of gene pool along with causing inbreeding depression.DNA was extracted and quantified with the standard protocol in Molecular Biology and Genomics Laboratory of Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore. Specific primers were designed by using special software i.e. Primer Fox for mitochondrial D-loop region from NCBI accession no. NC_006853.1.Primers optimization was done after primer designing and afterwards, PCR amplification was performed. Then sequencing of target fragments was carried out using Prism ABI 3130L sequencer and Analyser.Sequences were alligned with the help of software blast2sequence and SNPs were detected. It was found that ratio of transition mutation was higher than transversions i.e. 41 transition and 10 transversions. Sequences were analyzed and compared with already reported sequence of Mitochondrial DNA of Bosindicuss, Bostaurus, Bubalusbubalis, Canis lupus familiaris, Caprahircus, Equuscaballusisolate, Ovisaries and Cameliusdromedaries sequencesavailable at NCBI. Single Nucleotide Polymorphisms (SNPs) were then detected. A phylogenetic tree constructed using MEGA 5.1 software revealed that Pakistani, European and Asian cattle are genetically same but different from Buffalo.This work is very helpful about breed characterization of two cattle breeds (Sahiwal and Cholistani) and developing understanding about genetic architecture of cattle breeds as present study conclude that 52 SNPs were present in Sahiwal and Cholistani breed of Pakistan.
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Assessment Of Genetic Diversity In Balochi And Rakhshani Sheep Breeds Of Balochistan Using Microsatellite Dna
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Publisher: 2011 Dissertation note: Pakistan being agriculture based country has a great potential in livestock sector, it plays an important role in the economy of the country. Pakistan is rich in Animal Genetics Resource (AnGR) and has various breeds of sheep but lacking genetic data of these breeds which need to established data for their genetic identification. Customarily, classification of breed was based on phenotypic traits. In some cases, recent genetic studies have found differences in the structure proposed. Molecular characterization is a prevailing tool to consider the genetic variation existed within and among breeds. Characterization and evaluation of genetic differences among these breeds is necessary for their effective and meaningful improvement and conservation. The advent of molecular techniques has led to an increase in the studies that focus on the genetic characterization of domestic breeds using genetic markers. Due to their reliability and availability, the microsatellites have become preferred method for the genome mapping. Microsatellites or STRs are the 2-6 nucleotide tandem repeats present in both coding and non coding regions of both prokaryotes and eukaryotes. Microsatellites are powerful tools in genome mapping, forensic DNA studies, paternity testing, population genetics and conservation/ management of biological resources.
The present study was conducted on the molecular diversity analysis of two sheep breeds Balochi and Rakhshani of Balochistan using 11 FAO recommended microsatellites markers. Blood samples of unrelated true representative animals of sheep breeds were selected from their breeding tracts and from different Government Livestock Farms in Balochistan province. DNA was extracted with the standard protocol and amplification of DNA done with selected markers in Molecular Biology and Genomics Laboratory in the Institute of Biochemistry and Biotechnology. PCR products were examined on non denaturing Polyacralamide Gel Electrophoresis (PAGE). Genotyping results vanalyzed through the software POPGENE VERSION 1.31 and "POWER STATE" for calculating the observed and expected number of alleles, expected and observed heterozygosity, homozygosity, F-statistics (FST, FIT, FIS), Polymorphic Information Content (PIC), matching probability power of discrimination and power of exclusion. This work provided the genetic data which is useful in breed identification and making effective breeding policies and conservational activities in future according to FAO global Farm Animal Genetic resource data.
Average observed heterozygosity, average observed homozygosity, observed number of allels (na) and expected number of alleles for all loci and population in this study were 0.6055, 0.3945, 6.3636 and 4.2805 respectively. Almost all of the microsatellite markers showed significant variations in both breeds of sheep.
This work provided the genetic data which will be helpful in breed identification and making effective breeding policies and conservational activities in future according to FAO global Farm Animal Genetic resource data. Moreover this study can become the basis for further research investigations in sheep breeds in Balochistan and Pakistan.
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Production, Purification And Characterization Of Laccase From White Rot Fungus
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Publisher: 2014 Dissertation note: Laccase (oxidoreductase, EC 188.8.131.52) are blue copper dependent oxidases and the mainligninolytic enzyme produced by white rot fungus. Laccase catalyze the oxidation of large snumbers of phenolic compounds (Kunamneni et al. 2007; Poonkuzhali et al. 2011). These enzymes have a molecular weight 60-90 kDa and consist of 15–30% carbohydrate. Laccases are the earliest and maximum investigated enzymatic systems. Laccase was initially found by Yoshilda in 1883 in the sap of Japanese laquer tree named as Rhusvernicifera. After a while in 1896, Bertrand and Laborde determined that laccase is a fungal enzyme.(Shraddha et al. 2007; Giardina et al. 2010).
Laccases are present extensively in nature, originating from plants, bacteria and fungi (Poonkuzhali and Palvannan 2011). In fungi, laccases are widely distributed in ascomycetes, deuteromycetes and basidiomycetes. The laccase producing fungus include Trametes versicolor, Pleurotus ostreatus, Polyporus, Trametespubescens, Cerrenaunicolour,PhanerochaetechrysosporiumandFunaliatrogiietc (Dwivediet al. 2011). Laccases occur morein fungi, than in the higher plants. Laccases are also present in few bacteria such as S.lavendulae, S.cyaneus, and M.mediterranea(Viswanath et al. 2008; Arias et al. 2003). In vegetables laccases have been recognized in turnips, apples, pears, cabbages, potatoes, beets, asparagus and various other vegetables (Jhadav et al. 2007).
Enzymes are produced by every living organism, however enzyme produced by microbes have various benefits over the enzyme originated from plants and animal origins.Laccases by nature
are important because of its huge diversity of catalytic activities, economical in production and comparatively more stable than other enzymes.The field of biotechnology proposes expanding possibilities for the production of several enzymes from microorganisms. New methods and techniques have been advanced by using enzyme as biocatalysts to produce big added value products like growing food requirements,good quality chemicals and medicines. Moreover enzymes are also utilized for environmental actions and for diagnostic and analytical motives. (Buchholz et al. 2005). Microbial enzymes are used as cost effective and environmentally sensitive substitutes for chemical processing in several industries and bioremediation. Therefore the commercial demand for microbial enzymes is increasing (Radhika et al. 2013).
Fungal laccases have boundless biotechnological functions across the globe like the decolouration and detoxification of industrial effluent, bleaching of pulp, phenolicselimination from wines, in preparation of biosensors in detergents blockindye transfer- functions (Yaver et al. 2001).It is also used in the formation of anticancer drugs, and included in few cosmetics to lessen their toxicity (Couto and Herrera 2006).In recent years, laccase have been skillfully practiced to the field of nanobiotechnology due to its capacity to mobilize electron transfer reactions without further addition of cofactor(Shraddha et al. 2007).
Laccase is ample in several white- rot fungi that are involved in lignin metabolism (Bourbonnais et al. 1997, Leontievskyet al. 1997). Fungal laccases have immense redox potential (up to +800 mV) than bacteria or plant laccases. The action of these laccases seems to be appropriate in nature and also has significant applications in the field biotechnology. These laccases are associated with the deterioration of lignin and also in the elimination of conceivably lethal phenols appear during the breakdown of lignin (Thurston et al. 1994).
The white rot fungus is corporeal in preference to morphological and composes of those fungi that are adequate of degrading lignin, which is a heterogenous polyphenolic compound in huge amount within the lignocellulose wastes(Eaton and Hale. 1993).Theircapability to deteriorate cellulose, hemicellulose, these are the polysaccharides forming the essential part of lingo cellulose is the basic metabolic processbetween the fungi and happen under the span of environmental conditions.The degeneration of lignindoesn’tprovide net energy so it is degraded during the secondary metabolism in order to gain polysaccharides present in lignin and carbohydrate complexes, supplying energy to which the organisms don’t have access(Jeffrics. 1990).The white rot fungi varyingly secrete one or more three extracellular enzyme namely manganese peroxidase, lignin peroxidase and laccase that are fundamental for degradation of lignin, ant they are generally mentioned as lignin modifying enzymes LMEs (Pickard et al. 1999).
Laccase is the subjects of demanding research in the recent years, because of their several properties like extensive substrate relevance, doesn’t required the inclusion of cofactors because they use oxygen as cofactor which is frequently present in the environment (Eugenio et al. 2009). Maximum number of laccases produced by various organisms is excreted as extracellular enzymes and this makes the purification process quite accessible. Laccase commonly display appreciable extent of stability. Due to these properties laccases are ideally applicable in diverse biological processes such as the treatment of industrial effluent, biopulping and biobleaching (Eggert et al. 2006).
The huge potential of laccase requires advancement in its production and, with huge activities and low cost (Herrera et al. 2007). The use of lignocellulosic agricultural waste as substrates is a tradition for the production of enzyme like laccase because it is ligninolytic in nature (Niladeviet al.2011). It is highly crucial to optimize the fermentation parameters for the adequate production of laccase (Revankaret al. 2007). .
The advantages of agro-industrial leftovers for cultivation media is of immense concern as agriculture waste cut down the expenditure of enzyme production and enhance the understanding on energy protection and recycling (Mansuret al. 2003).These agriculture wastes are comparatively economical and also contain ample nutrients such as lignin, cellulose andhemicellulose. These nutrients serve as inducer to energize the production of enzyme (Vassil et al. 2000).Due to these properties these agricultural waste can be used as substrate for the production of ligninolytic enzymes during the process of fermentation.
Laccase can be produced at varying rates by using a wide range of organisms grown on different substrates and by using several methods of fermentation, such as solid state, semisolid state, and submerged (Rodriguez et al. 1999; Boran et al. 2011). However, for effective laccase production, it is very important to use efficient laccase-producing organisms, suitable fermentation methods, and cheap and widespread sources. Accordingly, one of the most suitable approaches for the production of this enzyme is to use the most efficient agricultural wastes for increasing the production of the ligninolytic enzymes (Elisashviliet al. 2008).
Pakistan is an agricultural country and each year manufactures tons of agricultural by products. These agricultural wastes are accessible in markets at a very reasonable price and can be utilized as substrates in fermentation technique (Minussi et al. 2007). Agricultural waste products like rice husk, wheat bran, corn cob, millet husk and cereal huskhave been utilized by various scientists for laccase production (Osma et al. 2011; jhadav et al. 2009).The chemical properties of these agricultural wastes make them important and economical fermentation medium for biotechnological purposes(Giardina et al. 2010).The cellulose and hemicellulose constituents of lignocellulose wastes are widely used by several organisms but lignin, which is the maximum contrary material to microbial degradation, is transformed conveniently by only few organism of thw white rot fungus (Dwivedi et al. 2011).Lignin serves as a barrier that protects cellulose and hemicellulose from enzymatic attack, however, white rot fungi can attack this barrier in order to obtain energy from cellulose. These fungi produce different extracellular ligninolytic enzymes such as laccase, manganese peroxidase, and lignin peroxidase (Couto et al. 2006).
Fermentation is a biological approach that is used for the transformation of complicated substrates into basic composites by different microorganisms like bacteria and fungi. In the procedure of this metabolic breakdown the microorganisms also release various added compounds like carbon dioxide and alcohol asidefrom the conventional products of fermentation. These added compounds are known as secondary metabolites (Pandey et al. 1999). These Secondary metabolites span from enzymes, antibiotics, peptides and growth factors (Balakrishnan and Pandey. 1996; Machado et al. 2004; Robinson et al. 2001). They are also known as bioactive compounds becausethey carry biological activity(Demain et al. 1999).
Submerged fermentation is a type of fermentation in which components are present in a liquid media like broths and syrup. The co-active composites are poured into the fermentation broth. In this media the substrates are employed quiet immediately, due to this reason the nutrients in the media are either fortified or regained continuously. This type of fermentation approach is optimum for microorganisms such as bacteria, fungi because they depend upon on immense moisture content. The increased benefit of this approach is that the purification of the desired products or enzymes is quiet effortless. Submerged fermentation is especially used in the abstraction of secondary metabolites that are utilized in liquid form (Subramaniyam et al. 2012).
Furthermore 75% of the commercial enzymes are made by using submerged fermentation, it also supports the usage of genetically modified organisms to a large expanse then solid state fermentation. Submerged fermentation is also used on large extent because it doesn’t require equipment concerning solid state. On the contrary solid state fermentation is a mechanism operated in absence of free flowing water by utilizing solid support in form of natural substance ( Poonkuzhali et al . 2011). .
The major purpose of conducting this research is to design optimized fermentation process which produces effective amount of enzyme by using agricultural wastes. The use of agricultural wastes as substrates is economical and increase awareness on energy conservation .The enzyme can be used further for bioremediation because it not substrate specific and can act on broad range of substrates.
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