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Gene Cloning And Expression Of Pectinases And Their Diversity Analysis In The Rumen Of Small Tail Han Sheep

Posted on:2013-01-30Degree:DoctorType:Dissertation
Country:ChinaCandidate:P YuanFull Text:PDF
GTID:1110330374457872Subject:Biochemistry and Molecular Biology
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Pectic substances are one of the major components of plant cell wall in middle lamella, and aremainly comprised of linear chains of α-1,4-linked D-galacturonic acid residues. Pectic substances areone type of anti-nutritional factor in monogastric animal feed, but are of great use in ruminants. Theynot only stabilize the pH of rumen but also provide energy to microbes. Pectinolytic enzymes orpectinases are a heterogeneous group of related enzymes that hydrolyze the pectic substances.Pectinases have been used in several conventional industrial processes. As feed additives, pectinases canalleviate the anti-nutritional role of pectin. They can also be used in juice extraction and clarification,textile, and paper making etc. In recent years, more and more attentions have been concerned onpectinases due to their wide range of applications.In this study, Small Tail Han sheep were selected to study the pectinase diversity in rumenenvironment, explore their distribution in rumen microbes and obtain pectinases with applicationpotentials from both rumen environment and other microbes. Two main types of pectinases,polygalacturonase and pectate lyase, were focused. Based on the Pfam database,1degenerate primer setof polygalacturonase and4sets of degenerate primer of pectate lyase were designed according to thesequences of conserved motifs. Using the DNA from pectin-degrading bacterial and fungalrepresentatives as templates, these primers were verified to be efficient and reliable and were used forpectinase diversity analysis and full-length gene cloning.Pectinase gene fragments were PCR amplified using the ruminal metagenomic DNA from theSmall Tail Han sheep as the template and the five primer sets mentioned above and were used toconstruct clone libraries. Only gene fragments of families PF00295, PF00544, and PF09492weresuccessfully amplified. As a result, a total of572pectinase gene fragments were identified, and103distinct fragments shared sequence identity of lower than95%. Phylogenetic analysis indicated thatfamilies PF00295and PF09492contained some highly abundant fragments. In the Smaill tail han sheeprumen, the pectinase gene fragments of family PF00295were highest in amount and diversity, followedby family PF00544, and least in family PF09492. The well-known ruminal cellulolytic andnon-cellulolytic bacteria Butyrivibrio, Bacteroides, Fibrobacter, and Prevotella were identified to havecapacity to produce pectinases. PF00295fragment sequences were highly similar to those fromFibrobacter, PF00544and PF09492fragment sequences were closely related to those of Butyrivibrio,Bacteroides and Prevotella.Without construction of metagenomic libraries or isolation of microorganisms,6completepectinase genes were cloned directly from the metagenomic DNA of rumen and1complete gene fromthe metatranscriptomic mRNA of an eukaryotic organism using TAIL-PCR method. It's the first time toobtain a gene by combination of cDNA reverse transcription of metatranscriptomic mRNA andTAIL-PCR. This method provides a new way to clone full-length genes from metatranscriptomic mRNA. One gene from each family of PF00544and PF09492were successfully expressed inEscherichia coli. Both recombinant enzymes had the optimal temperature of40°C, similar to thephysiological temperature of rumen. The enzymes showed maximal activities at pH4.0and9.0,respectively, and remained active under rumen conditions (pH6.0–7.0). These data facilitate moreunderstanding of ruminal pectinases.Five full-length genes were cloned from cultured bacteria or fungi and successfully expressedheterologously. Two polygalacturonase genes from Penicillium sp. CGMCC1669and Klebsiella sp. Y1(pg I, pg II) were expressed in Pichia pastoris and E. coli, respectively. The specific activity of PG Iwas815.5U/mg, and the yield of secreted protein reached1.97g/L. Its optimal pH ranged from3.5to4.0, which is as same as the pH of apple juice. Thus PG I can be used in food industry such as applejuice extraction and clarification and has application potentials in many fields requiring an acid andlow-temperature milieu. PG II produced in E. coli was highly active in simulated gastric and intestinalfluid. Thus it can be used as a feed addictive to alleviate the anti-nutritional effect in monogastric animalfeed, to promote the feed utilizition and increase the egg production.Three pectate lyase genes were cloned from Streptomyces sp. S27, Klebsiella sp. Y1andXanthomonas campestris ACCC10048, respectively. A gene cluster including a pectate lyase gene and akdgm controlling gene was first identified in Klebsiella sp. All the three recombinant enzymes producedin E. coli were characterized as alkaline pectate lyases. Application tests showed that these enzymes hadpotentials in the textile industry. Besides, they can also be used in clarification of waste watercontaining pectin, such as that from pulp and paper mill.In summary, a rapid and efficient culture-independent molecular method was developed to explorethe diversity and distribution of pectinase genes in complex environments based on the5sets ofdegenerate primer. The starting point of a large number of novel pectinase gene fragments and2full-length pectate lyase genes from Small Tail Han sheep rumen leads to a systemic study of pectinasediversity and distribution in rumen and casts new insight into to the importance of their roles in pectindegradation in rumen environment. Moreover, several pectinases with application potentials wereobtained from prokaryotic and eukaryotic organisms by cloning and heterologous expression. This studyprovided the fundamental basis for pectinase application in a wider range of fields.
Keywords/Search Tags:pectinase, rumen, metagenome, metatranscriptome, gene diversity
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