Improvement of anti-nutritional effect resulting from β-glucanase specific expression in the parotid gland of transgenic pigs

Abstractβ-Glucan is the predominant anti-nutritional factors in monogastric animal feed. Although β-glucanase supplementation in diet can help to eliminate the adverse effects, enzyme stability is substantially modified during the feed manufacturing process. To determine whether the expression of endogenous β-glucanase gene (GLU) in vivo can improve digestibility of dietary β-glucan and absorption of nutrients, we successfully produced transgenic pigs via nuclear transfer which express the GLU from Paenibacillus polymyxa CP7 in the parotid gland. In three live transgenic founders, β-glucanase activities in the saliva were 3.2, 0.07 and 0.03 U/mL, respectively, and interestingly the enzyme activities increased in the pigs from 178 days old to 789 days old. From the feed the amount of gross energy, crude protein and crude fat absorbed by the transgenic pigs was significantly higher than the non-transgenic pigs. Meanwhile the moisture content of the feces was significantly reduced in transgenic pigs compared with the non-transgenic pigs. Furthermore, in all positive G1 pigs, β-glucanase activity was detectable and the highest enzyme activity reached 3.5 U/mL in saliva. Also, crude protein digestion was significantly higher in G1 transgenic pigs than in control pigs. Taken together, our data showed that the transgenic β-glucanase exerted its biological catalytic function in vivo in the saliva, and the improved performance of the transgenic pigs could be accurately passed on to the offspring, indicating a promising alternative approach to improving nutrient availability was established to improve utilization of livestock feed through transgenic animals.

[1]  W. Schwarz,et al.  Structure of the Clostridium thermocellum gene licB and the encoded beta-1,3-1,4-glucanase. A catalytic region homologous to Bacillus lichenases joined to the reiterated domain of clostridial cellulases. , 1992, European journal of biochemistry.

[2]  M. Ikawa,et al.  FISH analysis of 142 EGFP transgene integration sites into the mouse genome. , 2002, Genomics.

[3]  I. Johnson,et al.  Effect of oat gum on the physical properties of the gastrointestinal contents and on the uptake of D-galactose and cholesterol by rat small intestine in vitro , 1989, British Journal of Nutrition.

[4]  A. Bensadoun,et al.  Assay of proteins in the presence of interfering materials. , 1976, Analytical biochemistry.

[5]  X. Tian,et al.  Cloning of pig parotid secretory protein gene upstream promoter and the establishment of a transgenic mouse model expressing bacterial phytase for agricultural phosphorus pollution control. , 2006, Journal of animal science.

[6]  R. Prather,et al.  Production of cloned pigs by using somatic cells as donors. , 2003, Cloning and stem cells.

[7]  Antoni Planas,et al.  Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering , 2000 .

[8]  M. M. Franco,et al.  Animal transgenesis: state of the art and applications , 2010, Journal of Applied Genetics.

[9]  C. Glover,et al.  Gene expression profiling for hematopoietic cell culture , 2006 .

[10]  S. Strauss,et al.  Stability of transgenes in trees: expression of two reporter genes in poplar over three field seasons. , 2008, Tree physiology.

[11]  L. Schibler,et al.  Position-independent and copy-number-related expression of a goat bacterial artificial chromosome alpha-lactalbumin gene in transgenic mice. , 1999, The Biochemical journal.

[12]  S. Feighner,et al.  Effect of dietary carbohydrates on bacterial cholyltaurine hydrolase in poultry intestinal homogenates , 1988, Applied and environmental microbiology.

[13]  M. Kay,et al.  Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo , 2004, Gene Therapy.

[14]  V. Brahmachari,et al.  Epigenetic regulation of cytomegalovirus major immediate-early promoter activity in transgenic mice. , 2009, Gene.

[15]  Y. Mu,et al.  Position effect variegation and epigenetic modification of a transgene in a pig model. , 2012, Genetics and molecular research : GMR.

[16]  H. Flint,et al.  A bifunctional enzyme, with separate xylanase and beta(1,3-1,4)-glucanase domains, encoded by the xynD gene of Ruminococcus flavefaciens , 1993, Journal of bacteriology.

[17]  K. Knudsen Carbohydrate and lignin contents of plant materials used in animal feeding , 1997 .

[18]  K. K. Thomsen,et al.  Hybrid Bacillus (1-3,1-4)-β-glucanases: engineering thermostable enzymes by construction of hybrid genes , 1991, Molecular and General Genetics MGG.

[19]  Adrian Bird,et al.  Perceptions of epigenetics , 2007, Nature.

[20]  M. Sunde,et al.  The viscosity interaction of barley beta-glucan with Trichoderma viride cellulase in the chick intestine. , 1981, Poultry science.

[21]  M. Bedford Mechanism of action and potential environmental benefits from the use of feed enzymes , 1995 .

[22]  D. Headon,et al.  Enzymes in the animal-feed industry. , 1993, Trends in biotechnology.

[23]  J. Phillips,et al.  Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control , 2001, Nature Biotechnology.

[24]  I. Johnson,et al.  Effect of gel-forming gums on the intestinal unstirred layer and sugar transport in vitro. , 1981, Gut.

[25]  R. R. Marquardt,et al.  Effect of enzyme supplementation on the performance and digestive tract size of broiler chickens fed wheat- and barley-based diets. , 1993, Poultry science.

[26]  Y. Mu,et al.  Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs , 2009, PloS one.

[27]  K. Jakobsen,et al.  Effect of β-glucanase supplementation on pancreatic enzyme activity and nutrient digestibility in piglets fed diets based on hulled and hulless barley varieties , 1998 .

[28]  J. D. Summers,et al.  The feeding value of rye for the growing chick: effect of enzyme supplements, antibiotics, autoclaving and geographical area of production. , 1969, Poultry science.

[29]  Ji-Yeon Kim Overproduction and secretion of Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in B. subtilis and B. megaterium , 2003, Biotechnology Letters.

[30]  J. Bishop,et al.  Expression of a foreign gene in a line of transgenic mice is modulated by a chromosomal position effect , 1990, Molecular and cellular biology.

[31]  Z. Yin,et al.  Transgene inheritance in plants. , 2004, Journal of applied genetics.

[32]  J. Thoma,et al.  Determination of reducing sugar with improved precision. , 1965, Analytical biochemistry.

[33]  J. Augustin,et al.  β-1,3-1,4-Glucanase in sporenbildenden Mikroorganismen: II. Bildung von β-Glucan-Hydrolasen durch verschiedene Bacillus-Arten , 1980 .

[34]  Mathias Currat,et al.  Impact of Selection and Demography on the Diffusion of Lactase Persistence , 2009, PloS one.

[35]  S. Moisyadi,et al.  Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer , 2013, Transgenic Research.

[36]  D. G. Cran,et al.  Transgenes as probes for active chromosomal domains in mouse development , 1988, Nature.

[37]  G. Shu,et al.  β-Glucanase specific expression in the parotid gland of transgenic mice , 2013, Transgenic Research.

[38]  K. K. Thomsen,et al.  Hybrid bacillus endo-(1–3, 1–4)-β-glucanases: Construction of recombinant genes and molecular properties of the gene products , 1989, Carlsberg research communications.

[39]  H. Classen,et al.  Reduction of intestinal viscosity through manipulation of dietary rye and pentosanase concentration is effected through changes in the carbohydrate composition of the intestinal aqueous phase and results in improved growth rate and food conversion efficiency of broiler chicks. , 1992, The Journal of nutrition.

[40]  J. O’Doherty,et al.  Effects of increasing the intake of dietary β-glucans by exchanging wheat for barley on nutrient digestibility, nitrogen excretion, intestinal microflora, volatile fatty acid concentration and manure ammonia emissions in finishing pigs. , 2007, Animal : an international journal of animal bioscience.

[41]  L. Samuelson Transgenic approaches to salivary gland research. , 1996, Annual review of physiology.

[42]  Cecil W. Forsberg,et al.  Pigs expressing salivary phytase produce low-phosphorus manure , 2001, Nature Biotechnology.

[43]  I. A. Preece,et al.  NON‐STARCHY POLYSACCHARIDES OF CEREAL GRAINS I. FRACTIONATION OF THE BARLEY GUMS , 1952 .

[44]  A. Bacic,et al.  Chemistry and Organization of Aleurone Cell Wall Components From Wheat and Barley , 1981 .

[45]  B. Kerr,et al.  Effect of β-glucanase supplementation of cereal-based diets for starter pigs on the apparent digestibilities of dry matter, crude protein and energy , 1996 .

[46]  Sam W. Lee,et al.  Hzf Determines Cell Survival upon Genotoxic Stress by Modulating p53 Transactivation , 2007, Cell.

[47]  J. Ingerslev,et al.  Tissue-specific expression in the salivary glands of transgenic mice. , 1992, Nucleic acids research.

[48]  Li-Chu Tsai,et al.  Crystal structure of a natural circularly permuted jellyroll protein: 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes. , 2003, Journal of molecular biology.

[49]  C. Biliaderis,et al.  Water extractable (1→3,1→4)-β-d-glucans from barley and oats: An intervarietal study on their structural features and rheological behaviour , 2005 .

[50]  Dongshan Yang,et al.  Use of the 2A Peptide for Generation of Multi-Transgenic Pigs through a Single Round of Nuclear Transfer , 2011, PloS one.