Cloning and expression of calpain and phytase genes for the improvement of animal growth and nutrition
Abstract
The calpain system is a critical protease system in protein accretion and animal muscle growth. The calpain cDNA's from pigs and cattle were cloned to study the roles of calpain in muscle growth in domestic animals. By using reverse transcriptase and polymerase chain reaction, porcine $\mu$- and m-calpain, and bovine $\mu$-, m-, skeletal muscle specific (skm) calpain, and calpastatin cDNA's were cloned. Sequence analysis indicated that porcine and bovine calpain sequences were about 90% identical to rabbit or rat calpain sequences. The transcriptional sizes of porcine and bovine calpains were 3.0 kb for $\mu$-calpain; 3.1 and 1.5 kb for m-calpain; 3.9 kb for skm-calpain; 3.8 and 5.0 kb for calpastatin. The expression of calpain genes were then studied in the steers treated with a $\beta$-agonist, cimaterol. The expression of $\mu$-, m-calpain and skm-calpain genes and activities were not changed by the cimaterol treatment. However, calpastatin mRNA and activity were increased by cimaterol (p $<$ 0.05), and calpastatin mRNA and the rate of protein accretion were highly correlated (p$ <$ 0.01). The indicator of protein synthesis, $\alpha$-actin mRNA was not changed by cimaterol. Therefore, the enhanced muscle accretion was attributed to the increased expression of calpastatin in response to cimaterol treatment. Further, skm-calpain was found not to be expressed in L6 and C2C12 muscle cell lines. Phytase from Aspergillus niger can increase the bioavailability of phosphorus in grain by degrading phytate, phytate phosphorus accounting for up to 80% total phosphorus in grain. Because purified phytase is too expensive to use in animal production, an attempt was made to produce phytase in yeast Saccharomyces cerevisiae as an economical source of phytase. The phytase gene from A. niger was cloned by PCR, and subcloned into a yeast expression vector with a constitutive promoter. The expression construct was transformed into a mutant strain of S. cerevisiae, CG 219-2. Expression of recombinant phytase was obtained at the level of 23 phytase units/g yeast, eleven fold higher than the untransformed control. Because yeast supplementation has already been shown to improve animal performance, phytase producing yeast will make phytase less expensive, and yeast more valuable to the feed industry and animal producers.
Degree
Ph.D.
Advisors
Patterson, Purdue University.
Subject Area
Livestock|Food science|Molecular biology
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