Abstract：Pigs (Sus scrofa) have a low utilization efficiency for dietary phosphorus that is stored as phytic acid, and they excrete large amount of undigested phosphorus in the faeces, which cause serious pollution to the environment. Transgenic expression of phytase in salivary glands is a new way to improve the digestion of phytic acid and reduce the output of fecal phosphorus pollutant for pigs. This study aimed to produce and analyze transgenic pigs expressing phytase specifically in their salivary glands. A plasmid which included a expression cassette of bacteria-derived phytase gene appA (acid phosphoanhydride phosphohydrolase) was constructed. The appA gene in the plasmid was controlled by the promoter of pig parotid secretory protein (PSP) gene, which was a salivary glands specific promoter. Transgenic pig fibroblasts were produced after transfection with the plasmid treated with G418 selection. Using transgenic fibroblasts as nuclear donor cells, 14 founder (F0) pigs were generated by the somatic cell nuclear transfer technique and 8 of them were identified as transgenic pigs by PCR. The PCR showed that foreign appA particularly expressed in the salivary glands but not in the liver, kidney, heart and duodenum of F0 transgenic pigs. The digestion trial indicated that transgenic pigs had higher apparent digestibility of dry matter (P=0.108), energy (P=0.117), Ca (P=0.059) and P (P=0.092) than that of wild-type pigs, although the difference between transgenic and wild-type pigs did not had statistical significance. The fecal phosphorus output was significantly (P＜0.05) decreased by 16% in transgenic pigs, as compared to that in wild-type pigs. This suggested that transgenic expression of appA in the salivary glands improved the utilization of feed phytic acid. The appA transgene was stably transmitted from F0 transgenic pigs to their transgenic offspring as it was also detected in the genome of F1 transgenic pigs. The Southern blot analysis demonstrated that only a single copy of appA gene was inserted into the genome of transgenic pigs. The appA also specifically expressed in salivary glands but not in the brain, heart, liver, spleen, lung, kidney and pancreas tissues of F1 transgenic pigs. Among the 3 salivary glands, the appA gene expressed the highest in submaxillary gland, followed by the parotid gland and sublingual gland in F1 transgenic pigs. The transgene express pattern in F1 transgenic pigs was similar with that in F0 transgenic pigs. In this study, transgenic pigs expressing phytase specifically in the salivary glands were successfully produced, which provides scientific foundation for breeding new pig breed with low phosphorus excretion in the future.
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