唾液腺特异表达植酸酶转基因猪的制备及分析  

Production and Analysis of Transgenic Pig(Sus scrofa) Specifically Expressing Phytase in Salivary Glands

作　　者：孙悦[1] 张献伟[1] 帅亮[1] 郑恩琴[1] 张然[2] 刘德武[1] 吴珍芳[1] 李紫聪[1] 

机构地区：[1]华南农业大学国家生猪种业工程研究中心,广州510642 [2]中国农业大学农业生物技术国家重点实验室,北京100193

出　　处：《农业生物技术学报》2016年第8期1147-1155,共9页Journal of Agricultural Biotechnology

基　　金：转基因生物新品种培育科技重大专项(No.2014ZX08006004;No.2016ZX08006002);广东省科技计划项目(No.2011A020102003)

摘　　要：猪(Sus scrofa)对饲料中植酸磷的消化利用效率很低,其粪便中排放大量未吸收的磷,对环境产生严重污染,在猪唾液腺中表达分泌植酸酶是解决猪粪磷污染的一条新途径。为了制备唾液腺特异表达植酸酶的新型减磷排放转基因猪,本研究构建了一条由猪腮腺分泌蛋白基因(parotid secretory protein,PSP)启动细菌源植酸酶基因app A(acid phosphoanhydride phosphohydrolase)表达的转基因载体,并利用体细胞核移植技术获得14头原代(F0)转基因克隆猪,经PCR鉴定,其中8头为阳性猪。PCR分析显示,app A基因在转基因猪的唾液腺组织特异表达,但在肾脏、肝脏、心脏、十二指肠等组织中不表达。营养代谢分析证明,转基因猪粪磷排放量比非转基因猪显著减少了16%(P<0.05),而转基因猪对干物质和能量的表观消化率以及对Ca、P的消化率均高于野生型猪,但未达到显著水平。外源基因从F0转基因猪稳定遗传至F1,并且Southern blot分析显示,外源基因是以单拷贝形式整合至转基因猪基因组。F1转基因猪的app A表达模式与F0转基因猪相似,其只在唾液腺中特异表达,且以颌下腺表达最高,其次是腮腺和舌下腺,在肾脏、心脏、肝脏等组织不表达。本研究成功获得唾液腺特异表达植酸酶转基因猪,为培育新型减磷排放的环保转基因猪新品种提供了科学依据。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 app A（acid phosphoanhydride phosphohydrolase） was constructed. The app A 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 app A 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 app A in the salivary glands improved the utilization of feed phytic acid. The app A 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 ap

关 键 词：磷 植酸酶 转基因猪 腮腺分泌蛋白基因(PSP)启动子 

分 类 号：S828[农业科学—畜牧学]