转β-甘露聚糖酶基因克隆猪的制备与分析

doi:10.3864/j.issn.0578-1752.2016.08.014

Abstract

Abstract: 【Objective】The objective of this study is to transfect the plasmid pPSPBGP-manA into porcine fetal fibroblast cells and screening of transgenic cell lines with G418. Transgenic pigs were generated by somatic cell nuclear transfer and identified by PCR and Southern blot analyses, beta-mannose mRNA protein were identified by RT-PCR and Western blot, so as to provide a scientific basis for the further study of the genetically modified pig.【Method】The plasmid pPSPBGP-manA was linearized with Not I and purified. The Duroc pig fetal fibroblast cells were cultured to the third generation and using liposome method to transfer plasmid into porcine fetal fibroblasts. Then the transgenic cells were selected by G418 and the transgenic pigs were obtained by somatic cell nuclear transfer. Genomic DNA was isolated from the new born porcine tail tissue by phenol-chloroform extraction. One microgram of each genomic DNA sample was used as the template for a single PCR and Southern blot analyses. Total RNA was isolated from frozen parotid gland, sublingual gland, mandibular gland, brain, heart, liver, spleen, lung, kidney and stomach tissues of transgenic pigs using Triol method. RT-PCR of pig total RNA was performed to identify the expression of manA gene in different tissues. The expression level of manA gene in different individuals and tissues was detected by relative quantitative PCR. The saliva beta-mannose activity analysis with DNS method and the content of nutrients in feces was determined. Moreover, the protein expression of exogenous manA gene in transgenic pig was detected by Western blotting. 【Result】 Stable transfection of the transgenic cell line was obtained by G418 screening and PCR identification. Twenty-one cloning pigs were produced by somatic cell nuclear transfer, PCR and Southern blotting showed that there were 16 transgenic pigs and the positive rate was 76%.Transgenic pig salivary beta-mannosidase enzyme activity was (0.092±0.003)U·mL^-1 and fecal crude protein content decreased significantly. RT-PCR and relative quantitative PCR analyses demonstrated that manA is strongly expressed in parotid gland and sublingual gland, but is not present in mandibular gland, brain, heart, liver, spleen, lung, kidney or stomach, and the manA expression level in parotid gland was higher than in sublingual gland. The beta-mannanase protein was detected in the parotid and sublingual gland by Western blotting. 【Conclusion】 The transgenic cell line and transgenic pig were successfully obtained and the exogenous manA gene could be specific ally expressed in parotid and sublingual gland.

Key words: beta-mannanase, transgenic clone pig, production, detection and analysis

ZHANG Mao, ZHANG Guan-guan, LIU De-wu, CAI Geng-yuan, WU Zhen-fang, LI Zi-cong. Generation of Beta-Mannanase Transgenic Clone Pig and Analysis[J].Scientia Agricultura Sinica, 2016, 49(8): 1567-1576.

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Generation of Beta-Mannanase Transgenic Clone Pig and Analysis

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