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

摘要/Abstract

摘要： 【目的】将前期构建的含有黑曲霉β-甘露聚糖酶基因manA的质粒pPSPBGP-manA转染杜洛克猪胎儿成纤维细胞，利用G418筛选出转基因细胞系，通过体细胞核移植方法生产出转manA基因猪，利用分子生物学方法对转基因猪进行阳性鉴定、基因及蛋白表达水平的检测，同时测定转基因猪唾液β-甘露聚糖酶酶活、粪便营养物质含量，旨在为生产及进一步研究转manA基因猪提供一些科学依据。【方法】用限制性内切酶Not I将质粒pPSPBGP-manA线性化并通过脂质体法将其转入杜洛克猪胎儿成纤维细胞，然后用不同浓度的G418进行筛选、维持培养，将筛选后的细胞进行体细胞核移植生产克隆猪；克隆猪出生后采取尾组织样用酚-氯仿法提取基因组DNA进行PCR及Southern blotting鉴定阳性转基因猪；收集转基因猪唾液并利用DNS法进行β-甘露聚糖酶活性测定，同时收集转基因猪粪便测定粪便中营养物质含量；取转基因猪的腮腺、颌下腺、舌下腺、心脏、肝脏等组织并用Triol法提取RNA，然后进行RT-PCR鉴定manA基因的表达，再进行相对定量PCR检测manA基因在不同个体及组织间的表达差异；通过Western blotting 技术分析转基因猪外源性manA基因的蛋白表达水平。【结果】经G418筛选后进行PCR鉴定得到阳性转基因细胞系；通过体细胞核移植方法生产出21头克隆猪，经PCR及Southern blotting鉴定出16头转基因猪，阳性率为76%；转基因猪唾液中β-甘露聚糖酶的活性为（0.092±0.003）U·mL^-1,粪便中粗蛋白含量明显降低；经RT-PCR、相对定量PCR鉴定，manA基因在转基因猪腮腺和舌下腺组织中特异表达，其它组织不表达，腮腺组织表达量高于舌下腺组织；Western blotting检测发现在腮腺和舌下腺组织中有manA的表达。【结论】通过G418筛选得到转manA基因细胞系并成功得到转manA基因猪，外源manA基因能够在转基因猪中腮腺和舌下腺组织特异性表达，为转manA基因猪的生产及进一步研究奠定了基础。

关键词: &beta, -甘露聚糖酶；转基因克隆猪；制备；检测分析

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

张茂，张冠冠，刘德武，蔡更元，吴珍芳，李紫聪. 转β-甘露聚糖酶基因克隆猪的制备与分析[J]. 中国农业科学, 2016, 49(8): 1567-1576.

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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