摘要
牛肉做为世界消费量第二的肉类食品得到越来越多消费者的青睐。培育出肉质好、营养高的肉牛品种，是育种工作者努力的方向。目前，转基因技术是肉牛改良的重要手段之一，寻找安全、高效的转基因载体及改良作用明显的目的基因成为转基因研究的重要课题。PiggyBac（PB）转座子在转基因昆虫、小鼠等的研究中充分体现了其优势，但在转基因牛中的研究还不多。脂肪酸结合蛋白3（Fatty acid binding protein3，FABP3）是影响肌内脂肪（Intramuscular fat，IMF）沉积的候选基因，fat-1基因编码的ω-3脂肪酸脱氢酶能促进ω-3多不饱和脂肪酸（Polyunsaturated fattyacids，PUFAs）的合成，对人类健康有重要作用。本研究探索了PB转座子在牛基因组中的转座特点，并在小鼠水平对FABP3和Bfat-1（牛源化fat-1）基因进行转基因研究，为生产转基因牛探索新方法、寻找新基因提供了理论依据。本研究共得到如下结果：
1．构建了PB[CMV-EGFP]、pcDNA-PBase二元转座体系，利用细胞核电转染技术转染牛耳组织成纤维细胞，发现在转座酶（PBase）存在时，有抗性的阳性克隆明显增多，且转染效率是对照组的9倍。
2．利用基因组步移技术检测实验组阳性细胞插入位置的5′旁侧序列，获得了8个有效的插入位点，且有5个位点定位到染色体1、2、11和X染色体上。序列分析发现，PB转座子特异性的插入到“TTAA”位置，且5′端倾向于插入到两个基因间非调控区的GC（62.5％）富集区。
3．采用DNA混池测序法，在西门塔尔牛和雪龙黑牛中检测到6个潜在的FABP3基因单核苷酸多态位点（Single nucleotide polymorphism，SNP）。对第1外显子T67C位点的研究发现：该位点与背膘厚、大理石花纹评分及剪切力均不显著相关（P>0.05）。
4．通过RT-PCR方法克隆了牛肌肉组织FABP3基因；依据11种牛肌肉蛋白相关基因密码子使用频率对线虫fat-1基因进行密码子优化，获得了牛源化的Bfat-1基因；并利用生物信息学方法对两个候选基因进行了核酸、蛋白结构和功能的分析及预测。
5．利用显微注射技术获得了4只FABP3转基因小鼠。分析转基因及野生型小鼠的尿样、血样生化分析结果及全身脂肪组织核磁共振成像（Magnetic resonance imaging，MRI）结果得出：转基因鼠的脂肪酸沉积略有增加，初步确定FABP3基因能够促进脂肪沉积。
6．利用尾静脉高压注射法将PB[CMV-EGFP-Bfat-1]质粒注入小鼠体内，Bfat-1基因在小鼠肝脏中瞬时表达，注射24h、48h和72h后，肝脏中18碳和20碳（48h除外）ω-6/ω-3PUFAs比值显著降低（P<0.05），肌肉中仅20碳ω-6/ω-3PUFAs比值有显著降低（P<0.05）。
7．利用细胞核电转染方法将PB[CMV-EGFP-FABP3]和pcDNA-PBase环状质粒导入牛胎儿成纤维细胞，经G-418筛选获得了FABP3基因过表达细胞系，为进一步生产FABP3转基因牛提供了实验材料。
As the world’s second consumption meat, beef get more and more pro-consumer’s gaze. Thus, it isof graet effort to improve the beef quality and nutrition for breeders. Currently, transgenic technology isone of the most important methods for beef cattle breeding. They are hot topics to find a safe andefficient transfer vector and useful target genes in transgenic research. PiggyBac（PB）transposons is auseful tool for transgenic insect, mouse and etc., however, the studies about PB transposons intransgenic cattle were few. Fatty acid binding protein3(FABP3) is a candidate gene for intramuscularfat (IMF) deposit; Ω-3fatty acid desaturase encoded by fat-1gene can catalyze the conversion of ω-6polyunsaturated fatty acids (PUFAs) to ω-3PUFAs that were good at improving the health of human.The study was to probe the transposition of PB transposon in bovine genome and verify the function ofFABP3and Bfat-1(optimized gene of fat-1) gene. The results of the study were as follows:
1. Donor plasmid of PB[CMV-EGFP] and helper-dependent plasmid of pcDNA-PBase wereconstructed and transferred into bovine ear tissue fibroblasts by amaxa basic nucleofector kit forprimary mammalian fibroblasts. The results showed that the cell colonies of experimental grouptransfected with PB co-transposition system were9times higher than the control group transfectedwithout PB transposase (PBase).
2. Genomic DNA of transgenic cells was extracted and integration sites of transposon5′Bac weredetected by genome walking technology. Eight integration sites were obtained in bovine genome and5sites were mapped on chromosomes1,2,11and X chromosome. The results showed that PB transposonwas inserted into TTAA location and5′tended to be inserted into intergenic non-regulatory regionthat rich in GC (62.5%).
3. Six potential single nucleotide polymorphisms (SNPs) were detected in Simmental andXuelong Black cattle populations for FABP3gene by DNA-pool sequencing. And T67C locus was notsignificantly associated with backfat thickness, marbling score and shear force in the experimentalpopulation (P>0.05).
4. FABP3gene was cloned by RT-PCR using bovine muscle tissue; Bfat-1gene that expresshighly in cattle genome was codon optimized gene of C. elegans fat-1gene based on the codonfrequencies of11cattle muscle protein genes. And the nucleic acid, protein structure and function ofthe two candidate genes were predicted by the bioinformatics methods.
5. Four bovine FABP3transgenic mice were made by microinjection. The blood and urinebiochemical analysis, the systemic fat tissue magnetic resonance imaging (MRI) scanning of transgenicmale mice and wild-type mice were tested. The results confirmed initially that FABP3gene can promotefat deposition.
6. PB[CMV-EGFP-Bfat-1] plasmid was injected into mice by hydrodynamic tail vein injectionmethod. Bfat-1can transient express in mouse liver. After24h,48h and72h, ω-6/ω-3PUFAs ratio of18and20carbon (except48h) decreased significantly in liver (P<0.05); only the ratio of20carbondecreased significantly in muscle (P<0.05).
7. PB[CMV-EGFP-FABP3] and pcDNA-PBase circle plasmide were transferred into bovine fetalfibroblasts by amaxa basic nucleofector kit for primary mammalian fibroblasts. And transgenic cellswere obtained by G-418. It will provide experimental material for the production of FABP3transgeniccattle in the future.
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