【作者】 王锦；

【导师】 石德顺；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 水牛是中国南方极具开发潜力的主要家畜,具有耐粗饲、乳品品质好等优点。但由于我国本地水牛产奶性能低下,致使奶水牛产业的发展缓慢。因此,如何应用现代生物技术培育出高产奶量的水牛新品种是目前要解决的主要问题。本研究首先分析了水牛泌乳期和非泌乳期乳腺组织基因表达谱,寻找差异表达基因,研究水牛的泌乳机制,并为培育高产奶量的转基因水牛提供丰富的候选基因素材；然后,探索转座子系统在水牛转基因中的应用,以期提高水牛转基因的效率；构建了乳腺特异性生长激素基因(GH)转基因表达载体,在乳腺细胞和乳腺组织中进行检测,分析外源GH基因的表达对乳蛋白等基因表达的影响；最后,采用随机整合、转座子主动整合等转基因技术将重组GH基因导入水牛基因组,生产GH转基因的水牛胚胎,以期培育出高产奶量转基因水牛新品种。本研究取得的主要结果总结如下：1.水牛泌乳期与非泌乳期基因表达谱及差异表达分析。构建水牛泌乳期和非泌乳期乳腺组织基因表达谱,分析差异表达和泌乳期高丰度表达的基因,探讨水牛泌乳的分子机制,也为转基因水牛提供外源基因乳腺泌乳期特异性表达启动子。基因表达谱序列拼接后获得114,014个单一的Unigenes,与牛的基因组信息比对注释了30290个基因,可以定位到270条相应的通路上(pathway),其中与乳脂肪、乳蛋白和乳糖合成代谢通路相关基因表达丰度较高。在泌乳期乳腺组织中特异性表达的基因有3053个,占到所有基因序列的2.68%。比较水牛乳腺两个时期基因表达量,泌乳期表达量上调的基因有1390个,下调的基因有5851个。在270条KEGG通路中有228个通路涉及到差异表达基因,如介导生长激素作用的JAK-STAT和MAPK信号通路。实时定量PCR (QRT-PCR)检测到水牛的非泌乳期乳腺组织中有少量的乳蛋白基因表达。K酪蛋白(CN3)基因表达量在非泌乳期与泌乳期乳腺中都是显著高于其它基因。与非泌乳期相比,泌乳期乳腺CN1S1基因表达量增加了6958倍,CN1S2增加了4800倍,kCN和αLA增加了1000倍左右,而β酪蛋白增加了244倍,与测序结果基本一致。泌乳期和非泌乳期的乳腺组织中检测至(?)GHR, PRLR(?)(?)GF1R的表达,说明GH、PRL(?)(?)IGF1可能参与了泌乳的分子调控过程。2.水牛乳腺上皮细胞(BME)基因表达分析。通过PB转座子携带的LT抗原永生化诱导水牛乳腺上皮细胞(BME),建立检测乳腺转基因表达载体的细胞模型。PB转座子为骨架的永生化载体pXL-BACII-bCN2/LT转染后的BME细胞的腺泡消失速度和成纤维样转变降低。在添加催乳素诱导培养后,第30代的永生化处理的BME细胞能检测到乳蛋白基因的表达。其中CN1S1基因表达量为非泌乳期乳腺的88.32倍,CN3为32.09倍,CN1S2和αLA基因表达量分别为2.8倍和3.3倍。结果表明,初步建立了细胞水平检测乳腺特异性表达载体的体外模型。3.水牛转座子转基因技术方法的建立。本研究通过构建piggyBac (PB), passport (PP)和sleeping beauty (SB)转座子转基因载体,转染水牛胎儿成纤维细胞(BFF),同时对转座子转基因系统进行优化,以期建立水牛转座子转基因技术体系。转座子转基因载体转染水牛的胎儿成纤维细胞(BFF)后,均能观察到标记基因EGFP的表达。应用p18T载体、PB、PP和SB转座子表达NEO基因,转染细胞后筛选获得的G418抗性细胞克隆形成数(CFN)差异不显著。与随机整合相比,加入相应的转座酶后,PB转座子系统抗性细胞克隆数量提高了22倍,PP转座子系统提高了8倍,SB转座子提高了14倍,PB转座子系统获得了最高的转基因效率。PB转座子在转座子和转座酶比例(Tn:Ts)为1：l的时候转基因效率最高,而PP和SB转座子为1：0.5。当添加的转座酶质粒过量时,PB转座子的转基因效率下降幅度没有PP和SB转座子大。通过QRT-PCR检测了不同转座子外源基因的整合拷贝数,结果显示：随机整合组每个单倍体基因组整合拷贝数从5个拷贝至51.9个拷贝不等。PB转座子为16.62拷贝,PP转座子为2.5拷贝。SB转座子整合到基因组中的转基因拷贝数最多,单倍体基因组中为98个拷贝。以上结果显示：三种转座子均能应用于水牛的转基因研究,转基因效率优于随机整合转基因。4.生长激素转基因载体构建与细胞水平检测。克隆分析了人、奶牛和水牛的生长激素基因(GH),PB转座子携带的GH转基因载体转染乳腺细胞和乳腺组织块,研究GH转基因的表达对其乳蛋白等基因表达的影响。结果显示牛、水牛和牦牛之间GH基因的同源性最高,与其它偶蹄目也有较高的同源性,生长激素氨基酸序列也较为保守。将分别携带有人和奶牛GH的piggyBac转座子转基因载体(pXL-BacⅡ-bCN2.8/hGH2.1-EGFP-Neo, PXL-BacⅡ-bCN2.8/hGH2.2-EGFP-Neo)转染人的乳腺癌细胞系(Bcap37),能观察至(?)EGFP的表达,RT-PCR和western blot均能检测到人和奶牛GH基因的特异性表达。分别用P18T-bGH和PB-bGH转基因载体转染永生化BME细胞后,检测到bGH转基因的表达。p酪蛋白、CN1S1酪蛋白和α乳清白蛋白基因表达量均有显著提高,GHR和IGF1R基因的表达量也有上调。pB-bGH载体转染的水牛乳腺组织块,CN1S1, CN1S2,p酪蛋白和K酪蛋白基因的表达量提高了50倍以上,初步证明GH转基因载体的转染可以显著提高乳蛋白基因的表达水平。5.转GH基因水牛克隆胚胎的生产与早产转基因克隆水牛的检测。使用受精卵胞质注射、卵胞质内单精子注射(ICSI)和转基因体细胞核移植的方法生产转GH基因的水牛胚胎,并对经胚胎移植后的获得的早产转基因克隆水牛进行了鉴定。水牛受精卵胞质注射和水牛卵母细胞内单精子注射(ICSI) PB转座子GH转基因载体和转座酶载体后,均获得EGFP表达的转基因阳性胚胎。以转GH基因BFF为核供体,制备了EGFP表达的转基因克隆水牛囊胚。经胚胎移植后获得了转GH基因克隆水牛的妊娠,于9月龄早产。特定蓝光照射下在早产水牛头侧部能观察到绿色荧光表达,解剖结果显示心、肺、肝、胃和脾发育正常,但大肠,肾脏,生殖系统等发育不良,而且有严重的组织粘连。激光共聚焦显微镜下观察到心、肝、脾、肺、膈肌和肌肉等组织冰冻切片均有较强的EGFP表达。通过PCR检测和染色体步移法检测到早产克隆水牛的各组织中均有外源基因的整合。这些工作为获得乳腺特异性表达GH基因转基因水牛奠定了较好的工作基础。更多还原

【Abstract】 The swamp buffalo, roughage resistance and good quality milk, is a great potential livestock for development in south of China. However, due to the poor milk yield performance of local buffalo, China’s buffalo industry is in a slow development. Therefore, how to use modern bio-technology to breed higher milk yield buffalo is the primary and major goal. In this study, gene expression profile construction of the buffalo lactation and non-lactating mammary gland was used to analyze differential expression genes, which could be useful in buffalo lactation molecular mechanisms and looking for the special promoters. These promoters could regulate exogenous gene expression in the mammary gland with the goal of higher milk yield buffalo. Then, transposon system was explored in buffalo’s transgenic reseach with a high transgenic efficency. The vectors expressing extraneous growth hormone gene (GH) were transfected buffalo mammary epithelial cells and mammary tissue to dectection milk protein gene expression. At the last, random integration and transposable active integration transgenic methods were used to carry recombinant growth hormone gene (GH) regulated by lactation mammary-specific expression promoter. Higher milk yield buffalo is the final target. The main results of this study are summarized as follows:1. Differential gene expression between buffalo lactation and non-lactation mammary gland. Gene expression profile construction of the buffalo lactation and non-lactating mammary gland was used to analyze differential expression genes, which could be useful in buffalo lactation molecular mechanisms and looking for the special promoters. These promoters could regulate exogenous gene expression in the mammary gland with the goal of higher milk yield buffalo. The high-quality reads obtained by RNA-seq were assembled and mapped to114,014single Unigenes, which were mapped to270pathways from KEGG database respectively. The genes associated with milk lipid, milk protein and milk lactose synthesis and metabolism pathways expressed in a high amount. Genes expressed only in the lactation mammary gland were3,053accounting for2.68%of all genes, which were37,162genes accounting for32.61%in non-lactation mammary gland. The expression up-regulated genes in lactation mammary gland had1390and down-regulated expression genes were5851. There were228of270pathways which have differential expressing genes, such as JAK/STAT and MAPK singnal pathway. Quantitative real-time PCR (QRT-PCR) was used to confirm the results of RNA-seq. Kappa casein (CN3) was the most expression at these two periods. The expression of CN1S1gene increased6958times in lactation period compared to non-lactating period. The gene expression of kCN and aLA increased about1000times,4800times for CN1S2and244times for beta casein respectively. There were expression of GHR, PRLR and IGF1R detected in two period’s mammary gland, which may indicate that GH、PRL and IGF1play roles in molecular regulation of milk secretion.2. Gene expression of buffalo immortalized mammary epithelial cells (BME). BME cells immortalized by pXL-BACII-bCN2/LT vector, which large T antigen gene regulated by buffalo’s beta casein gene promoter in apiggyBac transposon vector, reduced the loss of acinar-like cells and fibroblast-change process. With prolactin induced, milk protein gene expression could be detected in the30th generation immortalized BME cells. Comparison to gene expression of non-lactation mammary tissue, the increase gene expression times of CN1S1gene were88.32, which were32.09times for CN3gene and24.99times for a LA gene. These results indicate that the cell detection model of transgenic mammary gland expression vector was established preliminarily.3. Establishment of transposon transgene system in buffalo. Transposon transgene vector from piggyBac (PB), passport (PP) and sleeping beauty (SB) were constructed and transposon system was explored in buffalo’s fetal fibroblast for transgenic reseach with a high transgenic efficency. The maker gene expression of EGFP in buffalo fetal fibroblast (BFF) could be observed after transfected by transposon vector. The G418-resistent cell colony formation numbers (CFN) were not different among p18T vector、PB、PP and SB transposon, which carried the Neo maker gene. Comparison with CFN of random integration transgenic methods, CFN obtained of PB transposon system was22times, PP transposon system was8times and SB transposon system was14times. PB transposon system had the most transgenic efficiency. The transgene integration copy number was detected by QRT-PCR. The copy number of random integration group varied from5to51.9copies per haploid genome, which were16.62copies for PB transposon and2.5copies for PP transposon. SB transposon with98copies was the most number of transgene integration copies. These results indicate that three transposon transgene systems could applied in buffalo’s transgenic research, which transgenic efficency is better than random integration transgene.4. Construction and detection of growth hormone transposon vector. Full-length genomic DNA fragments of human GH (2180bp), cattle GH (2207bp) and buffalo GH (2192bp, Gi:JF894306) were amplified from blood genome DNA. GH gene and amino acid were conservative in mammary animal. Human breast cancer cells (Bcap37) were transfected pXL-BacII-bCN2.8/bGH2.1-EGFP-Neo or PXL-BacII-bCN2.8/hGH2.2-EGFP-Neo vector, which carried human GH gene or cattle GH gene, with transposase vector pCMV/PBase-bPGK/DsRed. Transgenic GH gene expressed in G418-resistent Bcap37cells confirmed by RT-PCR and western blot. Immortalized BME cells, which transfected p18T-bGH or PB-bGH transgenic vector, could be detected the bovine GH gene expression and the significant expression increase of beta casein gene, CN1S1casein gene and alpha-lactalbumin gene. The gene expression of GHR gene and IGF1R gene were up-regulation. Moreover, gene expression of CN1S1, CN1S2, beta casein and kappa casein increased more than50times in mammary tissue transfected with pB-bCN2.8/bGH2.1-EGFP-neo vector. These results showed that transfected transgenic vector could increase milk protein gene expression preliminary.5. Production of GH transgenic clone buffalo embryo and detection of premature birth transgenic buffalo. Transgenic embryos, produced by oocyte cytoplasm injection and inner cytoplasm sperm injection (ICSI) with pXL-BacII-bCN2.8/bGH2.1-EGFP-Neo and transposase vector, could be observed EGFP expression. BFF cells transfected vector and selected as positive cell colony by G418, which were the nucleus donor for somatic nuclear transfer. The nuclear transfer, p18T-bCN2.8/bGH2.2-hEF1a/EGFP-IRES-neo transgenic BFF cells as nuclus donor, reconstructed blastocyst could be observed EGFP expression. A premature buffalo offspring after embryo transplant was born at pregnant9months. The premature offspring could be observed green fluorescence at head side under the specific blue light. This offspring’s heart, lung, liver, stomach and spleen tissues were normal development. But large intestine, kidney and reproductive system were dysplasia, which were adhesions. Green fluorescence was observed in frozen sections of the heart, liver, spleen, lung, diaphragm and muscle tissue under laser scanning confocal microscope. Transgene were integrated in the premature offspring’s genome detected by PCR and chromosome walking. These results laid a good basis for breeding mammary-specific expression GH transgenic buffalo.更多还原