【作者】 张建斌；

【导师】 韩俊文； 李奎；

【作者基本信息】 山西农业大学 ， 动物遗传育种与繁殖， 2018， 博士

【摘要】 作为一个诱导基因表达调控系统,四环素(tetracycline,Tet)调控系统在基因工程中已开始应用于复杂的生物发生反应、机体的疾病及行为等。它已成为研究某一基因功能的有效工具,尤其在转基因小鼠的基因功能上,得到了广泛的应用。生长激素(growth hormone,GH)在不同物种中均具有促进性成熟,提高瘦肉率,改善饲料转化率等优点,但也存在副作用,在转生长激素基因的动物上尤为明显。这可能由于转GH基因动物,都是全身过表达GH基因,而且外源GH基因从胚胎期到成年都持续发挥生物学效应,其机体发育的异常很可能和GH在全身多个组织器官中的自分泌异常有关。在Tet调控系统中,诱导底物四环素的浓度与外源基因表达水平在一定范围内呈正相关,因此,可以通过控制四环素及其衍生物强力霉素(doxycycline,DOX)的有无、浓度高低来调控所导入的外源基因表达水平,进而认识所导入基因在不同水平及不同生长阶段的表达,了解其在动物生长发育中作用,进一步深入研究GH生物学功能。本研究采用分子生物学方法,构建了猪生长激素(porcine growth hormone,pGH)真核细胞可诱导表达载体,采用阳离子脂质体法转染到猪髋动脉内皮细胞(porcine iliac artery endothelial cells,PIEC),在细胞水平上对Tet调控系统的诱导效率和表达水平进行了验证。然后利用该载体以原核显微注射方法,将外源基因片段(pTTGH)直接注射到原核期胚,制备转基因小鼠并用蛋白质组学的方法对其功能进行了研究。主要结果如下:(1)成功构建了重组表达载体(pTTGH)。在成功构建重组载体pTRE-GH12的基础上,应用PCR扩增出TRE-GH12基因序列片段。然后,通过酶(SalI)切位点将其连接到pCAGGS-rtTA载体上,构建pTTGH载体。通过对酶切、测序,结果证明构建的pTTGH载体符合要求。将构建好的载体用阳离子脂质体法转染到PIEC细胞。经遗传霉素(G418)抗性筛选后,将强力霉素添加到培养液中进行诱导。然后,通过荧光定量PCR在不同时间检测猪髋动脉内皮细胞内pGH mRNA的表达水平;配制不同浓度的强力霉素,将其加入细胞培养液内,在mRNA水平上测定猪生长激素的表达效率及表达水平。结果说明,将强力霉素添加到细胞培养液内,经过12、24、36、48和60h的检测,外源生长激素的mRNA表达水平分别比对照组高4.21、6.79、73.27、34.29和30.07倍。在培养基中添加不同浓度(100、200、300、400、500、600、700、800、900、1000 μg/mL)的强力霉素,pGHmRNA表达量分别是对照组的9.87、11.45、26.87、30.09、36.77、47.84、46.87、41.23、35.27和29.87倍,处理组与对照组差异极显著(P<0.01)。(2)将pTTGH质粒以原核显微注射方法制备转基因小鼠。以Southern鉴定为阳性的Founder鼠为基础扩繁培育,对转基因后代鼠在3～10周龄期间通过饮水中添加诱导底物DOX进行诱导实验。结果表明,转基因小鼠体重在诱导期内持续增加,生长速度高于转基因小鼠无药物诱导组和非转基因对照组,转基因组小鼠血清中pGH水平远远高于对照组小鼠,差异显著(P<0.05)。ELISA方法测定血清样本中内源IGF-1的表达水平,结果表明,转基因小鼠血清中胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)水平明显上升,说明外源GH通过调节IGF-1调节动物机体的生长。通过对转基因小鼠的肝脏、脾脏、肺脏、肾脏、背肌、腿肌等组织进行形态学变化及病理学观察。苏木精和伊红染色法(hematoxylin-eosin,HE)切片结果表明:转基因小鼠加DOX诱导之后与同窝非转基因小鼠加DOX诱导之后相比,功能性器官肝脏、脾脏、肺脏、肾脏都没有发生可见病变。(3)通过对转基因小鼠和对照组小鼠大脑、肝脏和肌肉共6个组织样本,采用同位素标记相对与绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)技术进行质谱鉴定试验,再对获得的蛋白质组学数据进行生物信息分析,进而鉴定过表达GH转基因小鼠对机体的影响,进一步为GH高表达对大脑发育、能量代谢和生长性状的分子机制提供理论依据。转基因和对照组小鼠大脑组织共得到360个DEPs,转基因组高表达蛋白148个,低表达蛋白242个。肝脏组织共得到170个DEPs,转基因组高表达蛋白74个,低表达蛋白96个;肌肉组织比较共得到390个DEPs,转基因小鼠高表达蛋白156个,低表达蛋白234个。通过GO(Gene Ontology)注释和KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway分析差异蛋白主要富集在RNA剪切过程、细胞间蛋白转运过程、mRNA加工过程及胰岛素信号通路、AMPK通路、PI3K-Akt信号通路、PPAR信号通路等信号通路中。更多还原

【Abstract】 As a a regulatory system of inducible expression,Tet-on regulatory expression system has been applied to biogenic reactions which become an effective tool to study the function of genes,especially in mice.It has become a very effective induction model to study the gene function of transgenic animals.Growth hormone is naturally produced by the pituitary gland,a pea-sized organ at the base of the brain,which can promote maturation,improve percentage of lean meat and ameliorate feed conversion rate of different species,but there are also side effects.This may be due to overexpression in transgenic animals,and the exogenous GH gene has a biological effect from the embryo to adult,and the abnormal development of the organism is likely to be related to the autocrine abnormality of GH in multiple tissues.The concentration of doxycycline(DOX)was positively correlated with the level of exogenous gene expression in a certain range;therefore,expression level of exogenous gene was changed by controlling the concentration of tetracycline and DOX.It was helpful to grasp the transgene expression level at different levels and different growth stages,understand its role in animal growth and development stage,and further study the biological function.This study aims to explore the construction of eukaryotic expression vector carrying porcine growth hormone gene(pGH)by using molecular biology method.After transfected into PIEC cell lines(Porcine iliac artery endothelial cells)through cationic liposome successfully,the efficiency and expression level of inducible expression(Tet-on regulatory expression system)after adding DOX were verified at the cellular level.Then we used microinjection method for preparing transgenic mice and conducted a preliminary study on their functions.The results were showed as following:First,construction of recombinant expression vector(pTTGH)was carried out successfully.On the basis of the successful construction of recombinant vector pTRE-GH12,pTRE-GH fragment was amplified by PCR from pTRE-GH 12,then the ligation of Sal I digested fragment to Sal I digest vector pCAGGS-rtTA,the recombinant vector pCAGGS-rtTA-TRE-GH12(pTTGH)was construted successfully.The pTTGH vector DNA was transfected PIEC cells by way of Cationic liposome.After enriched by genetic resistance agent(G418),Quantitative fluorogenic real-time PCR(RT-qPCR)assay for pGH mRNA expression level when doxycycline(DOX)was added to the culture medium for induction.On the other hand,different concentrations of doxycycline prepared were added into the cell culture medium,RT-qPCR and Western Blotting assay for determining the expression efficiency of porcine growth hormone.The results of digestion,sequencing and restriction showed that the pTTGH vector was constructed successfully and could meet the requirement of the next experiment.RT-qPCR results showed that expression of mRNA of porcine growth hormone was increased 4.21,6.79,73.27,34.29 and 30.07 times when DOX adding into cell culture fluid after 12,24,36,48 and 60 h and compared with the control group(no DOX).Inducied with adding different DOX concentration of 100,200,300,400,500,600,700,800,900 and 1000 μg/mL in culture medium,the expression of nRNA of porcine growth hormone were 9.87,11.45,26.87,30.09,36.77,47.84,46.87,41.23,35.27 and 29.87 times than’ the control group(no DOX),and the difference was extremely significant(P<0.01).Results of gray intensity analysis(Western Blotting)showed that relative expression of protein of porcine growth hormone were 0.70、0.68、0.62、0.71 and 0.72,when doxycycline adding into cell culture fluid after 12,24,36,48 and 60 h compared with the control group was 0(no DOX).The results showed the vector has been constructed successfully and which can realize controllable expression of pGH.This study established a foundation for preparing for controllable expression of pGH in the transgenic animal and helps us to further study the function of growth hormone.The preparation of pTTGH transgenic mice was transfected through prokaryotic microinjection method.Based on Southern blot results,positive founders were bred for getting offsprings.When get the offsprings of transgenic mice,the induction experiment was carried out with the induction substrate doxycycline DOX in the drinking water of the transgenic mice at 3-10 weeks old.The results showed that the body weight of transgenic mouse continued to increase during the induction period,and the growth rate is higher than that of non transgenic mice and transgenic control group(no DOX),the level of pGH in serum of transgenic mice was significant higher than that of control mice(P<0.05).The expression level of endogenous IGF-1 in serum samples was detected by ELISA.The results showed that the level of IGF-1 in the serum of transgenic mice was obviously increased,indicating that exogenous GH regulates the growth of the organism by regulating the expression of IGF-1.The morphological and pathological changes of the heart,liver,spleen,lung,kidney,dorsal muscle and leg muscle of transgenic mice were observed,the results of HE showed that functional organs of the heart,liver,spleen,lung and kidney were no visible lesions of transgenic mice after adding antimycin DOX via drinking water,compared to wild-type mice sibbing with DOX.Meanwhile we also get the conclusion the foreign gene pGH has already integrated into mouse into mouse chromosome,and it can inherit in accordance with the laws of Mendelian inheritance.Compraed with the control group,there was no harm cause to the organism of offsprings of transgenic mice in vivo by the the expression of exogenous growth hormone after incuced by the DOX and the healthy condition of transgenic mice were not significantly different.The establishment of modle of pGH transgenic mouse contributed to explore the influence of the molecular mechanism and signal pathway of growth hormione secration.Meanwhile it would promote the application of GH in livestock production intensely;it also provides a theoretical foundation and technical support for the preparation of safe and efficient GH transgenic livestock.By using technology of isobaric tags for relative and absolute quantitation(iTRAQ),6 samples of brain,liver and muscle in transgenic mice and non-transgenic mice were used for mass spectrometry identification,and then the bioinformatics analysis was carried out.Then the effect of GH transgenic mice on the body was identified,which will further provide theoretical basis for the overexpression of GH on the molecular mechanism of brain development and energy metabolism.The result of functional enrichment analysis of differentially expressed proteins from brain tissues illustrate that a total of 360 DEPs were detected from transgenic and non-transgenic mice brain,148 highly expressed proteins and 242 low expressed proteins of transgenic group;a total of 170 DEPs were detected from transgenic and non-transgenic mice liver,74 highly expressed proteins and 96 low expressed proteins of transgenic group;a total of 390 DEPs were detected from transgenic and non-transgenic mice muscle,156 highly expressed proteins and 234 low expressed proteins of transgenic group.Through comparative analysis,we can come to conclusion that the effect of excessive GHacted on brain,liver and muscle after overexpressing GH.We found that differential proteins by GO analysis and K.EGG pathway analysis,which mainly enriched in the process of RNA shear,intercellular protein transport and mRNA process-cycle and insulin signaling pathway,the AMPK pathway,the PI3K-Akt signaling pathway,and the PPAR signaling pathway.更多还原