【作者】 孙杰；

【导师】 李大全； 罗军；

【作者基本信息】 石河子大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 乳腺为哺乳动物新生儿的生长发育提供营养均衡的乳汁,并且可以为人类生活提供不可缺少的乳制品。在奶山羊的生理周期中乳腺组织经历发育、泌乳和退化三个循环过程,与此同时乳腺上皮细胞也历经增殖、分化,凋亡的变化。在奶山羊一个泌乳周期的不同阶段,产奶量的高低和乳成分的变化存在很大的差异,随着基因组学研究的不断深入,人们逐渐认识到导致这些差异产生的原因,是由乳腺组织中诸多相关功能基因时空表达决定的,即在不同泌乳期乳腺组织中的功能基因差异表达的结果。本研究在西农萨能奶山羊泌乳生理变化规律的基础上,构建不同泌乳期乳腺差异表达基因文库,克隆筛选到的差异表达基因,进行生物信息学分析和预测,采用实时定量PCR分析其表达模式,并对可能影响乳成分变化的重要候选基因骨桥蛋白(OPN)进行初步的探讨。为进一步研究奶山羊乳腺泌乳的分子机制,以及产奶量和乳成分调控机理提供理论依据。1、利用抑制性消减杂交(SSH)技术构建西农萨能奶山羊泌乳初期和盛期乳腺组织正反(E-P和P-E)向差减cDNA文库,以管家基因GAPDH作为消减指标检测两个文库的消减效率分别为25和210倍,表明了泌乳初期和盛期乳腺组织中差异表达基因的富集效率也达到25倍以上；对E-P和P-E消减文库中部分差异表达基因进行测序,E-P文库测序分析后得到的差异表达基因按功能可大致分为：乳蛋白形成的相关基因(aS2酪蛋白、B酪蛋白、K酪蛋白、a乳白蛋白前体物),转录复合体相关基因(核糖体蛋白L10、核糖体蛋白L23、核糖体蛋白3B、Cbp/p300),能量代谢相关基因(NADH脱氢酶),细胞增殖分化相关基因(GHITM、OPN和H3F3B)；P-E文库中差异表达基因可分为：脂肪代谢相关基因(HFABP、PPARGClA),细胞生长代谢相关基因(SAA3、SSAT、SPARC和LPO),转录相关基因(剪接因子3B)；将奶山羊基因组中尚未克隆和研究的新基因PPARGClA、OPN、SPARC、GHITM和SSAT采用实时定量PCR进行验证,结果E-P文库中OPN、GHITM在泌乳初期乳腺组织中mRNA的表达丰度分别比泌乳盛期高5.04和4.20倍,P-E文库中PPARGCIA、SPARC、SSAT在泌乳盛期乳腺组织中mRNA的表达丰度分别比泌乳初期高3.58、12.13和8.0倍,均为阳性克隆。2、根据不同物种间相同基因编码区的保守性和同源性序列设计合成引物,采用RT-PCR技术克隆了奶山羊乳腺中PPARGClA、OPN、SPARC、GHITM和SSAT基因的编码区序列,并提交Genbank获得登录号；利用在线多个生物信息学分析软件对山羊、牛、人和小鼠的PPARGClA、OPN、SPARC、GHITM和SSAT基因编码的蛋白质进行结构和功能预测,结果发现五个基因在不同物种间氨基酸序列高度同源,所编码的蛋白质结构、跨膜螺旋、信号肽和潜在功能位点大致相同。其中PPARGClA是一种转录调控因子,各物种均预测含有N端的转录激活区、核激素相互作用的LXXLL基序及C端的RNA识别基序；OPN是亲水性的分泌型糖蛋白,在不同的物种OPN均普遍存在RGD基序,该基序与细胞聚集、黏附、增殖及组织重塑等有关,山羊OPN比人和小鼠多cAMP依赖的蛋白激酶磷酸化位点,凝集因子VLSPR重复位点；SPARC是一种小分子的糖蛋白,结构域N端274-286处含有EF-hand钙结合位点,可能与乳腺中钙的代谢有关；GHITM是一种跨膜蛋白,有8个强跨膜区域,推测该基因可能与乳成分的跨膜转运有关；SSAT是细胞内维持代谢平衡的关键酶,有1个Gcn5-乙酰胺基转移酶功能域与SSAT的催化作用密切相关。3、以β-actin为内标,采用实时定量PCR技术测定西农萨能奶山羊在一个泌乳周期(泌乳初期、盛期、中期、后期、末期和干奶期)中PPARGCIA、OPN、SPARC、GHITM和SSAT基因的表达丰度变化规律,结果表明：PPARGCIA和SSAT基因的变化趋势与泌乳曲线基本一致,推测它们是乳腺泌乳周期性变化的调节因子；OPN在泌乳初期的表达丰度相对值最高,依次是泌乳后期、末期、盛期、中期和干奶期,其中泌乳初期相对表达水平约为末期高3.9倍左右,进一步说明,在妊娠后期OPN基因就开始表达,OPN基因可能在乳成分变化中发挥重要的生理功能；SPARC在泌乳初期的表达丰度相对值最高,随后一直下降,在干奶期表达量最低；GHITM在泌乳初期的表达丰度相对值最高,依次是泌乳后期、盛期、末期和干奶期表达量最低。4、构建OPN基因真核表达重组质粒pcDNA3.1/myc-His(-)A-OPN,与空质粒同时转染MCF-7细胞,利用MTT试验检测OPN基因对MCF-7细胞生长的影响,结果表明：转染pcDNA3.1-OPN细胞比转染pcDNA3.1细胞和对照细胞组活性明显升高,细胞的生长速度明显加快(P<0.05),且转染目的基因组pcDNA3.1-OPN的细胞增殖率为111.4%,高于未转染目的基因组pcDNA3.1的101.3%,说明OPN基因的表达对MCF-7细胞的增殖具有促进作用。更多还原

【Abstract】 Mammary gland, which provide nutritious milk for growth and development of newborn mammals, while human life is indispensable to provide dairy products. The cycle of mammary gland has development, lactation and involution of the three processes, during each cycle mammary epithelial cell experience the process of proliferation, differentiation and cell apoptosis. The milk component and milk yield are greatly changed in the different lactation stages of dairy goat. With the study of knowledge on genomics, people gradually recognized that these changes are determined and regulated by a series of functional genes expression during the temporal and spatial in mammary gland. The paper is based of the lactation physiological changes in Xinong Saanen dairy goat. Screening, cloning and functional analysis of differentially expressed genes will elucidate the molecular of mammary gland. Suppression subtractive hybridization(SSH) method is used to isolate differently expressed genes in mammary gland of difference lactation stage stage of the Xinong Saanen goat(Capra hircus),further clone the differently expressed genes and predict their structure and function by bioinformatics,study the function of osteopontin (OPN) gene which is one of the different expression candidate genes. For further study of the molecular mechanisms of lactation, the milk production and milk composition provide a theoretical basis.1. Suppression subtractive hybridization(SSH) technology is used to isolate differently expressed genes in mammary gland of early lactation stage and peak stage of the Xinong Saanen goat(Capra hircus).Some differently expressed genes were sequenced and analyzed.The new genes of dairy goat were searched and studied. Forward and reverse subtracted differently expressed cDNA library (E-P and P-E) were successfully created in mammary gland of different lactation. The subtraction efficiency was confirmed by a housekeeping gene named GAPDH,the results indicated that GAPDH was subtracted efficiently at 25 and 210 folds for E-P and P-E subtracted cDNA library respectively. Partial clone of E-P subtracted cDNA library were sequenced, we found that E-P library mainly included four groups of functional genes:milk protein related genes (αs2,βand K-casein, a-lactalbumin precursors), transcription complex genes(ribosomal protein L10,L23,3B and Cbp/p300),cell energy supplement genes NADH,cell proliferation and differentiation genes (GHITM、OPN and H3F3B).P-E library mainly included three groups of functional genes: milk fat metabolism related genes (HFABP、PPARGC1A),cell growth metabolism related genes (SAA3、SSAT、SPARC and LPO) and transcription related genes (splice factor 3B). Real-time reverse transcriptase-polymerase chain reaction was used to analyse the change of mRNA expression level of OPN and GHITM form E-P subtracted cDNA library. The results showed that they had 5.04 and 4.20 folds higher in early lactation stage than that in peak lactation stage. The change of mRNA expression level of PPARGCIA、SPARC and SSAT form P-E subtracted cDNA library had 3.58、12.13 and 8.0 folds higher in peak lactation stage than that in early lactation stage.2. We designed primers based on the high homology sequence in gene coding region among different species and existed sequence for goat, the CDs region of PPARGC1A、OPN、 SPARC、GHITM and SSAT genes were cloned and sequenced from the goat mammary gland by RT-PCR, bioinformatics were employed to predict gene specific structure and function. The structure and function of PPARGC1A、OPN、SPARC、GHITM and SSAT were compared among goat, bovine, human, and mouse using bioinformatics software. The putative signal sequences, transmembrane domain, secondary and tertiary structure of five proteins were predicted by way of bioinformatics. The different species have their characteristics. The peroxysome proliferator-activated receptor gamma coactivator-lalpha(PGCla) is transcriptional coactivators consisting of N-terminal transcriptional activation domains and nuclear hormone receptor-interacting motif (LXXLL) and C-terminal RNA processing motifs that plays a central role in the regulation of cellular energy metabolism. OPN protein is secreted hydrophilic glycoprotein. In different species of OPN have RGD motif, which is correlated with the accumulation and cell adhesion, proliferation and tissue remodeling. Goat OPN had two additional potential binding motifs:cAMP-dependent protein kinase phosphorylation site and agglutination factor V LSPR repeat loci compared to humans’and mice. SPARC is a glycoprotein of small molecule, including N-terminal domain and EF-hand calcium-binding site, which may be related to calcium metabolism in mammary gland. GHITM is a transmembrane protein, there are eight strong transmembrane region, suggesting the gene may be associated with milk-related ingredients transmembrane. SSAT is the key enzyme of the cell metabolic balance and a Gcn5-acetyl-amino-transferase domain is closely related to SSAT of the catalytic effect.3. Using beta-actin gene as the internal control, the SYBR Green quantitative real-time PCR (QPCR) analysis were conducted to determine the mRNA expression of PPARGC1A、OPN, SPARC、GHITM and SSAT genes in mammary gland at 28th,60th,100th,190th,270th and 330th day after kidding. The results showed that the expression trends of PPARGCIA and SSAT genes were similar with goat lactation curve, the dairy goat mammary gland increased milk yield dramatically during the first time of lactation, as evident from the marked increase in expression during the onset of lactation. PGCla mRNA expression reached the highest level on day 100, and subsequently decreased to a very low level on day 330. The expression on day 100 is about 13 folds (P< 0.01) of that on day 330. The results indicated that OPN gene exhibited the higher expression level in early (28 d) and late (190 d) lactation and the lowest level in dry period (330 d), which demonstrated a high-low-high-low pattern.SPARC gene exhibited the highest expression level in early (28 d) and late (190 d) lactation and subsequently decreased to a very low level on day 330.GHITM gene exhibited the highest expression level in early (28 d) subsequently decreased during the lactation of late, peak, end and dry periods.4. Recombinant plasmid of pcDNA3.1-OPN was constructed by inserting the fragment of OPN gene into eukaryotic expression vector pcDNA3.1 and used to transfect the MCF-7 cell line following the restrictive endonuclease cleavage and sequence identification of the target gene segment, the effect of OPN gene on MCF-7 cell proliferation was assessed by MTT analysis. There was a significant difference (P< 0.05) in the proliferation between OPN gene transfect and non-transfect MCF-7 cells, it is suggested that the expression of OPN gene could stimulate the proliferation of MCF-7 cells. The growth rates of the OPN gene transfect is 111.4% while non-transfect MCF-7 cells is 101.3%.更多还原