小尾寒羊高繁殖力候选基因BMP15和GDF 9的研究

以控制Belclare和Cambridge绵羊高繁殖力的骨形态发生蛋白 15 (bonemorphogeneticprotein 15 ,BMP15 )基因和生长分化因子 9(growthdifferentiationfactor 9,GDF9)基因为候选基因 ,采用PCR RFLP技术检测BMP15基因和GDF9基因在高繁殖力绵羊品种 (小尾寒羊、湖羊 )以及低繁殖力绵羊品种 (多赛特羊、特克塞尔羊、德国肉用美利奴羊 )中的单核苷酸多态性 ,同时研究这两个基因对小尾寒羊高繁殖力的影响。结果表明 :在 5个绵羊品种中都没有检测到GDF9基因的G8突变 (C→T) ,也没有检测到BMP15基因的B4突变 (G→T)。高繁殖力的小尾寒羊在BMP15基因编码序列第 718位碱基处发生了与Belclare绵羊和Cambridge绵羊相同的B2突变 (C→T) ,而其余 4个绵羊品种则没有发生这种突变。对于BMP15基因的B2突变 ,在小尾寒羊中检测到AA、AB两种基因型 ,A等位基因频率为 0 734,B等位基因频率为 0 2 6 6。小尾寒羊与其余 4个绵羊品种间B2突变基因型分布差异极显著 (P <0 0 0 1)。突变杂合基因型 (AB)小尾寒羊平均产羔数比野生纯合基因型 (AA)多 0 6 2只 (P <0 0 1)。研究结果表明 ,BMP15B2突变对小尾寒羊高繁殖力影响作用十分明显 ,同时排除了GDF9G8突变和BMP15B4突变影响小尾寒羊高繁殖力的可能性     

小尾寒羊高繁殖力候选基因ESR的研究

利用PCR—SSCP技术对高繁殖力绵羊品种（小尾寒羊、湖羊、德国肉用美利奴羊）和低繁殖力绵羊品种（多赛特羊、萨福克羊）的雌激素受体（estrogen receptor,ESR）基因第一外显子部分序列进行单核苷酸多态性研究。结果表明：小尾寒羊、湖羊和德国肉用美利奴羊中存在3种基因型（AA、BB、AB）,而在多赛特羊和萨福克羊中只存在两种基因型（AA、AB）。统计结果表明：湖羊、德国肉用美利奴羊、小尾寒羊、萨福克羊和多赛特羊A等位基因频率分别为0．672、0．786、0．846、0．857和0．867,B等位基因频率分别为0．328、0．214、0．154、0．143和0．133。测序结果表明：BB型和AA型相比在外显子1第363位发生1处碱基突变（C→G）。独立性检验表明：小尾寒羊和湖羊之间基因型分布差异极显著（P〈0．01）,湖羊和多赛特羊之间基因型分布差异显著（P〈0．05）,其他各个绵羊品种之间基因型分布差异均不显著。A8基因型和BB基因型小尾寒羊产羔数比AA基因型分别多0．51只（P〈0．05）和0．7只（P〈0．05）。研究结果表明：ESR基因可能是控制小尾寒羊多胎性能的一个主效基因或与之存在紧密的遗传连锁。     

绵羊GDF9和BMP15基因多态性检测

以绵羊GDF9基因FecG^H突变和BMP15基因FecX^B和FecX^G突变为候选基因,采用PCR—RFLP方法研究其在湖羊、夏洛来、陶赛特、萨福克、中国美利奴肉用多胎品系、中国美利奴羊和罗米丽羊7个品种中的多态性．结果发现,湖羊群体中存在GDF和BMP15基因的FecG^H和FecX^B突变,但发生率极低,分别为0．645％(2／310)和0．968％(3／310)：而其它品种中则没有发现GDF9和BMP15基因的相应突变．这一发现对于建立绵羊基因标记辅助选择方法具有重要意义．     

解偶联蛋白基因(UCP)作为影响鸡脂肪性状候选基因的研究

解偶联蛋白基因是新近发现的能够增加能量的消耗,与脂肪代谢和能量调控有密切关系的一个基因,可以将其作为研究肉鸡体脂代谢的候选基因。以肉鸡和3个地方品种（北京油鸡,石岐杂,白耳鸡）以及海兰褐蛋鸡为实验材料,用2对引物对解偶联蛋白基因（UCP）的3‘非翻译区进行SNPs检测,探讨UCP基因作为影响鸡脂肪性状候原可能性。利用单链构像多态（SSCP）的方法进行SNPs的检测和基因型的判别。x^2检测表明,2对引物的突变产生的基因型和基因频率在各品种间差异极显著（P＜0．01）,但在肉鸡与北京油鸡之间,白耳鸡与海兰褐蛋鸡之间的差异不显著,初步推断是因为北京油鸡属于肉用性状较突出的地方品种,推断它与现代肉鸡的遗传基础类似,而白耳鸡是偏向于蛋用的地方品种,和蛋鸡的遗传基础类似。引的2的1197处突变产生的3种基因型与肉鸡屠体性状的最小二乘分析结果显示：BB基因型个体的腹脂重和腹脂率显著低于AB,AA基因型的个体。初步推断UCP基因为影响鸡脂肪性状的主效基因或与主效基因连锁。     

异育银鲫BMP15基因片段的克隆及序列分析

根据斑马鱼骨形态发生蛋白15(bone morphogenetic protein,BMP15)基因的保守区序列设计引物,采用RT-PCR法扩增出异育银鲫BMP15基因的部分序列,该片段长811 bp,编码270个氨基酸。经过BLAST比对,该氨基酸序列与斑马鱼、欧洲海鲈及人等动物BMP15基因的同源性分别为80%、38%及29%。本试验结果为进一步研究该基因的结构与功能提供了资料。     

肌内脂肪候选基因的研究

肌内脂肪含量是重要的肉质指标,而脂肪酸结合蛋白在动物体内代谢过程中的作用,已经被认为与肌间脂肪含量等肉 质性状呈显著相关,其中两个编码基因已被列为这些数量性状的候选基因,进行了重点研究。本文通过搜索NCBI Pulmed和 Gene Bank中的文献和有关数据,比较全面地综述了近年来肌内脂肪和两类脂肪酸结合蛋白基因的研究。     

大鼠脑皮层细胞低氧反应候选基因的研究

机体对低氧的应激反应与适应主要是通过对有关低氧反应基因 (ｈｙｐｏｘｉａｒｅｓｐｏｎｓｉｖｅｇｅｎｅｓ ,ＨＲＧ)的调控实现的 ,ＨＲＧ参与低氧的感受、低氧信号的传导及生理反应等。近年来对低氧反应基因的研究主要是关于己知基因的调控序列、调控环节及生物学功能的研究 ,尚未有新的、有明确生物学意义的低氧反应候选基因被发现。因此 ,我们采用ＤＤ ＰＣＲ技术 ,观察低氧大鼠脑皮层细胞在低氧时基因的差异表达 ,以期了解中枢神经系统在低氧过程中可能参与的相关基因。　Ｆｉｇ .13ｄｉｆｆｅｒｅｎｔｉａｌｌｙｅｘｐｒｅｓｓｅｄｈ…     

BMPR-IB基因对绵羊产羔数影响的回顾和Meta分析

为了验证澳大利亚美利奴绵羊的候选基因研究中骨形态发生蛋白受体IB型(BMPR-IB)基因与产羔数增加是否有关,及随后在不同的绵羊品种中进行的研究显示了不同的结果,特别是在亚洲地区的绵羊品种中更是如此的现象。因此,有必要对不同绵羊品种的各种研究进行Meta分析,合并加权均数差(WMD)和置信区间(CI)以评估这些关联的强度。试验共包括18项研究,其中10 895个样本用于BMPR-IB基因多态性。结果表明,观察到BMPR-IB基因与绵羊胎产羔数之间存在显著的相关性(BB vs.++:WMD=0.88, 95%CI=0.73～1.04,p0.01; B+vs.++:WMD=0.53, 95%CI=0.45～0.64,p=0.01),并且BMPR-IB基因的作用对于绵羊的产羔量是加性的(每个等位基因的一个拷贝增加约0.5个羔羊)。说明这种涉及非常大样本量的Meta分析意味着BMPR-IB基因与绵羊胎产仔数之间的显著关联,应进一步研究以确定这些不一致结果中常见因果变体的潜在机制。     

FTH1基因多态性与湖羊和小尾寒羊产羔数的关联分析

为了解FTH1基因在2个绵羊品种中的遗传变异及其与产羔数的关系,本研究以具有详细产羔记录的湖羊(n=424)和小尾寒羊(n=432)为研究对象,采用DNA-pooling结合PCR-RFLP的方法进行单核苷酸多态性(single nucleotide polymorphism, SNP)扫描,同时分析SNP位点与绵羊产羔数的相关性。结果表明,在FTH1基因第一内含子上发现一个g.1635GA的SNP位点,并在小尾寒羊和湖羊群体中均有AA、AG和GG三种基因型,其基因型频率分别为0.08、0.45、0.47和0.10、0.43、0.47,A和G等位基因频率分别为0.305、0.695和0.315、0.685,表明GG为优势基因型,G为优势等位基因;湖羊群体的基因He、Ho、Ne和PIC分别为0.431、0.569、1.759和0.338;在小尾寒羊中则分别为0.424、0.576、1.737和0.334。χ~2适合性检验表明,在此位点湖羊和小尾寒羊群体均不处于Hardy-Weinberg平衡状态(p0.05)。与产羔数的关联分析发现,在湖羊群体中,GG基因型个体的产羔数显著高于AA基因型(p=0.029),而在小尾寒羊群体中各基因型个体的产羔数差异不显著。综上所述,FTH1基因可以作为提高湖羊产羔数的分子辅助标记。     

鲤脑组织低温差异表达候选基因的筛选

采用双标准曲线相对实时荧光定量PCR法,分别以23℃常温对照组和6℃低温待测组的黑龙江鲤脑组织cDNA为模板,以18S rRNA为内参基因,检测26个候选基因的相对表达量。实验数据经显著性分析发现,有5个候选基因在低温条件下表达量显著上升(P<0.01),与对照组相比它们的表达量分别上升了2.11倍、13.9倍、2.52倍、7.38倍和1.83倍,基因功能比对结果表明其编码蛋白产物分别是脂肪酸链延伸蛋白、酰基辅酶A脱氢酶、转录起始因子IIB、肌醇-1-磷酸合成酶、血脑屏障HT7抗原;有7个候选基因在低温下表达量分别下降了21.8%、25.9%、16.6%、23.7%、15.8%、16.3%、42.5%,但对照组和待测组差异不显著(P>0.05),基因功能比对发现它们主要参与抑制糖酵解,促进细胞凋亡和干扰神经系统的重塑活动。上述低温下表达量显著上升的5个冷诱导候选基因的获得为今后进行不耐低温鱼类的基因工程育种提供了基因元件。     

小尾寒羊微卫星与RAPD标记的研究

小尾寒羊是世界上具有非季节性发情和多胎特性的高繁殖率绵羊品种之一。选择4个位于绵羊6号染色体上且与FecB基因紧密连锁的微卫星标记,对小尾寒羊基因组进行PCR扩增后,采用最小二乘法估计各等位基因片段对产羔数的影响。分析结果表明,等位基因OarJIA-5、OarJIA-10、BM143-12和OarHH55-11可以作为小尾寒羊多胎位点的分子标记。从100条随机引物中筛选出18条引物,对小尾寒羊、大尾寒羊、洼地绵羊、滩羊等4个绵羊品种和鲁北山羊的基因组进行扩增,共扩增出146条带,其中94条表现出多态性,占64．60%,同时扩增出每个品种的特异性条带。采用Nei氏标准距离和NJ聚类分析对不同品种的遗传关系进行分析,结果表明,4个绵羊品种亲缘关系很近,提示它们可能起源于共同的原始祖先。     

三种绵羊BMP15基因第二外显子多态性及与产羔数的相关性分析

绵羊存在影响多胎性状的不同主效基因,选择影响Romney Hanna绵羊和Cambridge绵羊高繁殖力的骨形态发生蛋白15 (bone morphogenetic protein 15, BMP15)为候选基因,采用PCR-SSCP的方法检测BMP15基因外显子Ⅱ第747位点(T747→C)和755位点(T755→C)在蒙古羊、甘肃高山细毛羊、小尾寒羊三种绵羊母羊中的多态性,同时还研究了上述两处突变对三种绵羊产羔数的影响。表明:(1)一共检测到野生纯合型AA、突变杂合型AB (T747→C)、AC (T755→C)三种不同的基因型,AA为优势基因型,A为优势等位基因;(2)三种基因型在甘肃高山细毛羊中均被检测到,而蒙古羊和小尾寒羊中未检测出AB基因型;(3)突变杂合型蒙古羊(AC)比野生纯合型(AA)的平均产羔数多0.27只(p<0.05)。(4)AC的基因型频率,双羔母羊和多羔母羊均高于单羔母羊。根据以上实验推测,BMP15第755位点发生的T→C突变(AC型)对蒙古羊一胎产双羔影响十分显著,甘肃高山细毛羊中AC基因型的绵羊其产羔数有比AA基因型和AB基因型多的趋势,因此该位点可能是一个影响绵羊高繁殖力潜在的DNA标记。     

Cryopreservation and multipotential characteristics evaluation of a novel type of mesenchymal stem cells derived from Small Tailed Han Sheep fetal lung tissue

Lung mesenchymal stem cells (L-MSCs) characterized by plasticity, reduced relative immune privilege and high anti-fibrosis characteristics play the crucial role in lung tissue regenerative processes. However, up to date, the multi-differentiation potentials and application values of L-MSCs are still uncertain. In the current study, the Small Tailed Han Sheep embryo L-MSCs line from 12 samples, stocking 124 cryogenically-preserved vials, was successfully established by using primary culture and cell cryopreservation techniques. Isolated L-MSCs were morphologically consistent with fibroblasts, could be passaged for at least 18 passages and more than 91.8% of cells were diploid (2n = 54) analyze by G-banding. The majority of cells were in the G0/G1 phase (70.5–91.2%), and the growth curves were all typically sigmoidal. Moreover, L-MSCs were found to express pluripotent genes Oct4, Nanog and MSCs-associated genes β-integrin, CD29, CD44, CD71, CD73 and CD90, while the expressions of hematopoietic cell markers CD34 and CD45 were negative. In addtion, the L-MSCs could be differentiated into cells of three layers with induction medium in vitro, which confirmed their multilineage differentiation potential. The secretion of urea and ALB showed the differentiated hepatocytes still possessed the detoxification function. These results indicated that the isolated L-MSCs displayed typical characteristics of mesenchymal stem cells and that the culture conditions were suitable for their maintenance of stemness and their proliferation in vitro.     

Prolactin Receptor as a Candidate Gene for Prolificacy of Small Tail Han Sheep

DNA polymorphism of the ovine prolactin receptor gene (PRLR) was investigated and used to study its effect on litter size in sheep. By means of PRLR gene sequence homology between sheep and human, three primer pairs were designed for polymerase chain reaction (PCR) amplification within intron 1 and exon 10 of the PRLR gene in sheep. In these parts of the gene the single nucleotide polymorphisms were detected by PCR-single strand conformation polymorphism (SSCP) in 314 Small Tail Han ewes. These poly-morphisms were used to study the associations with litter size. The results indicated that there were three genotypes (AA, AB and BB) detected by three primer pairs. For three primer pairs the frequency of allele A was 0.96, 0.79, 0.68; and the frequency of allele B was 0.04, 0.21, 0.32, respectively. The frequency of genotype AA was 0.93, 0.62, 0.51; the frequency of genotype AB was 0.06, 0.34, 0.34; the frequency of genotype BB was 0.01, 0.04, 0.15, respectively. The Small Tail Han ewes with genotype BB or AB had 0.64–0.76 or 0.44–0.54 more lambs than those with genotype AA, respectively. These results preliminarily showed that the prolactin receptor locus is either a major gene that influences the prolificacy in Small Tail Han sheep or is in close linkage with such a gene.     

四引物扩增受阻突变体系PCR快速测定绵羊 BMPR-IB基因型方法的建立

四引物ARMS PCR是检测SNP有效、快速、简便的方法.绵羊BMPR-lB基因是控制Booroola绵羊多胎性状的主效基因,此研究目的在于建立一种对BMPR-IB基因四引物ARMS PCR检测方法.根据四引物ARMS PCR技术原理,在绵羊BMPR-IB基因突变位点(A746G)设计一对特异性引物,并在突变点两侧设计一对参照引物,用来扩增含有突变点的DNA片段,可在一步PCR反应中根据电泳图谱准确判断绵羊个体的BMPR-IB基因型,对比PCR-RFLP检测结果表明,所建立的方法简单,操作简便,大大提高了检测效率.     

Novel Mutation in Exon 1 of the BMP15 Gene and its Association with Reproduction Traits in Sheep

The BMP15 gene is a growth factor and a member of the transforming growth factor β (TGFβ) superfamily, specifically expressed in oocytes. In the present study, polymorphism of BMP15 gene exon 1 was studied using single strand conformational polymorphism (SSCP) and direct DNA sequencing methods in 170 Mehraban and Lori sheep ewes. A 231-bp fragment in BMP15 exon 1 was amplified by PCR reactions. Two genotypes (GG and AG) with a new point mutation at position 121 bp of the studied fragment (c.379G>A in reference GenBank number AF236078.1 sequence), deducing an amino acid exchange in the codified amino acid sequence (p.Glu41Lys) were identified in the studied populations. The AG and GG frequencies were 74.4% and 25.6% in Mehraban and 44.7% and 55.3% in Lori sheep, respectively. Frequencies of the A and G alleles were 37.2% and 62.8% in Mehraban and 22.4% and 77.6% in Lori sheep, respectively. Two different secondary structures of protein were predicted for encoded precursor protein. The genotypes GG and AG did not have any significant association with the studied reproductive traits, but the AA genotype is likely to have a lethal or sterility effect.     

Effect of BMP15 and/or AMH during in vitro maturation of oocytes from involuntarily culled dairy cows

The high metabolic activity to which the dairy cattle are exposed to maintain milk production altered steroid metabolism that affects reproductive physiology and reduce oocyte competence. Our aims were (a) to characterize the competence of immature oocytes collected from dairy cattle based on the expression of genes in cumulus cells (CCs) and (b) to improve oocyte competence to support preimplantation embryo development by the supplementation of maturation medium with bone morphogenetic protein 15 (BMP15) and/or anti‐mullerian hormone (AMH). Oocyte donors were identified at the moment of ovary collection and grouped by involuntarily culled dairy cows (Holstein breed) or beef cattle. The embryo development speed to blastocyst of the cull dairy cattle versus beef cattle (control group) was lower. Besides, <10% of oocytes (with CC biopsies) derived from dairy cattle were able to develop to the blastocyst stage. In addition, a higher level of expression and a positive correlation were observed in the expression of most of the genes evaluated (LUM, KRT18, KRT8, CLIC3, BMPR1B, and SLC38A3) in the cumulus–oocyte complexes that produced blastocysts versus those which did not develop correctly (arrested development). Further, use of BMP15 in the maturation of oocytes from dairy cattle seems to increase competence, modulating the expression of OCT4, SOX2, CDX2, GATA6, and TP1 in resulting blastocysts.