MC1R是控制鸡黑色素形成的候选主效基因

黑素皮质素受体1 (melanocortin 1-receptor, MC1R)基因是控制动物黑色素合成的重要基因.采用多聚酶链反应-单链构象多态性分析(PCR-SSCP)以及DNA测序的方法,在由丝羽乌骨鸡与明星肉鸡为亲本建立的中国农业大学资源家系群体鸡MC1R基因的编码区检测到3个单核苷酸多态位点,并对该单核苷酸多态性进行了分析.结果显示,鸡MC1R基因编码区引物3扩增片段多态性是由G→A（867位）点突变引起的,引物5扩增片段的多态性是由C→T（1 292位）与C→G（1 377位）两个点突变引起的,最后对单核苷酸多态性与肤色、肉色、胫色与内脏膜色等黑色素性状进行了卡方独立性分析,结果显示,MC1R基因编码区867处突变与鸡的肤色性状显著相关（P＜0.05）,1 292处突变与鸡的活体胫色性状显著相关（P＜0.05）,1 377处突变与鸡的肉色性状显著相关（P＜0.05）.研究表明,MC1R基因可能是鸡黑色素性状的主效基因或者与鸡控制黑色素性状的主效基因连锁.     

甘肃肉羊新品种选育群GDF9基因外显子2多态性及其与产羔性状关联分析

根据GenBank发布的绵羊GDF9基因外显子2的序列设计4对引物,采用PCR-SSCP技术分析GDF9基因外显子2在甘肃内羊新品种选育群羊中的单核苷酸多态性,并与产羔性状进行关联分析.结果表明,GDF9基因的扩增片段在所检测的新品种群羊中存在PCR-SSCP多态性,检测到3种基因型(AA、AB和BB),而在32只无角陶赛特母羊群中只检测到AA和AB基因型.测序结果显示,GDF9基因编码区第978位碱基发生A→G突变,但没有导致氨基酸的改变;第994位碱基发生G→A突变,导致Ⅴ变成Ⅰ(缬氨酸→异亮氨酸).新品种选育群羊产羔数的最小二乘均值关系为AB＞ AA＞ BB,统计分析结果初步表明3种基因型之间差异不显著(P＞0.05).故该区域可能不是影响新品种群羊繁殖力的功能结构区城.     

鸡Myostatin基因单核苷酸多态性的群体遗传学分析

肌肉生长抑制素是控制骨骼肌生长发育的重要细胞因子,采用PCR－SSCP和测序的方法发现了5个位于Myostatin基因5′－和3′－调控区的单核苷酸多态性位点,对北京油鸡、白耳鸡、石歧杂、矮小黄鸡、小型黄鸡、惠阳胡须鸡、隐性白羽鸡、海兰、AA鸡等不同鸡种的该单核苷酸多态性分析结果表明：Myostatin基因的5′调控区引物P60／P61扩增片段多态性是由3个核苷酸的改变而产生的[分别是G→A（304位）、A→G（322位）、G→（344位）],引物P93／P94扩增片段的多态性是由G→A（167位）突变造成的,引物P117。P118PC扩增片段多态性是由T→C（177位）造成的。3′调控我引物P80／P81扩增片段多态性是由第7263位A突变为T造成的,引物P76／P77扩增片段多态性是由A→G（6935位）造成的。不同鸡种群体遗传学分析表明,5′－调控区引物60／P61扩增片段多态性片段多态性是由A→G（6935位）造成的。不同鸡种群体遗传学分析表明,5′－调控区引物P60／P61扩增片段多态性位点在北京油鸡的基因型频率分布与其他的品种有很大的差异,其BB型频率为0．700,AA基因型频率仅为0．033,而其他鸡种中以A基因优势;对于引物P93／P94,品种间的基因型频率差异极显著（P＜0．01）,北京油鸡和AA鸡的EE型频率鸡种中以A基因占优势;对于引物P93／P94,品种间的基因型频率差异极显著（P＜0．01）,北京油鸡和AA鸡的EE型频率低于其他品种,白耳鸡和海兰蛋鸡以EE型为主,其频率高于其他品种;3′－调控区引物P80／P81多态怀位点在9个鸡种中都是等位基因C占优势。引物P76／P77,总体上MM型的频率较低,杂合子MN型的频率较高。     

荷斯坦牛HSP70-1基因遗传多态性与乳腺炎抗性关系

以253头中国荷斯坦奶牛为研究对象, 检测HSP70-1基因的多态性, 并分析其多态性与中国荷斯坦牛体细胞评分(Somatic cell score, SCS)的相关性。首先以PCR-SSCP法寻找HSP70-1基因编码区的突变, 并通过测序确定突变的类型, 根据突变类型寻找合适的内切酶, 最终采用PCR-RFLP方法鉴定实验牛基因型; 然后分析基因多态性与中国荷斯坦牛SCS的相关性。结果表明HSP70-1基因的1 623 bp处产生G→A→C突变, 2 409 bp处产生G→A突变, 两位点都是沉默突变, 未引起氨基酸序列的改变; 经χ2 适合性检验, 中国荷斯坦牛在两个位点均未达到Hardy-Weinberg平衡状态; 同时, 群体基因座不同基因型与SCS相关分析的结果表明, 2409位点基因型与SCS相关性不显著(P>0.05), 1623位点基因型与SCS相关性显著(P<0.05), CC型SCS显著低于AG、GG型(P<0.05), CC基因型为乳腺炎抗性基因型。在中国荷斯坦奶牛群体中, HSP70-1基因CC基因型可作为改良奶牛乳腺炎抗性性状的分子遗传标记。     

乐至黑山羊PRLR基因外显子10多态性与产羔数的关系研究

设计2对特异性引物对乐至黑山羊PRLR基因第10外显子进行了PCR-SSCP检测,并研究该基因与产子性能的相关性。结果表明,P1引物扩增片段不存在多态性;P2引物扩增片段存在多态性,表现为AA,AB,AD和CD 4种基因型,测序结果表明,4种基因型都在该片段第89、94、146和157位存在C→T、A→C、C→G、G→C的突变;此外AA型还在61位发生C→T的突变;AD型还在175位发生A→G的突变;CD型还在24位发生T→C的突变,96位发生C→T的突变,通过统计分析发现AD型平均产羔数优于其他3种基因型,并且与AB型差异达到显著水平(P<0.05)。因此认为PRLR基因对于乐至黑山羊产子性能有一定的影响。     

藏獒MC1R基因多态性与毛色表型的关联研究

目的:通过检测藏獒黑素皮质激素受体1(MC1R)基因的单链构象多态性(SSCP)在不同毛色群体中的分布,探讨MC1R基因多态性与毛色表型的相关性。方法:采用DNA测序技术,选择不同毛色藏獒的DNA为样本,根据GenBank发布的荷斯坦牛MC1R基因序列设计一对引物,采用PCR-SSCP技术分析MC1R基因在藏獒中的SSCP。结果:MC1R基因在藏獒中具有PCR-SSCP多态性,分别检测到3种基因型(AA、AB和BB);对MC1R基因多态性片段DNA克隆测序后发现,MC1R基因在编码区第313位存在单碱基突变(G→A),该突变导致第105位氨基酸发生由丙氨酸向苏氨酸的改变(T105A)。结论:MC1R基因的多态性与毛色性状不存在显著的相关性。     

红鳍东方鲀(Takifugu rubripes)MC4R基因的多态性分析

采用PCR-SSCP(single strand conformation polymorphism)技术和DNA测序方法分析红鳍东方鲀MC4R(Melanocortin-4receptor)基因编码区多态性。在MC4R基因编码区48 nt和264 nt均发生了碱基的转换突变(G→A),两个突变位点分别位于M1和M2引物扩增产物中。引物M1扩增产物SSCP分析得到两种基因型:AA基因型和AB基因型,并且AA基因型和A等位基因频率明显高于AB基因型和B等位基因。引物M2扩增产物也得到两种基因型:CC基因型和CD基因型,CC基因型和C等位基因频率明显高于CD基因型和D等位基因。遗传变异结果分析表明,两个突变位点均属于低度多态性,而且群体遗传杂合度较低,反映了该群体的遗传一致性较高。     

催乳素受体基因外显子10多态性及其与济宁青山羊高繁殖力关系的研究

设计5对引物, 采用PCR-SSCP技术检测催乳素受体(prolactin receptor, PRLR)基因外显子10及部分3′非翻译区在高繁殖力山羊品种(济宁青山羊)和低繁殖力山羊品种(辽宁绒山羊、波尔山羊和安哥拉山羊)中的单核苷酸多态性, 同时研究该基因对济宁青山羊高繁殖力的影响。结果表明: 首次拼接出的山羊PRLR基因外显子10及部分3′非翻译区的核苷酸序列长度为1,118 bp, 与已公布的绵羊、牛、人PRLR基因mRNA相应序列的同源性分别为98.33%、93.92%、74.52%, 与已公布的羊驼PRLR基因部分序列的同源性为78.29%。引物P1、P2与P4扩增片段具有多态性, 其余2对引物的扩增片段不存在多态性。对于P1扩增片段, 在济宁青山羊和辽宁绒山羊中检测到AA型和AB型, 在安哥拉山羊中检测到AA型和AC型, 在波尔山羊中只检测到AA型; 克隆测序表明AB型与AA型相比有两处突变(186G→A和220T→C), 分别导致氨基酸由天冬氨酸变为天冬酰胺、亮氨酸变为脯氨酸; AC型与AA型相比有1处突变(140A→G), 该突变没有导致氨基酸变化; 济宁青山羊AA和AB基因型之间产羔数的最小二乘均值差异不显著(P>0.05)。对于P2扩增片段, 在济宁青山羊、辽宁绒山羊和波尔山羊中都检测到DD型和DE型, 而在安哥拉山羊中只检测到DD型; 克隆测序表明DE型和DD型相比有两处突变(52G→A和122G→A), 其中122 bp处的突变导致氨基酸由精氨酸变为甘氨酸; 济宁青山羊DD和DE基因型之间产羔数的最小二乘均值差异不显著(P>0.05)。对于P4扩增片段, 在济宁青山羊中检测到FF型和FG型, 在辽宁绒山羊中检测到FF型和GG型, 在波尔山羊中只检测到FF型, 在安哥拉山羊中检测到FF型、FG型和GG型; 克隆测序表明GG型和FF型相比在扩增片段的143 bp处发生1处碱基突变(A→G), 并导致氨基酸由蛋氨酸变为缬氨酸; FG基因型济宁青山羊产羔数最小二乘均值比FF基因型的多0.76只(P<0.05)。研究结果初步表明: PRLR基因可能是控制济宁青山羊多胎性能的一个主效基因或是与之存在紧密遗传连锁的一个标记。     

FSHβ 基因PCR-SSCP多态性及其与济宁青山羊高繁殖力关系的研究

采用PCR-SSCP技术检测促卵泡素b亚基(follicle-stimulating hormone β, FSHβ)基因5′调控区、外显子1和外显子2在高繁殖力山羊品种(济宁青山羊)和低繁殖力山羊品种(辽宁绒山羊、波尔山羊、安哥拉山羊)中的单核苷酸多态性, 同时研究该基因对济宁青山羊高繁殖力的影响。结果表明: 山羊与绵羊的FSHβ 基因该段核苷酸序列同源性为98%。9对引物中, 只有P9的扩增片段存在多态性。P9的扩增片段在济宁青山羊和辽宁绒山羊中检测到AA、AB和AC 3种基因型; 在波尔山羊中检测到AA、CC和AC 3种基因型; 在安哥拉山羊中检测到AA、BB、CC、AB、AC和BC共6种基因型。测序分析发现BB型与AA型相比在外显子2的第94 bp处有G→A突变, 并引起氨基酸改变(丙氨酸→苏氨酸); CC型与AA型相比在外显子2的第174 bp有一处C→T沉默突变。济宁青山羊AA、AB和AC基因型频率分别为0.686、0.137和0.177。AA基因型济宁青山羊产羔数最小二乘均值比AB基因型的多0.78只(P<0.05), 比AC基因型的多0.64只(P<0.05)。     

贵州地方山羊CAST基因外显子6、7和8的多态性分析

为分析山羊CAST基因的多态性,筛选出对山羊肉质性状有显著影响的SNPs位点,本研究以黔北麻羊和贵州黑山羊为试验对象,构建DNA池,采用PCR产物直接测序法对2个品种山羊该基因的外显子6、7和8进行单核苷酸多态性检测,估算各SNPs等位基因频率,并利用在线软件预测突变前后的m RNA二级结构及理化特性。结果显示,所设计引物的扩增片段发现1个同义突变,位于外显子8中的T81C。利用生物信息学软件对外显子8中的T81C位点进行分析,结果表明这个SNPs位点导致编码的m RNA二级结构以及理化特性的改变。     

牛FSHR基因第10外显子单核苷酸多态性及其与双胎性状的关系

以秦川牛和荷斯坦奶牛的双胎母牛和单胎母牛为实验材料 ,以牛的FSHR基因的第 10个外显子作为标记牛双胎性状的候选基因 ,用SNP法进行了多态检测 .结果发现 ,在秦川牛的双胎母牛中突变率 6 0 % (6 10 ) ,而在单胎母牛中突变率为 2 0 % (2 10 ) ;在荷斯坦奶牛中 ,双胎母牛突变率为31 2 5 % (5 16 ) ,单胎母牛突变率为 6 6 7% (1 15 ) ;由此可见双胎牛和单胎牛二者之间FSHR基因的第 10个外显子的突变率差异明显 .这表明 ,选择FSHR基因的第 10个外显子有可能作为双胎性状的候选基因 .序列分析发现 ,在FSHR基因的第 15 0 6位碱基发生了突变 (T→C) ,但氨基酸没有发生变化 .     

已建株鼻咽癌细胞的PLUNC基因启动子区序列多态性分析

目的检测人类标准鼻咽癌细胞中是否存在已知的PLUNC基因启动子-437bp-＋87bp区域的单核苷酸多态性（SNP）。以便进一步探索SNP与鼻咽癌的关系。方法采用PCR产物直接测序的方法,对7株体外培养的鼻咽癌细胞基因组DNA的PLUNC基因启动子区进行序列分析。结果发现7株PLUNC基因的启动子区皆存在已知的3个SNP位点（1888、2128和N2）和未知一个突变位点（N1）,其测观杂合度分别为85．7％、100％、100％和28．6％。其中3个已知SNP位点在筛查的细胞株中均存在T-C的突变,而且SUNE-1鼻咽癌细胞株的1888位点基因型为突变纯合子CC型。结论体外培养的标准鼻咽癌细胞株中存在已知的3个SNP位点（1888、2128和N2）的突变现象,且突变率为100％;1888位点鼻咽癌易患型（CC型）已在体外稳定建株;首次发现启动子-195bp区域N1突变位点。     

Mutations of C-Reactive Protein (CRP) -286 SNP,APC and p53 in Colorectal Cancer: Implication for a CRP-Wnt Crosstalk

C-reactive protein (CRP) is an established marker of inflammation with pattern-recognition receptor-like activities. Despite the close association of the serum level of CRP with the risk and prognosis of several types of cancer, it remains elusive whether CRP contributes directly to tumorigenesis or just represents a bystander marker. We have recently identified recurrent mutations at the SNP position -286 (rs3091244) in the promoter of CRP gene in several tumor types, instead suggesting that locally produced CRP is a potential driver of tumorigenesis. However, it is unknown whether the -286 site is the sole SNP position of CRP gene targeted for mutation and whether there is any association between CRP SNP mutations and other frequently mutated genes in tumors. Herein, we have examined the genotypes of three common CRP non-coding SNPs (rs7553007, rs1205, rs3093077) in tumor/normal sample pairs of 5 cancer types (n = 141). No recurrent somatic mutations are found at these SNP positions, indicating that the -286 SNP mutations are preferentially selected during the development of cancer. Further analysis reveals that the -286 SNP mutations of CRP tend to co-occur with mutated APC particularly in rectal cancer (p = 0.04; n = 67). By contrast, mutations of CRP and p53 or K-ras appear to be unrelated. There results thus underscore the functional importance of the -286 mutation of CRP in tumorigenesis and imply an interaction between CRP and Wnt signaling pathway.     

The Effect of Ser 128 Substitution on the Structure and Stability of cAMP Receptor Protein from Escherichia coli

Kinetic measurements of denaturation and renaturation of two mutants of cAMP receptor protein (CRP) at position 128, namely Ser→Ala and Ser→Pro, were performed in order to assess changes introduced by the mutation in the quaternary structure and protein stability. No significant changes were found in the unfolding/refolding reactions. However, small perturbations in the dissociation of CRP dimer can be seen, which indicate that subunit interactions are influenced by the mutation. Studies of intrinsic fluorescence quenching of these two mutants are also reported, showing changes compared with wild-type protein. Near-UV circular dichroism measurements indicate, however, that Trp residues remain in the same environment as in the wild-type CRP. It is proposed that Ser at position 128 is involved in maintaining the proper domain alignment within CRP subunits.     

Association study of CRP gene polymorphisms with serum CRP level and cardiovascular risk in the NHLBI Family Heart Study

Recent epidemiological studies have indicated that baseline C-reactive protein (CRP) levels may have value in prediction of cardiovascular risk. Using six tag single-nucleotide polymorphisms (SNPs) selected from our complete list of SNPs on the CRP gene, we investigated the association of CRP genotypes with plasma CRP levels and cardiovascular risk in the National Heart, Lung, and Blood Institute (NHLBI) Family Heart Study cohort (1,296 Caucasians, 48.5% male, 54.7 +/- 12.8 yr old). There was a significant trend toward association of CRP haplotypes with CRP levels (P = 0.045). SNP analysis indicated a highly significant association of SNP -757 (rs3093059, P = 0.0004) and SNP -286 (rs3091244, P = 0.0065) and a borderline association of SNP -7180 (rs1341665, P = 0.06) with CRP levels. Neither CRP haplotypes nor individual SNP genotypes were associated with intima-media thickness of the common carotid or internal carotid artery or the bifurcation of the carotid arteries. These results indicated a strong impact of local SNPs of the CRP gene on plasma CRP levels, but there was no direct evidence that these genetically controlled CRP elevations by local CRP SNPs contributed to cardiovascular disease phenotypes.     

Differential structural status of the RNA counterpart of an undecamer quasi-palindromic DNA sequence present in LCR of human β-globin gene cluster

Our previous work on structural polymorphism shown at a single nucleotide polymorphism (SNP) (A→G) site located on HS4 region of locus control region (LCR) of β-globin gene has established a hairpin→duplex equilibrium corresponding to A→B like DNA transition (Kaushik M, Kukreti, R., Grover, D., Brahmachari, S.K. and Kukreti S. Nucleic Acids Res. 2003; Kaushik M, Kukreti S. Nucleic Acids Res. 2006). The G-allele of A→G SNP has been shown to be significantly associated with the occurrence of β-thalassemia. Considering the significance of this 11-nt long quasi-palindromic sequence [5′-TGGGG(G/A)CCCCA; HP(G/A)11] of β-globin gene LCR, we further explored the differential behavior of the same DNA sequence with its RNA counterpart, using various biophysical and biochemical techniques. In contrast to its DNA counterpart exhibiting a A→B structural transition and an equilibrium between duplex and hairpin forms, the studied RNA oligonucleotide sequence [5′-UGGGG(G/A)CCCCA; RHP(G/A)11] existed only in duplex form (A-conformation) and did not form hairpin. The single residue difference from A to G led to the unusual thermal stability of the RNA structure formed by the studied sequence. Since, naturally occurring mutations and various SNP sites may stabilize or destabilize the local DNA/RNA secondary structures, these structural transitions may affect the gene expression by a change in the protein–DNA recognition patterns.     

小型猪葡萄糖转运子4外显子4a的基因多态性分析

目的分析我国特有的3个小型猪品系巴马小型猪、中国农大小型猪、五指山小型猪葡萄糖转运子4基因外显子4a的单核苷酸多态性（SNPs）分布特点,为我国小型猪在糖尿病和代谢性疾病研究中提供基础资料。方法以3个品系小型猪基因组DNA为模板,应用特异性引物进行PCR扩增,扩增产物纯化测序,进行BLAST比对分析。结果在小型猪GLUT-4基因外显子4a上有2个SNP位点：SNP1：GCT→GCC（Ala133 Ala）,3个品系均发生了变化,均为纯合突变,其突变率为（22/22,100%）。SNP2：GGC→GGT（Gly146 Gly）,巴马小型猪突变率为（6/6,100%）,均为纯合突变;五指山小型猪突变率为（6/6,100%）,均为纯合突变;中国农大小型猪突变率为（10/10,100%）,其中包括6例纯合突变（6/10,60%）和4例杂合突变（4/10,40%）。结论SNPs1,在所有测定的小型猪品系中检出,这可能是所测小型猪的共有特征。SNPs2可能是小型猪品系之间的差异。     

IL-6 gene variation is associated with IL-6 and C-reactive protein levels but not cardiovascular outcomes in the Cardiovascular Health Study

Interleukin-6 (IL-6) and C-reactive protein (CRP) levels increase with age and likely play a role in adverse health outcomes in older adults. The relationship between IL-6 gene tag single nucleotide polymorphisms (SNPs) and circulating IL-6 and CRP levels, cardiovascular disease (CVD) outcomes, and mortality in Caucasian (CA) and African American (AA) participants of the Cardiovascular Health Study (CHS) was evaluated using ANCOVA and Cox proportional hazards models. The minor allele of the promoter SNP 1510 and intronic SNP 3572 associates with significantly higher serum IL-6 and CRP levels in CA but not AA. The CRP association persisted after CA and AA populations were combined and after accounting for multiple comparisons. These associations did not carry through to cardiovascular disease outcomes. Decreased risk of stroke was identified in CA, with the minor allele of SNP 1111 (HRR 0.71, 95% CI 0.52, 0.95), P = 0.02, and increased risk of CVD and all-cause mortality (HRR 1.31, 95% CI 1.05–1.64) in AAs heterozygote for SNP 2989. While genetic variation in the IL-6 gene was associated with circulating IL-6 and especially with CRP concentrations in this study, there is little evidence for association between common IL-6 gene variation and adverse health outcomes in this population of older adults.     

高山美利奴羊INHA基因多态性生物信息学分析及与产羔数关联分析

本研究旨在研究高山美利奴羊INHA基因外显子多态性及其与产羔数的相关性。本实验通过比对高山美利奴羊全基因组测序结果中不同个体INHA外显子,分析高山美利奴羊INHA基因的单核苷酸多态性,应用生物信息学软件分析高山美利奴羊INHA基因突变前后不同等位基因的m RNA二级结构、蛋白质的二级结构及三级结构。通过上述分析,将引起高山美利奴羊INHA编码氨基酸变化的位点作为该基因的特异性候选位点,通过直接测序法分析高山美利奴羊特异性候选位点INHA基因多态性,并分析其与产羔数的相关性。结果发现,高山美利奴羊INHA基因外显子区域存在3个SNPs,分别为T206A (Met→Lys)、T387A(Thr→Thr)、G900A (Pro→Pro);INHA基因3个SNPS都改变了RNA的最小自由能以及二级结构,错义突变T206A (Met→Lys)引起蛋白质二级结构和三级结构的改变;高山美利奴羊INHA基因T206A突变表现出3种基因型分别命名为TT、TA、AA,基因型与产羔数关联分析发现TT、TA基因型个体的产羔数极显著高于TT基因型个体(p<0.01)。本研究初步表明INHA基因是影响高山美利奴羊产羔数的一个主效基因。