威宁绵羊和贵州半细毛羊STAT5b基因部分外显子SNP研究

为给贵州本地绵羊选种选育提供更好的科学依据,本试验利用威宁绵羊和贵州半细毛羊构建DNA池,设计4对引物扩增其STAT5b基因部分外显子及内含子序列。PCR产物经纯化后进行双向测序。利用DNAStar和BLAST分析确定多态性位点。利用生物信息学软件分析SNPs位点对STAT5b基因RNA二级结构、STAT5b蛋白二级及三级结构的影响。结果表明,在扩增的STAT5b基因中筛选到6个SNPs:exon5-G12A、exon8-G56A、exon8-C104T、intron2-A3164C、intron4-C1026T和intron5-T3323C,其中exon8-G56A为错义突变,导致编码的谷氨酸(Glu)变为赖氨酸(Lys);exon5-G12A和exon8-C104T多态位点均未改变氨基酸的编码,为同义突变;intron2-A3164C、intron4-C1026T和intron5-T3323C均在内含子区,不参与氨基酸编码。     

江口萝卜猪FABPs主要家族基因SNPs筛选及生物信息学分析

以江口萝卜猪为试验材料构建DNA池,采用直接测序技术对猪L-FABP、I-FABP、H-FABP、A-FABP基因进行SNPs快速筛查,共检测出7个SNPs,分别为intron1-T1745C、exon2-T125C、exon2-A131C、exon2-C153A、exon2-A65T、exon2-G40A、intron3-C65T。其中intron1-T1745C、intron3-C65T位于内含子上;exon2-T125C、exon2-C153A为同义突变;exon2-A131C、exon2-A65T、exon2-G40A为错义突变,分别使编码氨基酸发生Glu→Ala、Lys→Met、Glu→Lys的改变,对其进行生物信息学分析表明,突变前后的等位基因频率估算,mR NA二级结构预测,蛋白质二级、三级结构预测分析均有差异。     

贵州白山羊LEPR基因多态性

为探究LEPR基因多态性的遗传特性,本研究以贵州白山羊为试验材料,运用DNA池结合直接测序方法进行LEPR基因的SNPs位点的筛选,继而对LEPR基因RNA的二级结构以及其所编码蛋白质的二级结构和三级结构进行生物信息分析。结果表明,在试验群体LEPR基因中共发现4个SNPs,分别为exon4-G246A(Asp-Asn)、exon8-C39T(同义突变)、intron8-G59A(内含子突变)和exon18-C94T(Ser-Leu)。经生物信息学软件分析表明,exon4-G246A(Asp-Asn)和exon18-C94T(Ser-Leu)位点突变前后的等位基因频率、LEPR m RNA的二级结构、LEPR蛋白质二级结构和三级结构均有改变。     

家兔UCP2基因多态性的筛选和生物信息学研究

本文研究兔的UCP2基因的多态性,为地方兔种的选种和选育提供一定的依据。分别以比利时兔、加利福尼亚兔和新西兰兔构建其DNA池,设计6对引物扩增3个兔种UCP2基因的外显子序列和部分内含子序列,用PCR产物直接进行双向测序快速的筛选出兔的UCP2的多态位点。结果表明:在兔UCP2基因中筛选到8个多态性位点:intron2-G2217T、exon3-A63C、exon3-G169A、intron5-C61A、exon6-C11G、intron6-C7T、intron6-C46T、intron6-C73A,其中exon3-A63C和exon3-G169A位于第3外显子上,且exon3-A63C为错义突变,导致编码的酪氨酸(Tyr)变为丝氨酸(Ser)。exon6-C11G在第6外显子上,其余的多态位点都在内含子中,除了intron6-C7T外,其余的多态位点在三种兔种中都有。结论:通过生物信息学对兔UCP2基因的分析发现m RNA二级结构、二级结构和蛋白质的三级结构在突变前后都发生变化。     

家兔UCP3基因SNP多态性及生物信息学分析

以比利时兔、加利福尼亚兔和新西兰兔为实验对象构建其DNA池,直接测序筛选SNP位点,结果在兔UCP3基因中筛选到5个多态性位点:intron4-C1298T、exon6-G208A、exon6-G281A、exon6-T303C和exon6-G443A。其中exon6-G208A、exon6-G281A和exon6-G443A为错义突变,exon6-T303C为同义突变,而exon6-G208A导致编码的精氨酸(Arg)变为丝氨酸(Ser),exon6-G281A和exon6-G443A导致编码的丝氨酸(Ser)变为酪氨酸(Tyr)。通过生物信息学软件对兔UCP3基因突变前后RNA二级结构和蛋白质的二级结构进行预测分析,发现其结果均有差异。     

贵州地方黄牛IGF-1基因多态性及生物信息学分析

为分析黄牛IGF-1基因多态性,筛选出对贵州本地黄牛生长性状有显著影响的SNPs位点,本研究以贵州关岭牛、思南牛、威宁牛和黎平牛为试验对象,构建DNA混池,PCR扩增IGF-1基因所有外显子及部分内含子,采用直接测序法检测IGF-1基因多态性,利用生物信息学软件对IGF-1基因进行分析。结果表明:在贵州4个地方牛群体种中,共筛选出3个SNPS,分别为Intron2-A5123G、Exon3-G56419A、Intron3-C56464A,Exon3-G56419A属于同义突变。其中2个SNPS (Intron2-A5123G, Intron3-C56464A)为4个牛种所共有,而Exon3-G56419A突变仅存在黎平牛中。生物信息学分析表明,牛IGF-1编码蛋白为分泌蛋白,含有信号肽序列,相对分子质量为16 937.68,理论等电点为9.36,且突变前后IGF-1基因m RNA二级结构发生改变,自由能升高,稳定性降低。研究结果显示,4个牛种IGF-1基因具有较高的遗传多样性,可为贵州地方牛种的保种选育提供参考。     

贵州地方山羊品种H-FABP基因第3外显子SNP研究

[目的]研究3种贵州地方山羊品种H-FABP基因第3外显子的多态性以及进行该基因多态性生物信息学分析,以期筛选出适合的突变位点,研究其与山羊生长性状关联性影响。[方法]以3种贵州地方山羊品种为试验材料构建DNA池,结合直接测序技术筛选H-FABP基因SNPs,并利用在线软件分析H-FABP基因RNA二级结构及其蛋白二、三级结构。[结果]仅在黔北麻羊和贵州白山羊H-FABP基因第3外显子处分别筛选到T120A和G152C两个SNPs,其中exon3-T120A为导致编码氨基酸发生的Phe→Ile改变的错义突变,而exon3-G152C为苏氨酸(Thr)的同义突变。[结论]exon3-T120A位点影响黔北麻羊H-FABP基因RNA二级结构,最小自由能由-189.40 kcal/mol变为-190.00 kcal/mol,其蛋白二级结构无规则卷曲由49变为48,延伸链由36变为37,exon3-G152C位点仅影响贵州白山羊RNA二级结构,最小自由能变为-185.40 kcal/mol。     

三穗鸭DRD4基因多态性及生物信息学分析

本实验以三穗鸭为材料,采用PCR扩增并采用直接测序技术对三穗鸭DRD4基因进行SNP多态位点检测,共筛查出4个SNP位点:exon2-C12406T、exon3-A13144G、intron2-C12777T、intron2-T12789C。其中exon2-C12406T、exon3-A13144G位点突变为同义突变。对DRD4进行生物信息学分析表明,突变前后的等位基因频率估算有差异,但其m RNA二级结构预测,蛋白质二级、三级结构预测分析均无差异。     

贵州黑山羊和黔北麻羊LEPR基因第7、8、10和18外显子的多态性分析

为探究LEPR基因的遗传多态性,丰富山羊LEPR基因的研究,本研究以贵州黑山羊和黔北麻羊为试验材料,运用DNA池结合直接测序方法进行LEPR基因SNPs位点的筛选,从而对突变的SNPs位点进行RNA二级结构以及所编码蛋白质的二级结构和三级结构进行生物信息学分析。在试羊LEPR基因中共发现4个SNPs,分别为Exon7-T81C(同义突变)、Exon8-C39T(同义突变)、Exon10-A70G(Asn-Ser)和Exon18-C94T(Ser-Leu)。分析表明,4个位点突变前后的等位基因频率、mRNA二级结构的最小自由能、LEPR蛋白质二级结构和三级结构均有改变。结果表明,LEPR基因拥有较为丰富的遗传多态性。     

DNA池快速筛查牛STAM1基因SNPs及估算等位基因频率

目的:旨在对不同牛种STAM1基因进行SNPs筛查,为地方牛种选种选育提供一定理论依据.方法:选取生长发育性状明显差异的务川黑牛和贵州荷斯坦奶牛2个牛种构建DNA池,设计1对引物分别扩增2个牛种STAM1基因第14外显子序列总长898bp.切胶回收后对PCR产物进行双向测序.结果:在牛STAM1基因中快速筛查到5个SNPs:A33C、C66G、C356T、T523A、T652C,其中A33C(Tyr→Ser)、C66G(Pro→Arg)、C356T(Glu→Lys)为错义突变,T523A为同义突变,T652C位于内含子区.贵州荷斯坦奶牛在T523A和T652C两个位点基因频率为1.0000,而务川黑牛分别为0.6730和0.8106.生物信息学分析表明:突变前后STAM1的RNA二级结构和蛋白质二级、三级结构均有明显改变.结论:DNA池结合测序技术可快速筛选SNP位点,检测到STAM1基因第14外显子5个SNPs.     

Identification and assignment of base pairs in four helical segments of Bacillus megaterium ribosomal 5S RNA and its ribonuclease T1 cleavage fragments by means of 500-MHz proton homonuclear Overhauser enhancements.

Three different fragments of Bacillus megaterium ribosomal 5S RNA have been produced by enzymatic cleavage with ribonuclease T1. Fragment A consists of helices II and III, fragment B contains helix IV, and fragment C contains helix I of the universal 5S rRNA secondary structure. All (eight) imino proton resonances in the downfield region (9-15 ppm) of the 500-MHz proton FT NMR spectrum of fragment B have been identified and assigned as G80.C92-G81.C91-G82.C90-A83.++ +U89-C84.G88 and three unpaired U's (U85, U86, and U87) in helix IV by proton homonuclear Overhauser enhancement connectivities. The secondary structure in helix IV of the prokaryotic loop is completely demonstrated spectroscopically for the first time in any native or enzyme-cleaved 5S rRNA. In addition, G21.C58-A20.U59-G19.C60-A18.U61 in helix II, U32.A46-G31.C47-C30.G48-C29.G49 in helix III, and G4.C112-G5.C111-U6.G110 in the terminal stem (helix I) have been assigned by means of NOE experiments on intact 5S rRNA and its fragments A and C. Base pairs in helices I-IV of the universal secondary structure of B. megaterium 5S RNA are described.     

An approach to the structure determination of nucleic acid analogues hybridized to RNA. NMR studies of a duplex between 2'-OMe RNA and an oligonucleotide containing a single amide backbone modification.

The backbone modification amide-3, in which -CH2-NH-CO-CH2- replaces -C5'H2-O5'-PO2-O3'-, is studied in the duplex d(G1-C2-G3-T4.T5-G6-C7-G8)*mr(C9-G10-C11-A12-A13-C14-G15+ ++-C16) where . indicates the backbone modification and mr indicates the 2'-OMe RNA strand. The majority of the exchangeable and non-exchangeable resonances have been assigned. The assignment procedure differs from standard methods. The methyl substituent of the 2'-OMe position of the RNA strand can be used as a tool in the interpretation. The duplex structure is a right-handed double helix. The sugar conformations of the 2'-OMe RNA strand are predominantly N-type and the 2'-OMe is positioned at the surface of the minor groove. In the complementary strand, only the sugar of residue T4 is found exclusively in N-type conformation. The incorporation of the amide modification does not effect very strongly the duplex structure. All bases are involved in Watson-Crick base pairs.     

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。     

Novel SNPs of the Bovine PRLR Gene Associated with Milk Production Traits

The single nucleotide polymorphisms (SNPs) within exon 10 of the prolactin receptor gene (PRLR) were detected in Chinese Holstein cows using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods, and their genetic effects on milk production traits were evaluated in this study. Two newly detected SNPs (g.9206G→A and g.9681C→T) caused amino acid variations E378K and A536V, respectively, which were then preliminarily predicted at the topological level. Statistical results indicated that the two SNPs were significantly associated with milk yields, and cows with the combined genotype GGCC showed superior milk performance. A putative phosphorylation site was identified at residue 378K ([ST]-×-[RK]), which offers a partial explanation for the associations. These results suggest that the two novel SNPs within exon 10 of the PRLR gene associated with milk production traits are useful genetic markers in a selection program for Holstein dairy cattle.     

NMR structure of stem-loop SL2 of the HIV-1 psi RNA packaging signal reveals a novel A-U-A base-triple platform

The genome of the human immunodeficiency virus type-1 (HIV-1) contains a stretch of approximately 120 nucleotides known as the psi-site that is essential for RNA packaging during virus assembly. These nucleotides have been proposed to form four stem-loops (SL1-SL4) that have both independent and overlapping functions. Stem-loop SL2 is important for efficient recognition and packaging of the full-length, unspliced viral genome, and also contains the major splice-donor site (SD) for mRNA splicing. We have determined the structure of the 19-residue SL2 oligoribonucleotide by heteronuclear NMR methods. The structure is generally consistent with the most recent of two earlier secondary structure predictions, with residues G1-G2-C3-G4 and C6-U7 forming standard Watson Crick base-pairs with self-complementary residues C16-G17-C18-C19 and A12-G13, respectively. However, residue A15, which is located near the center of the stem, does not form a predicted bulge, and residues A5 and U14 do not form an expected Watson-Crick base-pair. Instead, these residues form a novel A5-U14-A15 base-triple that appears to be stabilized by hydrogen bonds from A15-H61 and -H62 to A5-N1 and U14-O2, respectively; from A5-H61 to U14-O2, and from C16-H42 to U14-O2'. A kink in the backbone allows the aromatic rings of the sequential U14-A15 residues to be approximately co-planar, adopting a stable "platform motif" that is structurally similar to the A-A (adenosine) platforms observed in the P4-P6 ribozyme domain of the Tetrahymena group I intron. Platform motifs generally function in RNA by mediating long-range interactions, and it is therefore possible that the A-U-A base-triple platform mediates long-range interactions that either stabilize the psi-RNA or facilitate splicing and/or packaging. Residue G8 of the G8-G9-U10-G11 tetraloop is stacked above the U7-A12 base-pair, and the remaining tetraloop residues are disordered and available for potential interactions with either other RNA or protein components.     

中国荷斯坦牛ABCG2基因编码区多态性检测及生物信息学分析

目的：研究中国荷斯坦牛ABCG2基因编码区（CDS）多态性,并进行生物信息学分析。方法：以中国荷斯坦牛为材料,利用PCR-SSCP技术对ABCG2基因CDS多态性进行检测,然后预测蛋白质序列的改变,并用生物信息学软件对蛋白质序列突变前后的结构及性质进行分析。结果：在外显子9中存在一个A→G碱基突变,导致氨基酸由酪氨酸突变为半胱氨酸,将此突变命名为Y367C;在外显子14中存在一个G→A突变,导致氨基酸由精氨酸突变为谷氨酰胺,将此突变命名为R578Q。2个突变一个位于功能区与跨膜区之间,一个位于跨膜区。生物信息学分析发现,蛋白质二级结构增加了1个卷曲（C）和2个转角（T）,同时减少了3个β折叠（E）,且ABCG2蛋白的组成和一些性质也发生了改变。结论：检测到的2个单核苷酸多态性引起了ABCG2蛋白性质和二级结构的改变;为进一步研究ABCG2蛋白对产乳性状的影响奠定了基础。     

β-LG基因外显子2多态性对荷斯坦牛乳成分的影响

目的：为了研究中国荷斯坦奶牛的β乳球蛋白（β-lactoglobulin,β-LG）基因外显子2多态现象对乳产量及成分影响。方法：本实验采用单链构象多态（PCR-SSCP）技术对中国荷斯坦奶牛β-LG基因（NCBI登录号：DQ489319）外显子2进行克隆及多态性研究。结果：8种SSCP带型：ab,abc,abed,abd,abe,abcde,abce和abde型,带型频率分别为：0．14,0．10,0．27,0．23,0．05,0．04,0．11和0．06（P〈0．05）;6个单核苷酸位点：位点1C〉T,位点2T〉C,位点3C〉T,位点4C〉G,位点5C〉A,位点6A〉T或C,且它们的遗传多态信息含量处于中度或高度多态（PIC〉0．25）。结论：中国荷斯坦奶牛β-LG基因外显子2区具有单核苷酸多态,单个核苷酸的改变影响奶牛的生产性能（牛乳产量、乳蛋白和脂肪含量等）。     

SNPs of CXCR1 gene and its associations with somatic cell score in Chinese Holstein cattle

Mastitis is one of the most prevalent diseases in dairy cattle; CXCR1 plays a key role in mastitis resistance via IL8 signaling pathway, with the CXCR1 SNPs showing a different degree of mastitis resistance. To investigate the situation of CXCR1 polymorphisms in Chinese Holstein cattle and determine the relationship between the CXCR1 SNPs and mastitis resistance, the CXCR1 SNPs in 610 Chinese Holstein cattle of 30 families were investigated using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. The results showed that four SNPs, -1830A > G, -1768T > A, -344T > C, and 783C > A were detected at 5' upstream and coding region. The correlation analysis demonstrated that -1830AA, -1768TT, and -344TT correlated significantly with the lowest SCS for each site, respectively. Haplotype analysis revealed Haplo2 (ATTA) correlated significantly with the lowest SCS. These findings indicated a prospect genetic marker of mastitis resistance in dairy cattle.     

Chromomycin dimer-DNA oligomer complexes. Sequence selectivity and divalent cation specificity

This paper reports on a solution NMR characterization of the sequence selectivity and metal ion specificity in chromomycin-DNA oligomer complexes in the presence of divalent cations. The sequence selectivity studies have focused on chromomycin complexes with the self-complementary d(A1-A2-G3-G4-C5-C6-T7-T8) duplex containing a pair of adjacent (G3-G4).(C5-C6) steps and the self-complementary d(A1-G2-G3-A4-T5-C6-C7-T8) duplex containing a pair of separated (G2-G3).(C6-C7) steps in aqueous solution. The antitumor agent (chromomycin) and nucleic acid protons have been assigned following analysis of distance connectivities in NOESY spectra and coupling connectivities in DQF-COSY spectra for both complexes in H2O and D2O solution. The observed intermolecular NOEs establish that chromomycin binds as a Mg(II)-coordinated dimer [1 Mg(II) per complex] and contacts the minor-groove edge with retention of 2-fold symmetry centered about the (G3-G4-C5-C6).(G3-G4-C5-C6) segment of the d(A2G2C2T2) duplex. By contrast, complex formation is centered about the (G2-G3-A4-T5).(A4-T5-C6-C7) segment and results in removal of the two fold symmetry of the d(AG2ATC2T) duplex. Thus, the binding of one subunit of the chromomycin dimer at its preferred (G-G).(C-C) site assists in the binding of the second subunit to the less preferred adjacent (A-T).(A-T) site. These observations suggest a hierarchy of chromomycin binding sites, with a strong site detected at the (G-G) step due to the hydrogen-bonding potential of acceptor N3 and donor NH2 groups of guanosine that line the minor groove. The divalent cation specificity has been investigated by studies on the symmetric chromomycin-d(A2G2C2T2) complex in the presence of diamagnetic Mg(II), Zn(II), and Cd(II) cations and paramagnetic Ni(II) and Co(II) cations. A comparative NOESY study of the Mg(II) and Ni(II) symmetric complexes suggests that a single tightly bound divalent cation aligns the two chromomycins in the dimer through coordination to the C1 carbonyl and C9 enolate ions on the hydrophilic edge of each aglycon ring. Secondary divalent cation binding sites involve coordination to the major-groove N7 atoms on adjacent guanosines in G-G steps. This coordination is perturbed on lowering the pH below 6.0, presumably due to protonation of the N7 atoms. The midpoint of the thermal dissociation of the symmetric complex is dependent on the divalent cation with the stability for reversible transitions decreasing in the order Mg(II) greater than Zn(II) greater than Cd(II) complexes.(ABSTRACT TRUNCATED AT 400 WORDS)