中国荷斯坦牛GlyCAM1基因遗传多态性的研究

以273头中国荷斯坦牛为研究对象,利用CRS—PCR、PCR—SSCP及DNA测序技术检测了GlyCAM1基因外显子3、内含子3的遗传多态性。结果表明：GlyCAM1基因分别在外显子3和内含子3的第2081（A／C）、2417（C／T）位存在突变,2个位点的等位基因频率A／B分别为0．7525,0．2475和0．9046,0．0954;经菇。适合性检验,中国荷斯坦牛内含子3的突变达到Hardy—Weinberg平衡状态（P〉0．05）,但其外显子3的突变未达到Hardy—Weinberg平衡状态（P〈0．05）。     

Identifying a new locus that regulates the development of rete ovarian cysts in MRL/MpJ mice

MRL/MpJ (MRL) is a mouse model for autoimmune disease and develops ovarian cysts with age. The ovarian cysts originate from the rete ovarii, which is considered to be the remnant of fetal mesonephric tubules. In a previous study, we analyzed the genetic background of ovarian cysts by using backcross progenies between MRL and C57BL/6N (B6) mice. By interval mapping, suggestive linkages were detected on several chromosomes (Chrs), and a significant linkage on Chr 14 was designated as MRL Rete Ovarian Cyst (mroc). In the present study, which evaluated 113 F2 intercross progenies, a significant linkage appeared on Chr 6 at the marker position D6Mit188 (likelihood ratio statistic = 18.5). In particular, the peak regions of Chrs 6 and 14, which contain major causative loci by backcross analysis, showed close reverse interaction. From these results, a locus on Chr 6 was identified as mroc2, the second major locus associated with ovarian cyst formation in MRL mice.     

Molecular cloning and tissue distribution of a short form chicken leptin receptor mRNA

In mammals, alternative splicing of the leptin receptor (LEPR) produces several C-terminal truncated isoforms that are believed to play a role in the transport, cellular internalisation and degradation of the hormone leptin. The chicken leptin receptor (chLEPR) is similar to its mammalian counterparts in terms of its intron/exon structure and conserved motifs. However, it is unknown whether the chLEPR also undergoes alternative splicing. To test this, structural analysis of intron 19 of the chLEPR, equivalent to the intron in which alternative splicing occurs in mammals, was combined with 3'-rapid amplification of cDNA ends (3'-RACE) to search for chLEPR splice variants. A 44-amino acid alternative exon 20 was identified that is spliced to generate a short isoform of the chLEPR (chLEPR-SF). Comparative sequence analysis of intron 19 identified two regions that are highly conserved between the chicken and mammals, indicating their possible importance as intronic elements in the regulation of alternative splicing of the LEPR in vertebrates. Tissue expression of the chLEPR-SF was lower and more restricted than that of the chLEPR long isoform. Collectively these data demonstrate that the chLEPR is alternatively spliced to produce at least one short isoform, as is the case in mammals.     

半番鸭Agouti基因的克隆与SSCP分析

以半番鸭血液基因组为模板进行PCR扩增,获得了半番鸭Agouti基因部分序列,该序列长为1178bp。序列分析表明该序列由部分第1外显子（92bp）、第1内含子（105bp）、完整的第2外显子（95bp）、完整的第2内含子（851bp）和部分第3外显子（35bp）组成;应用PCR-SSCP技术对扩增序列进一步研究发现位...     

山羊催乳素受体基因部分片段的克隆与序列分析

采用PCR技术扩增出济宁青山羊催乳素受体基因长892 bp的片段，该片段含有97 bp的部分外显子8序列（外显子8全长为100 bp）、683 bp的内含子8、70 bp的外显子9及42 bp的部分内含子9。将该片段克隆到pGEM-T Easy质粒中，重组质粒用PCR进行阳性克隆鉴定，然后测定核苷酸序列，并推导其氨基酸序列。该序列与绵羊、母牛、人、大鼠、小鼠的催乳素受体基因mRNA的对应序列的核苷酸同源性分别为99.4 %、97.01%、89.22%、89.22%、88.02%，氨基酸同源性分别为100%、94.55%、81.88%、81.82%、83.64%。     

德宏奶水牛DGAT1基因多态性及其与产奶性状的相关性分析

探讨德宏奶水牛（摩拉水牛×德宏水牛）DGAT1基因多态性与产奶性状的关系,以期为德宏奶水牛产奶性状标记辅助选择侯选基因的选择提供理论依据。以81头泌乳的德宏奶水牛为研究对象,利用PCR—SSCP方法结合候选基因直接测序法检测DGAT1基因第8外显子及第8内含子区的多态性,并采用最小二乘法分析DGAT1基因多态位点对产奶量、乳脂率及乳蛋白率的影响。结果在德宏奶水牛群体中共检测到CTGG,CCGG和CCGT三种基因型,测序结果显示,在检测的群体中未发现K232A位点的突变,而在DGAT1基因第8内含子的第14位检测到C→T突变,第35位检测到G→T突变。多重比较表明,CCGT基因型对德宏奶水牛的乳脂率有显著影响（P〈0．05）。     

京海黄鸡IGFBP-3基因外显子1和内含子1部分序列多态性及其与生长繁殖性状的相关性

以京海黄鸡母鸡为材料,采用PCR-SSCP技术对IGFBP-3基因外显子1及内含子1部分序列多态位点进行研究,并计算基因型频率、基因频率、卡方值和部分遗传多态性指标。结果表明,在外显子1上没有检测到多态位点,在内含子1上检测到1个多态位点,该位点为中度多态,在160 bp处发生T到G的突变,导致BB、AB型的56日龄体重显著大于AA型(P＜0.05),AA型的11月产蛋数显著大于BB、AB型(P＜0.05)。由此初步推断,内含子1对产蛋及生长发育很可能有一定的促进作用,A为产蛋的优势基因,B为体重的优势基因。     

Genetic variations of BRCA1 and BRCA2 genes in dogs with mammary tumours

Mammary tumours are the most common tumour type in female dogs. The formation of the mammary tumours is multifactorial but the high incidence of tumour disease in certain canine breeds suggests a strong genetic component. BRCA1 and BRCA2 are the most important genes significantly associated with mammary tumours. The aim of this study was to determine the association between the variations of these two genes and canine mammary tumours. 5′-untranslated region, intron 8 and exon 9 of BRCA1 and exons 12, 24, 27 of BRCA2 were sequenced in order to detect the genetic variations. In addition to six previously identified polymorphisms, six novel single nucleotide polymorphisms (SNPs) were detected. Five of the coding SNPs were synonymous and three of them were non-synonymous. The comparison of the sequences from 25 mammary tumour bearing and 10 tumour free dogs suggested that the two SNPs in intron 8 and exon 9 of BRCA1 and two SNPs in exon 24 and exon 27 of BRCA2, which are firstly identified in this study, might be associated with mammary tumour development in dogs. Especially one SNP in exon 9 of BRCA1 and one SNP in exon 24 of BRCA2 were found to be significantly associated with canine mammary tumours.     

山羊前列腺素内过氧化物合酶2基因的多态性检测及生物信息学分析

本试验以贵州省3大地方山羊品种贵州白山羊、贵州黑山羊、黔北麻羊和2个省外地方品种关中奶山羊和内蒙古绒山羊为研究对象,采用DNA池结合PCR直接测序法对山羊前列腺素内过氧化物合酶2(prostaglandin-endoperoxide synthase 2,PTGS2)基因进行单核苷酸多态性检测。结果表明,在5个山羊品种中共检测到6个SNPs位点:A96G、C20T、A239G、T192C、T164A和G91C,其中,A96G和T164A分别位于外显子7和外显子8中,且均为同义突变,其余4个变异位点位于内含子中。生物信息学分析显示,外显子7和8中的2个SNPs位点均导致了mRNA二级结构的改变。     

Genetic Characteristics of Frizzled and Incomplete Frizzled Feather and SNP Detection of KRT75 Gene

The study was mainly focused on the genetic characteristics of frizzled feather trait,the phenotype difference between frizzled feather（FF）and incomplete frizzled feather（IFF),and SNP analysis of the candidate gene KRT75 in the frizzle and incomplete frizzle chickens of different feather colors,which could provide scientific gist for the frizzle gene mapping,development and utilization of the two kinds of chicken.The resource population（homozygous Yellow feather Kirin chicken×Huaixiang chicken）of hybrid F2 was established.The back,neck,wing primary and wing-bow feather were observed and the scatter diagram of rachis bending degree was drawn.The blood of 10 white frizzle feather Kirin（WFF）chicken,10 Yellow frizzle feather Kirin（YFF）chicken,10 Royal,25 Yellow feather incomplete frizzle（YFIF）chicken and 15 Black feather incomplete frizzle（BFIF）chicken from homozygous WFF chicken×Royal chicken of hybrid F1 were extracted,then the DNA of those samples were extracted.The primer of KRT75 was designed,the PCR products were also sequenced after purification,Chromas 2.22 and DNAMAN software were used to analysis of sequence peaks photos and sequence alignment,respectively.The amount of orthogonal and reciprocals cross in hybrid F1 was 127 and 139, respectively, which all were the slight（incomplete）frizzled feather phenotype,with no different penetrance.The resource population of hybrid F2 were consisted of 55 frizzle chicks including some hens and cocks,106 incomplete frizzled and 68 contour feather chicken,the proportion of which was coincided with Mendelian segregation ratio of 1:2:1 by χ²-test（P>0.05),therefore it preliminary verified that the frizzle gene F was autosomal incomplete dominant inheritance.FF and IFF curved upward deviating from the skin,and the line slope of trend line of FF was 3.5 times than that of IFF.It found that there was no 69 bp deletion mutation in exon 5 region of KRT75 gene in WFF and YFF chicken,but 3 SNPs（T71C,T83C,C95T）were deleted in the 69 bp,which were homologous CC in WFF and YFF chicken,of two were heterozygous in YFIF chicken,while all were heterozygous in BFIF chicken.2 SNPs（T662C and T770C）were also deleted in exon 6,heterozygous of which were only deleted in BFIF chicken.The haplotype analysis indicated that 9 haplotypes were detected in 60 individuals,hap1/hap1 was specific genotype of WFF and YFF chicken.The haplotypes of two incomplete frizzle feather chicken were apparently different,hap4 was specific haplotype of YFIF chicken,while hap6,hap7,hap8 and hap9 were specific haplotypes of BFIF chicken.The frizzle gene F was autosomal incomplete dominant inheritance,there were obvious differences between FF and IFF traits,5 SNPs in exon 5 and 6 of KRT75 gene probably were the reference of molecular markers as distinguishing between frizzle and incomplete frizzle chicken.     

卷羽与半卷羽遗传特性分析及KRT75基因SNP位点检测

本试验通过对卷羽性状遗传特性和卷羽、半卷羽差异及对不同羽色卷羽、半卷羽鸡的候选基因KRT75序列SNP分析,为进一步对卷羽性状基因的定位和卷羽、半卷羽鸡的开发利用提供科学依据。以纯系黄色卷羽麒麟鸡和片羽怀乡鸡为素材,正反交构建F2代资源群体,对24周龄卷羽、半卷羽和片羽鸡背羽、颈羽、翅膀主翼羽和翼肩羽观察并绘制背羽羽轴弯曲程度散点图。随机抽取25周龄黄羽、白羽麒麟鸡和贵妃鸡各10只,F1代黄羽半卷羽鸡25只,以及白羽麒麟鸡×贵妃鸡杂交F1代黑羽半卷羽鸡15只的血样提取DNA,设计引物,PCR扩增纯化后测序,Chromas 2.22和DNAMAN软件用于分析测序峰形图和序列比对。结果显示,杂交F1代正交组127只,反交139只,不论公、母表型较一致,无外显率不同,均呈现轻度卷羽（半卷羽）,F1代间交配产生的F2代资源群体：卷羽鸡55只,半卷羽鸡106只,片羽鸡68只,公、母鸡均有卷羽,经过卡方适合性检验符合孟德尔遗传分离比1∶2∶1（P>0.05）,因此初步验证了控制卷羽基因F呈常染色体不完全显性遗传;卷羽、半卷羽鸡羽毛均背离皮肤向上弯曲,卷羽趋势线直线斜率是半卷羽的3.5倍;白羽和黄羽麒麟鸡外显子5处均不存在69 bp缺失,此69 bp发现3个SNPs：T71C、T83C、C95T,其中片羽鸡无突变,黄羽和白羽麒麟鸡均为纯合子,黄羽半卷羽鸡2个位点为杂合子,黑羽半卷羽鸡3个位点均为杂合子;外显子6处检测到了2个SNPs分别为T662C和T770C,仅在黑羽半卷羽鸡中为杂合子,其他均为纯合子,单倍型分析从60个个体中检测到了9种单倍型,hap1/hap1纯合型是黄羽和白羽麒麟鸡特异性的基因型,黄羽和黑羽半卷羽鸡的单倍型存在明显差异,hap4单倍型是黄羽半卷羽鸡特有的单倍型,hap6、hap7、hap8、hap9为黑羽半卷羽鸡特有的单倍型。卷羽性状基因F为常染色体不完全显性遗传,卷羽和半卷羽存在明显差异,KRT75基因序列外显子5和6处的5个SNPs可能作为区别卷羽和半卷羽鸡的分子标记参考。     

绵羊瘦素受体基因部分序列测定及其变异位点分析

试验旨在检测绵羊瘦素受体(leptin receptor,LEPR)基因序列突变位点,为进一步分析LEPR基因多态性与绵羊生长性状的关系奠定基础。选用美利奴羊与阿华西羊杂交群体作为试验材料,利用RT-PCR、测序等方法测定了4只绵羊的LEPR基因cDNA部分序列及内含子7序列,同时利用PCR-RFLP分析两个位点在群体(229只)中的多态性。测定出LEPR基因cDNA序列长2608 bp(包含外显子2-16完整序列及外显子1和17的部分序列)和LEPR第7内含子序列全长160 bp,共发现5个核苷酸变异位点,第2外显子中2个(T240C和A279G),第10、14外显子中各1个(A1683G,T2373C),内含子7中1个(1285+A73G),外显子中的4个变异位点均未引起编码氨基酸的改变。在研究群体中,A279G与A1683G中A的频率分别为0.415、0.467,后者处于非平衡状态,GG和AA是主要的单倍型。不同物种间序列一致性分值均比较高,但LEPR mRNA区序列一致性比内含子高,基于LEPR mRNA序列构建的系统发育树更符合实际情况,且可靠性值更高。结果表明,绵羊LEPR基因的保守性较强,突变形式主要是转换,A279G和A1683G可能是突变热点。     

白羽番鸭脂联素基因SNP与肌内脂肪和血清总胆固醇的关联分析

采用PCR-SSCP法对白羽番鸭脂联素基因全编码区和内含子进行多态性检测,并分析其多态对肌内脂肪(IMF)和血清总胆固醇(TC)的遗传效应,为番鸭分子标记辅助选择提供参考.结果表明:3个SNP分别在外显予1和外显子2的编码区发生A167G和G711A突变,为沉默突变,在内含子发生C290T突变;3个SNP位点均处于Ha...     

Involvement of CD28/CTLA4-B7 costimulatory pathway in the development of lymphadenopathy and splenomegaly in MRL/lpr mice

MRL/lpr mouse is an established animal model which develops autoimmune diseases including glomerulonephritis, sialoadenitis, hepatitis and inflammatory lung disease. Additionally, it has been reported that lpr strains uniquely accumulate CD3+ CD4- CD8- B220+ (double negative, DN) T cells in lymphoid organs leading to lymphadenopathy and splenomegaly. To investigate the role of CD28/CTLA4-B7 pathway in the development of lymphadenopathy and splenomegaly, MRL/lpr mice were treated with soluble form of CTLA4 molecules, CTLA4IgG, which efficiently blocks this pathway. It was demonstrated that (i) the development of DN T cells was independent of the CD28/CTLA4-B7 pathway, (ii) the CD28/CTLA4-B7 pathway was required for the development of lymphadenopathy and splenomegaly, (iii) the CD28/CTLA4-B7 pathway was important for the accumulation of various cell populations in the lymph node and spleen, (iv) composition of the accumulating cell populations was not altered by CTLA4IgG treatment, and (v) activation of conventional T cells and IL-4 production from conventional T cells were the CD28/CTLA4-B7 pathway dependent. Thus, we concluded that the CD28/CTLA4-B7 pathway was required for the development of full-blown lymphadenopathy and splenomegaly in MRL/lpr mice.     

巢湖鸭生长素基因和垂体特异性转录因子1基因的PCR-SSCP检测

以生长素基因Ghrelin和垂体特异性转录因子1 Pit-1基因为候选基因,采用PCR-SSCP和DNA测序技术检测2个候选基因在巢湖鸭群体中的单核苷酸多态性(SNPs)。结果表明,Ghrelin基因exon 3第54 bp位置有G→A碱基的点突变,在巢湖鸭群体中检测到AA、AB、BB 3种基因型,A等位基因频率为0.49,B等位基因频率为0.51;在exon 5第149位和166位发生了C→A和G→T的突变,检测到MM、MN、NN 3种基因型,M等位基因频率为0.19,N等位基因频率为0.81。Pit-1基因的2对引物的扩增片段均未检测到多态性,说明所检测的Pit-1基因外显子3和4序列比较保守。     

Study on the SNP of UCP1 Gene Exon 2 in Rabbit

In order to deeply understand the meat quality and provide the basic information and scientific proof for better heredity selection of rabbit.This experiment option New Zealand rabbit and Californian rabbit were used to construct own DNA pools, respectively for complete sequences amplification of exon 2 and part of intron 1 region of UCP1 gene, two-way sequencing the amplification of the product, BLAST and DNAStar analysis and determine the SNP of rabbit UCP1 gene.The results showed that six SNPs were found in UCP1 gene of the New Zealand rabbit:T135C, C232T, T332C, C360T, T550C and A560G, two synonymous mutation (T135C and C232T) and the other SNPs located in intron region respectively.Using RNA online prediction software forecast UCP1 gene RNA secondary structure prediction software minimum free energy changed from -917.51 kJ/mol to -866.10 kJ/mol for the polymorphisms led to the changes of RNA secondary structure.     

兔解偶联蛋白1基因第2外显子基因多态性研究

为了解兔肉质性状特征,给兔的品种选种选育提供理论依据。本试验选取新西兰兔和加利福尼亚兔分别构建各自的DNA池,对解偶联蛋白1(uncouplingprotein 1,UCP1)基因的第2外显子及第1内含子部分序列进行扩增,扩增产物进行双向测序,利用BLAST和DNAStar分析并确定其SNP。结果分析,在新西兰兔中发现6个SNPs:T135C、C232T、T332C、C360T、T550C和A560G,其中T135C和C232T为同义突变,T332C、C360T、T550C、A560G 4个突变位点位于内含子区。利用RNA在线预测软件预测UCP1基因RNA二级结构最小自由能由-917.51 kJ/mol变为-866.10 kJ/mol,说明SNPs对UCP1基因的RNA二级结构产生影响。     

SNPs Screening and Bioinformatics Analysis of SIM1 Gene in Congjiang Pigs

In this study, Congjiang pig was served as a research object, crossbred pigs (Congjiang pig×boar), outside crossbred pigs (Duroc×Landrace×Yorkshire) as controls.Using DNA pools and direct sequencing detected polymorphism of 7 exons, part of introns and 3'untranslated region (3'-UTR) sequences of SIM1 gene for three groups.Bioinformatics software predicted what impact polymorphic loci had to mRNA secondary structure and protein primary, secondary structure of SIM1 gene.The results showed that 12 SNPs were screened in SIM1 gene of three groups, C77T located in exon 5, T29186C and A29195C located in intron 9, C63T and C225T located in exon 10, and C107T, A426G, T583C, A586C, A605C and A615C located in exon 11, G267T located in 3'-UTR. C77T, T29186C, A29195C, C63T, C225T, C107T and G267T were silent mutations, A426G, T583C, A586C, A605C and A615C were missense mutations.The five missense mutations respectively led to isoleucine (Ile) into valine (Val), leucine (Leu) into proline (Pro), glutamate (Glu) into alanine (Ala), glutamic (Glu) into aspartic (Asp), asparagine (Asn) histidine (His), and according to online software forecast, mRNA secondary structure and protein primary, secondary structure of SIM1 gene changed in mutations before and after.