ZFX、ZFY基因与动物进化关系的分析

本文对China Holstein ZFY，ZFX基因外显子片段进行克隆、测序，并利用NCBI和BLAST^(5、6、7）与Japanese black、Sheep、Horse、Pig、Human的ZFY、ZFX的同一外显子同源序列进行对比分析，结果表明：本文分析的锌指蛋白基因ZFY和ZFX外显子片段在上述各物种间，种属距离越远，种属间SNP的差异越大。对China Hoolstein及Japanese black间的ZFY、ZFX对比分析表明：同种属内不同品种间的SNP也存在差异，但同源性高于种间。同种属或品种内ZFY与ZFX的同源比较ZFY的SNP高于ZFX，ZFY在进化过程中较ZFX更活跃，而ZFX较为保守，该结果与国外多数学者的研究结果一致^[2、3、13]，对ZFY与ZFX的SNP性质进行研究发现ZFY中的碱基颠换率为16．22％、ZFX为16％．本文结果可作为奶牛胚胎早期性别鉴定的基础方法。     

中国麦洼牦牛、美国荷斯坦奶牛的性别鉴定

根据中国黑白花奶牛的ZFX、ZFY基因序列设计引物ZFY1、ZFX2、ZFY3,对中国麦洼牦牛、美国荷斯坦奶牛ZFX、ZFY基因片段进行PCR体外扩增和性别鉴定.结果显示ZFY1、ZFX2引物对ZFX、ZFY都能扩增出一条652bp片段;ZFY基因特异性引物ZFX2、ZFY3只能对雄性扩增出一条359bp的片段;特异性引物ZFX2、ZFY3能对中国麦洼牦牛、美国荷斯坦奶牛进行性别鉴定.     

中国麦洼牦牛、美国荷斯坦奶牛的性别鉴定

根据中国黑白花奶牛的ZFX、ZFY基因序列设计引物ZFY1、ZFX2、ZFY3，对中国麦洼牦牛、美国荷斯坦奶牛ZFX、ZFY基因片段进行PCR体外扩增和性别鉴定。结果显示：ZFY1、ZFX2引物对ZFX、ZFY都能扩增出一条652bp片段；ZFY基因特异性引物ZFX2、ZFY3只能对雄性扩增出一条359bp的片段；特异性引物ZFX2、ZFY3能对中国麦洼牦牛、美国荷斯坦奶牛进行性别鉴定。     

会理黑山羊肌肉生长抑制素基因编码序列的克隆与多样性分析

为探究会理黑山羊遗传分化状况,试验参考普通山羊肌肉生长抑制素（MSTN）基因设计引物,采用PCR方法扩增、克隆会理黑山羊MSTN基因编码区,并与其他物种相应基因编码区核苷酸序列进行比对分析。结果表明,会理黑山羊MSTN基因完整编码序列长度为1128 bp,由外显子1（372 bp）、外显子2（375 bp）和外显子3（381 bp）组成;会理黑山羊与建昌黑山羊、黔北麻羊同源性最高,均达97.4%,聚为一簇,亲缘关系最近,与牛、恒河猴、狗等物种的同源性最低,亲缘关系远;会理黑山羊MSTN基因编码区共编码375个氨基酸,会理黑山羊MSTN基因编码的各种氨基酸含量相差较大,其中以亮氨酸含量最丰富,达8.80%。本研究为会理黑山羊的遗传资源保护、开发与利用提供了分子遗传学研究基础。     

双羔型辽宁绒山羊FSHR基因SNP分析的研究

分别选择遗传性能稳定的同一年龄的产双羔、单羔辽宁绒山羊母羊,对FSHR基因第10个外显子的1 487 bp至1 717 bp共230个碱基区段进行SNP分析,并以相同年龄的产双羔和单羔的萨能奶山羊为对照.从试验羊血液中提取基因组DNA,利用PCR法扩增FSHR第10个外显子的目的片段,利用PCR-SSCP法对PCR扩增的目的片段进行单核苷酸多态性分析.结果表明辽宁绒山羊FSHR基因第10个外显子突变率较大,并且其突变发生在第1 506个碱基,由C→T.说明FSHR基因的第10个外显子可以作为双羔性状的标记基因.     

山羊成纤维生长因子受体（FGFR1）基因的多态性检测

选择与羊同源性较高的牛FGFR1基因组序列（NM_001110207）设计3对特异性引物,采用DNA池PCR直接测序法快速检测黔北麻羊FGFR1基因的多态性,并以内蒙古白绒山羊和关中奶山羊比较,结果表明：在引PCR产物中只有黔北麻羊检测到多态,分别处于外显子5和内含子5的T133C和C211T的碱基突变,而其他2个品种...     

山羊CLAⅠα链基因的克隆及生物信息学分析

为探究山羊CLAⅠα链基因的多态性,本试验根据GenBank中山羊CLAⅠα链基因序列设计引物,采用RT-PCR技术从10个品系山羊白细胞中克隆山羊CLAⅠα链基因,测序后进行生物信息学分析。结果表明,CLAⅠα链基因长度为1 074 bp,编码357个氨基酸。经核苷酸序列分析发现,10个品系山羊CLAⅠα链基因同源性集中在82.8%~99.5%之间。将10个品系山羊CLAⅠα链基因核苷酸推导的氨基酸序列进行比对,发现突变集中在α1区域（22-110位氨基酸）与α2区域（111-203位氨基酸）。遗传进化分析表明,GenBank中山羊CLAⅠα链基因与红寺湖绒山羊、河西绒山羊、陕北绒山羊、阿尔巴斯绒山羊的CLAⅠα基因遗传关系较近,与其他6个品系山羊,即简阳大耳山羊、金堂黑山羊、西农萨能奶山羊、美姑山羊、大足黑山羊、努比亚山羊的CLAⅠα基因遗传关系较远。本试验结果为进一步研究CLAⅠα链基因的多态性奠定了理论基础。     

济宁青山羊BMP15基因外显子2的克隆与序列分析

本研究对高繁殖力的山羊品种济宁青山羊和低繁殖力的山羊品种内蒙古绒山羊共20只母羊的骨形态发生蛋白15（bone morphogenetic protein 15, BMP15）基因第2外显子的扩增产物进行了克隆测序及序列比较分析。结果表明：BMP15基因第2外显子的第3对引物扩增片段存在多态。济宁青山羊与内蒙古绒山羊相比，其BMP15基因外显子2差异由2个核苷酸和2个氨基酸改变组成，核苷酸同源性为99%。济宁青山羊与鸡、小鼠、人、猪、牛、绵羊之间BMP15基因外显子2的核苷酸序列同源性为71%～99%，氨基酸序列同源性为43%～99%。     

4个山羊品种BMP6基因部分片段的多态性分析

根据GenBank发表的绵羊骨形态发生蛋白6（bone morphogenetic protein 6,BMP6）基因部分序列所包含的外显子5、6、7和小鼠BMP6基因外显子5、6、7序列设计3对引物,采用PCR SSCP技术检测BMP6基因外显子5、6和7在济宁青山羊、安哥拉山羊、波尔山羊、内蒙古绒山羊4个山羊品种250个个体中的单核苷酸多态性。结果发现这3对引物的扩增片段在检测的4个品种中均无多态性,说明所检测的BMP6基因外显子5、6、7序列比较保守。     

内蒙古绒山羊BMP-2基因片段克隆与序列分析

该试验以内蒙古绒山羊基因组DNA为模板，利用聚合酶链式反应（PCR技术），对绒山羊BMP-2基因序列进行扩增，并进行序列分析。将质粒测序，所得结果表明，序列与GenBank数据库进行序列同源性比较，绒山羊BMP-2基因与绵羊BMP-2序列比较二者同源性达到99．4％，而与人BMP-2同源区段的同源性达到89％，从而说明最终获得的片段为绒山羊的BMP-2基因片段，并且绒山羊BMP-2基因序列和其他物种的同源性非常高。     

辽宁绒山羊BMP-2基因的克隆及序列比较分析

根据GenBank上绵羊的BMP-2基因序列设计特异性引物,以辽宁绒山羊基因组DNA为模板,利用聚合酶链式反应,成功克隆了常年长绒型和季节长绒型辽宁绒山羊BMP-2部分基因片段,丰富了绒山羊BMP-2基因序列。经与绵羊、牛、鼠、猪和人的BMP-2基因进行的比对结果表明,季节与常年长绒型辽宁绒山羊的BMP-2基因同源片段的同源性达到99．7％,二者与绵羊同源性为98．2％和98．4％;与牛同源性为98．2％和97．9％;与鼠同源性为86．3％和86％;与人同源性为88．1％和88．1％。结果表明,辽宁绒山羊BMP-2基因部分核苷酸序列与其他哺乳动物同源性很高,与绵羊、牛的同源性高达97％以上,这与它们的种属关系相近一致。与人、鼠的同源性也在86％以上,说明BMP-2基因在不同物种之间具有较高的保守性。     

黄淮山羊Leptin cDNA的克隆及鉴定

根据GenBank中公布的山羊Leptin基因部分基因组核苷酸序列，设计合成一对引物，以山羊脂肪组织总RNA为模板，采用RT—PCR法，扩增出Leptin成熟蛋白编码cDNA序列，将此扩增产物克隆入pMD18-T载体，进行PCR、双酶切鉴定及序列测定与分析。结果表明，扩增的山羊Leptin基因编码序列长为441bp，编码146个氨基酸。同源性分析表明，山羊Leptin基因成熟蛋白编码cDNA与小鼠、人、猪、牛及绵羊基因相应序列的同源性分别为82．22％、87．76％、92．97％、96．15％和98．64％，氨基酸同源性为84．25％、86．99％、92．47％、97．95％和100．00％，说明Leptin是一组在进化上高度保守的蛋白质。山羊Leptin成熟蛋白cDNA的成功克隆，为进一步研究黄淮山羊Leptin基因全结构、基因表达与调控奠定了基础。     

PCR扩增ZFY,ZFX序列鉴定奶牛性别的灵敏度研究

从克隆、测序所得奶牛ＺＦＹ、ＺＦＸ序列中设计１对高特异性引物（或探针），即分别特异于牛ＺＦＹ、ＺＦＸ的ＺＦ３、ＺＦ４，与克隆时使用的引物ＺＦ２搭配成ＺＦ２、ＺＦ３和ＺＦ２、ＺＦ４。利用这２对引物和ＺＦ１、ＺＦ２对奶牛基因组ＤＮＡ进行ＮｅｓｔＰＣＲ来判断性别，结果达到用超微量（８ｐｇ）ＤＮＡ，即可检出ＺＦＹ、ＺＦＸ序列而判定雌雄的灵敏度；用非同位素标记试剂盒（ＤＩＧ－Ｓｙｓｔｅｍ）标记ＺＦ３、ＺＦ４作为探针，对ＺＦ１、ＺＦ２扩增产物点杂交来判断性别，也达到同样的灵敏度     

阿尔巴斯绒山羊产绒量性状相关基因挖掘的研究

绒山羊产绒量主要由遗传决定,产绒量低是制约绒山羊产业化发展的瓶颈。在同一群体中选择高产绒量(>1 000 g)和低产绒量(<480 g)的山羊母羊各3只,其年龄均为3周岁,体重约34 kg;每只实验羊全基因组测序深度为30×,每个样本大约测得8×10⁶ SNP。对比高产组和低产组,利用选择性消除区域基因分析方法筛选出18个基因组区域可能与产绒量相关,从这些区域中筛选出选择信号较强的4个羊绒产量性状相关候选基因,分别是CUL1、FBXL3、YY1、EZH2,这些基因参与昼夜节律信号通路、Wnt/β-catenin信号通路和TGF-β信号通路,而这3个信号通路正是参与绒山羊次级毛囊发育的重要信号通路。研究结果表明,以上4个基因和相应的SNP可以作为高产绒量绒山羊的基因组辅助育种标记,有助于缩短高产绒量绒山羊品种选育进程。     

Effect of Light Controlling and Ventilation Design on Cashmere Growth and Ammonia Concentration of Cashmere Goat

This research was conducted to investigate the effect of light controlling and ventilation design on cashmere growth and ammonia concentration of cashmere goat,which would provide theoretical basis for high efficient and ecological feeding of cashmere goat during the cashmere non-growing period.A total of 56 non-pregnant Shaanbei White cashmere goat with similar age,weight and parity were selected and randomly divided into 3 groups.Group I was received natural light,group Ⅱ was received controlling (illumination at 09:30 to 16:30,and light control at 16:30 to 09:30 of next day),and group Ⅲ was received same light condition except for additional 15% concentrate feed supplemented.Different ventilation schemes of goat house under the light-controlled condition were set and cashmere wool growth traits and barn NH₃ concentrations were determined.The results showed that illumination condition had no significant effects on body weight of Shaanbei White cashmere goat (P>0.05),but average daily gain of groups Ⅱ and Ⅲwere slightly higher than group Ⅰ.Compared with group Ⅰ,the fluff mixtures weight of groups Ⅱ and Ⅲ were extremely significantly increased (P<0.01),the fluff ratio were significantly increased (P<0.05),and the weight of cashmere wool,which were increased by 68.40% and 78.78%,respectively,were extremely significantly increased (P<0.01).Less illumination resulted in longer cashmere fiber stretched length,while it caused an increasing fiber fineness of cashmere by increasing illumination.No significant differences were observed between groups Ⅱ and Ⅲ (P>0.05).Under light-controlled conditions,using four mechanical ventilators during non-light controlled time (09:30 to 16:30) could significantly reduce the concentration of NH₃ (P<0.05).The results indicated that light controlling could increase cashmere production during the cashmere non-growing period of cashmere goat,however,less nutrients intake should be done to prevent the increasing of fiber diameter.Proper ventilation and clearance rate should be considered to keep air quality and goat health according to feeding density and house space.     

猪链球菌9型cps9G基因的克隆与序列分析

根据GenBank中猪链球菌9型(SS9)基因的核酸序列,设计一对引物,采用PCR方法从确诊为猪链球菌的阳性样品中扩增cps9G基因片段,将其克隆到pMD18-T载体上,转化DH5a感受态细胞,提取重组质粒pMD18T-cps9G,经PCR和酶切鉴定后测序,并与GenBank上SS9相应序列进行同源性分析.结果表明,经PCR扩增,鉴定为SS9的有8株.序列分析发现,8株SS9的cpsgG片段的核苷酸序列较稳定,该基因片段长度均为562 bp,彼此间的核苷酸同源性达96.8%～99.8%,亲缘关系密切;与GenBank上已发表的SS9参考毒株的同源性介于96.0%～98.6%.     

河西绒山羊绒毛生长的季节性变化规律及其与生长激素关系的研究

随机选择6月龄、平均体重15.21 kg±0.61 kg的河西白绒山羊20只(公母各半),从5月份脱绒后开始对试验羊按月连续12个月采集血样和绒毛样品。用放射免疫法测定绒山羊血清生长激素(GH)浓度,用手排长度法测定各月绒毛生长长度。结果:①河西绒山羊绒毛在6月和7月开始萌发,生长期为8、9、10、11、12月五个月;8月份为生长高峰期,占其全年总生长量的31.5%;生长量从8—12月份呈线性下降趋势,至元月份停止生长,呈慢—快—慢增长模式。②河西绒山羊血清GH浓度呈现明显的季节性变化规律;经相关性分析,绒毛生长速度与血清GH浓度水平呈极显著正相关(r=0.81,P<0.01);性别对血清GH浓度没有影响。GH浓度对绒毛生长可能有促进作用。     

中蒙绒山羊主要生产性能比较分析

为比较分析内蒙古二郎山绒山羊与蒙古国棕色绒山羊主要生产性能的差异,每个品种选取60只绒山羊进行生产性能测定,并利用SAS软件和Excel进行统计分析。结果表明,二郎山绒山羊平均体斜长、胸围及产绒量极显著高于蒙古国棕色绒山羊（P〈0.01）,在平均绒细度和体重上,二郎山绒山羊显著高于蒙古国棕色绒山羊（P〈0.05）;2个品种绒山羊体高、体斜长、胸围与体重相关关系极显著（P〈0.01）,相关关系最大的是胸围;2个品种绒细度与产绒量之间呈正相关,但不显著（P〉0.05）,二郎山绒山羊绒长度与产绒量相关关系极显著（P〈0.01）。另外,2个品种绒山羊的体高、体斜长与绒长度之间存在负相关;蒙古国棕色绒山羊的胸围与绒长度之间也存在负相关。综合性能上,二郎山绒山羊品种优势比蒙古国棕色绒山羊明显。     

Reliable sex identification of dogs by modified PCR/RFLP analysis.

To find definitive RFLP sites for canine sex determination, DNA segments corresponding to parts of the canine ZFX and ZFY genes were amplified by PCR and were directly sequenced. According to the newly defined sequence data, the combination of Haelll and Cfr13I sites was found to be useful for not only identifying the sex of the canine DNA samples but also distinguishing them from the human DNA. Conveniently, these two enzymes worked simultaneously in the same single buffer. The double-digestion of the ZFX/ZFY PCR products with HaeIII and Cfr13I showed banding patterns unique to males and females in Canis familialis. This PCR/RFLP method was confirmed to be applicable to various breeds of dog.     

Cloning and Sequence Analysis of M Gene of Avian Infectious Bronchitis Virus Guangxi Strain

According to the M gene nucleotide sequence of avian infectious bronchitis virus (IBV) published in GenBank,one pair of primers were designed,the M gene fragments of IBV isolated from Guangxi province were amplified by PCR.Then the amplified fragments were cloned into pMD18-T vector and the positive recombinant plasmids were sequenced.The results showed that M gene from all of the IBV isolates consisted of 678 bp,coding for 225 amino acids.Two glycosylated sites were located nearby the N-terminal,three transmembrane domains were located in the 23 to 98 peptide region.Variations within the hydrophilicity region were easier than that in the hydrophobicity region.Compared with that of other published IBV strains,the homologies of nucleotide and amino acid sequences of the isolates were 83.6% to 92.5% and 82.7% to 95.1%,respectively.The phylogenetic tree analysis showed that it was closely related to SAIB20 and LX4,and clustered into one group;But it belonged to different branches with other reference strains,and had a distant relationship.These results suggested that the isolate was a new variant of IBV.