鸡ev21占位区和未占位区重复序列结构与羽速表型关系研究

本试验旨在探讨鸡羽速基因型Hae Ⅲ酶切鉴定方法的分子基础。以羽型明确的6个品系(太行鸡、坝上长尾鸡、大午粉鸡、海兰灰鸡、海兰褐鸡、海兰灰祖代四系鸡)313份鸡基因组为模板,利用Hae Ⅲ酶切的RFLP试验进行羽速验证,并对ev21占位区(OS)和非占位区(US)共有序列以及OS区特有序列进行酶切试验。结果表明:1)Blast分析发现1 450bp为鸡ev21的US区域片段。海兰灰及其祖代四系和大午粉祖代的羽速基因型与HaeⅢ酶切结果完全一致,但太行慢羽公鸡、慢羽母鸡、坝上长尾慢羽公鸡和海兰褐慢羽鸡的一致率分别为40.0%、27.6%、28.6%和0.0%;2)太行鸡和坝上长尾鸡ev21的OS和US区共有序列538bp酶切鉴定结果与表型一致率达到92%以上,其他品系(除海兰褐快羽公鸡为0.0%)均为100.0%;3)鸡ev21的OS区特异序列1 440bp的扩增阳性率在太行慢羽公鸡、慢羽母鸡和快羽公鸡中的阳性率分别为94.1%、65.5%和0.0%,在海兰灰祖代鸡中分别为100.0%和0.0%,并且1 440片段不能被Hae Ⅲ切开。综合分析6个品系ev21的OS和US区结构特征,认为US区Hae Ⅲ酶切位点变异不能作为慢羽和内源病毒ev21的鉴定依据,而OS区的ev21插入与1 440bp序列Hae Ⅲ酶切位点的A→G突变和八碱基重复是紧密连锁的。     

慢羽鸡ev21结合位点缺失个体的检测

内源病毒21(ev21)结合位点与慢羽基因K存在紧密连锁,检测ev21结合位点缺失的慢羽鸡个体对现代商品鸡生产力的提高具有重大意义。实验以商品代慢羽公鸡为研究对象,进行PCR扩增并用HaeⅢ对扩增产物进行消化,以此检测ev21结合位点缺失的慢羽鸡个体。结果表明:1 936只慢羽商品代公鸡个体中酶切结果为3条带的有1 846个,结果为2条带的2个,结果为1条带的23个,其余则没有明显条带。根据理论分析以及测序结果和性别鉴定的验证,推断酶切结果为2条带的2个个体为ev21缺失的慢羽鸡个体。     

乌骨鸡快慢羽的ev21和dSPEF2/dPRLR分子标记检测

鸡的快慢羽性状受Z染色体上的一对等位基因(Kk)所控制。据报道,慢羽K基因通常携带有插入的内源性病毒基因21(ev21基因)。同时,慢羽K基因还可能包含由SPEF2基因和PRLR基因的非编码区融合组成的重复片段(dSPEF2/dPRLR基因)。为明确乌骨鸡新品系的快慢羽分子结构特点,本试验选择HW1系黑羽乌骨鸡,在1日龄时,对756只健康雏鸡进行羽速观察,区分快羽和慢羽鸡。在90日龄时,分别采集快羽和慢羽鸡各40只血液,采用ev21基因和dSPEF2/dPRLR基因扩增引物及双重PCR方法扩增特异性片段。结果显示:HW1系乌骨鸡的快羽和慢羽鸡比例分别为72.2%和27.8%。慢羽乌骨鸡样本采用ev21基因引物扩增电泳后出现396 bp和341 bp两条带,而快羽乌骨鸡仅出现396 bp一条带。采用dSPEF2/dPRLR基因引物扩增电泳后,慢羽乌骨鸡样本出现1950 bp和1437 bp两条带,而快羽乌骨鸡仅出现1950 bp一条带。40只快羽鸡和40只慢羽鸡的ev21基因和dSPEF2/dPRLR基因PCR分型结果与出雏时的羽速观察分型结果一致。本试验表明,HW1系乌骨鸡中存在快慢羽性状,慢羽鸡的K基因上同时含有ev21插入基因和融合基因dSPEF2/dPRLR。因此后续研究中,将重点考虑以ev21和dSPEF2/dPRLR作为乌骨鸡快慢羽筛选的分子标记,达到准确区分纯合(Z~KZ~K)与杂合(Z~KZ~k)的慢羽公鸡,加快品系的选育速度。     

大恒优质肉鸡慢羽性状的分子鉴定方法研究

为了建立一种快速准确鉴定大恒优质肉鸡慢羽系公鸡纯合子的分子检测方法,以加快纯合慢羽系及其配套系的育种速度,利用鸡性连锁羽速基因K/k+中慢羽基因的断点连接序列以及快慢羽基因的SNP,选择200只(表型鉴定为慢羽)50日龄的大恒优质肉鸡慢羽系公鸡进行羽速基因的分子检测,检测结果与雏鸡羽速表型判定结果一致率达100%。采用限制性内切酶TaqⅠ法进一步检测发现,慢羽纯合子公鸡(197只)2条带,杂合子公鸡(3只)3条带。综上,可以利用该方法鉴定大恒优质肉鸡慢羽系公鸡基因型是否为纯合的慢羽个体。     

鸡内源白血病病毒ev21与慢羽非连锁分析和LTR区启动子活性分析

旨在分析鸡内源ev21病毒与慢羽的连锁关系及其结构特征和启动子活性,为建立无内源ev21病毒的慢羽种鸡提供检测方法,并对ev21的启动转录活性进行探索。长片段PCR扩增获得鸡内源ev21病毒基因序列,检测太行鸡、坝上长尾鸡、海兰褐、海兰灰及其祖代五个品系259份样本的病毒携带情况。利用NCBI数据库Blast比对分析并PCR获得病毒基因全长。构建ev21基因5′和3′LTR区的pGL3-basic重组质粒转染A375细胞检测其启动子活性。结果显示:获得完整ev21病毒基因组全长7 524bp,发现其反向插入在鸡Z染色体g.10681671-10681672之间,长片段7 590bp PCR检测发现ev21与海兰灰慢羽鸡完全连锁,但与太行鸡、坝上长尾鸡和海兰褐慢羽并不完全连锁,尤其海兰褐快羽公鸡ev21全部阳性。ev21基因5′LTR区具有高强度的启动子活性(P0.01),3′LTR区活性高于阴性对照,但差异不显著(P0.05)。综上,内源ev21病毒与鸡慢羽性状并不完全连锁,7 590bp长片段PCR为内源ev21病毒的筛查提供检测方法,ev21基因5′LTR区具有强启动子活性。     

清远麻鸡羽速基因的分子检测及其与体重和冠高相关性分析

本研究旨在探讨清远麻鸡的羽速分子检测技术鉴别的准确性以及在生产中的应用,以加快配套系的组建。基于内源性病毒21基因(ev21)结合位点与慢羽基因K紧密连锁,对清远麻鸡中249个个体进行ev21基因插入位点的PCR检测,并进一步对慢羽系80只公鸡进行PCR-RFLP分型,分析羽速与体重、冠高的相关性。结果表明:ev21基因插入位点的分子检测与表型鉴定一致率达到98.8%;分型发现32个为杂合子,19个为纯合子,29个为缺失体;在清远麻鸡的快羽系和慢羽系中,110日龄的体重和冠高呈高强度正相关,相关系数(r)分别为0.886和0.694(P<0.01);但快慢羽品系间体重和冠高差异不显著(P>0.05)。结果显示,清远麻鸡中应用快慢羽分子检测技术有利于选育计划的实施。     

鸡快慢羽分子鉴定方法的建立及其应用

利用找到的鸡快羽和慢羽基因,采用双重PCR方法建立了一种高效的鸡快、慢羽分子鉴定方法,在快慢羽自别雌雄鸡群体当中验证表明准确率达100%。采用该方法和经典表型鉴定方法对同一群鸡进行快慢羽判定,结果发现,除表型鉴定为微长型的个体与分子鉴定结果符合率为94.1%外,其它羽型符合率均达100%。对该鸡群快、慢羽个体早期生长速度比较研究发现,除出壳重差异不显著、第1周差异显著(P0.05),在其后各周,快、慢羽个体间体重差异均极显著(P0.01)。     

鸡OVR基因的一个片段PCR扩增克隆与鉴定

根据已知序列合成引物，并利用正交设计原理扩增出一个４２３ｂｐ的鸡ＤＮＡ片段，经酶切鉴定证明是鸡ＯＶＲ基因中含限制排卵突变的片段。这一结果为进一步研究ＯＶＲ基因的多态性打下了基础。     

广西10个地方优质品种鸡γ-干扰素基因的克隆与序列分析

根据基因库中鸡γ-干扰素的基因序列设计了1对特异性引物，应用反转录-聚合酶链式反应（RT-PCR）技术，从ConA诱导培养的广西10个地方优质品种鸡外周血淋巴细胞RNA中扩增了γ-干扰素基因，结果，均得到了大小为520 bp的特异性片段。将扩增产物纯化并克隆到pMD18-T载体上，获得重组质粒，经PCR和EcoRⅠ＋SalⅠ双酶切鉴定后测序。序列分析结果表明，广西10个地方优质品种鸡γ-干扰席基因均编码145个氨基酸的成熟蛋白，分子质量约为16．8ku，与哺乳动物和其他品种鸡的核苷酸序列同源性分别为38．8％～39．0％和99．2％～100％，氨基酸序列同源性分别为23．69／6～24．8％和97．6％～99．4％。     

广西10个地方优质品种鸡白细胞介素2基因的克隆与序列分析

根据基因库中鸡白细胞介素2基因序列设计一对特异性引物,应用反转录-聚合酶链式反应(RT-PCR)技术,从ConA诱导培养的广西10个地方优质品种鸡的外周血淋巴细胞RNA中扩增,均得到大小为470 bp的特异性片断。将10个品种鸡的RT-PCR产物纯化后克隆到pMD18-T载体上,得到重组质粒,经PCR和EcorⅠ、SalⅠ双酶切等方法鉴定后,测定鸡白细胞介素2基因序列。序列分析结果表明:广西10个地方优质品种鸡白细胞介素2基因都编码143个氨基酸的成熟蛋白,分子量约为16.3 ku,与哺乳动物和其他品种鸡核苷酸序列的同源性分别为24.8%-30.3%、98.8%-99.8%,氨基酸的同源性为14.6%-16.7%、96.5%-98.6%。     

A Genetic Diversity Analysis of Mitochondrial DNA D-loop Region in Four High Quality Chicken Breeds

This study was conducted to elucidate the genetic diversity of mitochondrial DNA (mtDNA) D-loop region in Qingyuan partridge chicken group 1,Qingyuan partridge chicken group 2,Yangshan chicken and Qingyuan Yellow feather black-bone chicken.The specific primers were designed according to mtDNA D-loop region of Gullus gullus spadiceus (accession No.:NC_007235.1) in GenBank.The sequence was analyzed after PCR amplification and sequencing,and the haplotype number,polymorphism number,haplotype diversity,nucleotide diversity and nucleotide mean difference were counted.The evolution divergence among breeds was calculated by Mega 5.10 software,and the phylogenetic tree was constructed.The results showed that the length of mtDNA D-loop region in four high quality chicken breeds was 591 bp,and 549 bp were used for subsequent analysis.The content of A,T,C and G were 27.2% to 27.3%,30.1% to 30.4%,29.5% to 29.8% and 12.8% to 12.9%,respectively,and the average content of G+C was 42.5%.There were 92 polymorphic sites which contained 14 singleton variable sites and 78 parsimony informative sites,and the percentage of transitions and transversions were 89.13% (82/92) and 10.87% (10/92),respectively.The haplotype diversity ranged from 0.682 to 0.835,and the nucleotide diversity ranged from 0.00849 to 0.01167.There were 32 haplotypes in all sequences,which could be divided into clades A,B,C and E,however,most of the individuals belonged to clades B (51.2%) and E (37.6%).The phylogenetic tree results showed that four high quality chicken breeds could be classified as 4 branches which were consistent with the haplotypes classification results.The results indicated that the four high quality chicken populations from Qingyuan had relatively high haplotype and nucleotide diversity and likely shared two or more common maternal lineages.     

传染性法氏囊病病毒VP2基因高变区序列分析

根据传染性法氏囊病病毒（ＩＢＤＶ）ＶＰ２基因ＣＤＮＡ序列,在ＶＰ２基因高变区设计一对引物,用ＲＴ－ＰＣＲ方法扩增ＩＢＤＶ分离株ＪＳ３和ＪＳ４。将扩增片段克隆后以双脱氧链末端终止法测定核苷酸旬。ＪＳ３和ＪＳ４的同源性最高达９８％。与已发表的ｖｖＩＢＤＶ,ＩＢＤＶ变异要ＢＤＶ经典株为ＩＢＤＶ弱毒株核苷酸序列的同尖拨天９２￣９８％之间,根据ＩＢＤＶ的大ＯＲＦ推导出该片段蛋白的氨基酸序更,ＪＳ３和ＪＳ４的     

Molecular characteristics of MC1R gene in tile-grey plumage of domestic chicken

ABSTRACT

1. Tile-grey plumage is a unique and rare feather type of local chicken breeds in China, but its genetic mechanism and corresponding genes remain unknown.

2. In order to identify the genetic basis and molecular characteristics of tile-grey plumage, this experiment investigated variations of melanocortin 1 receptor (MC1R) gene in Yunnan Piao chickens with typical tile-grey plumage characteristics in contrast with three Yunnan local breeds as well as two standard breeds with different plumage colour, and analysed the association between genic variation and tile-grey plumage.

3. Through sequencing and comparison of the entire coding region of the MC1R gene, a total of 10 SNP loci were detected, of which eight were non-synonymous mutations that cause amino acid changes. The gene frequency and genotype frequency of the MC1R mutation sites in different breeds and different plumage colour groups revealed that C69T, T212C and A274G were significantly associated with tile-grey plumage. Eighteen haplotypes of the MC1R gene were constructed based on 10 nucleotide variations and eight amino acid variations. Haplotype distribution and the median joining network in breeds and plumage colour groups revealed a main haplotype (hap2) for tile-grey plumage. Hap2 is unique to the tile-grey feather of Piao chicken, and the individuals carrying this haplotype account for 62.96% of the whole tile-grey chicken.

4. The results of this study are of significance for further analysis of the molecular basis of tile-grey plumage and the selective breeding of tile-grey plumage.     

绵羊KAP11.1基因克隆、原核表达及皮肤毛囊表达研究

【目的】 对毛囊角蛋白关联蛋白11.1（keratin associated protein 11.1,KAP11.1）基因进行克隆及原核表达,并对KAP11.1基因在不同品种绵羊皮肤毛囊中表达量进行比较,探究KAP11.1基因在南疆地方绵羊品种间表达差异及其对羊毛品质的影响。【方法】 以平原型和田羊、山区型和田羊和卡拉库尔羊体侧皮肤毛囊为研究材料,以GenBank中绵羊KAP11.1基因序列（登录号：HQ595347.1）为参照设计引物,对KAP11.1基因进行PCR扩增,构建pMD19-T-KAP11.1克隆质粒,双酶切鉴定后构建pET-28a （+）-KAP11.1原核重组表达质粒,经PCR和双酶切鉴定后测序并进行序列分析,转化大肠杆菌BL21（DE3）感受态细胞中表达,采用SDS-PAGE和Western blotting检测;利用实时荧光定量PCR技术检测KAP11.1基因在不同绵羊皮肤毛囊中的表达情况。【结果】 3种绵羊KAP11.1基因CDS区序列为480 bp,编码159个氨基酸,为不稳定的疏水蛋白。相似性比对结果发现,与参照基因相比,2种类型和田羊基因序列相似性均为99.79％,均在423 bp处发生突变,由C变为T,卡拉库尔羊基因序列相似性为99.38％,其69 bp处G变为T、93 bp处C变为T、423 bp处C变为T。系统进化树分析发现,3种绵羊和山羊亲缘关系最近,和瘤牛亲缘关系最远。KAP11.1蛋白二级结构主要由无规则卷曲组成。试验成功构建了pET-28a （+）-KAP11.1原核重组表达质粒,并纯化得到19 ku的KAP11.1蛋白。KAP11.1基因在山区型和田羊和卡拉库尔羊皮肤毛囊中的表达量均显著高于平原型和田羊（P<0.05）,在山区型和田羊和卡拉库尔羊中差异不显著（P>0.05）。【结论】 克隆获得480 bp的绵羊KAP11.1基因CDS区序列,成功构建了pET-28a （+）-KAP11.1原核重组表达质粒,并获得19 ku的KAP11.1蛋白,且KAP11.1基因在3个品种绵羊皮肤毛囊中均有表达。     

Sequencing of variable regions of the 16S rRNA gene for identification of lactic acid bacteria isolated from the intestinal microbiota of healthy salmonids

The aim of this study was to identify lactic acid bacteria (LAB) using polymerase chain reaction (PCR) amplification of variable regions of the 16S rRNA gene. Thirteen LAB strains were isolated from the intestinal microbiota of healthy salmonids. A approximately 500-bp region of the highly conserved 16S rRNA gene was PCR-amplified and following this, a portion of the amplicon (272-bp) including the V1 and V2 variable regions was sequenced. The sequence containing both the V1 and V2 region provided strong evidence for the identification of LAB. The LAB strains were identified as Carnobacterium maltaromaticum, Lactobacillus curvatus, Lactobacillus sakei, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, and Leuconostoc mesenteroides. The method described was found to be a very simple, rapid, specific, and low-cost tool for the identification of unknown strains of LAB.     

Cloning and Sequence Analysis of P97 Gene R1 Region of Mycoplasma hyopneumoniae in Guangxi Luchuan Pig

Through in-depth understanding of sequence characteristics, structure and genetic variation of adhesion factor P97 gene R1 region of Mycoplasma hyopneumoniae (Mhp) epidemic strains in Guangxi Luchuan pig, the assay was aimed to provide theoretical basis for mycoplasmal pneumonia of swine (MPS) integrated effective prevention and control measures of Guangxi local variety Luchuan pig, and lay a solid foundation for further study the characteristics of Mhp epidemic strains in Guangxi Luchuan pig.A pair of primers was designed to amplify P97 gene R1 region according to Mhp genome sequence, the MPS positive disease materials of Guangxi Luchuan pig from 2011 to 2014 as the object of study, after genomic DNA extraction and PCR amplification of P97 gene R1 region, the PCR products were sequenced.The base composition and deduced amino acid sequence of P97 gene R1 region of Mhp epidemic strains of Guangxi Luchuan pig were compared.The analysis of DNAStar showed that there were multiple base mutations in P97 gene R1 region of 15 strains of Guangxi Luchuan pig Mhp strains (GXLC-1, GXLC-2, GXLC-3, GXLC-4, GXLC-5, GXLC-6, GXLC-7, GXLC-8, GXLC-9, GXLC-10, GXLC-11, GXLC-12, GXLC-13, GXLC-14 and GXLC-15) and 3 strains of Guangxi other breeds of pigs Mhp strains (GX227, GX595 and GX674).The statistical repeat number of five amino acid (AAKPV/E) of P97 R1 region were 9 to 18, the average value was 12, TN repeats were 1 to 4.We found that Guangxi Luchuan pig Mhp strains mutation made its virulence and adhesion ability enhancement, and respiratory of Luchuan pig with the special structure of short and narrow, which could more easily lead to the occurrence of Guangxi Luchuan pig MPS.     

广西陆川猪猪肺炎支原体P97基因R1区克隆及序列分析

本试验通过深入了解广西陆川猪猪肺炎支原体(Mhp)地方流行毒株的黏附因子P97基因R1区的序列特征、基本结构及遗传变异等特点,为广西地方品种陆川猪猪支原体肺炎(MPS)有效的综合防控措施提供理论依据,并为进一步研究广西陆川猪Mhp地方流行毒株特性打下坚实的基础。参考Mhp基因组序列设计扩增P97基因R1区的1对引物,以 2011年-2014年广西陆川猪MPS阳性病料为研究对象,提取基因组DNA,PCR扩增P97基因R1区,对PCR产物进行测序,将广西陆川猪Mhp不同地方流行毒株的P97基因R1区序列的碱基组成和推导的氨基酸序列进行比对。DNAStar软件分析结果表明,所获得的15株广西陆川猪Mhp毒株(GXLC-1、GXLC-2、GXLC-3、GXLC-4、GXLC-5、GXLC-6、GXLC-7、GXLC-8、GXLC-9、GXLC-10、GXLC-11、GXLC-12、GXLC-13、GXLC-14、GXLC-15)和3株广西其他品种猪Mhp毒株(GX227、GX595、GX674)的P97基因R1区的多处碱基发生变异。通过推导氨基酸序列统计P97 R1区五氨基酸(AAKPV/E)重复数为9~18,平均值为12,TN重复数为1~4,结果发现广西陆川猪Mhp流行毒株变异使其毒力和黏附能力增强,再加上陆川猪呼吸道短小狭窄的特殊结构,更易导致广西陆川猪MPS的发生。     

Sex identification of Japanese black bear,Ursus thibetanus japonicus,by PCR based on amelogenin gene

A method for sex identification of the Japanese black bear was examined using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. This gene is located on the X and Y chromosomes, and there are 54 nucleotide deletions on the Y chromosome-specific gene. Forty-seven (26 male and 21 female) DNA samples and 23 (13 male and 10 female) DNA samples, respectively extracted from white blood cells and hairs of Japanese black bears were analyzed. The primers SE47 and SE48 from this X-Y homologous region were used in sex identification by PCR amplification. These primers amplified X- and Y-specific bands, which could be used to discriminate between sexes by a length polymorphism in all samples. We suggest that PCR amplification using the primers SE47 and SE48 is useful for sex determination of the Japanese black bear and could be applied to DNA analysis of small samples such as hairs.     

羽毛降解菌的筛选及其降解特性研究

本试验旨在从实验室保藏菌株中筛选出可用于羽毛降解的菌株并优化其培养条件,应用于羽毛的生物降解.以酪蛋白培养基进行菌株初筛,以羽毛为唯一有机营养源制作的羽毛培养基进行菌株复筛;通过细菌形态学观察和16S rDNA序列测定进行菌株鉴定,并对所筛菌株在羽毛培养基中的培养条件进行优化研究.结果表明:1)通过酪蛋白培养基筛选出8...     

四川盆地6个地方鸡群体遗传关系研究

随着交通便利与市场化杂交利用,我国地方鸡遗传资源面临血液混杂、品种来源和品种间的遗传关系不清晰的问题。为了给第三次全国畜禽遗传资源普查工作提供依据。本研究对位于中国大巴山脉和乌蒙山区的6个地方鸡群体（每个群体公、母各10只）共118个个体进行了简化基因组GBS测序,分析了核酸多态性（Pi）、观测杂合度（Ho）、期望杂合度（He）、多态信息含量（PIC）、观测等位基因数（Na）、有效等位基因数（Ne）、遗传分化（Fst）、遗传距离（DR）和基因流（Nm）等指标。研究发现,岩水鸡黑羽群体的遗传多样性较低（He=0.154;PIC=0.125）,大宁河鸡遗传多样性最为丰富（He=0.197,PIC=0.162）。城口山地鸡和大宁河鸡之间的遗传分化最低（Fst=0.053）,基因流最高（Nm=4.50）,丰岩乌骨鸡和岩水鸡黑羽群体之间的遗传分化最高（Fst=0.183）,对应的基因流最低（Nm=1.12）。系统发生树和主成分分析显示,岩水鸡白羽群体和黑羽群体聚为一类,城口山地鸡、大宁河鸡和旧院黑鸡聚为一类,丰岩乌骨鸡单独为一类。Structure分析表明,城口山地鸡与岩水鸡白羽群体血缘混杂。本研究探明了邻近区域地方品种的遗传关系,位于乌蒙山区的丰岩乌骨鸡与大巴山脉的旧院黑鸡、城口山地鸡、大宁河鸡、岩水鸡黑羽和白羽具有较远的遗传距离。本研究为地方品种的保护和开发利用提供了依据。