BALB／c等小鼠线粒体DNAD环区PCR—SSCP分析

用ＰＣＲ－ＳＳＣＰ方法分析ＢＡＬＢ／ｃ等７个近交系小鼠线粒体ＤＮＡ（ｍｔＤＮＡ）的多态性,探讨其遗传起源及亲缘关系,结果发现,这些小鼠ｍｔＤＮＡ的高变异性区,Ｄ－ｌｏｏｐ ５’及３’端的ＳＳＣＰ电泳带型完全相同,未发现多态性。表明ＴＡ２、６１５、Ｔ７３９、ＢＡＬＢ／ｃ、Ｃ３Ｈ、Ｃ５７ＢＬ／６Ｊ、ＤＢＡ／２等近交系小鼠的ｍｔＤＮＡ具有高度的同源性。     

栽培稻F1花粉不育基因座S—a的分子定位

以栽培稻品种台中６５及其等基因Ｆ１不育系ＴＩＳＬ４为材料,用ＲＦＬＰ和ＲＡＰＤ等技术,对Ｆ１花粉不育基因座Ｓ－ａ定位。通过用ＲＦＬＰ和ＲＡＰＤ方法对亲本间进行多态性分析,发现亲本间的多态性很低,说明经多代回交后,在等基因系基因组中供体亲本的ＤＮＡ片段所占的比例很小。通过连锁分析,将Ｓ－ａ定位在第１染色体。Ｓ－ａ与分子标记ＣＤＯ５４８、Ｏ１１－１０００、ＲＧ１４６和Ｙ１３－５００之间的遗传距离分别为     

斜茎黄芪根瘤菌的16S rDNA和23S rDNA PCR-RFLP比较分析

在表型性状数值分析和ＡＦＬＰ指纹图谱分析的基础上,选取５４株斜茎黄芪根瘤菌的代表菌株及已知根瘤菌参比菌株,进行１６ＳｒＤＮＡ和２３ＳｒＤＮＡ的ＰＣＲ－ＲＦＬＰ比较分析。结果表明斜茎黄芪根瘤菌具有极大的系统发育多样性,分别具有２４个１６ＳｒＤＮＡ遗传图谱类型和２２个２３ＳｒＤＮＡ遗传图谱类型,１６ＳｒＤＮＡ与２３ＳｒＤＮＡＰＣＲ－ＲＦＬＰ聚类分析树状图谱有较好的一致性,但也存在一些差异。在对较大类群的划分上,它们的结果与表型性状数值分析结果有较好的一致性。将１６ＳｒＤＮＡ和２３ＳｒＤＮＡＰＣＲ－ＲＦＬＰ分析     

斜茎黄芪根瘤菌的16SrDNA和23SrDNAPCR—RFLP比较分析

在表型性状数值分析和ＡＦＬＰ指纹图谱分析的基础上,选取５４株斜茎黄芪根瘤菌的代表菌株及已知根瘤菌参比菌株,进行１６ＳｒＤＮＡ和２３ＳｒＤＮＡ的ＰＣＲ－ＲＦＬＰ比较分析。结果表明斜茎黄芪根瘤菌具有极大的系统发育多样性,分别具有２４个１６ＳｒＤＮＡ遗传图谱类型和２２个２３ＳｒＤＮＡ遗传图谱类型,１６ＳｒＤＮＡ与２３ＳｒＤＮＡＰＣＲ－ＲＦＬＰ聚类分析树状图谱有较好的一致性,但也存在一些差异。在对较大类群的划分上,它们的结果与表型性状数值分析结果有较好的一致性。将１６ＳｒＤＮＡ和２３ＳｒＤＮＡＰＣＲ－ＲＦＬＰ分析数据合并在一起进行分析时,得出２６个综合遗传图谱类型和１个综合聚类分析树状图谱。很明显,１６ＳｒＤＮＡ与２３ＳｒＤＮＡ的合并,能够得出更可靠的系统发育结论。     

用PCR—RFLP方法研究藏族HLA0—DQA1和—DQB1基因多态性

应用目前ＨＬＡ研究领域中成熟的、有效的ＰＣＲ－ＲＦＬＰ基因分型技术,从ＤＮＡ水平对藏族健康群体进行了ＨＬＡ－ＤＱＡ１（４９人）和－ＤＱＢ１（４９人）基因分型,这在国内外属首次。所采用的ＰＣＲ－ＲＦＬＰ基因分型技术是在ＨＬＡ－ＤＱＡ１和－ＤＱＢ１各等位基因全部序列已知的情况下,对其第２个外显子碱基序列扩增进而进行ＲＦＬＰ分析的方法。这种方法得到的ＲＦＬＰ的所有片段都是已知序列,因而精确度很高,同时为     

采用PCR—RFLP和RAPD对球壳孢目真菌系统学的研究

对球壳孢目真菌首次采用ＰＣＲ－ＲＦＬＰ和ＲＡＰＤ进行了系统发育研究,以ＩＴＳ１和ＩＴＳ４为引物对４属１２种２４个菌株的核糖体ＤＮＡ转录间区进行了ＰＣＲ扩增,４种内切酶酶切,结果表明：主要属的ＩＴＳ区长度不同,同属不同种相同。Ｃｏｎｉｏｔｈｙｒｉｕｎ,Ｐｈｙｌｌｏｓｔｉｃｔａ,Ａｓｃｏｃｈｙｔａ,ＳｅｔｏｒｉａＩＴＳ区长度分别为６３０,５６０,５５０,５４０ｂｐ;酶切图各间差别明显,属内种间基本一致     

糖单孢菌16S rDNA的PCR-RFLP分析

ＰＣＲ－ＲＦＬＰ分析适用于种和种间水平的多相分类研究。文章对ＰＣＲ－ＲＦＬＰ方法应用于糖单孢菌属分类有具体方法进行了探讨;报道了一组适于糖单孢菌属ＰＣＲ－ＰＦＬＰ分析的限制性内切酶,并用这组酶对６株糖单孢菌属分离株进行了研究,进一步探讨了实验菌株在该属的分类地位。同时指出了ＰＣＲ－ＲＦＬＰ方法应用中应注意的问题。     

用RT—PCR和RFLP对禽传染性支气管炎病毒中国分离株的分型

用ＲＴ－ＰＣＲ方法获取了禽传染性支气管病毒标准毒株Ｍ４１,Ｈ５２,Ａ５９６８和国内分离株Ｄ４１,Ｆ,Ｇ的Ｓ１基因,对它们做ＲＦＬＦ分析,发现Ｄ４１,Ｆ株属于马萨诸塞血清型,Ｇ株为变异株。对用ＲＴ－ＰＣＲ和ＲＦＬＰ来区分ＩＢＶ的分型方法的应用理论和前景进行了论述。     

几种差别表达基因显示技术及其在植物方面的应用

９０年代以来,先后出现了ＤＤ,ＲＤＡ,ｃＤＮＡ－ＲＤＡ,ＳＡＧＥ,ＧＥＦ,ＲＦＤ,ＳＳＨ以及ＡＴＡＣ－ＰＣＲ等数种未知产物的差别表达基因显示技术,本文对这些技术的异同。各自的优缺点以及它们在植物方面的应用现状和前景作了简单综述。     

植物总DNA样品的快速制备

利用Ｑｉａｇｅｎ微量植物ＤＮＡ提取试剂盒,在１小时内即可从植物组织获得总ＤＮＡ,提取过程中勿需酚／氯仿和ＳＤＳ抽提,操作简便、快捷。所得ＤＮＡ样品的ＯＤ２６０／ＯＤ２８０值在１．７－１．９之间。样品纯度高;该样品不含ＰＣＲ反应抑制剂及其他酶反应抑制剂,可被各种限制性内酶完全降解,适合于ＰＣＲ、印迹、ＲＡＰＤ、ＡＦＬＰ和ＲＦＬＰ分析等各种下游应用。     

Unique mutations in mitochondrial DNA of senescence-accelerated mouse (SAM) strains

Mitochondrial DNA (mtDNA) is exclusively inherited maternally and hence could offer a good method for tracing the lineage of mouse strains. We examined the mtDNA sequence of senescence-accelerated mouse (SAM) strains as well as other laboratory strains of inbred mice to deduce the ancestral strain of SAM. Four unique mutations were identified at bases 2256, 10,847, 11,181, and 13,053 in SAM strains. The mutations were not found in other mouse strains including AKR/J, one of the parental strains of SAM. Comparison of the mtDNA sequences also led to the consensus mtDNA sequence of laboratory strains of inbred mice. The seven laboratory strains of common inbred mice showed polymorphisms at base 9348, thymine repeat from base 9818, and adenine repeat from base 9821, and could be classified into five types by combination of the differences. Although we could not identify mouse strains with the same type of mtDNA as SAM in this study, the polymorphisms would provide a promising clue to ascertain the ancestral strain(s) of SAM. The polymorphism in mtDNA could be used to ascertain the genealogy of other mouse strains as well.     

Maternal contamination of some NZB sublines and genetic profiles of NZ-strains

Six sublines of NZB mice bred in Japan were collected and their mitochondrial DNA (mtDNA) was examined by restriction analysis. The phenotypes of at least three of these sublines (NZB/Nrs, NZB/Nga and NZB/KlJms) differed from a standard one (NZB/BlWehi). Since mtDNA is inherited maternally, all sublines of a single inbred strain should share the same mtDNA phenotype. Therefore, b-type of mtDNA should be observed in all NZB sublines. Nevertheless, the above-mentioned sublines showed d-type mtDNA. These results suggested a genetic contamination of these sublines. This was confirmed by the finding that six aberrant alleles were detected also in their nuclear genomes using biochemical markers. For elucidation of the cause of contamination, we characterized the genetic profiles of four standard NZ-strains, NZB/BlWehi NZO/BlWehi, NZC/BlWehi and NZX/BlWehi, and of common inbred strains with black coat color, C57BL/6J, C57BL/10Sn, C57BL/Ks, C58/J and AU/SsJ. We found that five of the six aberrant alleles most strongly corresponded with those of C57BL/Ks. These results suggest that this contamination was ascribable to cross of NZB mice with a certain C56BL strain. We also deduced that NAB/BlPt and NZB/Füll also probably were contaminated strains, suggesting that this contamination was not restricted to Japan.     

中国三种实验用小型猪线粒体DNAD-loop多态性分析

分析中国三种实验用小型猪线粒体DNA（mtDNA）D-loop的多态性,建立各品种品系猪的遗传标记,为各品种、品系猪的鉴别提供依据。应用PCR技术分别对西双版纳近交系小耳猪、广西巴马小型猪、贵州小型香猪和长白猪的血液总DNA样品中mtDNAD-loop进行扩增,用23种限制性内切酶消化,观察其酶切多态。PCR扩增其mtDNAD-loop5′端227bp高变区域,应用PCR?SSCP和PCR直接测序分析,观察其单链构象多态和序列多态。结果显示：三种小型猪之间未见酶切长度多态、单链构象多态和序列多态。与长白猪之间表现出单链构象多态和序列多态。本研究认为：三种实验用小型猪之间mtDNA多态性贫乏,证明其亲源关系很近,在母系起源和进化上有一致性,应用PCR RFLP、PCR SSCP和PCR直接测序分析,尚不能作为三种实验用小型猪品种、品系鉴定的依据,但与长白猪等欧系猪比较有一定差异。 Abstract：the present study is to analyze the polymorphism of the mtDNA D-loop in three breeds of laboratory miniature pigs in China , and to establish its cytoplasmic DNA markers to distinguish among them . The polymorphism of mtDNA D-loop and its 5′-end high variable regions were detected by PCR-RFLP , PCR-SSCP and PCR-direct sequencing on Xishuangbanna Small-ear inbred pig, Guizhou miniature Xiang pig , Guangxi Bama miniature pig and Landrace.There was no polymorphism obtained among or within three breeds of Chinese laboratory miniature pigs besides Landrace. It is concluded that the polymorphism of mtDNA D-loop within the three breeds of Chinese laboratory miniature pigs is poor , These methods cannot be used to distinguish among them , but it can be used to distinguish them from Landrace.     

An investigation of genetic variation within a series of congenic strains of mice

2 congenic strains of mice, B6N.AKN-Ahk and D2N.B6N-Ahb, imported from the USA, were found to be either segregating or fixed for an incorrect allele at a number of biochemical loci. B6N.AKN-Ahk, supposedly congenic with C57BL/6N, had the wrong genotype at 6 out of 12 biochemical loci; D2N.B6N-Ahb, supposedly congenic with DBA/2N, was segregating at 3 out of 9 loci. There was genetic variation in mandible shape within the 2 strains but no abnormal coat colours were found and no hybrid vigour in breeding performance was detected. Analyses in the USA confirmed these results and showed that 2 other congenic strains, C3N.D2N-Ahd and AKN.B6J-Ahb, were also segregating at a number of loci. Some of the alleles found in the C3N.D2N-Ahd mice must be the result of a genetic contamination. The simplest explanation for this breakdown in the backcrossing programme is genetic contamination with other congenic strains or recombinant inbred lines under development in the same laboratory. These findings emphasize the importance of continual genetic monitoring of all genetic stocks at regular intervals and in particular during the development of congenic and recombinant lines.     

Genetic profile of alloxan-induced diabetes-susceptible mice (ALS) and-resistant mice (ALR)

Two inbred strains from a foundation stock derived from Crj: CD-1 (ICR) mice were established after more than 20 generations of full-sib mating, and by simultaneous selective breeding for developing and not developing diabetes after alloxan administration (45 mg/kg in males, 47 mg/kg in females). To elucidate the genetic background of the two inbred strains, i. e., alloxan-induced diabetes-susceptible (ALS) strain and alloxan-induced diabetes-resistant (ALR) strain, their biochemical genetic markers and immunogenetic markers were examined. 1) For both strains, investigation of biochemical genetic markers at 19 loci and immunogenetic markers at 11 loci revealed no variation in any gene within the same strain, showing to be homogeneous, thus indicating establishment of the inbred strains. 2) The two strains differed from each other at 2 loci of biochemical genetic markers and at 5 loci of immunogenetic markers. 3) The ALS and ALR strains can be regarded as new inbred strains derived from ICR mice. 4) The results show that the marker genes of the two strains are different at 7 loci, but it remains unclear whether or not these genes are involved in the difference between the two strains in susceptibility to alloxan.     

Mouse phenome research: implications of genetic background

Now that sequencing of the mouse genome has been completed, the function of each gene remains to be elucidated through phenotypic analysis. The "genetic background" (in which each gene functions) is defined as the genotype of all other related genes that may interact with the gene of interest, and therefore potentially influences the specific phenotype. To understand the nature and importance of genetic background on phenotypic expression of specific genes, it is necessary to know the origin and evolutionary history of the laboratory mouse genome. Molecular analysis has indicated that the fancy mice of Japan and Europe contributed significantly to the origin of today's laboratory mice. The genetic background of present-day laboratory mice varies by mouse strain, but is mainly derived from the European domesticus subspecies group and to a lesser degree from Asian mice, probably Japanese fancy mice, which belong to the musculus subspecies group. Inbred laboratory mouse strains are genetically uniform due to extensive inbreeding, and they have greatly contributed to the genetic analysis of many Mendelian traits. Meanwhile, for a variety of practical reasons, many transgenic and targeted mutant mice have been created in mice of mixed genetic backgrounds to elucidate the function of the genes, although efforts have been made to create inbred transgenic mice and targeted mutant mice with coisogenic embryonic stem cell lines. Inbred mouse strains have provided uniform genetic background for accurate evaluation of specific genes phenotypes, thus eliminating the phenotypic variations caused by mixed genetic backgrounds. However, the process of inbreeding and selection of various inbred strain characteristics has resulted in inadvertent selection of other undesirable genetic characteristics and mutations that may influence the genotype and preclude effective phenotypic analysis. Because many of the common inbred mouse stains have been established from relatively small gene pools, common inbred strains have limitations in their genetic polymorphisms and phenotypic variations. Wild-derived mouse strains can complement deficiencies of common inbred mouse strains, providing novel allelic variants and phenotypes. Although wild-derived strains are not as tame as the common laboratory strains, their genetic characteristics are attractive for the future study of gene function.     

Hippocampal commissure defects in crosses of four inbred mouse strains with absent corpus callosum

It is known that four common inbred mouse strains show defects of the forebrain commissures. The BALB/cJ strain has a low frequency of abnormally small corpus callosum, whereas the 129 strains have many animals with deficient corpus callosum. The I/LnJ and BTBR T+ tf/J strains never have a corpus callosum, whereas half of I/LnJ and almost all BTBR show severely reduced size of the hippocampal commissure. Certain F1 hybrid crosses among these strains are known to be less severely abnormal than the inbred parents, suggesting that the parent strains have different genetic causes of commissure defects. In this study, all hybrid crosses among the four strains were investigated. The BTBR × I/Ln hybrid expressed almost no defects of the hippocampal commissure, unlike its inbred parent strains. Numerous three‐way crosses among the four strains yielded many mice with no corpus callosum and severely reduced hippocampal commissure, which shows that the phenotypic defect can result from several different combinations of genetic alleles. The F2 and F3 hybrid crosses of BTBR and I/LnJ had almost 100% absence of the corpus callosum but about 50% frequency of deficient hippocampal commissure. The four‐way hybrid cross among all four abnormal strains involved highly fertile parents and yielded a very wide phenotypic range of defects from almost no hippocampal commissure to totally normal forebrain commissures. The F2 and F3 crosses as well as the four‐way cross provide excellent material for studies of genetic linkage and behavioral consequences of commissure defects.     

Genetic analysis of endogenous xenotropic murine leukemia viruses: association with two common mouse mutations and the viral restriction locus Fv-1.

We have defined 40 endogenous xenotropic virus (Xmv) loci from several common inbred strains of mice by examining provirus-cell DNA junction fragments in recombinant inbred mice. Some inbred strains carried unique proviruses, but most Xmv loci were present in several strains, indicating that many Xmv integration events preexisted modern inbreeding. It was also clear that most Xmv junction fragment variation between inbred strains resulted from independent integration events and not modification or restriction site polymorphism following integration. Chromosomal assignments were determined for 32 Xmv loci by comparing their recombinant inbred strain distribution patterns to those of known genetic markers. The Xmv loci were generally dispersed throughout the genome, but several chromosomal regions contained more than one provirus. Furthermore, several close genetic associations with cellular genes were discovered. Four Xmv loci were closely linked to Fv-1b, a dominant viral resistance gene present in C57BL/6J, BALB/cJ, A/J, and several other strains. Xmv-28 was closely linked to rd (retinal degeneration), and Xmv-10 was closely linked to a (non-agouti), both of which are old mutations as inferred from their broad distribution in mice. We suggest that Xmv integration contributed to genetic diversity in the past and that much of this diversity exists today in common laboratory strains.     

昆明小鼠4个可能近交系的基因分型验证

在我国,昆明小鼠作为一种实验动物广泛应用于药理和遗传学相关的研究领域。但由于昆明小鼠属于远交群,而且不同地区的种群间已经出现了严重分化,缺乏具有显著特征的近交系,这使得它在生物学上的应用受到了很大的限制。研究人员已经以昆明小鼠为背景培育出了几个可能的近交系,但由于缺乏可靠的遗传检测,至今未得到广泛的认可和应用。文章收集昆明小鼠的4个已经60代以上兄妹交配繁殖的可能近交系,并以两个标准近交系BALB/c和C57BL/6为参照,利用30个微卫星标记对每个品系的5只小鼠进行了微卫星基因分型,进而分析其遗传纯度。结果发现,品系A1和品系N4在本研究所用的30个位点均呈纯合状态;而T2和N2均在D15Mit16位点呈杂合状态。本研究第一次为我国昆明小鼠近交系的遗传学纯度提供了可靠的分子水平证据。今后应当加强昆明小鼠近交系的标准化,以扩大其在遗传学方面的应用。     

应用40对微卫星引物对6种近交系小鼠遗传背景进行监测的研究

采用40对引物的微卫星DNA PCR遗传质量符合要求的BALB／C－nu0nu-,DBA/2,SCID,T739,TA2,615等6种近交系小鼠进行遗传监测,结果26对引物有稳定的扩增结果,5对引物表现为单态性,21对引物表现出多态性,其中D2Nds3,D3Mitl5,D3Mitl7,D3Mit18,D16Mit7等6对引物表现出显著的多态性,反映了各品系小鼠独特的遗传背景,可应用于区别小鼠品系,监测系间的遗传污染,为有关小鼠品系积累了遗传背景资料,有助于将实验动物的遗传监测从表墼这度到DNA水平。