黑麂Y染色体的鉴别和Sry基因的克隆及定位

以流式细胞仪分离小麂(Muntiacus reevesi)Y染色体和黑麂(Muntiacus crinifrons)Y1,Y2,X+4和1号染色体,利用DOP-PCR技术富集了分离的各单条染色体。然后,将小麂的Y染色体的DOP-PCR产物经Cy3标记后直接作为涂染探针,应用染色体涂染技术与雌雄黑麂的核型标本进行杂交,确认了黑麂真正的Y染色体为Y2染色体。再以黑麂的Y1,Y2,X+4和1号染色体的DOP-PCR产物为模板,用人的特异性的SRY（sex determining region of the Y chromosome）基因引物对其进行扩增,结果表明黑麂只有Y2染色体出现了SRY扩增片段。然后扩增产物克隆和测序,比较它与人的同源性,初步把黑麂的Sry基因定位在Y2染色体上。最后提取雄性黑麂的基因组DNA,并用同一对引物对其进行扩增,亦得到Sry基因的片段,对此扩增片段进行克隆,测序,结果表明其与Y2染色体得到的Sry基因片段完全一样,与人SRY基因的同源性均为83%。 Abstract：The single Y chromosome of Muntiacus reevesi and Y1,Y2 ,X+4,1 chromosome of Muntiacus crinifrons were obtained by flow-sorting ,then they were amplified through DOP-PCR . After that, the metaphase karyotype of Muntiacus crinifrons were painted by using the product of the DOP-PCR of the Y chromosome of Muntiacus reevesi as a special probe and the result showed that Y2 chromosome was the real Y chromosome of Muntiacus crinifrons. Secondly the product of the DOP-PCR of Y1,Y2,X+4,1 chromosome of Muntiacus crinifrons were used as the templates of the next amplification using the special primer devised according to the human SRY gene .One band was obtained only from Y2 chromosome, then it was cloned to the T-vector and sequenced. The Sry gene sequence of Muntiacus crinifrons was acquired and the conclution was that there are 83% homology between the human and Muntiacus crinifrons. It was testified that in all mammal Sry gene is consertive. On the other side the Sry gene was located to the Y2 chromosome of the Muntiacus crinifrons.     

浣熊SRY-HMG box的克隆和序列分析

We amplified the 197 bp HMG box sequence by using Polymerase Chain Reaction (PCR) with primers according to the known SRY sequence from male raccoon genomic DNA, and then cloned and sequenced. The nucleic acid and amino acid sequence comparison between raccoon and other mammals revealed high conservation of the SRY HMG box in mammals (about 80%), which implied that the DNA binding activity was crucial to mediate the action of SRY in sex determination. The variation of raccoon HMG box sequence mainly occurred on purine by replacement and missense mutation, which implicated that only the HMG box protein with advanced structure had efficient activity. The homology and difference between mammals HMG box was accorded to the evolution systematic tree.     

染色体显微切割与DOP—PCR结合对赤麂Sry基因克隆，测序及初步定位

应用显微切割技术获得赤麂1号,Y1,Y2染色体,通过DOP－PCR增加模板DNA拷贝数,然后用人的性别决定基因（Sex-tetermininig Region of the Chromosome Y,SRY)中HMG框内设计1对引物,对DOP－PCR产物进行扩增,在雄性赤麂Y2染色体DOP－PCR产物中扩增出与人SRY基因同源的Sry基因片段,克隆,测序,首次在分子水平上证明赤麂Y2染色体是真正的Y染色体,同时对赤麂Syr基因进行了初步定位。     

大熊猫苦味受体T2R2 基因的克隆与序列分析

Based on bitter taste receptor T2R2 gene sequence of domesticated dog(AB249685), one pair of primers were designed and used to amplify an approximately 1.1 kb DNA fragment from genomic DNA sample of giant panda by using PCR. The PCR products were ligated into the pMD-18T vector, and then transformed into competent cells of E.coli DH5α. The identified positive clone was sequenced. The result showed that the T2R2 gene of giant panda was 1 008 bp in length, and contained complete exon, and 915 bp, encoding 304...     

人载脂蛋白AⅠ基因的克隆表达及功能

To identify, clone ,sequence and highly express the mature peptide gene of ApoA Ⅰ, total RNA was prepared from human fetal liver tissue. cDNA fragment encoding human ApoA Ⅰ was amplified by RT-PCR using specific primers, and then was inserted in pGEM-T vector. DNA sequencing indicates that the fragment is 729 base pairs in length and has 100% nucleotide homology with that of reported ApoA Ⅰ cDNA gene previously. The ApoA Ⅰ gene was cloned into pGEX 5X-1.The recombinant protein was expressed in E.coli DH5α, purified by glutathione-Sepharose 4B affinity chromatography and confirmed by SDS-PAGE. It was shown that the recombinant ApoA Ⅰ was expressed in E.coli, and the target protein amounted to 36% of total bacteria proteins. Cholesteryl ester transfer experiment showed that the recombinant ApoA Ⅰ was capable of promoting transfer of CE from HDL to LDL. Western blotting showed that the protein could react specifically with anti-ApoA Ⅰ antibodies.     

水牛SRY基因的克隆及序列分析

根据荷斯坦牛SRY基因设计一对引物,采用聚合酶链式反应（PCR）技术,以中国沼泽型水牛（Swamp Buffalo）基因组DNA为模板,扩增得到SRY（Sex Deterimation region of Y chromosome）全序列约2005bp,其中1-504bp为5’启动子区,1196-2005bp为3’侧翼序列,在505-1195bp为SRY的外显子,编码229个氨基酸。在SRY HMG box区域设计探针,用地高辛标记后分别与雄性、雌性水牛基因组DNA进行Southern 杂交,结果显示该段序列只在雄性DNA样本中有杂交信号,证明SRY基因为雄性特异。BLAST比对结果显示与牛属动物SRY基因的同源性为96％,其中SRY基因HMG box区域同源性高达99%,说明SRY基因具有高度的进化保守性     

46,XY女性患者SRY基因启动子区域的突变分析

大约15%的46,XY女性患者中发现SRY基因编码区突变,其他患者可能是SRY基因的调节区, 包括启动子区域发生了突变,或者其他相关基因发生突变所致。本文采用限制性酶切、PCR-SSCP及银染检测技术,对7例患者SRY基因的启动子区域进行了突变筛查, 结果未发现异常,提示这些患者的病因与SRY基因启动子区域本身无关,结合对患者SRY基因HMG基序DNA的突变分析结果,表明除SRY基因异常外还存在其他导致46,XY女性性反转综合征的遗传机制。 Abstract:Using restriction endonuelease digestion and PCR-SSCP with silver staining,we analyzed the promotor region of SRY gene in seven 46,XY femalcs.The results showed no abnormality,thus ruling out the mutations in the promotor region of the SRY gene as a possible cause of sex reversal in these XY females.In view with the absence of the mutations in the HMG regions of the SRY genes of several patients,it is suggested that SRY gene is not the only gene responsible for testicular development but is one of many hierarchical genes involved in a genetic cascade for sexual differentiation.     

野油菜黄单胞菌S-152内切葡聚糖酶基因在大肠杆菌中的克隆与表达

以质粒pUC9为载体,大肠杆菌JM83为宿主菌,用鸟枪法克隆到纤维素降解细菌野油菜黄单胞菌S-152的内切葡聚糖酶(CMCase)基因,克隆到的CMCas e基因位于2.7kb的HindⅢ片段上,该重组质粒命名为pUC9H-1。Southern印迹杂交分析结果显示所克隆到的内切葡聚糖酶基因与野油菜黄单胞菌染色体DNA有亲和性。对克隆株的酶学性质分析表明,其CMCase活力为0.310μmol Glu/mg蛋白·分钟,最适作用pH为6.4,最适作用温度为55℃。 Abstract:Using plasmid pUC9 as vector,Escherichia coli JM83 as host strain,an endoglucanase(CMCase)gene has been cloned by Shoot-gun method from cellulose-degrading bacteria Xanthomonas campestris S-152,The recombinant plasmid pCU9H-1 was isolated from the positive transformant producing CMCase.The CMCase gene located in a 2.7kb HindIII DNA fragment.The result of Southern hybridization indicated that the CMCase gene cloned has affinity to chromosome DNA of Xanthomonas campestris S-152.The analysis of enzymatic activity of CMCase positive clone JM83(pUC9H-1)was carried out and the result indicated that the CMCase activity was about 0.310μmol Glu/mg pro·min,the optimum pH was 6.4 and the optimum temperature was 55℃.     

载脂蛋白E基因多态性与动脉粥样硬化性脑梗塞关系的研究

应用聚合酶链反应(PCR)扩增ApoE基因外显子4中编码112位和158位氨基酸的 DNA片段,将该长为292bp的PCR产物以HhaI酶切,根据其限制性片段长度多态性图谱确定ApoE基因型。对广东汉族人群的50例动脉粥样硬化脑梗塞(ACI)患者和50例健康对照者的ApoE基因多态性频率的研究结果表明:所研究的人群中,ApoE基因多态性频率与ACI没有关联。ACI患者中各基因型亚组之间血清TG和TC浓度无显著性差异。 Abstract:The polymerase chain reaction(PCR)and restriction fragment length polymorphism techniques(RFLPs)were used to study the relation between apolipoprotein E gene polymorphism and atherosclerotic cerebral infarction(ACI).A 292 bp DNA fragment containing codes for the 112 and 158 amino acid residues in the fourth exon of ApoE gene was amplified by PCR.The PCR products were digested with HhaI.The polymorphism patterns of Apo E gene,allele frequencies in 50 Chinese healthy and 50 patients with ACI,and the serum levels of TG and TC in the different subgenotypes of 50 patients with ACI were detected.,no statistical significant differences of alleles frequencies were found between the cases with ACI and the control,and no statistical significant differences of the serum levels of TG and TC were found among the different subgenotypes of 50 patients with ACI.These results suggested that ApoE gene polymorphism was not associated with the development of ACI.     

中国人SRY基因的分离、克隆和核苷酸序列分析

睾丸决定因子基因(Testis-determining factor,TDF)位于Y染色体短臂上,它的表达产物诱导睾丸组织的发生。SRY基因(Sex-determining Region of the Y)位于Y染色体的性别决定区内,许多特征显示SRY就是TDF。我们选用与SRY基因相应的引物,用PCR技术对正常人男女各10例的基因组DNA进行扩增。将特异扩增的男性SRY基因片段重组到质粒PUC12中,得到含有中国人SRY基因片段的克隆,命名为PSY-1、PSY-2。用[~(32)p]标记重组质粒中的SRY基因片段作探针,与PCR结果进行Southern杂交,男性样品均显示特异杂交带,女性样品为阴性。用末端终止法测定克隆的SRY基因片段的全部核苷酸序列为299bp,含有SRY基因中高度保守及功能特异性区域的240bp,我们对此进行了讨论。     

大熊猫SRY基因的PCR扩增和克隆

本文采用人ＳＲＹ基因的一对引物,通过ＰＣＲ扩增获得了雄性大熊猫ＳＲＹ基因片段。表明大熊猫存在与人ＳＲＹ基因同源的相应基因,将ＰＣＲ产物与载体ｐＵＣ－Ｅｃｏ－Ｔ连接后,用以转化ＪＭ１０９菌,经过与人ＳＲＹ基因探针菌落杂交筛选获得了大熊猫ＳＲＹ苈在克隆,命名为ｐＡＭＹ０．６,其插入片段为相应于人ＳＲＹ基因保守区在内的一段约６０９ｂｐＤＮＡ。此外,还制作和比较分析了人和大熊猫基因片段的限制酶图谱。     

牛SRY同源基因的PCR扩增和克隆

本文采用人SRY基因的一对引物,通过PCR扩增获得了雄性牛(Bos taurus)SRY同源基因片段。进一步证实牛存在与人SRY基因同源的相应基因。将PCR产物与载体pUC—Eco—T连接后,用以转化JM109菌,经过与人SRY基因探针菌落杂交,筛选获得了牛SRY同源基因克隆pBosY O.6后者的插入片段为相应于人SRY基因保守区在内的一段约609bp DNA。此外还比较分析了人和牛SRY同源基因片段限制酶图谱。     

赤麂SRY基因的测序及其与部分偶蹄目动物SRY基因序列的比较

采用人SRY基因的一段保守序列的引物,通过PCR在雄性赤麂中扩增出了赤麂SRY基因的特异片段,通过DNA斑点杂交证实其扩增产物与人SRY基因探针进行菌落杂交筛选出赤麂SRY基因的阳性克隆,并对其进行了,将其序列与基因库中录入的所有偶蹄目动物的SRY基因序列进行同源性比较,用UPGMA法构建了其系统进化树,从分类和进化上对赤麂SRY基因进行分析。     

乌龟Sox gene的PCR扩增及其条件

参照人SRY gene HMG-box保守区的序列,设计一对引物,扩增了乌龟的Sox gene,并对扩增条件进行了优化。结果显示乌龟Sox gene的扩增片段与人SRY gene扩增片段大小相同,为220bp左右,且雌雄个体间无差异;最佳扩增条件为：Mg^2 2.0mM、dNTP120μM、引物0.3μM及退火温度52℃。本研究为探索乌龟的性别决定机制以及Sox gene进货的保守性提供分子资料。     

两种蛇Sox基因的PCR-SSCP分析 *

采用PCR技术,以特异扩增人SRY基因HMG-box保守区的一对引物,扩增了乌梢蛇和赤链蛇的Sox基因。结果 两种蛇雌雄个体与人一样,均能扩增出大小为220bp左右的基因片段。对扩增产物进行的SSCP分析发现两种蛇之间以及种内雌雄个体间单链迁移率略有差异,而与人有较大差异。本文为研究蛇类的性别决定机制及Sox基因的演化提供了分子生物学资料。     

两种泥鳅中Sox基因的检出(英文)

性别决定基因ＳＲＹ都具有一个高度保守的基因序列──ＨＭＧ－ｂｏｘ,编码Ｓｏｘ蛋白,在胚胎发育过程中起重要作用。本文利用两种引物检测了泥鳅和大鳞副泥鳅的Ｓｏｘ基因。第一对引物特异扩增人类ＳＲＹ基因的保守区。在扩增泥鳅的基因组ＤＮＡ时可见长度分别为２００、５５０、９４０和１０００ｂｐ的四条扩增带。在大鳞副泥鳅中可以扩增出２００、５５０、９００ｂｐ三条带（Ｆｉｇ．１）。Ｓｏｕｔｈｅｒｎ杂交表明２００和５５０ｂｐ两条带可以和ＳＲＹ基因探针杂交（Ｆｉｇ．２）。第二对引物是兼并引物,可以特异扩增Ｓｏｘ基因的ＨＭＧ－ｂｏｘ区域。以基因组ＤＮＡ为模板可以在大鳞副泥鳅中扩增出２２０、５５０、７００和１５００ｂｐ四条带（Ｆｉｇ．３）;而在泥鳅中则为２２０、５３０、５７０和１５００ｂｐ四条带（Ｆｉｇ．４）。ＰＣＲ产物的长度差异被认为是由于部分Ｓｏｘ基因的ＨＭＧ－ｂｏｘ区域中存在着内含子。没有发现性别特异扩增带。以上结果说明哺乳动物与性决定有关的组分在脊椎动物中是广泛存在的。但这些组分在鱼类中是否与性决定有关,或仅是哺乳动物性决定因子的进化前体尚不得而知。无论如何广泛深入研究鱼类的Ｓｏｘ基因对于揭示性决定系统和因子的进化方式是十分