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.     

毛冠鹿ZFY、ZFX基因片段的克隆与性别鉴定

根据人和鼠性别分化相关的ZFY、ZFX基因序列设计引物,以雌雄毛冠鹿的基因组DNA为模板进行PCR扩增,将扩增产物克隆到pMD18T上,获得ZFY、ZFX重组克隆,并测定了ZFY、ZFX基因片段的序列,序列比较显示两者同源性达 91%,仅在少数位点有差异,以此确定AvaⅡ为ZFX上特异酶切位点,通过PCR扩增和AvaⅡ特异酶切对毛冠鹿性别进行鉴定。 Abstract: According to the human sex differentiation related ZFY and ZFX genes, a pair of primers were designed , and fragments were amplified from the genomic DNA of male or female tufted deer. Subsequently the amplified fragments were cloned into the vector pMD18T and were sequenced. It is found that the sequences of ZFY gene and ZFX gene have 91% homology. Based on the different nucleotides, restriction site of AvaⅡ was found to be specific to ZFX gene. The results show that the combination of PCR with AvaⅡ digestion is a simple and sensitive way to identify the tufted deer sex.     

麂亚科动物钾离子通道基因片段及其内含子 序列的克隆与进化分析

运用PCR扩增、T-A克隆、测序等技术,获得黑麂,小麂,赤麂和毛冠鹿4种麂亚科动物基因组DNA的钾离子通道部分序列。序列分析表明:麂属动物之间的外显子区域序列差异为0.90%～1.44%,毛冠鹿与麂属动物之间的差异为0.90%～1.26%,可见这一段序列的同源性较高。而内含子部分序列差异在麂属动物之间的差异为0～1.22%,毛冠鹿与麂属动物的差异为1.83%左右。由NJ法和最大简约法（Mp法）构建的进化树表明黑麂与赤麂亲缘关系较近,小麂与他们同为一属但关系较远,而毛冠鹿与它们之间的分化程度已达到属间水平。研究表明基因组的内含子序列能够较真实反映近缘动物之间的关系,是进行分子进化比较分析的较理想标记。 Abstract: In this study, partial fragments of potassium ion channel gene were amplified using the genomic DNA of muntjak, reevesi, crinifrons, and Elaphodus cephalophus. The PCR products were ligated to the plasmid of pMD18-T Vector by the method of direct T-A cloning. The positive clones were identified by colony PCR. The sequences of the recombinant clones were determined using M13-47/RV-M universal primers and aligned by the software CLUSTALW. The nucleotide divergences of exon were 0.90%-1.44% among three species of Muntiacus, 0.90%-1.26% between E. cephalophus and each of Muntiacus deer. In the nucleotide of intron there is 0%-1.22% difference among these muntjac deers, and the divergene reached about 1.83% between E. cephalophus and the three species of Muntiacus. Using the software of MEGA to analyse molecular phylogeny, Phylogenetic trees were constructed with neighbor-joining method and maximum parsimony method. The result showed Muntiacus, crinifrons is most closely related to muntjak, with reevesi as their sister species. E. cephalophus is in the other genus.     

毛冠鹿大脑组织全长cDNA文库构建

运用SMART技术构建了毛冠鹿(Elaphodus cephalophus)大脑组织全长cDNA文库。提取大脑组织总RNA,Oligotex mRNA Kit纯化、获得poly(A) RNA,以CDSⅢ/3′PCR引物进行逆转录,LD-PCR扩增获得全长双链cDNA,经SfiⅠ酶切及柱层析分离后,500 bp以上的片段与载体λTripIEx2连接,体外包装得到cDNA文库。经鉴定原始文库滴度为5.1×105pfu/ml,扩增后文库滴度为1.5×109pfu/ml,重组率达到85%以上,插入片断平均长度约为1.0 kb,说明构建文库质量符合要求,可用于大脑特异表达基因的筛选。从该文库中克隆到了rig基因全长,包含5′和3′非编码区,从第43至477个核苷酸为一完整阅读框(ORF),此阅读框可编码一个145氨基酸的rig蛋白。