家猪13/17罗伯逊易位染色体着丝粒的研究 第2页
着丝粒是细胞有丝分裂和减数分裂过程中，保证染色体正常分裂、分离到子细胞的结构、功能元件。它参与减数分裂中同源染色体的配对、姊妹染色单体的连接与细胞分裂后期的分离及细胞分裂后期启动的调控。染色体运动和分离的分子机制的研究是遗传学的热点课题。因此，着丝粒的研究也是染色体功能研究的焦点之一。研究认为染色体的分离机制在真核生物中是保守的。过去认为这种保守性来自于DNA序列的特化，现在研究显示着丝粒的功能是保守的，但组成着丝粒的DNA和蛋白质在不断进化。
猪着丝粒DNA重复序列分为两个家族，即MC1和AC2。MC1是由340bp的重复单体串联组成，分布于猪的中或亚中着丝粒染色体（1-12）和X染色体上，而AC2主要是由14bp单体构成的重复序列组成，位于所有的端着丝粒染色体（13-18）上，目前还没有获得猪Y染色体着丝粒DNA重复序列的报道。
本研究采用微量全血培养法制备13/17罗伯逊易位猪染色体标本片，通过显微切割方法获得13/17罗伯逊易位染色体着丝粒区域。经两轮DOP-PCR扩增获得100bp~2000bp左右的连续带。扩增产物经纯化后用T载体克隆方法构建该区域DNA文库。用NCBI及DNAMAN等软件分析该文库序列分析。用克隆质粒作为探针前体，采用PCR法将Dig-dUTP随机插入DNA中，而使DNA被标记，通过荧光原位杂交方法从文库中筛选出猪13/17罗伯逊易位染色体着丝粒特异性序列。
获得猪13/17罗伯逊易位染色体着丝粒特异DNA探针，可为今后研究家猪染色体畸变、构建精细的家猪连锁图谱和物理图谱奠定基础，为13/17罗伯逊易位所产生的遗传效应和表型效应提供理论依据。通过对家猪13/17罗伯逊易位的研究，将会进一步加深对真核生物染色体罗伯逊易位分子机制的探讨和研究。
关键词：猪；着丝粒；罗伯逊易位；显微切割；DOP-PCR；荧光原位杂交
The Research of Centromere in Swine 13/17 Robertsonian translocation
Abstract
Centromere is the frame work and functional element in karyokinetic division and reduction mitosis. It was used to guarantee chromosome was separated into daughter cell normally. In maturation division it play important role in matching homologous chromosome to pairs and controling sister chromatid conjunction and separation during anaphase of cell division.The molecule mechanism of chromosome movement and separation is hot topic in genetics. The research of centromere is one of the focus in chromosome function study .Separation mechanism of chromosome is conservative in eukaryotic organism. The conservatism was caused by DNA specialization. And now the research showed that the function of centromere is conservative but DNA and protein of centromere is evolving.
The swine centromeric heterochromatin was characterised by two distinct satellite DNA families designed MC1 and AC2. The MC1 family was comprised of the metacentric chromosomes (1-12) as well as X chromosome, and their centromeres had been shown to be composed of divergent 340 bp monomer units. The AC2 family was comprised of 14 bp monomer units consisting of the acrocentric chromosomes (13-18).
13/17 Robertsonian translocation chromosome specimen was prepared by micro whole blood cultivation. Centric region of translocation chromosome was obtained through microdissection. After DOP-PCR 100bp~2000bp successive sequence was amplified. The product was purified to construct this region DNA library with the T vector clone method. Sequence analysis was done by NCBI and DNAMAN ,and so on. Then prepared probes through PCR with Dig-dUTP。Specificity sequence was sieved by fluorescence in situ hybridization after plasmid sequence was labeled . Thus it could provid theory basis to the research of molecule mechanism and inherit regular of 13/17 Robertsonian translocation.
The specificity probe of 13/17 Robertsonian translocation was used to establish foundation on researching chromosomal aberration and constructing subtle linkage map and physical map of susscrota domestica and supply theory of hereditary effect and phenotypic effect. The research of molecule mechanism of Robertsonian translocation in eukaryotic organism was deepened during the research of swine 13/17 Robertsonian translocation.
Keywords: swine; centromere；Robertsonian translocation; microdissection; DOP-PCR; fluorescence in situ hybridization
英文缩写词表
英文缩写 英文全称 中文全称
Amp Ampicillin 氨苄青霉素
bp Base pair 碱基对
ConA Concanavalin A 刀豆素A
DAPI 4,6-diamidino-2-phenylinodule 4, 6-二氨基-2-苯基吲哚
ddH2O Double distilled water 双蒸水
DIG Digoxin 地高辛
DNA Deoxyribonucleic acid 脱氧核糖核酸
dNTP Deoxy Nucleotide TriPhosphate 脱氧核苷酸
EB Ethidium Bromide 溴化已锭
EDTA Ethylene diaminetetra acetic acid 乙二胺四乙酸
FITC Fluorescein Isothiocyanate 异硫氰酸荧光素
IPTG Isopropyl- β-D-thilgalactoside 异丙基-β-D-硫代半乳糖
LB Luria-bertani medium LB－培养基
OD Optical dendity 光密度
PBS Phosphate buffered saline 磷酸盐缓冲液
PCR Polymerase Chain Reaction 聚合酶链式反应
PHA Phytohemagglutintin 植物血球凝集素
SDS Sodium dodecyl sulfate 十二烷基磺酸钠
SSC Sodium chloride/sodium citrate 柠檬酸盐缓冲液
TAE Tris-acetic acid-EDTA buffer Tris 乙酸缓冲液
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