【作者】 李湘阳；

【导师】 周坚；

【作者基本信息】 南京林业大学 ， 植物学， 2003， 博士

【摘要】 本研究以杉木为材料,利用单染色体的微分离、微克隆技术,对杉木单染色体进行了分子标记的定位及特异片段筛选工作的探索。研究取得了以下几个方面重要进展: 1.单染色体的微分离及体外扩增 用微细玻璃针及毛细管都可以对染色体单分离,用玻璃针分离单染色体的优点在于:能准确定向获取目标染色体并能对染色体进行定点切割以获得染色体特定片段;缺点是:操作难度大,易被细胞质污染。用毛细管吸取单染色体的优点是:分离染色体不易丢失,可避免细胞质的污染,且操作相对容易;缺点是:不能对染色体进行定向切割,易被非目标染色体污染。分离单染色体扩增采用简并核苷酸引物直接扩增法(DOP-PCR,degenerate oligonucleotide-primed-PCR),进行两轮扩增。DOP-PCR扩增片段长度为200～1000bp之间,多数片段集中在300～600bp之间。Southern杂交分析证明扩增片段来自杉木基因组DNA。 2.单染色体之间的RAPD多态性研究 杉木有11对染色体,只有第四对染色体带随体。定向分离杉木第四号具随体染色体及其它两条非随体形态单染色体,用DOP-PCR扩增放大各单染色体DNA信号,再对扩增后各单染色体的产物进行RAPD研究。证明不同染色体间存在明显的多态性,为染色体专化序列的获得及在分子水平上对杉木染色体进行编号奠定基础。 3.杉木第四号染色体特异片段的筛选 分别分离杉木同一细胞中的第四号具随体及剩余20条非随体两类染色体,将这两类染色体都经过DOP-PCR扩增后,用组合随机引物对对DOP-PCR扩增产物进行RAPD分析。在用引物对OPB07+OPB10对随体染色体及非随体染色体的DOP-PCR产物的扩增中,发现在500bp～250bp之间,随体染色体有四条特异扩增带;用引物对OPB07+OPB18对随体染色体及非随体染色体的DOP-PCR产物的进行扩增,在900bp左右获得一条随体染色体特异带;而用引物对D07+D05对随体染色体及非随体染色体的DOP-PCR产物的扩增中,发现随体染色体在250bp左右有一条特异扩增带。将其中的900bp左右特异RAPD带进行克隆、测序,得到两个同源性为99%的907bp片段,并将该RAPD标记成功地转换为稳定性和重复性更好的SCAR标记。该SCAR标记既可以用于鉴定微分离的杉木第四号染色体,也为杉木已构建的遗传图谱中连锁群与染色体进行对应奠定基础。更多还原

【Abstract】 The microdissection and microcloning of single chromosome of Cunninghamia lanceolata were carried on. In this study by means of chromosome microdissection, microcloning and random amplified polymorphic DNA (RAPD) locating molecular markers on chromosome and screening specific DNA fragments of single chromosome were investigated. The main results were highlighted in the following:1.Single chromosome microdissection and DOP-PCRChromosome microdissection can be finished by glass needle and glass capillary. The virtues of glass needle were as follows: It was nicety and directional to get single interest chromosome or specific chromosome segments by glass needle. But the process was difficult and it was easy contaminated by cytoplasmic DNA. The virtues using glass capillary were as follows: The process was easy. It can avoid loss of microdissected chromosome. And it was not easy to be contaminated by cytoplasmic DNA. But it was impossible to get specific chromosome segments and it could be contaminated by non-target chromosome. DOP-PCR amplification was carried out on isolated chromosome. Two rounds amplification were performed for each sample. The range of DOP-PCR products was between 200bp to 1000bp, with predominant fragments at 300bp~600bp. The Southern hybridization showed that the PCR products were derived from the Cunninghamia lanceolata DNA.2.Study on polymorphism among different single chromosome of Cunninghamia lanceolata by RAPDCunninghamia lanceolata has a symmetric karyotype comprising 11 pairs of metacentric and submetacentric chromosomes, one pair of which has satellite. After a satellite chromosome and other two different non-satellite chromosomes were isolated, DOP-PCR amplification was carried out on isolated chromosomes. Then the DOP-PCR products from three different single chromosomes were tested with RAPD. The results indicated: the polymorphism among different single microdissected Cunninghamia lanceolata chromosome was evident. This research provides a new method to identify microdissected Cunninghamia lanceolata chromosome on molecular level.3.Screening satellite chromosome specific fragments of Cunninghamia lanceolataA pair of satellite chromosomes and 20 other non-satellite chromosomes in one cell was isolated. The two kinds of chromosomes were amplified by DOP-PCR. And the DOP-PCR products were analyzed by RAPD-PCR using pairwise combinations of primers. There were fourspecific fragments between 500bp~250bp which were belong to satellite chromosomes in the RAPD profile with pairwise primers OPB07 and OPBIO. Another specific about 900bp fragment of satellite chromosomes was obtained in the amplified products with pairwise primers OPB07 and OPB18. And a specific about 250bp fragment was also belonging to satellite chromosomes, which was get in the RAPD profile using pairwise primers OPD07 and OPD05. It was got two specific homologous (about 99%) 907bp fragments after the approximate 900bp specific RAPD fragment was cloned and sequenced. It was successful to convert the specific 907bp RAPD marker to SCAR marker and locate it on satellite chromosomes. This research will make it possible to identify the satellite chromosome on molecular level and correspond a genetic linkage group of Cunninghamia lanceolata to a specific chromosome.更多还原