银杏第1染色体DNA文库的构建

观察了50个银杏根尖的染色体,发现在约半数根尖中,最大的1对染色体(第1染色体)随体有差异,其中1条随体较大而明显,另1条则很小.采用玻璃针分离法,通过显微操作系统成功地分离了随体较为明显的那条第1染色体.将分离到的单条染色体去蛋白,Sau3A酶切,并在染色体DNA片段两端加上Sau3A人工接头,进行2轮PCR扩增,得到了大小为300-3000bp的扩增片段.用第2轮PCR产物构建质粒文库,得到了约含有75000个重组子的该染色体DNA文库.随机挑取66个重组子进行分析,发现插入片段大小主要分布在500-2000bp,平均为800bp.该文库为银杏第1染色体特异探针的筛选、遗传图谱的构建、重要基因的克隆以及性染色体的鉴别等研究提供了基础.     

Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-Specific DNA Sequences

The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) was identified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes were microisolated and collected. After two rounds of PCR amplification, the PCR products were ranged from 150 - 3 000 bp,with predominant fragments at about 200 - 2 000 bp. Using Ag.intermediumgenomic DNA as a probe, Southern blotting analysis confirmed the products originated from Ag. intermediumgenome. The products were purified, ligated to pUC18 and then transformed into competence E.coli DH5α to produce a 2Ai-2 chromosome DNA library. The microcloning experiments produced approximately 5×105 clones, the size range of the cloned inserts was 200- 1 500 bp, with an average of 580bp. Using Ag. intermediumgenomic DNA as a probe, dot blotting results showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences in the library. Four Ag. intermedium clones were screened from the library by RFLP, and three clones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitive sequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitive DNA sequence.     

植物染色体微分离、微切割与微克隆技术的研究进展

介绍了染色体微分离、微切割与微克隆技术的发展历史;综述了植物染色体微分离、微切割和微克隆技术的研究进展及其应用与展望。     

染色体研究的新方法与新进展

从染色体显微分离与显微克隆、染色体连锁图谱、染色体原位杂交、染色体涂染、染色体的基因定位以及染色体的片段转移等几个方面,分别综述了染色体研究的新方法和新进展,并展示了分子细胞遗传学的发展前景。     

亚洲棉7号染色体分离及文库构建

 采用前低渗、酶解、后低渗和盖片轻压相结合的方法,建立了激光法分离二倍体亚洲棉石系亚1号单条染色体技术。单染色体经去蛋白、酶切和PCR扩增后,产物以Southern杂交、简单重复序列(Simple sequence repeats, SSR)引物扩增和荧光原位杂交技术(Fluorescence in situ hybridization, FISH)进行验证,构建了棉花单条染色体文库。克隆片段长度在150~1000 bp,平均为550 bp;文库包含1.38×10⁵个克隆,覆盖染色体长度1倍左右,空载率为1%,滴度为1.3×10⁶ pfu·mL^-1,单一拷贝和低拷贝比例达到59%以上,为该条染色体分子标记的筛选、重要基因克隆和定位、遗传图谱的饱和奠定基础。     

染色体微切割、微克隆技术及其应用

介绍了染色体微切割、微克隆技术的原理及主要方法，并讨论了该技术在植物中的应用。     

中间偃麦草2Ai-2染色体DNA文库构建及特异性序列的克隆

 以二体异附加系Z2与其普通小麦亲本宛7107杂交F1的花粉母细胞为材料,用原位杂交方法确定其中的单价体为中间偃麦草2Ai-2染色体;在此基础上,以显微分离、回收、LA-PCR (Linker-adaptor PCR) 扩增了该条染色体,扩增产物长度在0.15～3 kb之间,主要分布在0.2～2 kb。以α-32P-dCTP标记的中间偃麦草的基因组DNA为探针,对LA-PCR扩增产物进行杂交,证实扩增产物来自中间偃麦草。将扩增片段纯化后连接到质粒载体pUC18上,构建了2Ai-2染色体DNA文库。该文库包含约     

植物染色体微分离微克隆技术研究进展与展望

染色体微分离与微克隆技术是细胞遗传学与分子遗传学相结合的一项桥梁技术 ,近年来在植物中的应用日渐活跃 .本文综述了该技术在植物中的研究进展 ,并对其应用前景作了展望     

西瓜第一染色体显微分离及其特异文库的构建

采用玻璃针分离法,通过显微操作系统成功地分离到西瓜(2n=22)的第一单染色体,将分离到的西瓜染色体放入0.2 mL Eppendorf管中,经去蛋白,Sau3A酶切,并在染色体DNA片段两端加上Sau3A人工接头后,进行两轮PCR扩增,得到0.4～2.0 kb之间的DNA片段.用西瓜的基因组标记成探针,与单染色体扩增产物进行southem杂交,表明这些西瓜单染色体扩增片段与西瓜基因组DNA之间有同源性,从而证明单染色体DNA确实已被成功地扩增.将第2轮PCR产物构建质粒文库,得到约20 000个重组子.这是染色体微分离、微克隆技术首次在瓜类作物中的应用,该方法为直接从西瓜单条染色体DNA文库中筛选分子标记奠定了基础,说明利用染色体显微分离、克隆技术对西瓜等瓜类作物基因组进行研究是可行的.     

Microdissection of a single chromosome and construction of the microclone library from soybean

A single chromosome was microdissected from the metaphase chromosomes of soybean by using a glass needle. After two rounds of Sau3A linker-adaptor-mediated PCR, smear DNA fragments ranged from 0.3–2.5 kb were obtained. Southern hybridization showed that the PCR products from the dissected chromosome were homologous with the soybean genomic DNA. In situ hybridization of the chromosome PCR products to the mitotic metaphase spreads showed that the PCR products were hybridized to the soybean, but the chromosomal signals were not restricted to the microdissected chromosome. The second-round PCR products from the microdissected chromosome were cloned into plasmid vector to generate a chromosome-specific microclone library, which included approximately 200,000 recombinant clones. 178 randomly selected clones were estimated to range in size from 0.3∼1.8 kb, with an average size of 830 bp, of which approximately 44% were high/medium copy clones, while 56% were low/unique copy clones. This revised version was published online in August 2006 with corrections to the Cover Date.