【作者】 刘玉玲；

【导师】 王坤波；

【作者基本信息】 中国农业科学院 ， 作物遗传育种学， 2010， 硕士

【摘要】 棉花是世界上主要的天然纤维作物,也是重要的经济作物。其中,亚洲棉成熟早、抗逆性强、抗虫性、纤维强度高等优点,在生产中具有极高的经济价值。但是棉花细胞具有特殊性,不仅染色体较小、相互之间的差异小,而且棉花细胞含有多糖、酚、丹宁等物质,所以棉花染色体制片相比较是困难的,直接导致了棉花的分子细胞遗传学研究相对滞后。本研究针对棉花细胞的特点,探索二倍体棉花单条染色体的分离建库技术,并在此基础上进行棉花单条染色体RGA克隆,以期为深入进行棉花基因组研究提供基础资料。研究结果如下:1.单条染色体的分离、去蛋白、酶切、扩增和验证技术体系的完善。采用酶解前的前低渗、酶解、酶解后的后低渗和盖片轻压相结合的方法获得适于激光切割仪切割的高质量亚洲棉石系亚1号中期染色体的膜制片,分离单条染色体后去蛋白、酶切和PCR扩增,以Southern杂交、SSR引物扩增和FISH验证扩增产物,形成了完整的分析验证体系。完善了棉花单染色体分离技术体系。2.染色体涂色研究。根据棉种间的单染色体涂色研究,发现亚洲棉和草棉之间的同源程度高,两者几乎相似;在与5个四倍体棉种进行单染色体涂色研究表明,亚洲棉与毛棉的同源性最差,与黄褐棉、达尔文棉的同源性较低,而与陆地棉、海岛棉的同源性较高,其中,与陆地棉的同源性最高。说明棉花四倍体之间进化程度或者速度可能也存在着差异。3.单条染色体文库的构建。构建了亚洲棉石系亚1号的第7号染色体文库,文库包含38000个克隆重组子,插入片段长度在150¹000bp,平均长度为550bp。对文库进行分析可以看出,文库覆盖为1.9倍,空载率为1%,滴度为1.3×106pfu/mL,单一和低拷贝达到59%以上。文库建立为该条染色体重要基因克隆和定位、分子标记的筛选、遗传图谱的饱和奠定了坚实的基础。4.单条染色体抗病基因同源序列的克隆。利用同源序列法扩增亚洲棉石系亚1号的第7号单染色体DOP-PCR第二次扩增池,获得30余条序列,将这些序列进行酶切归类并测序后,获得L3、L11、L15、L20和L27等5个代表性序列。将此序列与已有的抗病基因同源序列（RGA）进行比对,结果表明,这5条序列均为NBS-LRR类的RGA序列,含有RGA所特有的保守序列。将此5条序列与几种植物已经克隆的RGA序列进行聚类,得出序列L3、L11和L15聚成很窄的一类,与毛果杨的一个RGA序列NBS-LRR,mRNA （gi|224102274| ref|XM₀02334160）相近,说明它们之间的同源性非常高,L20和L27聚成另一类,与柑橘的两个RGA序列RGA17（gi|53680927|gb|AY746413.1）和RGA12 （gi|53680917|gb|AY746408-.1）同源性较高,聚在相近的类别中。更多还原

【Abstract】 Cotton is one of the main natural fiber and major cash crops in the world. Among them Gossypium arboreum has a very economic high value in production because of its advantage about early maturity, stress resistance, insect resistance, high fiber strength. It is difficult to prepare good chromosome preparations of cotton, not only because of the cotton chromosomes are small and similar, but also there are much substance such as polysaccharide, tannins and gossypol in cotton cell. The study was aim at constructing the technique about chromosome microdissection, and a single chromosome library construction. Forthermore, cloning resistance gene analogs (RGAs) in a single chromosome pool from second Diolig-prime-polymerase chain reaction (DOP-PCR). We were looking forword to accumulating basic cotton genome research data in the end. The major findings are as follows:1. Complete the system of a single chromosome microdissection, protein removed, enzymolysis, PCR and verification of production in Gossypium arboreum. The high qulity mitosis metaphase chromosome membrane preparations of Gossypium arboreum suitable for laser cutting were obtained after dealed with the method of pre-hypotonicity, enzymolysis, post-hypotonicity and squashed with cover slide. Amplified production was obtained after the single chromosome was removed protein, enzymolysis and PCR. A verified system was constructed after integrating the method of Southern blotting, SSR primer amplification and fluorescence in situ hybridization. A highly efficient technique of cotton chromosome microdissection was completed.2. Studying on cotton chromosome painting. According to the study of chromosome painting, Gossypium arboreum has high homology with Gossypium herbaceum, it is speculated that the two species has close relationship. Compared with five tetraploid cotton, it was found that Gossypium arboreum had the lowest homology with Gossypium mustelinum, had lower homology with Gossypium tomentosum, Gossypium darwinii, but had higher homology with Gossypium hirsutum and Gossypium barbadense, and had highest homology with Gossypium hirsutum, which indicated that the degree or speed of evolution between the tetraploid cotton may different.3. Single chromosome library construction. A single chromosome library of the 7th chromosome of Gossypium arboreum Shixiya 1 was constructed. There were approximately 38,000 recombinant clones in the library. After analying the library it was found that the size of the inserted DNA sequences varied from 150bp to 1000bp and the average size was about 550bp, the coverage of library was about 1.9 times, the rate of no-load was 1%, the titer was 1.3×106pfu/mL, single and low copy sequences were over 59%. The construction of the 7th chromosome library of G. arboreum Shixiya 1 would facilitate the cloning and mapping of important genes, specific probe screening, genetic map saturation on this chromosome.4. RGA Cloning from a single cotton chromosome. About 30 DNA sequences were obtained after amplified with the second round DOP-PCR as plate and with disease-resistant homologous sequence as primer. Five classic nucleotide sequence L3, L11, L15, L20 and L27 were selected after cluster and sequencing among the 30 DNA sequences. Sequences alignment with other resistant gene analog homologous showed that the five sequences were the typical NBS-LRR-type resistant gene analog. Clustering analysis with several plant RGA sequences indicated that the sequences of L3, L11 and L15 in the same cluster and were highly homologous with one RGA sequence from Populus trichocarpa, NBS-LRR, mRNA (gi|224102274| ref|XM_ 002334160), L20 and L27 was in the other cluster and homologous with two RGA sequences, RGA17 (gi|53680927|gb|AY746413.1) and RGA12 (gi|53680917|gb|AY746408-.1) from Citrus reticulata Blanco.更多还原