【作者】 彭仁海；

【导师】 王坤波；

【作者基本信息】 中国农业科学院 ， 作物种质资源学， 2009， 博士

【摘要】 棉花是世界上最主要的天然纤维作物,棉花基因组测序已进入实质性阶段,因此积累棉花基因组测序的基础性资料和解决关键依赖技术已经迫在眉睫。本研究根据棉花细胞富含棉酚和丹宁等次生代谢物质,棉花染色体小、形态相似、制片困难的特点,探索建立完整的棉花FISH技术体系,同时针对棉花多倍体的特征,探索棉花单染色体分离技术,建立单染色体文库,为棉花基因组测序、基因克隆、染色体涂色乃至进化研究积累基础性资料。主要研究结果如下:1.棉花染色体微分离、微克隆技术体系的建立。采用前低渗、酶解、后低渗和压片相结合的方法获得适于切割的中期染色体膜制片,进而形成一套行之有效的单染色体切割流程。棉花单染色体扩增后,通过Southern杂交、SSR引物扩增和荧光原位杂交验证,形成了完整的分析验证体系。首次建立了一套高效、快捷、易于操作的棉花染色体微分离、微克隆技术体系。2.棉花染色体涂色研究。根据棉种间染色体涂色研究,发现两个二倍体棉种亚洲棉和草棉之间的同源性很高;在五个四倍体棉种中,亚洲棉与陆地棉、海岛棉的同源性较高,而与黄褐棉、达尔文棉的同源性较低,与毛棉的同源性最差,说明棉花四倍体属多系起源可能更可信。3.棉花单染色体文库的构建。建立了亚洲棉石系亚1号的第5号染色体文库,含有37000个克隆重组子,插入片段大小为500～1800bp,平均为750bp。该文库的建立为该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棉花基因组测序的最终完成积累了基础性研究资料。4.棉花单染色体RGA克隆。以水稻的抗病同源引物P1和P2对亚洲棉石系亚1号的第5号染色体接头介导PCR(LA-PCR)的第二次扩增池进行扩增,获得P7、P12、P19和P23等4个序列。序列比对表明,这4条序列均为NBS-LRR类棉花的抗病基因同源序列(RGA)。进一步对它们进行聚类,序列P7,P12,P19聚成很窄的一类,它们之间的同源性很高,估计它们来源于同一基因簇,P23聚成另一类,与黑松的RPS2和油菜的RGA30同源性较高。5.棉花FISH技术系统的建立。首次在棉花中探索成功高效制备DNA纤维技术,建立起一套完整的棉花FISH技术系统。该技术方便快捷,成功率高,成本低,容易被掌握和快速推广,必将为深入进行棉花基因组研究乃至棉花基因组测序的最终完成提供强有力的技术支持。6.棉花FISH技术系统的应用研究。45S rDNA在亚洲棉石系亚1号中期和粗线期染色体上有两对大的信号,在DNA纤维上信号为非连续的念珠状,多个拷贝串联排列,每个拷贝长度大约在3～11μm,推测每个拷贝的实际长度为11～30kb;端粒序列在亚洲棉石系亚1号中期和粗线期染色体端部都有双点信号,在DNA纤维上信号也为非连续的念珠状,不同染色体上信号长度大约在1～9μm不等,推测实际长度为4～27kb;gDNA信号几乎布满整个亚洲棉石系亚1号中期和粗线期染色体,在粗线期染色体上能明显区分出常染色质区和异染色质区,DNA纤维上的信号均为非连续的念珠状。Pima90的两个BAC克隆150-D-24、182-F-9在雷蒙德氏棉中期染色体上两信号中心点距离为0.21μm,在粗线期染色体上为3.8μm,在DNA纤维上荧光信号为典型的非连续的念珠状,其中150-D-24长度为32.7μm,182-F-9长度为37.9μm,两个克隆之间的距离128.4μm。按照DNA纤维伸展程度为3.27kb/μm,则150-D-24实际长度为107kb;182-F-9实际长度为124kb;两个克隆之间的实际距离为419.8kb。但DNA纤维存在着堆积和断裂,实际长度可能会更长些。更多还原

【Abstract】 Cotton is one of the main natural fiber crops in the world.Cotton genome sequencing has entered the substantive procedures,so it is urgent to accumulate basic data of cotton gene sequencing and to resovle critical techniques.There are rich secondary metabolites in cotton,such as tannins and gossypol,so it is difficult to make good chromosome slides.What’s more,the cotton chromosomes are small and similar.The aim of this study was to explore and establish the system of DNA FISH,which may resolve the cotton genome sequencing completely in the end.At the same time,we explored the single chromosome microdissection,constructed a single chromosome library and accumulated basic data for the cotton genome sequencing,gene cloning,chromosome painting and even the evolution of the Gossypium.The followings are main results:1.Establishment of chromosome microdissection and microcloning system in cotton.After pre-hypotonicity, enzymolysis,post-hypotonicity and squashed with cover slide,the mitosis metaphase chromosomes slides suitable for cutting was obtained.Based on these,we established an efficient procedure of micro-dissecting a single chromosome.Amplified production of single chromosome were verified by Southern blotting,SSR primer amplification and fluorescence in situ hybridization,and a complete system of verification and analysis was formed.We established a highly efficient,fast,easy-to-use system of chromosome microdissection and microcloning in cotton for the first time.2.Studying on cotton chromosome painting.After carrying out chromosome painting between G.arboreura with six other cotton species,it was found that the diploid cotton G.arboreum has high homology with diploid cotton G.herbaceum.Compared with five other tetraploid cotton,it was found that G.arboreum had higher homology with G.hirsutum and G.barbadense,but had lower homology with G.tomentosum,G. darwinii,and G.mustelinum,the lowest is G.mustelinura,which illustrated that the theory of tetraploid cotton polyphyletic origin may be more believable.3.Constructing a single cotton chromosome library.We constructed a single chromosome library of the 5th chromosome with large satellite of G.arboreum Shixiya 1.The library consisted of approximately 37,000 recombinant clones.The size of the inserts varied from 500～1800bp with an average of 750bp.The construction of the 5th chromosome library of G.arboreum Shixiya 1 would facilitate the specific probe screening,genetic map construction,important gene cloning and accumulate of basic data for sequencing on the chromosome.4.Cloning RGA from a single cotton chromosome.Four nucleotide sequence P7,P12,P19 and P23 were obtained after amplified in second round LA-PCR pool of the 5th chromosome in G.arboreum Shixiya 1 with disease-resistant homologous primers P1 and P2 of rice.The blast results showed that the four sequences were the NBS-LRR-type resistant gene analog(RGA) in cotton.Clustering analysis indicated that the sequences of P7,P12,P19 were high homologous and in the same cluster,so we speculated they originated from the same gene cluster,but the P23 was in other cluster and homologous with RPS2 gene of Brassica nigra and RGA30 gene of Brassica napus.5.Establishing cotton FISH system.The cotton DNA-fiber was well extracted in cotton for the first time. An integrated cotton FISH system was formed based on the DNA fiber-FISH which was successfully combined with the mitosis metaphase chromosome FISH and pachytene chromosome FISH.The technique is so simple,rapid,low cost and easy-to-use that it is bound to provide powerful technique for cotton genome sequencing.6.Researching on application of cotton FISH systems.There are two-pair large signals on the mitosis metaphase chromosomes and pachytene chromosomes of G.arboretum Shixiya 1 hybridized with 45S rDNA probe.The signals look like noncontinuous beads on the DNA fiber,which consist of multi-copy and arrange tandemly.Fiber-FISH results with 45S rDNA probe showed that an average signal length is about 3～11μm in many tandem copy sequence(measure number,n=8),so we estimated the size of each copy to be approximately 11～30kb in cotton genome.There are dual signals on each end of the mitosis metaphase chromosomes and pachytene chromosomes hybridized with telomere DNA probe.The signals also look like non-continuous beads on the DNA fiber,the length is about 1～9μm,so we estimated the size is about 4～27kb.The signals almost covered the whole mitosis metaphase chromosomes and pachytene chromosomes of G.arboretum Shixiya 1 hybridized with gDNA probe,the euchromatin zone and heterochromatin zone were identified clearly on pachytene chromosomes,and the signals also look like non-continuous beads.Two BAC clones 150-D-24 and 182-F-9 in DNA BAC library of Pima90 were selected as probe to hybridize with mitosis metaphase chromosomes,pachytene chromosomes and DNA fiber of G.raimondii.The results indicate that the distance between the two signals of BAC clone is 0.21μm on mitosis metaphase chromosomes,3.8μm on pachytene chromosomes.The signals on DNA fibers are also non-continuous-like beads.The length of 150-D-24 is about 32.7μm and that of 182-F-9 is about 37.9μm. The distance of two BAC clone is 128.4μm.According to a stretching degree of approximately 3.27 kb/μm, the size of 150-D-24 is about 114kb,the size of 182-F-9 is about 107kb,and the distance between 150-D-24 and 182-F-9 is about 419.8kb.Due to accumulation and fracture of DNA fiber,the actual size may be larger.更多还原