【作者】 姜淑梅；

【导师】 李文雄；

【作者基本信息】 东北农业大学 ， 作物栽培学与耕作学， 2003， 博士

【摘要】 本研究利用染色体微切割、微克隆技术，建立小冰麦异附加系TAI-27中异附加中间偃麦草染色体文库。中间偃麦草具有抗病、抗逆等多种特性，小冰麦异附加系TAI-27附加一对中间偃麦草染色体，具有大麦黄矮病抗性，抗病基因位于异附加中间偃麦草染色体上。本研究在染色体文库建立的基础上，以克隆抗病基因为目的进行了一系列研究。 利用染色体微切割、微克隆技术，采用微玻璃针法，成功分离小冰麦异附加系TAI-27中异附加的染色体，通过PCR扩增获得足够附加染色体DNA，成功创建了库容量比较大，对染色体的覆盖比较全的小冰麦异附加系TAI-27中附加染色体的文库。该染色体文库的建立对于进一步建立这条染色体的高密度遗传连锁图谱，获得与重要性状连锁的分子标记，进而分离重要基因及研究基因组进化有重要意义。小冰麦异附加系TAI-27中异附加染色体的成功分离也为本研究后继工作奠定了良好基础。 在小冰麦异附加系TAI-27中异附加染色体文库构建的基础上，首次利用同源杂交和抑制PCR原理，通过改进同源片段特异扩增技术(Hybrid specific amplification，HSA)，成功分离小冰麦异附加系TAI-27附加小染色体上的表达序列，建立一个快速分离特定染色体表达序列的方法。试验证明杂交特异扩增(HSA)分离染色体cDNA是可行的。并且由于使用单链cDNA可以大幅降低成本，操作也更为简便。研究对分离特定染色体的表达序列标签(EST)及克隆新基因有重要参考价值。获得的染色体上表达序列能为小麦抗性育种提供分子标记。同时进一步拓宽了染色体微分离、微切割与微克隆技术的应用。 利用蚜虫饲咬小冰麦异附加系TAI-27，接种大麦黄矮病病毒GAV株系，对接种大麦黄矮病毒的TAI-27与对照进行了差异表达的研究，克隆到与因外界环境胁迫表达的EST同源的序列，并进行了遗传背景分析和染色体定位。这一结果对最终克隆和应用中间偃麦草这对染色体上的抗性基因及分析TAI-27的抗性机制有重要意义。有9个cDNA序列为TAI-27中首次克隆(已在GenBank登记注册，注册号BQ788525-BQ788533)。 根据抗病基因保守的结构域设计简并引物，利用PCR方法结合染色体显微分离技术，从TAI-27中微分离的异附加单条染色体及其父母本中扩增到若干NBS序列。经过与Genebank数据库进行核酸及氨基酸序列比较，得到的片段与抗病基因或抗病基因同源序列同源，证明得到的片段为NBS(nucleotide binding site，NBS)类型的抗病基因同源片段(Resistances gene analog，RGA)。对微分离的异附加单条染色体中获得的一条RGA进行了Southern杂交和Northern杂交分析，结果证明它位于TAI-27中来源于中间偃麦草的附加染色体上。这一结果为克隆TAI-27中附加的中间偃麦草染色体上的R基因全长提供了特异性探针。 小冰麦TAI-27中异附加染色体文库的构建是本研究的工作基础。本研究有助于进一步了解附加染色体上的遗传信息，为最终获得附加的中间偃麦草染色体上的抗病基因及建立附加染色体EST数据库奠定基础。更多还原

【Abstract】 Thinopyrum intermedium possesses the properties of disease resistance, drought resistance, cold resistance and other strict environmental stresses. Wheat-Thinopyrum alien additional line TAI-27 created by hybridization between common wheat and Thinopyrum intermedium has a pair of chromosomes derived from Th. Intermedium. The additional chromosomes carry the resistant gene to BYDV. In this paper several strategies were used for research on resistant genes located on the additional chromosomes.Firstly, one alien additional chromosome was isolated and collected successfully under an inverted phasecontrast microscope using a microneedle. Chromosome DNA of the alien additional chromosome was amplified by two rounds of PCR. Consequently, a chromosome specific DNA library with a higher cloning efficiency and relatively large insert size was constructed, which is very useful to help us to set up chromosome physical maps and genetic maps, screen molecular markers linked with important economical traits, clone key genes encoding important proteins and study the evolution of the genome.Secondly, a method to clone expressed sequences of specific chromosome was created by combining chromosome microdissection and microcloning and HSA (hybrid specific amplification) techniques. The expressed sequences of the alien additional chromosome of Wheat-Thinopyrum alien additional line TAI-27 were cloned and sequenced. The method established in this research can help us to construct EST database of the specific chromosome, clone important genes and provide molecular markers for wheat breeding. Additionally, it starts a new area of application of the plant chromosome microdissection and microcloning technique.Thirdly. A subtractive library was constructed using the leaves of Wheat-Thinopyrum alien additional line TAI-27 infected by aphid carried GAV strain of BYDV in the developmental stage of three leaves and the control. Some of differentially expressed gene sequences, which matched with ESTs expressed in wheat under certain stress, were screened out. It is significant to clone resistant genes located on the alien additional chromosome derived from Thinopyrum intermedium and understand resistant mechanism of TAI-27.Fourthly, Resistant gene analogs (RGAs) were amplified by PCR with the degenerated primers designed based on the NBS domain of R genes from the DNA of the alien additional chromosome of TAI-27, DNA and cDNA of Thinopyrum intermedium, TAI-27 and 3B-2. Sequence homology analyzing in GenBank showed that those sequences amplified with degenerated primers were high homologous with known resistant genes or resistant gene analogs. The results of Southern hybridization and Northern hybridization showed that one of the RGAs, which come from the alien additional chromosome, was the specific probe of the alien additional chromosome of TAI-27 which could be used for cloning of the complete resistant gene located on the alien additional chromosome. This research supplied a convenient method for resistant gene cloning by combining chromosome microdissection and microcloning and homology-based cloning techniques.This research based on chromosome microdissection and microcloning provided much molecular information about the alien additional chromosome of TAI-27, which would beuseful for further cloning resistant genes located on the alien additional chromosome derived from Thinopyrum intermedium and creating EST database of the specific chromosome.更多还原