番茄基因组文库及突变体库的构建与分析
摘要
番茄(Solanum lycopersicum L.)是我国重要的蔬菜作物之一,然而番茄在种植过程中较易遭受病害侵蚀,影响产量,如番茄疮痂病、番茄黄化曲叶病毒病、霜霉病等。为了快速寻找抗病基因以及控制重要农艺性状的基因,培育持久抗病综合性状优良的品种,本论文在构建细菌人工染色体文库和突变体库两个方面开展工作,取得了以下结果。
参考已经成熟的BAC文库构建方法,以PCC1BAC为载体,构建了野生醋栗番茄PI128216的基因组BAC文库,获得了61,440个单克隆。从384孔单克隆板的文库中随机挑取2,000个克隆,用碱裂解法提取质粒并用脉冲场凝胶电泳检测文库的质量,结果显示平均插入基因组DNA片段大小为94kb,空载率和重组率为5%,据此计算该文库包含约6,000Mb大小的DNA片段,是番茄基因组大小(950Mb)的6倍以上,这个文库为染色体步移及染色体登陆方法的图位克隆筛选基因提供可能性。
为了丰富番茄遗传研究材料,本研究同时应用相同的野生醋栗番茄材料PI128216构建了甲磺酸乙酯(EMS)诱变的突变体库。经过预实验确定了构建突变体库所用化学诱变剂的两个处理浓度,利用这两个EMS诱变浓度处理14,000粒番茄种子(0.8%处理9,000粒种子,1.0%处理5,000粒种子),收获M1代种子种植自交获得了M2代种子,对M2代突变体库生育期内的所有能观察到的性状进行了相关调查,根据番茄种植资源描述规范和数据标准划分等级,将性状分为16个级和33个亚级,包括对种子、植株大小、植株习性、叶片形态、叶片颜色、开花期、花序、花形态、花颜色、果实大小、果实颜色、果实数量、不孕、胎萌以及抗病性等几个级别进行观察统计。结果显示,本研究所构建的突变体库中含有大量表型突变体,且会出现一个单株上出现多种突变性状现象,为利用番茄材料进行相关遗传研究提供了保障。
鉴于PI128216是一个抗番茄疮痂病的材料,为了研究其抗性机理,期望从上述突变体库获得感病突变体。因此,采用注射接种的方法对M2代突变体库每个单株进行了抗番茄疮痂病菌T3小种的鉴定,在注射番茄疮痂病T3小种24h后观察发病情况,两次接种都没有发现感病突变单株,推测可能是由于突变群体较小导致的。
总之,本研究构建了醋栗番茄材料PI128216的基因组BAC文库和EMS突变体库,既为番茄的遗传研究提供了突变体材料,也为基因的图位克隆提供了基础。
Tomato (Solarium lycopersicum), as one of the most popular vegetable crops in China, is suffering serious diseases, including bacterial spot, tomato yellow leaf curl virus, downy mildew and so on. In order to facilitate identifying genes conferring diease resistance and important horticultural traits for developing elite varieties with disease resistance, the work described here focus on constructing a BAC library and developing mutants.
The S. pimpinellifolium accession PI128216was used for genomic BAC library construction. High weight molecular DNA was isolated, digested and ligated into the vector PCC1BAC. A total of61,440clones were obtained. The quality of the library was examined by isolating plasmid from two thousand randomly selected clones and Pulse field gel electrophoresis. The results showed that the average size of DNA fragment was94kb. The rate of empty vector and recombinant was5%. Thus, the total size of DNA fragments in the library was about6,000Mb, which covered more than6X of the tomato genome. This library will be useful for positional cloning and chromosomal landing of functional genes.
In order to enrich the resources for research materialsgenetic study, a mutant library was also constructed using the tomato accession PI128216. Two concentrations of ethylmethane sulfonate (EMS) were adapted for seed treatment based on preliminary experiments. A total of9,000seeds were treated with0.8*EMS, and15,000seeds were treated with1.0%EMS. Morphological variations were recorded in the M1and M2generations using the descriptors and data standard for tomato. Sixteen classes and33sub-classess including seed, plant size, plant growth habit, leaf morphology, leaf color, blooming stage, inflorescence, flower morphology, flower color, fruit size, fruit color, fruit amount, infertility, viviparity and resistance. A large amount of mutants were obtained and some plants showed multiple mutations. These mutants provide a source for genetic study.
The M2plant of mutant were subjected to evaluation of resistance to bacterial spot race T3via infiltration inoculation. Unfortunately, none of the1,924mutants were susceptible to the pathogen, which might be due to the population size was not enough to discover the change of resistance.
In conclusion, a BAC library and a mutant library were constructed using the tomato accession PI128216. These materials will provide bases for genetic study, map-based cloning, and breeding.
参考已经成熟的BAC文库构建方法,以PCC1BAC为载体,构建了野生醋栗番茄PI128216的基因组BAC文库,获得了61,440个单克隆。从384孔单克隆板的文库中随机挑取2,000个克隆,用碱裂解法提取质粒并用脉冲场凝胶电泳检测文库的质量,结果显示平均插入基因组DNA片段大小为94kb,空载率和重组率为5%,据此计算该文库包含约6,000Mb大小的DNA片段,是番茄基因组大小(950Mb)的6倍以上,这个文库为染色体步移及染色体登陆方法的图位克隆筛选基因提供可能性。
为了丰富番茄遗传研究材料,本研究同时应用相同的野生醋栗番茄材料PI128216构建了甲磺酸乙酯(EMS)诱变的突变体库。经过预实验确定了构建突变体库所用化学诱变剂的两个处理浓度,利用这两个EMS诱变浓度处理14,000粒番茄种子(0.8%处理9,000粒种子,1.0%处理5,000粒种子),收获M1代种子种植自交获得了M2代种子,对M2代突变体库生育期内的所有能观察到的性状进行了相关调查,根据番茄种植资源描述规范和数据标准划分等级,将性状分为16个级和33个亚级,包括对种子、植株大小、植株习性、叶片形态、叶片颜色、开花期、花序、花形态、花颜色、果实大小、果实颜色、果实数量、不孕、胎萌以及抗病性等几个级别进行观察统计。结果显示,本研究所构建的突变体库中含有大量表型突变体,且会出现一个单株上出现多种突变性状现象,为利用番茄材料进行相关遗传研究提供了保障。
鉴于PI128216是一个抗番茄疮痂病的材料,为了研究其抗性机理,期望从上述突变体库获得感病突变体。因此,采用注射接种的方法对M2代突变体库每个单株进行了抗番茄疮痂病菌T3小种的鉴定,在注射番茄疮痂病T3小种24h后观察发病情况,两次接种都没有发现感病突变单株,推测可能是由于突变群体较小导致的。
总之,本研究构建了醋栗番茄材料PI128216的基因组BAC文库和EMS突变体库,既为番茄的遗传研究提供了突变体材料,也为基因的图位克隆提供了基础。
Tomato (Solarium lycopersicum), as one of the most popular vegetable crops in China, is suffering serious diseases, including bacterial spot, tomato yellow leaf curl virus, downy mildew and so on. In order to facilitate identifying genes conferring diease resistance and important horticultural traits for developing elite varieties with disease resistance, the work described here focus on constructing a BAC library and developing mutants.
The S. pimpinellifolium accession PI128216was used for genomic BAC library construction. High weight molecular DNA was isolated, digested and ligated into the vector PCC1BAC. A total of61,440clones were obtained. The quality of the library was examined by isolating plasmid from two thousand randomly selected clones and Pulse field gel electrophoresis. The results showed that the average size of DNA fragment was94kb. The rate of empty vector and recombinant was5%. Thus, the total size of DNA fragments in the library was about6,000Mb, which covered more than6X of the tomato genome. This library will be useful for positional cloning and chromosomal landing of functional genes.
In order to enrich the resources for research materialsgenetic study, a mutant library was also constructed using the tomato accession PI128216. Two concentrations of ethylmethane sulfonate (EMS) were adapted for seed treatment based on preliminary experiments. A total of9,000seeds were treated with0.8*EMS, and15,000seeds were treated with1.0%EMS. Morphological variations were recorded in the M1and M2generations using the descriptors and data standard for tomato. Sixteen classes and33sub-classess including seed, plant size, plant growth habit, leaf morphology, leaf color, blooming stage, inflorescence, flower morphology, flower color, fruit size, fruit color, fruit amount, infertility, viviparity and resistance. A large amount of mutants were obtained and some plants showed multiple mutations. These mutants provide a source for genetic study.
The M2plant of mutant were subjected to evaluation of resistance to bacterial spot race T3via infiltration inoculation. Unfortunately, none of the1,924mutants were susceptible to the pathogen, which might be due to the population size was not enough to discover the change of resistance.
In conclusion, a BAC library and a mutant library were constructed using the tomato accession PI128216. These materials will provide bases for genetic study, map-based cloning, and breeding.
引文
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