晋A棉花线粒体基因组BAC文库的构建及重要功能基因的筛选
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摘要
本研究以棉花晋A细胞质雄性不育系及其保持系为研究材料，构建了相应的线粒体基因组细菌人工染色体(BAC)文库，并以线粒体功能基因为探针得到阳性克隆。线粒体基因组文库的构建将为克隆相关基因和研究线粒体基因组结构提供重要的技术平台。其目的是克隆棉花线粒体基因组相关基因，了解棉花线粒体基因组的结构，进而克隆与不育性相关的基因。
研究获得以下结果：
1．本实验在借鉴了各类作物BAC文库的构建方法后，建立了一套高效的线粒体基因组BAC文库构建的技术体系，包括线粒体的分离、高质量大片段DNA的获得、大量部分酶切最佳条件的确定、片段大小的选择、高效的连接与转化体系等。
2．分别构建了棉花晋A细胞质雄性不育系及其保持系线粒体基因组的BAC文库。
(1)不育系BAC文库的平均插入片段为22.29kb，空载率为0.9％；保持系BAC文库的平均插入片段为21.36kb，空载率为0.8％。据棉花线粒体基因组大小约为700kb计算，两个文库覆盖率分别为线粒体基因组的79和76倍，从理论上筛选出任何一个线粒体基因或序列的概率几乎为100％。
(2)插入片段可以在宿主大肠杆菌中稳定繁殖至少100代，无缺失、重组等现象发生，说明BAC克隆的插入片段可以在宿主大肠杆菌中稳定保存。
3．根据其它植物线粒体的功能基因设计引物，通过PCR扩增到相应的线粒体功能基因的片段orfB、coxI、atpA、nad4L和atp6，将这些片段分别克隆到pGM-T载体上，进行序列测定和对比确认。
4．通过不育系与保持系的orfB，coxI和nad4L3个基因的全序列的比较，证明晋A不育系与保持系在orfB、coxI和nad4L基因全序列上无差异。
5．利用棉花的线粒体功能基因或片断：orfB、coxI、atpA、nad4L和atp6对所构建的BAC文库进行菌落原位杂交，均筛选到了阳性克隆。
6．对棉花线粒体基因组的几个亚克隆进行序列测定表明：棉花线粒体基因组同样含有重复序列和来源于细胞核基因组叶绿体基因组序列。
Cytoplasmic male sterility (CMS) is a widespread phenomenon observed in＞150 flowering plant species. CMS is a maternally inherited trait and is often associated with unusual open reading frames (ORFs) found in mitochondrial genomes. Two kinds mitochondrial genome BAC library were constructed using JA cytoplasmic male sterile and its maintainer lines. The library construction of mitochondrial genome is a necessary step to clone gene and research the structure of the genome.
The results can be summarized as follow:
1. According to many methods of crops, a high-efficient technology system of BAC library construction was established. This system contains that isolation of mitochondrion, preparation of high-molecular weight (HMW) mtDNA, partial digestion of megabase DNA with a restriction enzyme, selection of digested fragment and transformation with high efficiency.
2. The sterile line and maintainer line of JA-type cotton were used as materials, and BAC libraries of their genome were constructed.
(1) Each library consisted of about 2500 clones with average insert size 22.29kb(or 21.36kb), ranging from 10.3kb to 37.5kb. It was calculated that each library contains approximate 79 and 76 times of cotton mitochondrial genome and provide 100% possibility to isolate any interested cotton genes or sequences in each library. The deep coverage and the large insert size of the libraries will facilitate physical mapping, isolation, and cloning of cotton mitochondrial genes.
(2) The BAC clone could be amplified by culturing for 100 generations without changing the structure of BAC clone.
3. We designed a set of primers based on the conserved sequences of the orfB、coxⅠ、atpA、nad4L and atp6 coding region. These genes locus were subjected to PCR analysis using these primers. The PCR products using mitochondrial cotton DNA as a template were further sequenced after cloning.
4. It was testified that orfB, coxⅠand nad4L were conversed between Jin-A cytoplasmic male sterile and its maintainer lines.
5. We obtained all positive clones from two BAC library by colony in situ hybridization of orfB, coxⅠ, atpA, nad4L and atp6.
6. It is documented that plant mtDNAs contain many repeated sequence and sequences of foreign origin. Chloroplast DNA (cpDNA) sequences and sequences of nuclear origin are found in cotton mitochondrial genome.
研究获得以下结果：
1．本实验在借鉴了各类作物BAC文库的构建方法后，建立了一套高效的线粒体基因组BAC文库构建的技术体系，包括线粒体的分离、高质量大片段DNA的获得、大量部分酶切最佳条件的确定、片段大小的选择、高效的连接与转化体系等。
2．分别构建了棉花晋A细胞质雄性不育系及其保持系线粒体基因组的BAC文库。
(1)不育系BAC文库的平均插入片段为22.29kb，空载率为0.9％；保持系BAC文库的平均插入片段为21.36kb，空载率为0.8％。据棉花线粒体基因组大小约为700kb计算，两个文库覆盖率分别为线粒体基因组的79和76倍，从理论上筛选出任何一个线粒体基因或序列的概率几乎为100％。
(2)插入片段可以在宿主大肠杆菌中稳定繁殖至少100代，无缺失、重组等现象发生，说明BAC克隆的插入片段可以在宿主大肠杆菌中稳定保存。
3．根据其它植物线粒体的功能基因设计引物，通过PCR扩增到相应的线粒体功能基因的片段orfB、coxI、atpA、nad4L和atp6，将这些片段分别克隆到pGM-T载体上，进行序列测定和对比确认。
4．通过不育系与保持系的orfB，coxI和nad4L3个基因的全序列的比较，证明晋A不育系与保持系在orfB、coxI和nad4L基因全序列上无差异。
5．利用棉花的线粒体功能基因或片断：orfB、coxI、atpA、nad4L和atp6对所构建的BAC文库进行菌落原位杂交，均筛选到了阳性克隆。
6．对棉花线粒体基因组的几个亚克隆进行序列测定表明：棉花线粒体基因组同样含有重复序列和来源于细胞核基因组叶绿体基因组序列。
Cytoplasmic male sterility (CMS) is a widespread phenomenon observed in＞150 flowering plant species. CMS is a maternally inherited trait and is often associated with unusual open reading frames (ORFs) found in mitochondrial genomes. Two kinds mitochondrial genome BAC library were constructed using JA cytoplasmic male sterile and its maintainer lines. The library construction of mitochondrial genome is a necessary step to clone gene and research the structure of the genome.
The results can be summarized as follow:
1. According to many methods of crops, a high-efficient technology system of BAC library construction was established. This system contains that isolation of mitochondrion, preparation of high-molecular weight (HMW) mtDNA, partial digestion of megabase DNA with a restriction enzyme, selection of digested fragment and transformation with high efficiency.
2. The sterile line and maintainer line of JA-type cotton were used as materials, and BAC libraries of their genome were constructed.
(1) Each library consisted of about 2500 clones with average insert size 22.29kb(or 21.36kb), ranging from 10.3kb to 37.5kb. It was calculated that each library contains approximate 79 and 76 times of cotton mitochondrial genome and provide 100% possibility to isolate any interested cotton genes or sequences in each library. The deep coverage and the large insert size of the libraries will facilitate physical mapping, isolation, and cloning of cotton mitochondrial genes.
(2) The BAC clone could be amplified by culturing for 100 generations without changing the structure of BAC clone.
3. We designed a set of primers based on the conserved sequences of the orfB、coxⅠ、atpA、nad4L and atp6 coding region. These genes locus were subjected to PCR analysis using these primers. The PCR products using mitochondrial cotton DNA as a template were further sequenced after cloning.
4. It was testified that orfB, coxⅠand nad4L were conversed between Jin-A cytoplasmic male sterile and its maintainer lines.
5. We obtained all positive clones from two BAC library by colony in situ hybridization of orfB, coxⅠ, atpA, nad4L and atp6.
6. It is documented that plant mtDNAs contain many repeated sequence and sequences of foreign origin. Chloroplast DNA (cpDNA) sequences and sequences of nuclear origin are found in cotton mitochondrial genome.
引文
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