【作者】 冯大领；

【导师】 申书兴； 李广敏；

【作者基本信息】 河北农业大学 ， 作物遗传育种， 2011， 博士

【摘要】 大白菜为十字花科芸薹属植物中重要的蔬菜作物,也是我国种植面积最大的叶用蔬菜。随着分子生物学的发展,基因组结构和功能、基因表达调控的分子机制等成为了蔬菜专家研究的热点问题。大片段基因组文库的构建是基因组学研究必不可少的工具。尽管国际上已经构建了大白菜的基因组文库,但有关芸薹属基因组结构和功能研究依然处于初级阶段,为了给我国大白菜基因组结构及功能研究提供方便,构建我国优良自交系的BAC文库也是当务之急。大白菜主要以营养器官为产品,早期抽薹是大白菜春季生产的主要障碍,严重影响着它的产量和品质,因此,研究培育大白菜晚抽薹品种,满足市场的周年供应成为蔬菜育种工作者的重要任务。研究大白菜开花相关基因及调控的分子机制将为培育抗抽薹新品种提供理论基础及分子鉴定手段。本文以我国优良的大白菜自交系‘85-1’为材料,利用pIndigoBAC5为载体,以HindⅢ为限制性内切酶,经过高分子量DNA的制备、DNA与载体的连接、转化等过程,成功构建了大白菜BAC文库。通过构建混合池,用PCR方法筛选了大白菜含有CO相关基因的BAC克隆,并对扩增产物进行了序列分析,为研究大白菜抽薹开花相关基因的序列特征及与其他芸薹属植物基因序列特征的差异性奠定了基础。主要研究结果如下:(1)该文库共含有57600个克隆,以单克隆的形式保存在150个384孔板中;以大白菜单倍体基因组总长度550 Mb计算,本文库重组子的覆盖率约为大白菜基因组的10.3倍,该文库得到任意单拷贝DNA序列的概率为99.99 %,文库的覆盖率符合要求,能够满足基因图位克隆、物理图谱构建、基因定位的需要;随机挑取200个BAC克隆,经过检测,其插入片段平均大小为98.4 Kb,插入片段多集中在90~120 Kb之间。85%的BAC克隆插入片段在90 Kb以上,证明片段大小的均一性较好;在200个检测的克隆中,有3个克隆没有插入片段,文库的空载率为1.5%;随机挑取6个BAC克隆进行连续5 d的培养,HindⅢ完全酶切的结果表明,各个克隆经过约125代左右的繁殖,DNA指纹图谱没有变化,证明DNA插入片段能够在大肠杆菌中稳定遗传,克隆的稳定性较好;本试验的连接转化效率为:100~200 Kb的DNA与载体连接转化后,一次转化获得5000个左右的克隆,200~300 Kb的DNA与载体连接转化后,一次转化只获得800个克隆,因此连接转化效率与片段的大小密切相关,片段越大,连接转化效率越低;(2)通过拟南芥CO基因的同源序列设计6对引物,对47个384孔板的18048个克隆进行PCR筛选。通过47个一级混合池,48个二级混合池,72个单克隆的三步PCR筛选,共167个PCR反应筛选到3个阳性克隆,分别命名为BrCO97A1、BrCO140N21、BrCO147H21;(3)经过NCBI网站的blastn比对,本试验筛选的BAC克隆含有编码constans-like蛋白的基因序列,序列相似性与Brassica rapa(白菜,AA),Brassica napus(甘蓝型油菜,AACC),Brassica juncea(芥菜型油菜,AABB)最接近,其次为Brassica oleracea(甘蓝,CC),序列相似性最小的为Brassica nigra(黑芥,BB)。因此,该克隆上的CO相关基因序列与C基因组的同源性高于B基因组。(4)通过与http://brassicadb.org网站上查找的CO相关基因的18个序列进行比对,结果表明:PCR产物序列与Bra008668有96%的最大序列相似性。在与Bra008668的比对结果中,查找到12个SNP位点,其中C/T变异6个,G/A变异2个,A/T变异2个,A/C变异1个,T/G变异1个;平均每51 bp即含有一个SNP位点。尽管这个数字代表性不强,但也说明在大白菜基因组中SNP位点是丰富的。更多还原

【Abstract】 As an important vegetable belonged to the genus Brassica of the family Brassicaceae, Brassica rapa subsp. pekinensis acts as the most widely planted leaf vegetable in China. With the development of plant molecular biology, the structure and function of genome and the mechanism of gene expression regulation have become hot topics which attract a lot of vegetable researchers. The construction of genomic libraries containing large segments is an essential tool for genomic studies. The BAC library of Chinese cabbage has been constructed internationally, however, the research about the genome structure and function of the genus Brassica is still in initial stage. Therefore, it is important to construct a BAC library of Chinese cabbage cultivar. The edible part of Chinese cabbage is its nutritorium. Early bolting is a major problem of cultivation in spring which seriously decreases the amount and quality of Chinese cabaage. So it is a big task to cultivate the late bolting variety to meet the need of vegetable market. The research on the bloom-related genes and mechanism of gene expression regulation will provide a theoretical basis for cultivating new Chinese cabbage cultivars.In the study, using Chinese cabbage inbred line‘85-1’as the material, pIndigoBAC5 as the vector, HindⅢas the restriction enzyme, a Chinese cabbage BAC library was successfully constructed through the production of high molecular weight DNA, connection of DNA and vector, and transformation process. By constructing first- and second-level pools, we screened the BAC clone with CO gene by the method of PCR and analysed SNP loci in the sequence of amplified product. The study lay solid foundations on analysing the sequence character of CO gene and difference with other Brassica plants.The results are as follows:1) The BAC library contains 57600 clones in total and the clones were stored in 150 384-well plates in the form of monoclone; According to the haploid genome size of 550 Mb, the library represented approximately an equivalent of 10.3 fold size of Chinese cabbage haploid genome. Therefore the possibility to find a single-copy gene in this library is 99.99%. Two hundred clones were selected randomly and digested by NotⅠrestriction enzyme. The average insert size is 98.4 Kb and the majority clones are during 90~120 Kb. 85% of BAC clones is longer than 90 Kb, which showed a good uniform of segment size. The clone numbers without insert segment are three among 200 clones, so the rate of empty clone is 1.5%; We selected six BAC clones randomly which were cultured for five days successively. The plasmid was completely digested by restriction enzyme HindⅢ. The result shows that DNA fingerprints of every clone have not changes after about 125 generations. Therefore the genetic stability of insert segment is perfect. Connecting the 100~200 Kb DNA with vector and transforming, we got about 5000 clones, while only about 800 clones were abtained though connecting the 200~300 Kb DNA. So we concluded that the efficiency of connection and transformation is closely related to the length of DNA segment. The longer the segment is, the lower the connection efficiency is.2) We designed six pairs of primers according to homologous sequence of the CO gene from Arabidopsis thaliana, and screened the clones from 18048 clones in 47 384-well plates by PCR amplification. In total three positive CO clones were obtained through three steps of PCR screening with 167 reactions. We named them BrCO97A1, BrCO140N21, BrCO147H21 respectively.3) The BAC clones contain CO gene which could code constans-like protein. Comparing the sequence of amplification product in the website NCBI, we found that the sequence similarity among the PCR product and Brassica rapa(AA), Brassica napus(AACC) and Brassica juncea(AABB) was high, next to Brassica oleracea(CC), and the third Brassica nigra(BB). So the similarity with genome C is higher than genome B.4) By comparing with 18 sequences containing CO gene found in the website http://brassicadb.org, the rusults show that the sequence similarity is 96% between the PCR pruducts and Bra008668. In the readable sequences, we also found 12 SNP locus, in which there are six C/T variants, two G/A variants, two A/T variants, one A/C variants, one T/G variants. There is one SNP locus per 51 bp. It is not a very representative number, but reflected the richness of SNP locus in genome.更多还原