【作者】 刘宏高；

【导师】 丁毅；

【作者基本信息】 武汉大学 ， 遗传学， 2010， 博士

【摘要】 全基因表达谱方法是后基因组时代里生命科学研究领域的核心方法之一,发展性价比高和适用性广的全基因表达谱方法非常必要。目前的全基因表达谱方法可分为基于测序、基于杂交和基于凝胶电泳策略三大类。测序类方法可提供样本的序列及丰度,但全长测序成本太高,而标签测序难以准确定位。杂交类的cDNA微阵列法获得了广泛的应用。虽然很成功,但它仅适用于具备基因组或cDNA数据库的少数物种,且只能检测已知的基因或转录本(”封闭性”)。这两类方法都需要昂贵的实验平台。凝胶类方法相对测序类和杂交类方法具有诸多的优点：如它只需要RT-PCR、DNA测序胶电泳、克隆和测序等常规分子生物学技术和常规仪器和试剂,因而适用面广；它不需要已知序列信息,因而适用于任何真核物种和任何转录本(”开放性”)；借助PCR可获得高灵敏度；通过一定数量的引物组合可达到高通量要求；两个或多个样本的多态可在同一凝胶相邻泳道中直观显示和比较；差异转录本衍生片段(Transcript-derived fragment, TDF)被回收、重扩增和克隆测序后可用于后续实验和分析等。(1)本研究基于凝胶电泳的基础建立了一种鲁棒有序的mRNA差异显示新方法。它通过巧妙的引物设计、poly(dT:A)置换技术、磁珠法分离技术和依赖于4碱基的覆盖率解决方案,充分结合两大基于凝胶的全基因表达谱方法mRNA差异显示和cDNA-AFLP方法的优点,并以有序的、无冗余的方式,获得了较高覆盖率。其基本原理为：利用5’-biotin-MlyI-AcuI-T16-V引物(bmaT16V引物)和样本的总RNA反转录合成双链cDNA。MspI酶切双链cDNA,连接MspI接头,利用磁珠法选择性地分离poly(A:T)端带生物素的酶切片段,洗脱其余片段以去除冗余。然后用Acul再次酶切分离出的片段,并利用磁珠法吸附新产生的酶切片段中带生物素的的部分即poly(dT:A)端,而释放出来的含不带生物素的酶切片段的上清被收集起来并连接AcuI假接头。以该连接产物作为常规cDNA-AFLP方法的模板进行预扩增和选择性扩增,并利用DNA测序胶电泳分离扩增片段,再通过银染的方法显示基因表达图谱,通过比较获得差异TDF片段。差异TDF片段可被切下回收、重扩、克隆和测序,供进一步分析。和mRNA差异显示方法相似,RoDD方法锚定1mRNAA的3’-UTR。从实验技术的角度而言这一区域的可靠性最好,从差异表达研究方法的角度而言非翻译区的多态性最为丰富。而RoDD的基本原理与实验步骤几乎同于基于磁珠法的cDNA-AFLP方法,因而它具有PCR效率高,PCR可靠性好、重复性好,假阳性少和无冗余等优点。鉴此,我们把它命名为鲁棒有序的mRNA差异显示方法(Robust ordered mRNA differential display, RoDD)。(2)为了对该方法进行评估,我们建立了简化的数学模型计算,利用perl语言编写的计算机软件进行模拟,还利用基因组、cDNA信息较完备的栽培稻日本晴(Oryza sativa L. ssp.japonica cv. Nipponbare)幼苗的叶和根为材料进行简单的验证实验。数学模型和计算机软件模拟结果显示RoDD方法分别覆盖了己发表的人类转录本92.79%和82.13%,水稻转录本的92.75%和84.56%,高于任一同类的方法。在以日本晴幼苗的叶和根为材料的RoDD方法验证实验中,我们通过32个PTT接头引物组合的选择性扩增在叶和根中分别得到1650(其中含552差异TDF)和1576(其中含478差异TDF)扩增条带,共计2128；据此推测通过全部的196个PTT引物组合可分别覆盖叶和根中的10106和9653扩增条带,共计13000种；同理,30个PNN组合选择性扩增分别在叶和根中得到1274(其中含368差异TDF)和1260(其中含354差异TDF)扩增条带,共计1628种条带,据此推测通过全部的240个PNN引物组合可分别覆盖叶和根中的7803和7717条带,共计13000种。合并起来,我们推测在水稻的叶和根中分别可获得17909和17370条带,并进一步推测在叶和根中分别有17909和17370个基因表达；同时通过432个引物组合可覆盖两种组织中的26000种转录本。这证实了RoDD确实具有很高的覆盖率。我们随机挑取了40个转录本衍生片段(TDF)中,BLAST结果显示39个被定位在日本晴的cDNA数据库,半定量RT-PCR结果确定34个TDF片段呈阳性。这说明RoDD方法具有较高的可靠性。值得注意的同源搜索失败的唯一TDF片段却在多次半定量RT-PCR重复实验中表现阳性,因而它可能是尚未在日本晴发现或注释的新基因。另外,本研究中还设计了引物效率比较实验和缩微版的RoDD实验,证明了RoDD引物策略中PCR效率高、可靠性好等优势。(3)RoDD方法的应用。云南紫稻细胞质雄性不育系是本实验室育成的一种新的细胞质源的水稻雄性不育系,但其细胞质效应、雄性不育和育性恢复机理尚缺乏深入研究。利用同核异质系是研究细胞质差异最有效的途径之一,所以我们构建了云南紫稻细胞质源的雄性不育系梅香A与6种与梅香A细胞核背景相近、但细胞质类型(野败型、K型、D型、冈型、印水型和马协型)不同的近等核异质雄性不育系,并将RoDD方法应用于它们的全基因表达谱比较研究中,同时RoDD方法还被应用于珍汕97A和K17A与梅香B核置换过程中历代杂种全基因表达谱差异的比较。结果发现不同类型的细胞质对近等核的全基因表达影响的差异很小,核置换历代杂种表达谱差异也很小。我们推测这可能是因为它们同属孢子体细胞质雄性不育系统,具有相同的起源和协同进化历程。实验中我们还发现了几个梅香A、B相对其6种近等核异质雄性不育系以及梅香A、B两者之间的特征谱带,对这些谱带的深入研究可能在分子水平上为云南紫稻细胞质的独特性提供一些佐证。更多还原

【Abstract】 The global gene expression profiling plays a pivotal role in biological research in post-genomic era and therefore the development of cost-effective and universal methods for global gene expression profiling is of great necessity. The current global gene expression profiling tools, principally, may fall into three categories:sequencing-based, hybridization-based and gel-based. The sequencing-based methods offer the sequence information and its abaundance. But the fl-cDNA sequencing suffers from formidable costs, while the tag sequencing from ambiguous tag mapping. Microarray is the leading method of hybridization-based category that has been widely used. Although great success, it is only a "close" system that is limited to species with available genome or transcriptome sequence. In addition, both sequencing and microarray need sophisticated platforms. In comparison, the gel-based category has many advantages:it needs only routine technologies that consists of RT-PCR, DNA sequencing gel electrophoresis and cDNA cloning, which can be readily performed with conventional instruments and reagents; it is applicable to any eukaryotic species and transcripts; the input RNA can be detected by PCR as sensitive as 20 ng; high-throughput can be achieved by a limited number of primer combinations; polymorphisms of many samples can be visualized and compared side by side in the same gel; the transcript-derived fragments (TDFs) of interest can be cloned and sequenced for further analyses.(1) We have established a relatively cheap method to profile global gene expression. It incorporates the advantages of elaborately designed primers, poly(dT:A) replacement, bead-based isolation and a 4-bp-site-dependent coverage solution, combining the virtues of two popular gel-based GGEP methods, mRNA differential display (DD) and complementary DNA amplified fragment length polymorphisms (cDNA-AFLP). Briefly, ds-cDNA was synthesized using a 5’-biotin-MlyI-AcuI-T16-V primer (bmaT16V) and total RNA of the leaves and roots of rice (Oryza sativa), followed by digestion using Mspl and ligation to the MspI adapters. The biotinylated restriction fragments were trapped by streptavidin-coated magnetic beads and digested using Acul. Newly-generated biotinylated fragments were arrested by the magnetic beads while the released non-biotinylated restriction fragments were isolated and ligated to the pseudo adapters, which served as templates for pre-amplification, followed by selective amplification, DNA sequencing gel electrophoresis and cDNA cloning. It anchors the 3’-UTR of mRNA just like DD. However, its procedures are identical to those of bead-based cDNA-AFLP. Therefore, it is robust, holding the virtues of high polymorphism, high PCR reliability, less false positives, low redundancy and low cost. Furthermore, it has higher coverage because of the dependence on the orderly presence of only one 4-bp restriction site on each cDNA. Thus, this method was named "Robust Ordered mRNA Differential Display (RoDD)".(2) To evaluate the coverage of RoDD, a draft mathematical modeling, an in silico simulation were performed, and a simple RoDD validation experiment was carried out using the leaves and roots of one-week-old seedlings of rice (Oryza sativa spp. japonica cv. Nipponbare). The results of the mathematical modelling and the in silico simulation showed the RoDD method has coverage of 92.79% and 82.13% on the published human transcripts, and 92.75% and 84.56% on the published rice transcripts, respectively, higher than any methods of the same categories.1,650 (containing 552 TDF) and 1,576 (containing 478 TDF) bands were obtained in the rice leaves and roots by PCRs using 32 PCs for the PTT adapters, respectively. Summing up,2,128 transcripts were covered. Assuming that 196 PCs for the PTT adapters were completely used, an estimated 10,106 and 9,653 transcripts of the rice leaves and roots could be covered, respectively, summing up to 13,000.1,274(containing 368 TDF) and 1,260(containing 354 TDF) bands were obtained in the rice leaves and roots by PCRs using 30 PCs for the PNN adapters, respectively. Summing up,1,628 transcripts were covered, Assuming 240 PCs for the PNN adapters were completely used, an estimated 7,803 and 7,717 transcripts of the rice leaves and roots could be covered, respectively, summing up to 13,000. In all,17,909 and 17,370 (summing up to 26,000) transcripts could be covered in the leaves and roots of rice if a total number of 432 PCs were completely used. These results indicated high coverage of the RoDD method. Forty TDFs were randomly picked for sequencing and BLAST analysis, of which 39 TDFs were well mapped on O. sativa (japonica cultivar-group) cDNA, and 34 TDFs were confirmed to be true positives, indicating a high reliability. It was interesting to note that the only TDF with no significant similarity found was confirmed to be true positive in many repeated semi-quantitative RT-PCR assays using newly-prepared root samples, indicating a new candidate transcript. In addition to low cost and high coverage, experimental data also showed that the RoDD method has the characteristics of low redundancy, less false positives and high efficiency of polymerase chain reactions (PCRs). A primer efficiency comparason assay and a RoDD miniature assay were performed, which showed the advantages of the high primer efficiency and reliability of RoDD. Therefore, the RoDD method is a cost-effective and universal alternative for the state-of-the-art GGEP methods, sequencing and microarray.(3) The application of the RoDD method. The cytoplasmic male sterile line of Yunnan purple rice A (CMS-P) represents a new cytoplasm source, but it lacks the details of the cytoplasmic effect and mechanism of male sterility and fertility restoration. For it is one of the most effective ways to use the isogenic alloplasmic CMS lines to explore the cytoplasmic characteristics, we constructed 6 isogenic alloplasmic CMS lines (each contains the cytoplasm of CMS-WA, CMS-K17A, CMS-D, CMS-G, CMS-ID, CMS-MA, respectively) of Meixang A, a CMS line of CMS-P type, though hybridization and subsequently recurrent backcrossings to Meixiang B, the isogenic alloplasmic maintainer of Meixiang A. The RoDD method was used to comparatively profiling the global gene expression of seven near-isogenic alloplasmic CMS lines of rice. Moreover, the RoDD patterns of the F1, BC1F1, BC2F1 of Zhenshan 97A/Meixiang B and K17A/Meixiang B were compared so as to monitor the dynamic alterations during the nuclear replacement. However, results showed little polymerphysm. We speculated that it is due that they belong to sporophytic CMS system, which have the same origin and coordinated evolution process. Several characteristic bands of Meixiang A, B or both were found. With further study, these bands may provide some molecular evidence for the "unique" cytoplasm of Yunnan purple rice.更多还原