Determination of the entire structure of wheat chloroplast genome

目前仅有水稻和玉米两种禾本科植物叶绿体基因组的完整核苷酸序列被报道。在本项目中，我们成功地确定了小麦叶绿体基因组全序列，这是第三个案例。我们使用普通小麦（Triticum aestivum） cv。中国春作为材料，因为这个品种在世界范围内的遗传研究中使用最广泛。此外，我们还构建了小麦叶绿体DNA的连续无性系库。此外，设计了pcr引物组，用于扩增除两个小麦叶绿体基因外的所有基因的编码区，并成功扩增了它们的编码区。主要结果如下。(1)小麦叶绿体基因组全序列：小麦叶绿体基因组大小为134,540 bp，比玉米（140,387 bp）小4.2%，与水稻（134,525 bp）基本一致。大、小单拷贝区（LSC和SSC）和反向重复区（IR）大小分别为80,347,12,789和20,702 bp。该基因组包含109个基因，其中4个（重复）编码4种rRNA， 30个编码trna（对应20个氨基酸），70个编码蛋白质亚基，5个功能未知。(2)连续克隆库：构建了覆盖LSC、SSC和IR整个区域的55个连续克隆（部分重叠）组成的叶绿体DNA库。克隆的平均大小为2246 bp。(3)小麦叶绿体基因扩增的PCR引物：设计了107个叶绿体基因的PCR引物。利用这些方法，制备了少量扩增的dna，用于除两个小麦叶绿体基因外的所有编码区域。

Complete nucleotide sequence of chloroplast genome has been reported only in two Gramineae species, rice and corn. In this project, we succeeded determination of the complete sequence of wheat chloroplast genome as the third case. We used a common wheat (Triticum aestivum) cv. Chinese Spring as the material, because this cultivar has been used most extensively worldwide in genetic investigations. In addition, we constructed a contig clone bank of wheat chloroplast DNA. Also, PCR-primer sets were designed to amplify coding regions of all but two wheat chloroplast genes, using which their coding regions were successfully amplified. Main results are given below.(1) Complete sequence of wheat chloroplast genome : Size of wheat chloroplast genome was 134,540 bp, that was 4.2% smaller than that of corn (140,387 bp) but almost the same as that of rice (134,525 bp). Sizes of three regions, large and small single copy regions (LSC and SSC) and inverted repeat (IR), were 80,347, 12,789 and 20,702 bp, respectively. This genome contained 109 genes, 4 (in duplication) coding for 4 rRNA species, 30 for tRNAs (corresponding to 20 amino acids), 70 for protein subunits and 5 of unidentified functions.(2) Contig clone bank : A chloroplast DNA bank consisting of 55 contig clones (some are partially overlapping) that cover entire regions of LSC, SSC and IR was constructed. Average size of the clones was 2,246 bp.(3) PCR primer sets for amplification of wheat chloroplast genes : A pair of PCR primers for each of 107 chloroplast genes were designed. Using these, small amounts of amplified DNAs were prepared for the coding regions of all but two wheat chloroplast genes.