体细胞重组是细胞维持基因组稳定的重要机制，与细胞的正常生长密切相关。但是植物中，由于缺少基因组信息明确、内源性的、与体细胞重组有关的突变体，在体细胞重组方面的研究受到很大的限制。大豆种质资源中发现了两个表型相似的孪生斑点突变子y11和cd-5。它们的纯合突变体呈黄化致死表型，杂合子植株叶片呈黄绿色，且常出现黄绿相邻、大小相似的孪生斑点。初步研究显示这些孪生斑点与体细胞重组有关，但还缺少分子生物学上的证据。本项目拟通过基因精细定位、转座子标签等方法来克隆y11和cd-5基因；然后用生物信息学、功能基因组学的方法分析其基因及蛋白的功能，以阐明y11和cd-5导致大豆发育、叶绿体异常的原因；再结合分子遗传学与细胞学手段，分析这两基因在杂合子孪生斑点的行为，探索孪生斑点产生的分子机理。此项目将拓展对植物体细胞重组、及核基因组对叶绿体调控方面的认识。

Somatic recombination (SR) is an important cellular process for maintaining genome stability of an organism. However, its basic mechanism is poorly understood, because it occurs rarely in one mitosis division compared to the recombination during one meiosis division; and normally, only one out of two reciprocal products can be recovered. Research on the mechanism of plant SR is very limited due to lack of useful SR mutants. No research is reported in soybean in this area. Two mutants discovered in soybean, y11 and cd-5, brought light on soybean SR research. Mutant y11 and cd-5 have similar phenotypes. Homozygous mutants are lethal and yellowish. Heterozygous mutants are viable and green-yellow. And interestingly, there are always some "twin sectors" emerging on the green-yellow leaves of heterozygous plants, one yellow and one green adjacent with similar size. Early investigations implied that twin sectors are the results of somatic recombination, but there are no direct evidences from cytology or molecular biology. Since two mutants show similar phenotypes and their genes reside in homeologous chromosome regions, y11 and cd-5 could be homologues genes. Mutant cd-5 generates through soybean transposon Tgm9 tagging experiment, so it can be cloned by TAIL-PCR. In addition, recently released soybean complete genome sequence with massive physically mapped molecular markers will give the gene cloning project a big help. In this project, we aim to 1) clone soybean cd-5 and y11 genes through accurate physical mapping followed by positioning mapping methods like transposon tagging or a simplified next generation sequencing method; 2) analyze their gene functions to reveal how they affect the development of soybean and the function of chloroplast with bio-informatics and functional genomics tools; 3) identify the molecular mechanism of twin sectors by using cytology and molecular genetics methods. Results of the project shall make a significant contribution to the research areas on plant somatic recombination, and nucleus-chloroplast communication network.

我们在大豆中发现了两个表型相似的孪生斑点突变子y11和cd-5。它们的纯合突变体呈黄化致死表型，而杂合子植株叶片呈黄绿色，且常出现黄绿相邻、大小相似的孪生斑点，但作用机制还不清楚。为此，本项目克隆了y11和cd-5基因并进行了遗传学验证，确认两者互为同源基因，均编码镁离子螯合酶的I亚基。我们将其编码的蛋白分别命名为GmChlI1a和GmChlI1b。这两个蛋白的序列相似对高达98%以上，应该从一次近期的基因重复分化而来。更巧合的是，y11和cd-5都发生了相同的单碱基置换突变，导致所编码蛋白的Q275突变成了R。Q275R突变导致I亚基ATP酶活性降低，但却不影响其参与镁离子螯合酶复合体的形成，因此可减低叶绿体中镁离子螯合酶活性，降低镁离子螯合酶的稳态水平，影响叶绿素的合成。因此，IQ275R突变蛋白的含量与镁离子螯合酶的活性成负相关：纯合突变体中叶绿素含量只有野生型的~1/10，植株为黄色致死；杂合突变体叶绿素的含量为野生型的1/2，呈黄绿色。叶绿素含量减半导致植株叶绿体类囊体垛叠减少、光耗散降低，但是光合效率并不减弱，有利于植物的生物量积累，为育种提供新的方向。不过，大豆的镁离子螯合酶组成十分复杂，其基因组编码了4个I亚基、2个D亚基、3个H亚基，且各同似物之间的氨基酸序列非常相似，任意I、D、H组合都能形成复合体。生物信息学、实时定量RT-PCR、及功能互补实验均指向各亚基同似物之间有功能分化现象，为优化大豆镁离子螯合酶活性提供了新线索。本项目的结果还为研究镁离子螯合酶的组装提供了关键证据。镁离子螯合酶由1个I亚基六聚体、1个D亚基六聚体、和1个H亚基垛叠而成，但是组装顺序不清楚。IQ275R突变导致大豆叶绿体内I亚基含量大量减少，D亚基次之，H亚基含量基本不变。这清楚地说明在镁离子螯合酶的组装过程中，I可以先行成六聚体，然后再参与后期组装。最后，y11和cd-5虽然不参与体细胞重组，但是它们可作为植物体细胞重组的报告基因，为筛选体细胞重组突变体、阐释体细胞重组机制提供了的难得的遗传材料。