大豆叶片孪生斑点突变现象的分子机制及与体细胞重组的关系

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.

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。