水稻是一种重要的粮食作物，已有8000-10000年的栽培驯化历史。水稻产量作为一个复杂的农艺性状，由单株有效穗、每穗颖花数和千粒重组成，属于典型的数量遗传性状，由多个数量性状位点（Quantitative trait loci, QTLs）调控。随着基因定位、基因组测序和功能基因组研究的发展，许多控制产量的QTL被鉴定出来，尤其是调控分蘖和穗粒数的基因。截止目前，被鉴定的调控分蘖和穗粒数的基因已有二十多个。在这些基因中，有些属于正向调控，而有些属于负向调控。本研究中是选择负向调控穗粒数的两个基因—Gn1a和DEP1,借助本实验室前期研发的CRISPR/Cas9的单子叶敲除体系PLANTCRISPR-CLONING ，对两个基因进行高效编辑突变，以期获得比自然等位基因对产量贡献率还要突出的新等位基因。我们以日本晴水稻为受体，经过了T0代、T1到T2代的种植。在T1代中，针对DEP1基因共鉴定出24中纯合敲除的基因型，同时Gnla基因鉴定出12种纯合敲除的基因型。对这些基因型进行序列比对分析，发现大多突变类型都呈现翻译提前终止的现象，产生截断的蛋白质。通过T2代基因型鉴定，发现T1代产生的突变类型可以在T2代中稳定遗传。通过T2代产量表型数据统计和分析，在Gn1a突变体中获得4种穗粒数增加的突变类型，其中Gn1a-M8增产效果最大，尤其在穗粒数、主穗二次枝梗数增加上呈非常显著水平，被鉴定为Gn1a所获得的最优良等位基因；在DEP1突变体中获得3种穗粒数增加的突变类型，其中DEP1-M18增产效果最大，尤其在二次枝梗数增加上呈非常显著水平，被鉴定为DEP1所获得的最优良等位基因。; As a vital cereal crop, rice has been domesticated for 8000-1000 years. Now rice is the global staple food, so improving the grain yield of rice keeps being the hottest topic in the field of rice research. Grain yield of rice is a complex agronomic trait, which is mainly determined by three traits: number of panicles per plant, number of grains per panicle and the mass of 1000 grains. All the three traits are typical quantitative traits controlled by multiple quantitative trait loci (QTLs), making it hard to locate related genes with traditional gene mapping experiments. With the development of gene mapping and genome sequencing as well as functional genome research, many QTLs have been identified, especially genes related to tillers and number of grains per panicle. So far, more than 20 genes have been identified to regulate tilling and grain number, being either positive control factors or negative control factors, which provides precious materials for our research.In our study, two negative regulation genes, DEP1 and Gn1a, were selected to be edited using the CRISPR/Cas9 system “PLANTCRISPR-CLONING”, a system developed in our lab for editing genes of monocots and dicotsefficiently. We hope to obtain novel alleles performing better than the natural alleles. We chose Nipponbare as the receptor and cultivated from T0 to T2. In the T1 generation, 24 homozygous knock-out genotypes for DEP1 gene were identified,and 12 homozygous knockout genotypes from Gn1a individuals were identified. These mutations were found to be stably hereditary in progeny by determining T2 rice plants’ genotype. With our present work, we have acquired artificial four mutants which are in Gn1a plants and in which Gnla-M8 is identified to be the best excellent allele due to the effects of improving rice yield, especially on increasing grains and secondary branches are very significant level. Meanwhile, three artificial mutants are identified to be relation to increase the number of panicle grains in DEP1 plants in which DEP1-M18 is identified to be the best excellent allele due to the effects of improving rice yield, especially on increasing the secondary branches is very significant level.
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