水稻功能型滞绿DGP1基因的克隆与功能研究

Stay-green mutants are increasingly drawing breeders’ attentions due to their potential in crop yield increase. We previously screened and identified an unique stay-green mutant, dark green panicle 1(dgp1), which showed great breeding values with characteristics of stay-green leaves and panicles, increased photosynthesis efficiency and grain yield. As we have cloned and functionally confirmed the DGP1 gene, we propose to (1) pinpoint the DGP1 functions using combined approaches of gene expression analysis, subcellular localization, and transgenic gain-of-function studies; (2) identify DGP1-intreacting proteins (DGP1-IPs) and subsequently characterize the functions of the confirmed positive DGP1-IPs; (3) pinpoint the internodes of DGP1 signal transduction pathway and ultimately uncover (or dissect) the gene networks, in which the DGP1 functions in regulation of chlorophyll metabolism based on the information obtained from and (1) and (2) combined with the transcriptome analysis on dgp1 mutant; (4) identify the elite DGP1 mutant alleles via sequence analysis on DGP1s from various sources of rice germplasm in combination with techniques including TILLING and evaluate their breeding values in rice. Upon the completion of this proposed project, we shall unravel the molecular mechanism of chlorophyll metabolism regulated by DGP1 in rice leaf and panicle and provide important genetic resources and breeding materials for molecularly designed breeding in rice.

功能型滞绿突变体在作物育种利用中具有增产潜力，受到育种家的广泛关注。我们前期获得一个罕见的深绿穗（dark green panicle 1, dgp1）突变体，其叶片滞绿、光合效率增强、促进籽粒生物量的累积，是一个具重大育种价值潜力的功能型滞绿突变。本项目拟在克隆DGP1基因的基础上，通过基因表达、亚细胞定位、转基因过表达等分析DGP1基因功能，鉴定DGP1互作蛋白DGP1-IP（DGP1-interacting protein）及其功能，结合dpg1突变体的转录组分析，寻找DGP1信号转导中的网络节点，最终解析DGP1调控叶绿素代谢的基因网络和途径；通过对不同稻种资源DGP1基因的序列分析，结合TILLING等技术，发掘水稻DGP1基因的优异等位变异，并进行深入的育种价值评估。本课题的完成将揭示水稻叶片及颖壳叶绿素代谢调控的分子机理，亦为水稻分子设计育种提供优异基因资源与中间材料。

水稻是重要的粮食作物，高产育种是确保我国粮食安全和农业可持续发展的有效保障。水稻抽穗后，功能叶（剑叶、倒二叶、倒三叶）中光合同化物累积效率与高光效时期持续性对籽粒产量具有决定性的作用。但是，水稻在籽粒发育后期，功能叶片就开始衰老，导致光合速率迅速下降，籽粒中物质累积率减少，严重影响其光合效率。因此，提高水稻功能叶光合累积率，延长光合持续期，使叶片吸收更多光能，是培育高光效品种的有效手段。功能型滞绿突变体在作物育种利用中具有增产潜力，受到育种家的广泛关注。我们前期获得一个罕见的功能型滞绿突变体dgp1，其叶片滞绿、光合效率增强、促进籽粒生物量的累积，具重大育种价值潜力。本研究在克隆DGP1基因的基础上，分了析dgp1突变基因对叶绿体发育、叶绿素合成与光合作用的影响，发现dgp1基因突变促进了叶绿体的发育、叶绿素生物合成与光合作用的效率；研究DGP1基因的表达模式与亚细胞定位，发现该基因在地上部分高表达，在根中不表达，其编码蛋白定位在细胞核和细胞质中；通过酵母双杂交和BiFC鉴定了DGP1的互作蛋白，解析了其调控叶绿体与光合作用的分子机理；并通过CRISPR/Cas9技术获得多个水稻主栽品种的dgp1突变体，选育了多个高产新品系。该研究的完成有助于揭示DGP1调控水稻叶绿素代谢与光合分子机理，亦为水稻分子设计育种提供优异基因资源与中间材料。