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首页> 外文期刊>Agronomy >Development of High Yielding Glutinous Cytoplasmic Male Sterile Rice ( Oryza sativa L.) Lines through CRISPR/Cas9 Based Mutagenesis of Wx and TGW6 and Proteomic Analysis of Anther
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Development of High Yielding Glutinous Cytoplasmic Male Sterile Rice ( Oryza sativa L.) Lines through CRISPR/Cas9 Based Mutagenesis of Wx and TGW6 and Proteomic Analysis of Anther

机译：通过基于CRISPR / Cas9的Wx和TGW6诱变和花药的蛋白质组学分析开发高产糯质细胞质雄性不育水稻品系

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Development of high yielding and more palatable glutinous rice is an important goal in breeding and long-standing cultural interaction in Asia. In this study, the TGW6 and Wx , major genes conferring 1000 grain weight (GW) and amylose content (AC), were edited in a maintainer line by CRISPR/Cas9 technology. Four targets were assembled in pYLCRISPR/Cas9Pubi-H vector and T 0 mutant plants were obtained through Agrobacterium mediated transformation with 90% mutation frequency having 28% homozygous mutations without off-target effects in three most likely sites of each target and expression level of target genes in mutant lines was significantly decreased ( P 0.01), the GW and gel consistency (GC) were increased, and the AC and gelatinization temperature (GT) were decreased significantly and grain appearance was opaque, while there was no change in starch content (SC) and other agronomic traits. Mutations were inheritable and some T 1 plants were re-edited but T 2 generation was completely stable. The pollen fertility status was randomly distributed, and the mutant maintainer lines were hybridized with Cytoplasmic Male Sterile (CMS) line 209A and after subsequent backcrossing the two glutinous CMS lines were obtained in BC 2 F 1 . The identified proteins from anthers of CMS and maintainer line were closely associated with transcription, metabolism, signal transduction, and protein biosynthesis. Putative mitochondrial NAD + -dependent malic enzyme was absent in CMS line which caused the pollen sterility because of insufficient energy, while upregulation of putative acetyl-CoA synthetase and Isoamylase in both lines might have strong relationship with CMS and amylose content. High yielding glutinous CMS lines will facilitate hybrid rice breeding and investigations of proteins linked to male sterility will provide the insights to complicated metabolic network in anther development.
机译：发展高产，可口的糯米是亚洲育种和长期文化互动的重要目标。在这项研究中，TGW6和Wx是赋予1000粒重（GW）和直链淀粉含量（AC）的主要基因，是通过CRISPR / Cas9技术在一个维持系中进行编辑的。在pYLCRISPR / Cas9Pubi-H载体中组装了四个靶标，通过农杆菌介导的转化获得了T 0突变体植物，该突变体具有90％的突变频率，具有28％的纯合突变，在每个靶标的三个最可能位点和靶标的表达水平均无脱靶作用突变株系中的基因显着降低（P <0.01），GW和凝胶稠度（GC）增加，AC和糊化温度（GT）显着降低，谷物外观不透明，而淀粉含量没有变化（SC）和其他农艺性状。突变是可遗传的，一些T 1植物被重新编辑，但T 2世代是完全稳定的。花粉育性状态是随机分布的，并且突变体维持系与细胞质雄性不育（CMS）系209A杂交，并且在随后回交后，在BC 2 F 1中获得了两个糯CMS系。从CMS和维持系花药中鉴定出的蛋白质与转录，代谢，信号转导和蛋白质生物合成密切相关。 CMS系中不存在假定的线粒体NAD +依赖性苹果酸酶，这是由于能量不足导致花粉不育，而两种系中假定的乙酰辅酶A合成酶和异淀粉酶的上调可能与CMS和直链淀粉含量密切相关。高产糯CMS不育系将促进杂交水稻育种，对与雄性不育有关的蛋白质的研究将为花药发育中复杂的代谢网络提供真知灼见。

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