敲除OsNramp5基因创制低镉优质粳稻新材料的应用评价  

Evaluation of New japonica Rice Lines with Low Cadmium Accumulation and Good Quality Generated by Knocking Out OsNramp5

作　　者：裴峰 王广达 高鹏 冯志明 胡珂鸣 陈宗祥 陈红旗[3] 崔傲 左示敏 PEI Feng;WANG Guangda;GAO Peng;FENG Zhiming;HU Keming;CHEN Zongxiang;CHEN Hongqi;CUI Ao;ZUO Shimin(Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding/Key Laboratory of Plant Functional Genomics of Ministry of Education,College of Agriculture,Yangzhou University,Yangzhou 225009,China;Jiangsu Collaborative Innovation Center for Modern Industrial Technology of Grain Crops/Jiangsu Key Laboratory of Crop Genetics and Physiology,Yangzhou University,Yangzhou 225009,China;State Key Laboratory of Rice Biology,China National Rice Research Institute,Hangzhou 311400,China;Zhenjiang Hexiatu Agricultural Science and Technology Co.,Ltd,Zhenjiang 212008,China;Joint International Research Laboratory of Agriculture and Agri-Product Safety,Ministry of Education of China,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学江苏省作物基因组学和分子育种重点实验室/植物功能基因组学教育部重点实验室,江苏扬州225009 [2]扬州大学江苏省粮食作物现代产业技术协同创新中心/江苏省作物遗传生理重点实验室,江苏扬州225009 [3]中国水稻研究所水稻生物学国家重点实验室,杭州311400 [4]镇江禾下土农业科技有限公司,江苏镇江212008 [5]扬州大学教育部农业与农产品安全国际合作联合实验室,江苏扬州225009

出　　处：《中国水稻科学》2023年第1期16-28,共13页Chinese Journal of Rice Science

基　　金：江苏省重点研发计划资助项目(BE2019339);江苏省农业重大新品种创制项目(PZCZ201703);扬州市重点研发计划资助项目(现代农业YZ2020031)。

摘　　要：【目的】探究在粳稻中敲除OsNramp5基因对镉等金属元素积累、产量和品质的影响,为科学高效地生产优质健康粳米提供新材料和理论参考。【方法】利用CRISPR/Cas9技术,在优良食味高产粳稻品种南粳46背景下对OsNramp5进行基因敲除,结合标记辅助选择,获得了无外源基因的OsNramp5敲除突变系,考查其产量和品质性状变化特征,以及在不同镉含量土壤中和锌肥、硒肥喷施后的籽粒镉、锰、硒等七种金属元素的含量变化。【结果】在T_(0)代获得了5种OsNramp5不同变异类型的转基因植株,设计开发了其中3种突变型的特异分子标记,进而结合标记辅助选择,在各植株的T_(2)代中获得不含潮霉素选择标记基因和CAS9蛋白基因的纯合敲除系。与野生型相比,无论在高浓度还是低浓度镉含量土壤中,敲除系整株包括籽粒中的镉含量和地上部组织中锰含量均显著低于南粳46;敲除系的株高略微下降,穗粒数显著降低并导致其单株产量明显下降,但在低分蘖肥处理中产量下降未达显著。敲除系稻米外观总体好于对照,蛋白质和直链淀粉含量明显高于对照,导致其食味值明显降低。敲除系籽粒中铜、锰和硒的含量显著低于对照,但在施用锌肥和硒肥后可以显著提高锌和硒的含量,同时有利于进一步降低籽粒镉的含量。施硒有利于改善稻米外观但施锌则相反,喷施两种肥料都会降低稻米食味值,不利于食味品质提升。敲除系在极端高/低温条件下的结实率变化与对照无异,但对纹枯病的抗性显著降低。【结论】在优质高产粳稻品种中获得了无标记OsNramp5基因敲除系,证明其在生产低镉富硒富锌安全营养健康型稻米中具有重要价值,但在产量和食味品质上则有不同程度的下降。【Objective】It is crucial to elucidate the effects of OsNramp5 gene knockout on cadmium and other metal elements accumulation,yield and quality in japonica rice,which can provide new materials and theoretical references for scientific and efficient production of high-quality and green japonica rice.【Methods】Using CRISPR/Cas9 technology,the OsNramp5 was knocked out under the background of Nanjing 46(NJ46),a japonica rice variety with good taste and high yield.Combined with molecular marker-assisted selection,some OsNramp5 knockout lines without exogenous genes were obtained.The changes in yield and quality traits and seven metal elements contents such as cadmium,manganese and selenium in grains after spraying zinc fertilizer or selenium fertilizer and cultivated in soils with different cadmium content were investigated.【Results】Five transgenic plants with different mutations of OsNramp5 were obtained in the T_(0) generation,and specific molecular markers of three of them were designed.Then,combined with marker-assisted selection,homozygous knockout lines without hygromycin selective marker gene and CAS9 protein gene were obtained in the T_(2)generation of each plant.Compared with the wild type,the cadmium content in the whole plant including the grain of the knockout lines and the manganese content in shoots were significantly lower than those of the wild type in both high and low concentrations of cadmium soil.The plant height and grain number per panicle of the knockout lines decreased slightly,and the yield per plant decreased significantly in the knockout lines at high and medium tillering fertilizer levels while no changes were observed at the low tillering fertilizer level.The appearance of the knockout lines was better than that of the control,and the contents of protein and amylose were significantly higher than those of the control.The contents of copper,manganese and selenium in grains of knockout lines were significantly lower than those of the control NJ46,but zinc and selenium fertilizer co

关 键 词：粳稻 低镉 基因编辑 品质 产量 育种价值 

分 类 号：S511.22[农业科学—作物学]