利用CRISPR/CAS9基因编辑技术创制香型郑稻19新种质

Breeding Fragrant Rice Zhengdao19 Using CRISPR/Cas9 Mediated Gene Editing Technology

有特殊香味的稻米深受我国消费者欢迎,是优质水稻的重要指标之一,本研究通过基因编辑创制香型优质水稻新种质。水稻中甜菜碱醛脱氢酶基因Badh2是控制香味的关键基因,以适于直播品种郑稻19为供体材料,利用CRISPR/CAS9技术定点突变水稻Badh2基因。获得T0代转基因阳性植株11株,其中9株子粒有香味,检测6个香型T1株系靶点序列,6个株系均发生突变,发现7种突变类型,5种是缺失类型,2种插入类型,其中3个株系(T1-2、T1-3和T1-7)出现双等位突变。6个T1代株系共测序22个单株,检测到纯合突变10株;T2代检测28株,其中纯合突变21株。以潮霉素抗性基因引物检测T1-2、T1-6和T1-7株系后代单株载体脱落情况,T1代3个株系未获得无载体纯合突变单株,T2代中获得8株无载体纯合突变单株,来源于T1-2株系有3株,来源于T1-7株系5株。T0代9株香型单株子粒2-AP含量1.259±0.072μg/g,T1代8个突变株系2-AP含量0.537±0.111μg/g,均显著高于对照郑稻19。考察中选T1-2、T1-7株系的农艺性状,与郑稻19无显著差异。这些无载体突变单株可作为香型种质在种质创新和育种中应用。

Fragrant rice is favored by Chinese consumers. In this study, Fragrance rice mutant lines were developed with CRISPR/CAS9 by editing gene Badh2, a key gene determining rice fragrance of Zhengdao 19, a rice variety suitable for direct-sowing as transformation donor. Totally, 9 fragrant mutants were obtained out of 11 positive transgenic T0 plant individuals. And twenty-two individuals of six T1 lines were confirmed by editing region sequencing. Seven mutation types were discovered, including five kinds of delete, two kinds of insert. Totally, three biallelic mutation lines(T1-2, T1-3 and T1-7) and ten homozygous mutants were found. Meanwhile sequencing twenty-eight T2 individuals revealed twenty-one homozygous mutants. Utilization of selectable marker screening to T2 progeny(from T1 lines T1-2, T1-6 and T1-7) eight marker-free homozygous mutants were found, and three were from T1-2, five from T1-7, respectively. Compared with the wild type, the 2-acetyl-1-pyrroline content increased from 0.003μg/g(in the wild type) to 1.259±0.072μg/g for T0 mutants planted in field, from 0.002μg/g(in the wild type) to 0.537±0.111μg/g for T1 mutants planted in greenhouse, without significant alternation in mainly agronomic traits. All selected marker-free homozygous individuals was available to be applied in germplasm enhancement and fragrance rice breeding practice.