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作物杂志,2018, 第6期: 36–42 doi: 10.16035/j.issn.1001-7283.2018.06.006

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

利用CRISPR/CAS9基因编辑技术创制香型郑稻19新种质

王付华1,薛华政1,王亚1,王生轩1,王越涛1,付景1,杨文博1,白涛1,李俊周2,尹海庆1   

  1. 1 河南省农业科学院粮食作物研究所,450002,河南郑州
    2 河南农业大学农学院,450002,河南郑州
  • 收稿日期:2018-08-24 修回日期:2018-11-06 出版日期:2018-12-15 发布日期:2018-12-06
  • 作者简介:王付华,副研究员,研究方向为水稻遗传育种;薛华政为共同第一作者,助理研究员,主要从事作物遗传育种研究
  • 基金资助:
    国家“七大农作物育种专项”长江中下游优质高产高效粳稻新品种培育项目(2017YFD0100400/3);河南省水稻产业技术创新团队首席专家项目(S2012-04);河南省重大科技专项(171100110300);河南省农业科学院自主创新专项(2018ZC11)

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

Wang Fuhua1,Xue Huazheng1,Wang Ya1,Wang Shengxuan1,Wang Yuetao1,Fu Jing1,Yang Wenbo1,Bai Tao1,Li Junzou2,Yin Haiqing1   

  1. 1 Institute of Cereal Crop, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
    2 Agronomy College, Henan Agricultural University, Zhengzhou 450002, Henan, China
  • Received:2018-08-24 Revised:2018-11-06 Online:2018-12-15 Published:2018-12-06

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摘要：

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。

关键词: 水稻, 郑稻19, 香味, Badh2, CRISPR/CAS9

Abstract:

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.

Key words: Rice, Zhengdao19, Fragrance, Badh2, CRISPR/CAS9

图1

靶点序列及测序验证 方括号内序列为靶位点,方框内序列为编辑靶点检测引物"

表1

研究所用引物"

引物名称Primer name 引物序列Primer sequence 用途Usage
Badh identify_F CCATCGGTACCCTCCTCTTC 靶点序列扩增Amplification of target region
Badh_identif_R ATCGATCGATTTGGGGCTCA
Vector_identify_F CGATTCCGGAAGTGCTTGAC 载体验证 Vector identification
Vector_identify_R CGTCTGCTGCTCCATACAAG

表2

T1和T2代靶位点序列突变分析"

T1株系号
No. of
T1 line		 T1测序株Sequenced of T1 T2测序株Sequenced of T2 靶点序列
Target sequence		 突变情况
Mutation
总株数/突变体
Total/Mutant		 纯合/杂合
Homozygote/Heterzygote
in mutants		 总株数/突变体
Total/Mutant		 纯合/杂合
Homozygote/Heterzygote
in mutants
WT CAAGTACCTCCGCGCAATCGCGGCC
T1-2 3/3 0/3 6/6 6/0 CAAGTACCTCCGCGC--TCGCGGCC -2
CAAGTACCTCCGCG-----CGGCC -5
T1-3 3/3 2/1 6/6 3/3 CAAGTACCTCCGCG--------GCC -8
CAAGTACCTCCGCGCA-TCGCGGCC -1
T1-6 5/4 2/2 5/4 2/2 CAAGTACCTCCGCGCAAaTCGCGGCC +1
T1-7 5/5 3/2 5/5 5/0 CAAGTACCTCCGC------------ -12
CAAGTACCTCCGCGCAAaTCGCGGCC +1
T1-8 3/2 0/2 3/3 3/0 CAAGTACCTCCGCGCAAacacttgat +9
T1-9 3/3 3/0 3/2 2/0 CAAGTACCTCCGCGCAAaTCGCGGCC +1
小计Total 22/20 10/10 28/26 21/5

图2

转基因T1、T2代单株编辑位点基因型及载体PCR检测"

图3

T0、T1代株系香味物质2-AP的含量 **:在0.01水平上显著"

图4

中选T1株系植株表型及产量相关农艺性状"

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