中国农业科学 ›› 2020, Vol. 53 ›› Issue (8): 1501-1509.doi: 10.3864/j.issn.0578-1752.2020.08.001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用CRISPR/Cas9技术编辑水稻香味基因Badh2

祁永斌,张礼霞,王林友,宋建,王建军()   

  1. 浙江省农业科学院作物与核技术利用研究所,杭州 310021
  • 收稿日期:2019-09-02 接受日期:2019-11-30 出版日期:2020-04-16 发布日期:2020-04-29
  • 通讯作者: 王建军
  • 作者简介:祁永斌,E-mail: qi_yongbin@hotmail.com。
  • 基金资助:
    浙江省科技厅公益技术研究项目(2017C32007);浙江省粮食新品种选育重大科技专项(2016C02050);国家重点研发计划“七大作物育种”重点专项(2017YFD0100302);浙江省农业科学院人才培养项目(2018R16R08E01)

CRISPR/Cas9 Targeted Editing for the Fragrant Gene Badh2 in Rice

QI YongBin,ZHANG LiXia,WANG LinYou,SONG Jian,WANG JianJun()   

  1. Institute of Crop Science and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021
  • Received:2019-09-02 Accepted:2019-11-30 Online:2020-04-16 Published:2020-04-29
  • Contact: JianJun WANG

摘要:

【目的】稻米香味是水稻品质改良的一个重要内容,其性状主要受1个隐性基因Badh2的控制。利用CRISPR/Cas9基因编辑技术将常规品种中的Badh2进行编辑,从而获得Badh2发生突变的基因编辑株系,使香味性状得到改良。【方法】利用CRISPR/Cas9基因编辑的原理,在水稻Badh2的第2和第7外显子处设计靶点,通过BLAST分析确定其特异性并构建到CRISPR/Cas9表达载体。以浙江省主要推广的水稻品种嘉58和秀水134的愈伤组织为受体,利用农杆菌介导的遗传转化法,通过潮霉素抗性筛选获得阳性转基因植株。转基因株系经测序明确其在Badh2的突变类型,经PCR分析与鉴定获得Badh2发生突变并无转基因标记成分的稳定株系。利用气相色谱质谱联用仪(GC-MS)检测基因编辑株系糙米米粉中2-AP的含量,明确其香味成分与非转基因对照之间的差异。【结果】在水稻第2和第7外显子处设计靶点构建表达载体,通过遗传转化转基因株系的Badh2完成了定向突变。共获得T0代嘉58基因编辑的株系15株,在第2外显子处发生突变的有8株5种不同的突变类型,均为不同单碱基插入不同位点;在第7外显子处发生突变的有7株5种不同的突变方式,均为碱基或片段缺失。获得秀水134基因编辑的株系11株,在第2外显子处共有5株,均为单碱基插入;在第7外显子处有6株,均为片段缺失。48株T1代秀水134基因编辑株系中,共获得16株无转基因标记的基因编辑株系,其中5株在第2外显子发生突变,11株在第7外显子处发生突变。4个T2代基因编辑株系的米粉中2-AP平均含量分别为0.309、0.347、0.332和0.295 μg·g -1,极显著(P<0.01)高于非转基因对照(0.046 μg·g -1)。【结论】利用CRISPR-Cas9技术可对水稻香味基因Badh2进行定向编辑,并且可获得无转基因成分的基因编辑株系,其香味性状得到明显改良。

关键词: 水稻, CRISPR/Cas9, 基因编辑, Badh2, 香味

Abstract:

【Objective】Rice fragrance, a very important trait for quality improvement, is mainly controlled by a recessive gene Badh2. In this study, CRISPR/Cas9-mediated editing were used to generate the gene-edited rice plants with the fragrant Badh2 in the conventional elite rice varieties, and the trait of fragrance was improved.【Method】 The targeted sequences were designed according to the sequence of exon2 and exon7 of the Badh2 by the principle of CRISPR/Cas9 gene editing. Its specificity of the targeted sequence was determined by BLAST analysis, and then constructed into CRISPR/Cas9 expression vector. The callus of Jia58 and Xiushui134 which are widely cultivated in the Zhejiang Province were selected as explants to transform by Agrobacterium-mediated genetic transformation, and the positive transgenic plants were obtained by the screening of hygromycin resistance. Sequencing analysis of transgenic lines was used to detect the presence of the mutation type on the loci of Badh2. Stable marker-free gene-edited lines carrying the mutation on Badh2 were obtained by PCR analysis and identification. The content of 2-AP in the brown rice flour were measured by GC-MS, and the difference between the gene-edited lines and non-transgenic control was determined. 【Result】 Badh2 of the transgenic lines was directionally mutated by the genetic transformation using the expression vector on which the target sequence of the exon2 and exon7 were designed and constructed. A total of 15 T0 gene-edited lines were obtained from Jia58. Eight of them were generated mutation on the exon2 with five different mutations types in which different single base was inserted into different position. Seven of them were generated mutations on the exon7 with five different mutation types in which the base or fragment deletion was produced. A total of 11 T0 gene-edited lines were obtained from Xiushui134, of which five lines were generated mutations on the exon2 with the single base insertion and six lines on the exon7 with the fragment deletion. A total of 16 marker-free gene-edited lines were obtained from 48 T1 Xiushui134, of which five lines were generated mutations on the exon2, and 11 lines on the exon7. The average 2-AP content in brown rice flour of four T2 gene-edited lines were 0.309, 0.347, 0.332 and 0.295 μg·g -1 respectively, which were significantly higher (P<0.01) than that of the non-transgenic control (0.046 μg·g -1). 【Conclusion】 The Badh2 which controlled the rice fragrance trait was directionally edited by using CRISPR/Cas9-mediated technology, and the marker-free gene-edited lines were obtained, of which the fragrance of Badh2 edited lines were significantly improved.

Key words: rice, CRISPR/Cas9, gene editing, Badh2, fragrance