棉花学报 ›› 2019, Vol. 31 ›› Issue (1): 31-39.doi: 10.11963/1002-7807.zxylxd.20181212
藏旭阳(),代培红,李继洋,蒲艳,顾爱星*(
),刘晓东*(
)
收稿日期:
2018-04-09
发布日期:
2019-01-15
通讯作者:
顾爱星,刘晓东
E-mail:1506409442@qq.com;xiaodongliu75@aliyun.com;594903711@qq.com
作者简介:
藏旭阳 (1990―),女,硕士研究生,
Zang Xuyang(),Dai Peihong,Li Jiyang,Pu Yan,Gu Aixing*(
),Liu Xiaodong*(
)
Received:
2018-04-09
Published:
2019-01-15
Contact:
Gu Aixing,Liu Xiaodong
E-mail:1506409442@qq.com;xiaodongliu75@aliyun.com;594903711@qq.com
摘要:
【目的】对已克隆的海岛棉U3和U6启动子进行功能鉴定,为构建棉花CRISPR/Cas9多位点基因编辑技术体系提供更多可用的U3和U6启动子。【方法】分别构建以GbU3-2P和GbU6-7P为启动子驱动sgRNA,以抗旱负调控基因GGB为靶序列的CRISPR/Cas9基因编辑载体,然后在新海16的棉花叶片原生质体中进行功能鉴定。通过Polymerase chain reaction方法富集构建好的CRISPR/Cas9基因编辑载体的核心片段,并利用PEG瞬时转化法将核心片段转入原生质体中。提取转化后的原生质体基因组DNA,采用酶切/Polymerase chain reaction法分析棉花GGB基因的突变情况并测序验证。最后绘制靶基因突变的频率分布图来计算该CRISPR/Cas9系统的编辑效率和确认突变的真实性。【结果】靶基因测序结果显示靶标位点序列突变的类型全部为碱基替换。【结论】以GbU3-2P和GbU6-7P为启动子的CRISPR/Cas9基因编辑体系可以成功地定点编辑棉花GGB基因的序列,引起基因突变。
藏旭阳,代培红,李继洋,蒲艳,顾爱星,刘晓东. 棉花U3和U6启动子在CRISPR/Cas9基因组编辑体系中的功能鉴定[J]. 棉花学报, 2019, 31(1): 31-39.
Zang Xuyang,Dai Peihong,Li Jiyang,Pu Yan,Gu Aixing,Liu Xiaodong. Functional Identification of Cotton U3 and U6 Promoters in the CRISPR/Cas9 Genome Editing System[J]. Cotton Science, 2019, 31(1): 31-39.
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