摘要
DUFs家族是一类未知功能结构域蛋白家族,转录谱和蛋白谱分析发现,DUFs蛋白在生物的生长和发育中发挥着至关重要的作用,因此对DUFs基因功能的研究将有助于了解生物体复杂的生长发育机制。目前该家族基因功能仍鲜见报道。为了探究水稻(Oryza sativa)OsDUF393(Domains of Unknown Function 393)基因的生物学功能,利用CRISPR/Cas9技术构建了水稻DUFs家族基因OsDUF393的载体,同时利用农杆菌介导的遗传转化将构建的CRISPR/Cas9载体转化水稻品种中花11中,经潮霉素抗性基因鉴定,获得了100株T0代转基因植株。对转基因植株的靶位点进行测序分析后发现有单碱基插入和大片段DNA序列缺失2种类型。通过RT-PCR分析突变体中OsDUF393基因的表达水平,结果表明,目的基因在不同纯合编辑突变体中转录水平均有下降。上述结果表明,CRISPR/Cas9重组载体成功实现了对OsDUF393基因的定向编辑。这些编辑突变体材料为后续该基因功能研究奠定了基础。
DUFs protein families are a large of uncharacterized protein families with unknown functional domain. Transcription profile and protein profile revealed that DUF protein play a crucial role in the biological process of development and growth. Therefore, the study of DUFs gene function will be helpful to understand the complicated mechanism of growth and development of organisms. To investigate biological function of the rice gene OsDUF393(Domains of Unknown Function 393), the CRISPR/Cas9 gene editing technique was employed. In this study, we constructed a CRISPR/Cas9 vector editing OsDUF393 of rice and introduced it into rice variety Zhonghua11 to produce transgenic plants by Agrobacterium-mediated plant transformation method. Totally 100 T0 transgenic plants were obtained. Sequencing analysis of the target sites showed that there are two types of mutations in the transgenic plants, including single base insertions and long fragment deletions. The expression level of OsDUF393 gene in the mutant was analyzed by RT-PCR,and the results showed that the transcription level of the target gene was decreased in different homozygous editing mutants. The results indicated that the OsDUF393 gene was edited successfully by CRISPR/Cas9 system. Taken together, these mutants are suitable materials for OsDUF393 function study.
作者
王荃
石雨鹭
邳瑞雪
孔晓聪
靳亚军
梁闪闪
张泗举
栾维江
WANG Quan;SHI Yulu;PI Ruixue;KONG Xiaocong;JIN Yajun;LIANG Shanshan;Zhang Siju;LUAN Weijiang(College of Life Science,Tianjin Normal University/Tianjin Key Laboratory of Animal and Plant Resistance,Tianjin 300387,China)
出处
《中国稻米》
2019年第2期40-45,共6页
China Rice
基金
天津市自然科学基金重点项目(16JCZDJC33400)
天津市自然科学基金一般项目(17JCYBJC30000)
天津市自然科学基金绿色通道项目(17JCYBJC41300)
天津市水稻产业技术体系-水稻种质创新岗IV(ITTRRS2018006)
天津师范大学博士基金(52XB1611
52XB1612)
