水稻<i>OsGRAS39</i>基因的表达特征及基于CRISPR/Cas9技术的突变体创建

doi:10.7668/hbnxb.20191602

摘要/Abstract

摘要： 旨在探究水稻OsGRAS39（LOC_Os10g22430.1）基因的功能，采用qRT-PCR技术分析OsGRAS39在水稻不同组织中及不同非生物胁迫条件、ABA和GA₃处理下的表达情况，并基于CRISPR/Cas9技术创建OsGRAS39基因敲除突变体。qRT-PCR分析结果表明，OsGRAS39在所有检测的组织中均表达，其mRNA丰度在水稻幼苗中最高，然后依次是幼根、叶片，而在抽穗期水稻的叶鞘、穗、根和茎中相对较弱；OsGRAS39的表达受高温胁迫抑制，受低温、NaCl和ABA诱导；在10% PEG6000处理下，其在所有处理时间点的表达水平无显著差异；在20 μmol/L的GA₃处理下，其表达水平在2~8 h下调而在24 h上调。另外，利用无缝克隆技术成功构建了OsGRAS39基因的CRISPR/Cas9敲除载体pP1C.3-OsGRAS39-gRNA，并通过农杆菌介导的遗传转化获得30株转基因水稻株系。靶位点测序结果表明，30株转基因阳性水稻苗中有8株检测到不同类型的突变，包括1个纯合突变体，5个双等位突变体和2个杂合突变体。综上，OsGRAS39可能参与水稻对高温、低温和NaCl胁迫抗性的调控；成功创建了OsGRAS39基因敲除突变体。

关键词: 水稻, OsGRAS39, 表达特征, CRISPR/Cas9, 基因编辑

Abstract: In order to investigate the function of OsGRAS39 (LOC_Os10g22430.1) in rice, the expression patterns of OsGRAS39 in different tissues and under diverse abiotic stresses as well as phytohormone ABA and GA₃ treatments were analyzed by qRT-PCR; and the OsGRAS39 gene knockout mutants were constructed based on CRISPR/Cas9 system. The results of qRT-PCR analysis showed that OsGRAS39 was expressed in all tissues tested. The mRNA abundance of OsGRAS39 was highest in rice seedlings, followed by young roots and leaves, while lesser in leaf sheaths, panicles, roots and stems at the heading stage. The expression of OsGRAS39 was inhibited by high temperature, while induced by low temperature, NaCl and ABA. Under the treatment of 10% PEG6000, there was no significant difference in the expression level of OsGRAS39 at all treatment time points. Under the treatment of 20 μmol/L GA₃, the expression level of OsGRAS39 was down-regulated at 2-8 h and up-regulated at 24 h. In addition, the OsGRAS39 gene knockout vector pP1C.3-OsGRAS39-gRNA was successfully constructed by seamless cloning. Thirty transgenic rice lines were obtained by Agrobacterium-mediated genetic transformation. The sequencing results of target site showed that 8 out of 30 transgenic rice seedlings were found to have different mutation types, including 1 homozygous mutant, 5 biallelic mutants and 2 heterozygous mutants. In conclusion, OsGRAS39 may be involved in the regulation of rice resistance to high temperature, low temperature and NaCl stress. The OsGRAS39 knock-out mutants were successfully created.

Key words: Rice, OsGRAS39, Expression characteristics, CRISPR/Cas9, Gene editing

中图分类号:

Q78
S511.03

邹杰. 水稻OsGRAS39基因的表达特征及基于CRISPR/Cas9技术的突变体创建[J]. 华北农学报, 2020, 35(6): 1-8. doi: 10.7668/hbnxb.20191602.

ZOU Jie. Expression Characteristics of Rice OsGRAS39 Gene and Creation of Its Mutants Based on CRISPR/Cas9 Technique[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(6): 1-8. doi: 10.7668/hbnxb.20191602.

http://www.hbnxb.net/CN/Y2020/V35/I6/1

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水稻OsGRAS39基因的表达特征及基于CRISPR/Cas9技术的突变体创建

Expression Characteristics of Rice OsGRAS39 Gene and Creation of Its Mutants Based on CRISPR/Cas9 Technique

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水稻OsGRAS39基因的表达特征及基于CRISPR/Cas9技术的突变体创建

Expression Characteristics of Rice OsGRAS39 Gene and Creation of Its Mutants Based on CRISPR/Cas9 Technique

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