利用CRISPR/Cas9基因编辑技术构建水稻ospin9突变体

doi:10.3864/j.issn.0578-1752.2021.18.002

Abstract

Abstract:

【Objective】Auxin efflux protein family PIN-FORMED (PIN) is a key protein family in controlling polar auxin transport (PAT). OsPIN9 is one of the monocot-specific PIN genes in rice, while its biological function still needs to be further elucidated. In this study, OsPIN9 was edited and ospin9 homozygous mutants were obtained using CRISPR/Cas9 genome editing technology. The resultant ospin9 mutant lines could provide a basis for further research on the function of OsPIN9.【Method】The specific target sequence was designed according to OsPIN9 genome sequence and OsPIN9 genome editing vector was constructed. Nippobare (Oryza sativa japonica) was used as the material and the hygromycin-resistant rice was obtained by Agrobacterium-mediated transformation. The positive transgenic lines were screened by PCR. The mutation sites were confirmed by the combination of PCR and subsequent analysis of sequencing results, the homozygous mutants were obtained and the difference of amino acid sequence and tertiary structure of OsPIN9 protein was analyzed between WT and ospin9 mutants. The expression of OsPINs genes in mutant roots was performed by quantitative real-time PCR (qRT-PCR), and the phenotype of ospin9 mutants was analyzed at the seedling stage. The effects of 1-naphthaleneacetic acid (NAA) treatment on seedling development were also analyzed under 0.05 μmol·L ^-1 NAA for 7 d.【Result】The target site sequence was designed based on the sequence of exon1 of OsPIN9 and, subsequently, the OsPIN9 genome editing recombinant vector was constructed. A total of 18 independent transgenic lines were obtained by transformation. Sequencing analysis revealed that three different mutation types were present in 7 T₀ generation lines, including 3 lines with T insertion, 3 lines with G insertion and 1 line with C insertion, and all the mutation sites happened at the 18 ^th base of the target sequence. Two homozygous mutation lines were further identified in the T₁ generation. BLAST analysis showed that the two types of OsPIN9 mutations caused frame-shift mutation and premature termination of translation, and the mutation protein was shortened from 426 aa in WT to 172 aa, thus leading to the complete disappearance of the transmembrane helices. qPCR analysis indicated that the transcription abundance of OsPIN9 significantly decreased in ospin9 mutants compared with WT, OsPIN1a and OsPIN5b were up-regulated, while OsPIN5a was down-regulated in ospin9 mutants. Both the shoot height and the number of adventitious roots of ospin9 mutants were reduced significantly than that of WT, while its root length was comparable to that of WT. The plant growth was inhibited and the adventitious root number was still less than that of WT under NAA treatment, but no significant difference was found between ospin9 mutants and WT plants. 【Conclusion】 Auxin efflux carrier OsPIN9 was directionally edited by using CRISPR/Cas9 technology, and two transgene-free homozygous ospin9 mutants were obtained. The mutation of OsPIN9 affected the expression level of other OsPINs genes, the shoot and root development were inhibited in ospin9 mutants at the seedling stage and NAA treatment partially rescued the development of adventitious roots in ospin9 mutants.

Key words: rice, OsPIN9, polar auxin transport, CRISPR/Cas9

WU ShiYang,YANG XiaoYi,ZHANG YanWen,HOU DianYun,XU HuaWei. Generation of ospin9 Mutants in Rice by CRISPR/Cas9 Genome Editing Technology[J].Scientia Agricultura Sinica, 2021, 54(18): 3805-3817.

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	用途 Usage
PIN9-CRISPR-F	TGTGTTTCTCCAACGAGCAGTGCGC	CRISPR/Cas9载体构建 CRISPR/Cas9 vector construction
PIN9-CRISPR-R	AAACGCGCACTGCTCGTTGGAGAA	CRISPR/Cas9载体构建 CRISPR/Cas9 vector construction
M13-F	GTTGTAAAACGACGGCCAGTGCC	筛选阳性克隆 Screening for positive clones
HPT-F	CTGAACTCACCGCGACGTCTGTC	筛选阳性植株 Screening for positive transgenic plants
HPT-R	TAGCGCGTCTGCTGCTCCATACA	筛选阳性植株 Screening for positive transgenic plants
PIN9-Assay-F	CGACCTGGCTTACGAACGAA	扩增OsPIN9基因组片段 Amplification of OsPIN9 genome fragment
PIN9-Assay-R	CCATGTCGAAGATGAGCACC	扩增OsPIN9基因组片段 Amplification of OsPIN9 genome fragment
PIN1a-qF	CCTGAAATCCATCTCCATCCTC	OsPIN1a表达分析 Expression analysis of OsPIN1a
PIN1a-qR	AACGTCGCCACCTTGTT	OsPIN1a表达分析 Expression analysis of OsPIN1a
PIN1b-qF	GAATCGTGCCCTTTGTGTTTG	OsPIN1b表达分析 Expression analysis of OsPIN1b
PIN1b-qR	TGTAGTAGACGAGGGTGATAGG	OsPIN1b表达分析 Expression analysis of OsPIN1b
PIN1c-qF	GAGCAATCAGCATCCCGAATA	OsPIN1c表达分析 Expression analysis of OsPIN1c
PIN1c-qR	GAGCAATCAGCATCCCGAATA	OsPIN1c表达分析 Expression analysis of OsPIN1c
PIN2-qF	CGTCTCCTTCAGGTGGAATATC	OsPIN2表达分析 Expression analysis of OsPIN2
PIN2-qR	AGAGCCATGAACAAGCCTAAG	OsPIN2表达分析 Expression analysis of OsPIN2
PIN5a-qF	CCCTACCTCAATCCATCACATC	OsPIN5a表达分析 Expression analysis of OsPIN5a
PIN5a-qR	GTAGGGAGACAAGCATTCCAA	OsPIN5a表达分析 Expression analysis of OsPIN5a
PIN5b-qF	GCAAAGGAGTATGGGCTTCA	OsPIN5b表达分析 Expression analysis of OsPIN5b
PIN5b-qR	GCAATCAGAATCGGCAGAGA	OsPIN5b表达分析 Expression analysis of OsPIN5b
PIN9-qF	GAGGACTCTCTGTTCACCATTC	OsPIN9表达分析 Expression analysis of OsPIN9
PIN9-qR	GAGAACGACGCTATCTTGTATCC	OsPIN9表达分析 Expression analysis of OsPIN9
OsACTIN1-qF	CTTCATAGGAATGGAAGCTGCG	qRT-PCR内参基因 Internal control for qRT-PCR
OsACTIN1-qF	CACCTTGATCTTCATGCTGCTA	qRT-PCR内参基因 Internal control for qRT-PCR

Generation of ospin9 Mutants in Rice by CRISPR/Cas9 Genome Editing Technology

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