利用CRISPR/Cas9基因编辑技术构建水稻ospin9突变体

[目的]生长素输出载体蛋白(PIN-FORMED,PIN)是控制生长素极性运输的关键蛋白,水稻OsPIN9是单子叶植物特有的PIN基因,但其生物学功能仍有待研究.利用CRISPR/Cas9基因编辑技术对OsPIN9进行编辑,获得OsPIN9发生突变的基因编辑株系,对进一步深入研究OsPIN9功能提供依据.[方法]根据O...

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

【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.