Construction of qSH1 Mutants in Rice (Oryza sativa) Using CRISPR/Cas9 and Characteristic Analysis of Mutagenesis
SHENG Xia-Bing1,2, WANG Xue-Feng2, TAN Yan-Ning2, SUN Zhi-Zhong2, YU Dong1,2, YUAN Gui-Long2, YUAN Ding-Yang2*, DUAN Mei-Juan1*
1 College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China; 2 Hunan Hybrid Rice Research Centre, Changsha 410125, China
Abstract：Seed shattering is one of the major agronomic traits closely related to rice (Oryza sativa)production. Easy shattering is not good for seeds harvest, nor suitable for mechanized production of rice. qSH1 (QTL of seed shattering in chromosome 1; LOC_Os01g62920) is one of the major genes to control rice seed shattering. Higher expression of qSH1 in the abscission zone (AZ) would promote the formation of AZ, and lead to easy shattering. The CRISPR/Cas9 system was used to edit qSH1 in this study. Three CRISPR/Cas9 vectors, which contain 5 targets for qSH1 gene or the key SNP site at its 5' regulatory region, were constructed and transformed to Oryza sativa ssp. indica HR1128 respectively. 31 T0 transgenic plants were obtained and confirmed by T-DNA specific PCR. The mutant efficiency for 3 vectors amounted to 85.71%, 76.92%, 63.64%. Through sequencing analysis of 10 T0 mutant plants, it was be found that they would mutate in a way of deletion, insertion or substitution, etc. As a consequence of variation in qSH1, it would either terminate translation in advance for a frameshift in CDS, or change its transcriptional level for an influenced binding ability for ABI3 transcription. In this study, the qSH1 gene or its regulatory region were targeted-edited successfully and obtained mutant plants with various types. These results will be used for analysing new functional or key regulated sites of qSH1, and rice suitable seed-shattering breeding.
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