关键词: 水稻, 基因编辑, OsIAA11, CRISPR/Cas9技术, osiaa11突变体

Abstract:

【Objective】Auxin signaling pathway regulated by OsIAA11 plays an important role in the plant growth and response to various environment stresses, impacting on the final grain yield during the late stage. To explore the effect of OsIAA11 mutation on the yield, the OsIAA11 mutant plant was obtained using CRISPR/Cas9 system, and the agronomic characters of mutant plants were investigated in the field. 【Method】According to the principle of CRISPR/Cas9 technology, two 20 bp targeted sequences were designed in the first exon and second exon of the OsIAA11 genome sequence. The gene shuffling of non-specific target sites was eliminated using the blast analysis. The oligonucleotides of two target sites were inserted into the pYLgRNA-U6a and pYLgRNA-U6b plasmid vector, and then were amplified twice by PCR technology to construct U6a-IAA11-T1 and U6b-IAA11-T2 expression-boxes. The recombinant pYLCRISPR/Cas9-IAA11-T12 vector was obtained by linking two expression-boxes to pYLCRISPR/Cas9-MT vector. The pYLCRISPR/Cas9-IAA11-T12 vector was transformed into the callus ofjaponica rice ZH11 through Agrobacterium-mediated method. The T₀ generation of transgenic plants was obtained by tissue culture, and the positive transgenic plants were selected through PCR method. The target sites of each T₂ generation from T₀ positive plants were determined via PCR and sequencing method, and finally the mutated genotypes were identified. Meanwhile, agronomic traits of the T₂ generation were investigated in the field. 【Result】The pYLCRISPR/Cas9-IAA11-T12 expression vector was successfully transformed into the callus of ZH11. 25 transgenic lines were obtained, and 20 transgenic lines were identified as the positive plants. After analysis of T ₂ transgenic plants via PCR and sequencing the target locus, 17 different homozygous mutations were identified in the two target sites of the OsIAA11 genomic sequence. Except for the single base insertion mutation of osiaa11-20-1, osiaa11-21-2, osiaa11-23, and osiaa11-25 in target 1 and osiaa11-22-2 in target 2, other genotypes mainly deleted small fragment base in target 1 and mutated a single base in target 2. Agronomic traits results showed that 17 osiaa11 mutants did not present a significant difference in plant height, panicle length, grain number per panicle, seed setting rate, 1000-grain weight, harvest index, and grain-straw ratio in comparison with the wild type. However, the osiaa11 displayed a significant decrease in effective spike rate, suggesting a more ineffective tiller phenotype. 【Conclusion】CRISPR/Cas9 technology successfully editedOsIAA11 and 17 different homozygous mutations genotypes were obtained. The mutant showed the reduced effective spike rate and increased ineffective tillers, indicating that OsIAA11 was related to the tiller bud appearance dominated by auxin.

Key words: rice, gene editing, OsIAA11, CRISPR/Cas9 technology, osiaa11 mutant