水稻LOC_Os10g05490位点冷胁迫条件下表达分析及CRISPR/Cas9定向编辑

CRISPR/Cas9 editing and expression analysis of LOC_Os10g05490 in rice under cold stress

为探明LOC_Os10g05490位点冷胁迫条件下的表达特性和耐冷性功能,以耐冷东乡野生稻和冷敏感水稻93-11为试验材料进行了半定量分析,结果表明,LOC_Os10g05490在冷敏感水稻93-11冷处理前后表达丰度相当,而在耐冷东乡野生稻中冷处理后表达丰度下调,这一结果与前期转录组测序(RNA-seq)结果相符,进一步验证了该位点可能与水稻耐冷相关,随后利用CRISPR/Cas9基因组编辑技术对LOC_Os10g05490编码区于同样具有较强耐冷性的粳稻品种台北309中实施定向基因敲除,并成功获得了26株转基因阳性植株。

To explore the expression and cold tolerance function of LOC_Os10g05490 in rice under cold stress,semiquantitative analysis was performed for Dongxiang wild rice and 93-11. The results showed that expression level of LOC_Os10g05490 in 93-11 was the same before and after cold stress,but it was down regulated in Dongxiang wild rice after cold stress. This was consistent with RNA-seq data and further study indicated that this gene might be associated with cold tolerance in Dongxiang wild rice. Plant expression vectors with CRISPR / Cas9 genome editing technique were subsequently constructed to target the LOC_Os10g05490 locus,and were transformed into the japonica rice variety TP309. A total of 26 transgenic positive plants were obtained. This study may lay the foundation for subsequent screening of mutant plants and identification of genes associated with cold tolerance in rice.