基于OsSPL10编辑创制沿黄光叶耐盐水稻新种质

Creation of new glabrous and salt-tolerant rice germplasm along the Yellow River by CRISPR-Cas9-mediated editing of OsSPL10

摘要:

OsSPL10基因能够调控水稻表皮毛发育，并与抗盐、耐干旱等性状相关，但该基因是否可用于基因编辑创制光叶耐盐水稻新种质尚不明确。本研究利用CRISPR/Cas9技术，以河南省沿黄稻区3个粳稻品种‘新丰2号’‘新科稻31’和‘新稻25’为材料，对OsSPL10基因进行编辑，经筛选鉴定获得6种spl10突变体。肉眼观察结合扫描电镜检测证实6种spl10突变体的叶片和颖壳均出现无毛性状，光叶标记基因OsHL6、OsGL6和OsWOX3B的表达量显著低于野生型；剑叶生理特征检测显示，6种spl10突变体的净光合速率、气孔导度和蒸腾速率均极显著高于对应的野生型水稻。苗期耐盐性鉴定结果表明，200 mmol/L NaCl处理7 d后，突变株的存活率远高于野生型。实时荧光定量PCR (quantitative real-time polymerase chain reaction,qRT-PCR)检测结果显示，与野生型相比，盐胁迫相关基因OsGASR1表达量显著降低，OsNHX2和OsIDS1的表达量显著升高。农艺学性状分析发现，与野生型相比，突变体的株高显著增加，产量等相关性状均无明显变化。本研究创制的6种spl10突变体不仅具有光叶、光颖壳特征，而且耐盐能力显著提高，为沿黄水稻定向育种提供了新的种质资源。

关键词:CRISPR/Cas9技术;沿黄水稻;OsSPL10;光叶;抗盐

Abstract:

The OsSPL10 gene has previously been reported to positively regulate trichome development and negatively regulate salt and drought stress tolerance in rice. However, it is not clear whether this gene can be used for gene editing to create new germplasm of glabrous leaf and salt-tolerant rice. In this study, we created six rice mutants by CRISPR/Cas9-mediated editing of OsSPL10 from ‘Xinfeng 2’, ‘Xinkedao 31’, and ‘Xindao 25’, the main rice cultivars along the Yellow River. Visual observation and scanning electron microscopy verified that the mutants lacked trichomes on the leaves and glumes, and the expression of glabrous marker genes OsHL6, OsGL6, and OsWOX3B in mutants was down-regulated compared with that in the wild type. The net photosynthetic rate, stomatal conductance, and transpiration rate of flag leaves in the mutants were significantly higher than those in the wild type. In addition, the survival rates of the mutants were much higher than that of the wild type after 7 days of treatment with 200 mmol/L NaCl. The results of quantitative real-time polymerase chain reaction (qRT-PCR) further verified that compared with the wild type, the mutants demonstrated down-regulated expression of the salt stress-related gene OsGASR1 and up-regulated expression of OsNHX2 and OsIDS1. Statistical analysis of agronomic traits showed that the mutants had increased plant height and no significant changes in yield-related traits compared with the wild type. The six spl10 mutants created in this study not only had glabrous leaves and glumes but also demonstrated enhanced tolerance to salt stress, serving as new germplasm resources for directional breeding of rice along the Yellow River.

Key words:CRISPR/Cas9;rice along the Yellow River;OsSPL10;glabrous leaf;salt tolerance