利用CRISPR/Cas9创建osarf7突变体及其农艺性状调查

Construction of osarf7 Mutants in Rice Based on CRISPR/Cas9 Technology and Investigation on Their Agronomic Traits

【目的】探究OsARF7基因在水稻生长发育中的生物学功能及其对水稻农艺性状的影响。【方法】利用CRISPR/Cas9技术,在粳稻中花11背景下对OsARF7进行定向编辑,获得OsARF7基因突变植株,并考查其农艺性状。【结果】获得22株T0代转基因株系,经PCR扩增潮霉素基因鉴定出20株阳性植株。提取T2代转基因植株的基因组DNA,通过对OsARF7的2个编辑位点区域DNA片段进行PCR扩增及测序分析,筛选到15种突变类型的纯合基因型植株。基因表达分析显示,osarf7突变体的OsARF7和生长素转运关键基因OsPIN1b、Os PIN2和OsLAX2的表达量均显著低于野生型。田间调查发现,与野生型相比,osarf7突变体的分蘖成穗率显著降低,其中,分蘖数比野生型增多的osarf7突变体,有效穗数基本保持不变,而分蘖数与野生型相近的osarf7突变体,有效穗数少于野生型,最终表现为无效分蘖明显增多,分蘖成穗率降低。【结论】OsARF7基因与生长素调控水稻分蘖发生相关,并影响分蘖芽的生长发育及成穗;OsARF7基因突变会导致迟发分蘖芽不能正常发育成穗;粳稻OsARF7突变体的创建对揭示OsARF7基因生理功能和探索生长素促控分蘖发生与成穗机理等具有积极意义。

【Objective】The purpose is to offer a deep insight into the biological function of Os ARF7 and its effect on agronomic traits during the growth and development stage of rice.【Method】CRISPR/Cas9 technology was employed to edit the gene sequence of OsARF7 and to obtain the mutants of japonica variety Zhonghua 11.The agronomic traits of the mutants were investigated in a field experiment.【Result】Twenty-two T0 transgenic lines were obtained through Agrobacterium-mediated infection of japonica rice Zhonghua 11 callus,and twenty positive transgenic lines were distinguished by screening the gene encoding hygromycin.For T2 transgenic lines,the regions of genomic DNA including two target sites were detected and identified by PCR amplifying and sequencing,and fifteen homozygous mutations were confirmed.Gene expression results displayed that the expression levels of OsARF7,Os PIN1b,Os PIN2,and OsLAX2 in the osarf7 mutants were significantly lower than those in the wild type.In comparison with the wild-type plants,osarf7 mutants showed a significant decrease in the productive panicle percentage.Among them,the osarf7mutants with more tillers than the wild type showed a similar effective panicle number to the wild type,and the osarf7mutants with the same tiller number as the wild type presented a decrease in the effective panicle number per plant in paddy field conditions,resulting in increased non-productive tillers and decreased effective panicle percentage.【Conclusion】OsARF7 was involved in the emergence and development of tiller bud dominated by auxin,and the successful construction of osarf7 mutants plays a positive role in revealing the biological function of OsARF7 and the mechanism of tiller formation and development dominated by auxin.