Knock-out Efficiency Analysis of Pi21 Gene in Rice using CRISPR/Cas9

【作者】 王芳权； FAN Fang-jun； LI Wen-qi； ZHU Jin-yan； WANG Jun； ZHONG Wei-gong； 杨杰；

【Author】 WANG Fang-quan;FAN Fang-jun;LI Wen-qi;ZHU Jin-yan;WANG Jun;ZHONG Wei-gong;YANG Jie;Institute of Food Crops,Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement/Jiangsu High Quality Rice R & D Center;Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops,Yangzhou University;

【机构】 Institute of Food Crops,Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement/Jiangsu High Quality Rice R & D Center； Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops,Yangzhou University；

【摘要】 Rice blast,bringing a great harm in rice yield and quality,is one of the main diseases in rice.Pi21 is a significant gene with broad-spectrum resistance to rice blast.The mutation of Pi21 gene improves the blast resistance observably.In this study,we constructed a CRISPR/Cas9 vector,which contains two targets（Target1 and Target2） of Pi21 gene,and transformed to rice variety Nanjing9108.Twenty-eight T₀ transgenic lines were obtained and confirmed by T-DNA specific PCR.By restriction digestion analysis,we detected a high mutant ratio,showing 78.57% mutant ratio in Target1 site,92.86% mutant ratio in Target2 site,and there is 78.57% ratio of lines containing both Target1 and Target2 mutant in transgenic lines.After sequencing analysis,the mutant types of targets,such as deletion of bases,insertion of bases,insertion behinds deletion of bases,and long fragment deletion of DNA,etc.,were found.Moreover,we found that most transgenic lines displayed frameshift mutation and caused lost function of Pi21,and some of transgenic lines shown amino acid deletion.In this way,a series of Pi21-knock-out rice lines are obtained,and further,can use for functional analysis of Pi21 and rice blast broad-spectrum resistant breeding.更多还原

【Abstract】 Rice blast,bringing a great harm in rice yield and quality,is one of the main diseases in rice.Pi21 is a significant gene with broad-spectrum resistance to rice blast.The mutation of Pi21 gene improves the blast resistance observably.In this study,we constructed a CRISPR/Cas9 vector,which contains two targets（Target1 and Target2） of Pi21 gene,and transformed to rice variety Nanjing9108.Twenty-eight T₀ transgenic lines were obtained and confirmed by T-DNA specific PCR.By restriction digestion analysis,we detected a high mutant ratio,showing 78.57% mutant ratio in Target1 site,92.86% mutant ratio in Target2 site,and there is 78.57% ratio of lines containing both Target1 and Target2 mutant in transgenic lines.After sequencing analysis,the mutant types of targets,such as deletion of bases,insertion of bases,insertion behinds deletion of bases,and long fragment deletion of DNA,etc.,were found.Moreover,we found that most transgenic lines displayed frameshift mutation and caused lost function of Pi21,and some of transgenic lines shown amino acid deletion.In this way,a series of Pi21-knock-out rice lines are obtained,and further,can use for functional analysis of Pi21 and rice blast broad-spectrum resistant breeding.更多还原