Genome Editing Of Rice OsMADs15 And OsMYC2 Genes By Using CRISPR/Cas9 System

Rice is not only a kind of major crops feeding a large population in the world,but also has been studied as a model monocot plant.In recent two decades,after the completion of rice genome sequencing and the development of biotechnologies,researchers have gained a series of progress in rice functional genomics study,which depends on large amount of rice mutants’isolation by all kind of methods.Rice floral organs are the reproductive organs of rice,and as a basic unit of yield,the development mechanism of spikelets have become one of the most important objects for researchers all over the world.In 2012,the newly developed CRISPR/Cas9system has become a widely used targeted genome editing tools,and it has obvious advantageous compared to that of traditional mutagenesis such as natural,physical,chemical,methods or T-DNA insertional method because of its features on high editing efficiency,low experiment costs and easy operation.To investigate the regulation mechanism of JA signaling gene OsMYC2 in rice reproductive development,we used the CRISPR/Cas9system to edit two targets of OsMYC2,one is in the N terminal and the other in C terminalj.We totally obtained 4 different heterozygous lines of9522^OsMYC2 mutants in the T₀ generation.But we couldn’t find any homozygous line in the offspring of heterozygous 9522^OsMYC2 lines,and further research demonstrated that the mutant pollens possessed abnormal vegetative nucleus,resulting in failure to transmit male gametes.On the other hand,in order to study the function of A-class MADS-box gene OsMADS15,we designed one target on the first exon of OsMADS15 and obtained 3 biallelic lines of 9522^Osmads15 mutants in the T₀ generation by using CRISPR/Cas9 system.In all of the 3 9522^Osmads15 mutants,all the allele-1 had frame-shift mutations in the OsMADS15 open reading frame,resulted in premature translation termination,and all the biallele-2 also had different affects in the translation of OsMADS15.And these mutational plants grew longer and leafy-like empty glumes,showing abnormal in lemma and palea development,and their inner whorl floral organs changed into seedling plants.In our research,we have successfully created heterozygous 9522^OsMYC2 mutants and preliminarily studied the function of OsMYC2 in the transmission of male gametophytes which provided a basis of further research of OsMYC2 in rice reproductive development;and9522^Osmads15 mutants also provided new materials for further genetic and regulatory network research on OsMADS15 function in rice spikelet development.