MutMap Cloning And Function Analysis Of A Novel Heading Date Gene ZM17 In Rice

Rice is an important food crop,and the heading date of rice plays an important role in ensuring stable rice production and high yield.Although the molecular mechanism of flowering regulation in the long-day plant Arabidopsis thaliana has been basically verified,the mechanism of flowering in short-day plant rice is still not very clear,and it needs further researches.In this study,large-scale mutagenesis of late-heading mutant lvp1 of Japonica rice variety Nipponbare is induced by chemical mutagen methyl ethylsulfonate(EMS),after screening and identification,a heading date mutant zm17 is obtained from the mutant library.Genetic analysis shows that the mutant zm17 is controlled by a pair of recessive nuclear gene.The mutant gene ZM17 in zm17 is cloned using the Mut Map method,and a ZM17 single gene mutant z17 is constructed.Then the heading date and major agronomic traits of z17 are investigated,and the differential expression analysis and transcription of the genes related to heading date are investigated.Transcriptome sequencing experiments are performed and the experimental results are obtained following:1?Heading date mutant zm17 identified from the mutant library,is used to clone ZM17 gene by Mut Map.And the research shows that gene ZM17 located on eighth chromosome in rice,and ZM17 is considered as a new gene of rice heading date and encoding a transcription with MYB domain.Furthermore,through experiments of genetic complementation and RNAi and CRISPR/Cas9,we verified the validity of gene ZM17.2?Expression pattern analysis showes that in the leaves,the expression level of ZM17 gene is higher,relatively higher at the vegetative stage,but the expression level of ZM17 gene significantly decreases after it enters the reproductive stage,suggesting that ZM17 might play an important role in rice flower conversion.3?The KEGG analysis of transcriptome sequencing data showes that ZM17 significantly affectes the rice circadian clock pathway.To further verify the rhythm expression of the clock genes in z17,it is found that ZM17 gene expression is rhythmic under different photoperiods,and the rhythmic expressions of the circadian clock genes Os LHY,Os PRR1,Os PRR37,Os PRR59,Os PRR73,Os PRR95,Os ELF3,Os GI,and Os CAB1 R are significantly affected under continuous light conditions,suggesting that ZM17 might be an important part of circadian clock pathway in rice.4?Given 10 h and 14 h light conditions,compared with the control Nipponbare,the heading date of z17 is earlier at 10 h light conditions and delayed at 14 h light conditions.Further analysis of the expression of related gene rhythms,in 10 h light conditions,the expression levels of Os GI,Hd1,Ehd1,Hd3 a and RFT1 increases in mutant z17.Within 14 h light conditions,Os GI,Hd1 increases,but Ehd1,Hd3 a,and RFT1 decreases,suggesting that ZM17 might regulate rice heading through Os GI-Hd1-Ehd1-Hd3a/RFT1 pathway.5?Under 10 h?11 h?12 h?13 h and 14 h light conditions,the investigation of the heading date of mutant z17 indicates that,the heading date of z17 advances at 10 h and 11 h light conditions,while the heading date delays at 12 h,13 h,and 14 h light conditions.The results of heading-related gene expression in light for 2 hours in z17 shows that the expression levels of Ehd1,Hd3 a and RFT1 in the mutant z17 are significantly increased compared to Nipponbare when the light is 11 h,while the duration of light is 12 h,13 h and 14 h,the expression levels of Ehd1,Hd3 a,and RFT1 in mutant z17 are significantly decreased compared with Nipponbare,suggesting that there may be a critical day lengh of ZM17 role reversal between 11-12 h light conditions.