Functional Identification Of Rice Polycomb Group Proteins OsFIE1 And OsFIE2 And Screening Of Interacting Proteins

Rice is an important food crop,and heading stage and endosperm development are important traits that affect rice production.The key genes in heading stage,identification of key genes in rice endosperm formation,and analysis of the regulation mechanism are the keys to accurately control rice production.Polycomb protein family(Pc G)plays an important role in inhibiting gene expression by modifying chromatin.Pc G proteins typically form multiple protein complexes,such as multicomb inhibitory complex 1(PRC1)and polymeric inhibitory complex 2(PRC2),which inhibit gene expression through lysine 27 trimethylation on histone3.It has been reported that PRC2 complex has been identified as essential for normal plant development in Arabidopsis thaliana,and is involved in the regulation of flowering,differentiation,endosperm formation and other key developmental processes in Arabidopsis thaliana.However,its homologous functions and regulatory mechanisms in rice remain to be explored.Os FIE1 and Os FIE2,encoding genes of Esc core components in rice,are part of FIS-PRC2 in rice PRC2 complex,and have been previously reported to be involved in regulating endosperm development and seed size.In this study,CRISPR/Cas9 gene editing mutations of Os FIE1 and Os FIE2 were created by reverse genetics and studied by phenotypic observation,quantitative PCR,yeast two-hybrid and other methods.The results are as follows:1.The CDS sequences of Os FIE1 and Os FIE2 genes were obtained,primers were designed,and the CRISPR/Cas9 gene editing vector was constructed.The rice variety SN9816 was transformed by agrobacterium infection method,and the homozygous mutant lines of T0 generation were obtained.The mutant showed earlier heading stage,shorter plant height and lower yield.2.The yield-related traits of wild-type SN9816 and osfie1 mutants were measured.The results showed that the seed setting rate of osfie1 was significantly lower than that of wild type,and there were some differences in grain length and grain width between osfie1 and wild type.The panicle length and 1000-grain weight of osfie1 were lower than those of wild type,and the yield of osfie1 mutant was lower than that of wild type.3.We selected osfie1 and SN9816,which were sown for 45 days in short sunshine and55 days in long sunshine,to further explore the pathway of osfie1 regulating rice heading stage by using q RT-PCR technology.The expression of Ghd7 in osfie1 was down-regulated under short-day conditions,but up-regulated in osfie1 under long-day conditions.The expression of Ehd1 was up-regulated in long and short sunshine.We speculated that the target gene affecting osfie1 regulation might be upstream of Ghd7 and Ehd1,which needs further exploration4.A total of 130 positive clones of Os FIE1 gene were screened and 60 candidate interaction proteins were identified by sequencing.A total of 162 positive clones of Os FIE2 gene were screened and 55 candidate interaction proteins were identified by sequencing.The interaction protein Os MT2 b was screened for Os FIE1 and Os FIE2.It provides a new direction for further exploring the mechanism of Os FIE1 and Os FIE2.In conclusion,Os FIE1 and Os FIE2 are most likely to interact with Os MT2 b to affect hormone levels in plants and regulate the growth and development of rice.This result lays a good foundation for further studies on gene functions.