Study Of Rice (Oryza Sativa L.) Heading Date Gene Ehd1 And Its Interacted Protein OSK3

Heading date(Oryza sativa L.)is an important agronomic trait in rice.Appropriate heading date is crucial to maintain high and stable yield of rice.Hd1 and Ehd1 are the key heading date genes.Ehd1 have no ortholog in the Arabidopsis genome.Ehd1 can upregulate downstream florigen genes RFT1 and Hd3 a to promote rice heading.Ehd1 encodes a B-type response regulator,with Receiver domain at the N terminal,and the GARP DNA-binding domain at the C terminal.In previous work,OSK3 was idtified as Ehd1 interaction protein.OSK3 is a member of sucrose non-fermenting-1-related protein kinases family.OSK3 located in rice chromosome 3,and its homologous gene OSK4 located on chromosome 8,which can regulated heading date.This study consists of three parts.The first part: functional study of Ehd1.Pull down assay verified that Ehd1 can form homodimer.Material that transgenic co-inhibition of silence of Ehd1 delays heading.Material that overexpress Ehd1 fused with FLAG(OE-Ehd1-FLAG-DJ)showed early heading,while material that silence of Ehd1 delays heading..This proved Ehd1 promote rice heading.42 new downstream genes were determined by RNA-sequencing data analysis.The second part: modified REPSA(Restriction Endonuclease Protection Selection and Amplification)method and Chromatin Immunoprecipitation followed by Sequencing was used to identify the DNA elements that bind preferentially by Ehd1.The third part: functional study of Ehd1 interactor OSK3 and OSK4.Pull down assay verified that OSK3 interacts with Ehd1.The CRISPR/Cas9 knocked out plants of OSK3 or its homologous gene OSK4 were obtained and its heading date were earlier than NIL-nhld,indicating that OSK3 is an inhibitor of flowering.This study lay foundation to elucidate the molecular mechanism of Ehd1.