Cloning And Characterization Of A Rice Pleiotropic QTL,Ghd6

Heading date of rice is defined as the days from sowing to the emergence of the first panicle,which influences rice regional and seasonal adaptability and yield.In this study,a pleiotropy QTL,named Ghd6,was detected to regulate heading date,plant height and yield in BC₄F₂ developed by crossing Teqing（TQ）with ZhenShan97（ZS）and following 4continuous backcross with ZS.Ghd6 promoted heading date in long days and short days and was insensitivity to photoperiod.We cloned Ghd6 by map-based cloning and studied its molecular function.The main results were presented below:1.Hd1 was the candidate gene for Ghd6:In BC₄F₂,heading date（HD）,plant height（PH）and spikelets per panicle（SPP）exhibited co-segregation under natural long-day conditions（NLD）.The segregation ratio of HD and PH fitted a 3:1 expected for a single dominance gene,which was called Ghd6.NIL-TQ of Ghd6 was heading later with taller culm and more SPP.3000 plants of BC₄F₂ population of Ghd6 was investigated and 576extremely late heading and tall plants were selected for fine mapping.Ghd6 was mapped to the region delimited by Ghd6-12 and S56 marker,where Hd1 was located.Sequences analysis of Hd1 showed there was a 4 bp deletion in CCT domain of TQ allele resulting frame shift.So Hd1 was candidate gene for Ghd6.2.Functional verification of Hd1/Ghd6:ProHd1:Hd1:GFP was transformed into NIL-TQ.Co-segregation analysis in T₁ and T₂ generations showed positive transgenic plants headed earlier with shorter culm and less SPP as ZS.We also knocked out Hd1 of ZS and the mutant headed later consistent with NIL-TQ.So,Hd1 was the pleiotropy QTL Ghd6.3.Hd1 function conversion in LD:In long days（LD）and short days（SD）,NIL-ZS always headed earlier with shorted culm and less SPP,which meant Hd1 promoted heading in both LD and SD with photoperiod insensitivity.Real-time PCR analysis showed Hd1upregulated Ehd1,Hd3a and RFT1 in LD and SD.However,in previous study,Hd1 was recognized to delay heading in LD while promote heading in SD with strong photoperiod sensitivity.Thus,Hd1 possessed alternative functions in LD which termed as Hd1 function conversion.4.Hd1 function conversion was dependent on Ghd7:Under MH63（MH）background,Hd1 separately delayed and promote heading in LD and SD with repressing and promoting expressions of Ehd1,Hd3a and RFT1,separately.Investigation of the heading dates of NILs of Ghd7 and Hd1 in ZS and MH backgrounds and found Ghd7 determined Hd1 function conversion in LD.5.Ghd7.1 and Ghd8 triggered Hd1 function conversion:Analysis of HD for NILs of Ghd7,Ghd8 and Hd1,Ghd8 extremely enhanced Ghd7 to convert Hd1.The HD of NILs of Ghd7,Ghd7.1 and Hd1 were surveyed,it indicated that Ghd7.1 interacted with or enhanced Ghd7 to convert Hd1 function in MH or ZS backgrounds,respectively.Moreover,Ghd8 jointly with Ghd7.1 to convert Hd1 function.6.Photoperiod sensitivity（PS）of NILs of Ghd7,Ghd8,Ghd7.1 and Hd1:We measured PS of NILs of Ghd7,Ghd8,Ghd7.1 and Hd1 by comparing HDs in LD and SD.Ghd7Ghd8Hd1 and Ghd7Ghd8Ghd7.1Hd1 had strongest PS.Ghd7Hd1,Ghd8Hd1,Ghd7.1Hd1 and Ghd7Ghd7.1 showed strong PS.Ghd7,Ghd8 and Hd1 alone or non-functional combination of these 4 genes were not sensitive to photoperiod,while Ghd7.1alone had a weak PS under MH backgrounds but strong under ZS backgrounds.7.Physical interactions between Ghd7,Ghd8 and Hd1:Ghd7 physically interacted with Hd1 through the CCT domain of Ghd7 binding to Zn finger（32-111aa）and 112-337aa of Hd1.OsHAP5A,OsHAP5B,OsHAP5D,OsHAP5E,OsHAP5T and OsHAP5O were identified to interact with Ghd8 by screening a yeast two hybrid library.Further study showed Hd1 interacted with OsHAP5A,OsHAP5B,OsHAP5D and OsHAP5E separately,while Ghd7 interacted with OsHAP5A,OsHAP5D and OsHAP5E weakly respectively.8.Hd1 acted as transcription activator:Hd1 showed powerful transcriptional activation activity conferred by 112-337aa.However,mutantion in CCT of Hd1 disrupted this activity.Ghd7 repress transcriptional activation activity of Hd1 but could not convert it to repression activity.9.Ghd7 and Hd1 significantly enhanced Ghd8 binding to DNA in genome:Under ZS background,ProGhd8:Ghd8:GFP transgenic plants was constructed with different combinations of Ghd7 and Hd1,which heading date were consistent with NILs.Immunoprecipitation-Mass Spectrometry（IP-MS）of Ghd7-Ghd8:GFP-Hd1 was also used to identify proteins interacting with Ghd8,and another HAP5 protein,OsHAP5C,was detected.Then chromatin immunoprecipitation（ChIP）assays with sequencing（ChIP-Seq）was used for ProGhd8:Ghd8:GFP transgenic plants to uncover genomewide binding sites of Ghd8.When presence of Ghd7 or Hd1,Ghd8 bond to more sites,which indicated complexes of Hd1/Ghd8 or Ghd7/Ghd8 possesssing stronger DNA binding ability.