Map-based Cloning And Functional Analysis Of The Strigolactones-related Genes In Rice (Orzya Sativa L.)

Strigolactones (SLs) is a type of novel plant hormones which inhibit plant branching. The SLsbiosynthesis and signal transduction pathway is still unclear, especially the signaling pathway poorlyunderstood. In this study, we used several rice high-tillering dwarf mutants as the experimentalmaterials, including the SLs biosynthesis pathway mutants Cong-Ai No.2(cl2), S1-40, gsor23andsignaling pathway mutants gsor300079and gsor300097. Using the map-based cloning approach, weidentified the mutated genes in the mutants mentioned above. Through yeast two-hybrid screening, weidentified interacting proteins with D3and D14, the two known signaling factors of SLs so far.Through EMS mutagenesis of gsor300079, we obtained7supressors of the d14mutant. The mainresults are as follows:1. Genetic analysis showed that the high-tillering dwarf phenotype of the standard variety of riceDUS test Cong-Ai No.2is controlled by a single recessive nucleus gene, which was mapped betweentwo markers C4-CL5and C4-CL4on the long arm of chromosome4. Sequencing analysis of theOsCCD7allele in cl2revealed that the1796thbase was substituted from C to T, changing the599thamino acid from proline to leucine. Exogenous application of GR24, a synthetic analogue of SLs,restored the tillering phenotype of cl2. Real-time RT-PCR showed OsCCD7is expressed in all ricetissues examined, with the highest expression in stems. Expression of the SLs biosynthetic gene D10and D17were upregulated, while the D14gene probly involved in SLs signaling was downregulated inthe cl2mutant.2. Sequencing analysis of the OsCCD7/D17allele in S1-40revealed that the3' splicing site of the3ndintron of D17was substituted from AG to AA, which caused two kinds of incorrect alternativesplicing forms of pre-mRNA in S1-40. Therefore, S1-40is a novel allellic mutant of D17。3. Genetic analysis of the gsor23mutant showed that high-tillering dwarf phenotype was controledby one single recessive gene, which was mapped within a physical distance of386kb between twomarkers C1-WT2and C1-WT4on the long arm of chromosome1. Sequencing analysis of the D10allele in gsor23revealed that the base C at the404thposition in the coding region was deleted, whichwould cause frameshift mutation after the134thamino acids. So gsor23is a novel allellic mutant of D10.After treatment with GR24, the high-tillering phenotype of gsor23was restored to normal. Real-timeRT-PCR analysis showed that D10expression was high in root, but low in leaves. Expression of theSLs biosynthetic gene D10was upregulated, while genes likely involved in the SLs signal transductionsuch as D3and D14was downregulated in the gsor23mutant.4. Maping and sequencing analysis of the D3allele in gsor300097revealed that the1583thbase inD3coding region was substituted from G to A, changing the528thamino acid from tryptophan to apremature stop codon TAG. So gsor300097is a novel allellic mutant of D3. After treatment with GR24,the synthetic anolog of SLs, the high-tillering phenotype of gsor300097was not inhibited. Real-timePCR and histochemical GUS staining showed that the D3gene is widely expressed in the rice tissues, with the highest expression in panicle and relatively low in internode. Subcellular localization assayshowed that D3was located in the nuclear.5. Yeast two-hybrid experiments showed that D3F-box region (N-terminal1~64aa) interactedwith the rice OSK1,5,20proteins, which showed high similarity with the Arabidopsis ASK1. The LRRdomain (71~670aa) of D3did not interact with the OSK1, OSK5, OSK20proteins; We also found thatD14, a protein likely involved in the SLs signaling pathway, did not interact with the D3, D3F-box, D3LRR domain in yeast. We had also obtained transgenic rice plants with restored tillering phenotype aftertranspformation of the rice Actin1promoter drived D3gene with3×Flag-3×HA dual tags intogsor300097. In the future we can use these transgenic plants to identify new interaction proteins withD3in plants.6. Subcellular localization experiment showed that D14was located in the nuclear. Yeasttwo-hybrid screening found the NAC1/WRKY/MYB family transcription factors and RNA-bindingprotein (RB) interact with the D14protein. BiFC experiments showed that NAC1and D14co-localizated in the nuclear. The NAC1RNAi transgenic rice plants showed the reduced tillersphenotype. We had also obtained transgenic rice plants with restored tillering phenotype aftertranspformation of the rice Actin1promoter drived D14gene with3×Flag-3×HA dual tags intogsor300079. The MBP-D14fusion protein was also expressed and purified from E.coli.7. Through EMS mutagenesis of gsor300079, we obtained7supressor mutants of d14. Five of themstill retained the original transposon nDART inserted in D14, while the other two only have thefootprint left after nDART transposition out of D14. These suppressor mutants would be used to identifynew components in SLs signaling pathway downstream of D14.