Functional Analysis Of Rice (Orzya Sativa L.) Tillering Regulator Gene OsIAA16 And Development Of Genetic Modifiers Of The D14 Mutant

Two subjects were studied in the thesis:1,Cloning and mechanism study on the tiller controlling gene OsIAA16 in rice;2,Characterization of a weak d14 allelic mutant sd33 and development of d14genetic modifers.Tillering is very important agronomic trait in rice and plays important roles in yield composition.Strigolactone?SL?is a novel plant hormone which regulates plant tillering.Up to now,the SL biosynthesis and signaling pathway is not fully understood,meanwhile the mechanism underlying the interaction between SL and auxin is still inconclusive.OsIAA16 and D14 were map-based cloned respectively from two high-tillering dwarf mutants hd103 and sd33.We carried out functional study of the OsIAA16 gene and developed genetic modifiers of the d14 mutant.The results were as follows:1.Compared with the wild type,tiller number was significantly increased and plant height significantly decreased in the hd103 mutant.Meanwhile,the root length was reduced and adventitious root number increased.The hd103 mutant exhibits typical phenotype of strigolactone-related mutants.Through map-based cloning,we found that the 185^th nucleotide in the coding region of OsIAA16?LOC_Os05g09480?was mutated from C to T,changing the corresponding amino acid from proline?P?to leucine?L?.The mutated amino acid was located at the conserved GWPPV domain,which is responsible for the degradation of the Aux/IAA protein.The mutated IAA16 protein was predicted to be more stable,which was consistent with the semi-dominant phenotype of the hd103 mutant.The RNAi construct of OsIAA16 was transformed into hd103.The transgenic plants showed increased height and reduced tiller number.The result indicated the mutation in OsIAA16 caused the dwarf and high-tillering phenotype of hd103.2.The results of qRT-PCR and histological staining of the transgenic rice plants harboring the OsIAA16 promoter-driven GUS reporter gene showed that OsIAA16 was expressed widely in various tissues,with the highest level in root and young leaf,then in young panicles,flower,tillering bud and young sheath.Transient expression of the GFP::OsIAA16 fusion gene in the rice protoplast showed OsIAA16 was located in the nucleus.3.The content of epi-5DS?the precursor of SLs?was increased in hd103.Exogenous application of GR24?a synthetic analog of SLs?could partially restore the tillering phenotype of hd103.Compared with d27?a mutant defective in SLs biosysthetic pathway?,hd103 showed reduced sensitivity to GR24.These results indicated that OsIAA16 was related to SLs signaling pathway.Expression level of the SL-related genes D10,D17,D27,D3,D14 and TB1 were down-regulated significantly in hd103,and D3 and D14 were most down-regulated.Yeast two-hybrid assay showed OsIAA16 did not interact with SL signal transducers D3?D14?D53?TB1 and MADS57.Based on these results,we speculated that OsIAA16 regulated the expression of these SLs-ralated genes,especially D3 and D14.4.The IAA level in hd103 was higher than that in the WT.The callus induction rate of hd103 was much lower than WT when the seeds was treated with 2 mg/L 2,4-D,and the callus of hd103 was smaller and irregular.Compared with the WT,the responding ability of hd103 to low-concentration of various kinds of auxin was decreased obviously.The expressed level of the auxin-responsive marker gene IAA20 was decreased obviously in hd103.Yeast two-hybrid assay showed that OsIAA16 could interact with the auxin receptors AFB2,and auxin response factors ARF6 and ARF17.These results indicated OsIAA16 was related to the auxin pathway.5.The model of auxin signaling pathway showed that Aux/IAAs interact with and inhibit the transcriptional activity of ARFs in the absence of auxin.In the presence of auxin,Aux/IAA was degraded and ARFs was released to activate the expression of auxin-responsive genes.ARFs could bind to the auxin-responsive element AuxRE?TGTCTC?in the promoter region of auxin-responsive gene and activate its transcription.Bioinformatic analysis showed that 1 or 2 AuxRE elements were present in the promoter region of D10,D17,D27,D3 and D14.We speculate that OsIAA16 could regulate rice tillering through regulating the expression levels of SLs-related genes.6.The sd33 mutant showed weak dwarf and high-tillering phenotype.Through map-based cloning,we found that the 887^th nucleotide in the coding region of D14?LOC_Os03g10620?was mutated from G to A,which changed the 296^th amino acid from glycine?G?to aspartic acid?D?.The mutant amino acid was located on the C-terminal loop of D14,which was not very important for the structure of D14protein.7.In order to obtain genetic modifiers of the d14 mutants,we did EMS mutagenesis of the SD33mutant.In the M₂ generation,one enhancer mutant sd33E1 and 3 supressor mutants sd33S1,sd33S2,sd33S3 were obtained.Sequence analysis revealed a second intragenic mutation in the D14 gene in sd33E1 while no intragenic mutation was found within sd33S1,sd33S2 and sd33S3.These three supressor mutants could provide good materials for cloning novel genes related to D14.