| Rice(Oryza sativa L.)grain yieldsare mainly determined by the number of panicles,the grain number of per panicle,and the weight of a thousand seeds,and the number of rice panicles directly depends on tillering.Rice tillering is one of the most important agronomic trait that decides yeilds.In recent years,studies have shown that the number of tillers in rice is regulated by a class of small RNA molecule callded miR156.Further revealing the molecular mechanism of rice tillering regulated by miR156 can provide important theoretical basis for breeding new rice cultivar of high-yielding.In our previous study,we ide ntified a T-DNA inserted rice mutant dmt2(dwarf and more tillers 2)in zhonghua11(ZH11)background,which showed the similar phenotype ofdwarf and high tillers of the mutant of a series of D gene mutants that found in recent years,all of which were deficient in strigolactones(SLs)pathway.We found that the mutant dmt2 are hypersensitive to SLs by treating the mutant dmt2 with GR24,an artificial synthetic analogue of strigolactones.In this study,sequence analysis confirmed that T-DNA was inserted at about 3.2 Kb upstream of the gene DMT2,whose final coding product is the mature mi R156 d.The function of the gene DMT2 were verified by research on its overexpression plants.The relationship between the mutant dmt2 and SLs are explored,and the main results were as follows:1.Molecular cloning of DMT2 was carried out and sequence analysis showed that T-DNA was inserted into the non-coding region of chromosome 2,3225 bp upstream of transcription initiation site of DMT2(Os02g0180800),which is a non-protein-coding gene and the final coding product is the mature mi R156 d.2.The initial transcription product of mi R156 d gene were detected in dmt2 by real-time quantitative reverse transcription PCR(q RT-PCR),which showed that the expression of pri-miR156 d increased significantly.3.The expression analysis of mi R156-targeted SQUAMOSA Promoter-binding Like(SPL)genes of dmt2 was carried out.The results showed that SPL2,SPL3,SPL4 and SPL11 showed significant down-regulation in dmt2.Therefore,we infer that SPL2,SPL3,SPL4 and SPL11 are candidate target genes of mi R156 d.4.The expression of several key genes in SLs synthesis and signal transduction pathway indmt2 was detected.The results showed that D10 gene in SLs synthesis pathway was significantly reduced,mi R156d/SPL may influence SLs pathway through D10 in SLs synthesis pathway.5.pre-miR156 d OE and pri-miR156 d OE were constructed,and the transgenic plants obtainedwere identified by PCR detection.And the phenotype analysis showed that the pri-miR156 d OE transgenic plantsobviously showed the phenotype of dwarfing and high tillering,whereasthe phenotype changes of the pre-miR156 d O E transgenic plants were relatively temperate.6.The expression of the candidate miR156 dtargeted genes(SPL2,SPL3,SPL4 and SPL11)and D10 in SLs synthesis pathway were analyzed in the pri-miR156 d OE plants,and the results showed that all these genes decreased obviously.In summary,this study provided experimental evidence and theoretical basis for further revealing the regulation mechanism of miR156 d gene in rice tillering,and shed new lights on the possible regulatory mechanism that mi R156d/SPLcorelated with the SLs pathway in regulating rice tillering,which may provide us with an idea for breeding new rice cultivarof high productivity. |
