Study The Functions Of Tiller Angle Genes In Rice

Tillerings,specialized branches of monocotyledon,which is one of the most important factors in ideal rice plants foundation and crop genetic improvement.The studies of rice tillering mainly exhibited in two aspects of the tiller angle and tiller number.Mi RNAs as a kind of small RNA which have an important regulation function to regulate plant growth and development by mainly regulating the important transcription factors in plants,providing new genetic resources for genetic improvement of rice.There are two objectives of this research:（1）Using rice over-expression of MIR160b/MIR167a transgenic plants to verify the targets of mi R160b/mi R167a by experimental methods,further illuminating the molecular mechanism of rice tiller Angle regulated by mi R160b/mi R167a;（2）Screening three tiller-related mutants from our T-DNA insertion mutant library to locate tiller-related genes by generating F₂ mapping population and taking map-based cloning approach,further exploring the molecule mechanism of rice tillering.The main results are as follows:1.The tiller angle increased 1.7 times,and the effective tiller numbers decreased35.2%and seed setting rate decreased 52.1%in MIR160b-overexpressing plants,compared with negative control.The tiller angle increased 1.5 times,the height decreased39.6%,and the effective tiller numbers increased 2 times,the seed setting rates decreased73.6%,but was not caused by pollen sterility.2.QRT-PCR results showed MIR160b-overexpressing plants inhibited the expression of OsARF13,OsARF18 and OsARF22 genes,and MIR167a-overexpressing plants inhibited the expression of OsARF12,OsARF17 and OsARF25 genes,and the expression of OsARF genes was inversely related with mi R160b and mi R167a.3.The RLM-5’RACE analysis showed that the cleavage sites of OsARF13,OsARF18 and OsARF22 genes by mi R160b-RISC and OsARF12,OsARF17 and OsARF25 genes by mi R167a-RISC were respectively located in 10^th and 11^th of mature mi R160b and mature mi R167a,indicating that the six OsARF genes of rice ARF family are respectively targets of mi R160b and mi R167a.When over-expression the mi RNA-resistant OsARF genes,although the expression levels of OsARF genes were obviously increased,there were not appeared expected mutant phenotype.4.We constructed Ubi::MIM160b and Ubi::MIM167a vectors of mature mi R160b and mi R167a by target mimicry technology.Phenotypic observation found that the Ubi::MIM160b and Ubi::MIM167a vectors restored the mutant phenotype of MIR160b-overexpressing plants and MIR167a-overexpressing plants,respectively.QRT-PCR results showed that the expression level of OsARF target genes of mi R160b and mi R167a increased respectively in MIM160b and MIM167a over-expressing plants.5.To verify the functions of OsARF genes in regulating rice tiller angle,we screened four OsARF mutants,including osarf12,osaf13,osarf17 and osarf18 mutants.Expression analysis showed that the expression level of OsARF12,OsARF13,OsARF17 and OsARF18 decreased in their mutants,compared with the wild-type,but these single mutants don’t appear mutant phenotype in tiller angle.The phenotype of osarf12osarf17double mutant was similar to MIR167a-overexpressing plants,and the phenotype of OsARF12-RNAi transgenic plants between osarf12 and osarf12osarf17.These results demonstrated that OsARF genes may functions redundancy in regulating rice tiller angle.6.QRT-PCR tested the expression level of nine genes,including tiller angle-related genes,LAZY1,LPA1,TAC1 and auxin-related genes,GH3.1,GH3.4,GH3.8,PIN1,IAA4and IAA21 in mi RNAs over-expressing plants.Compared with negative control,MIR160b over-expressing plants repressed the expression level of GH3.4,IAA4 and IAA21,and MIR167a over-expressing plants repressed the expression level of GH3.4 and GH3.8 and increased the expression level of LAZY1.Preliminary confirmed these genes may be involved in the mi RNA-mediated OsARF repression in regulating the rice tiller Angle.7.We screened three tiller-related mutants from T-DNA insertion mutation library,including two increased tiller angle mutants（ul88 and pm72）and one reduced tiller number mutant（az01）,and we used map-based cloning approach to locate the three genes and generated the F₂ mapping populations by hybrids between the three homozygous mutant and indica rice varity Zhenshan97.The segregation ratio of wild type and mutant obey 3:1Mendel ratio,indicated that these three mutants traits were controlled by a single recessive gene,respectively.Genetic mapping results indicated that AZ01 gene was located between molecular markers ID53 and ID57 of chromosome 10,with physical distance about 355kb through massive planting F₂ mapping populations and developing newly molecular markers.UL88 and PM72 genes have the same linked molecular makers which were very close to markers of LAZY1.