Map-based Cloning Of Key Gene SHORT PALEA 1 Regulating Floral Organ Palea Development In Rice(Oryza Sativa L.)

Rice is the staple food of more than 3 billion people in the world and a model crop for studying monocots.As a major food crop,the normal development of rice flower organs is critical to the formation of grain yield and quality.As a monocotyledonous plant,rice has a unique inflorescence structure compared to a dicotyledonous plant.Inflorescence of rice belongs to panicle,which is mainly composed of cob,primary branch,secondary branch and spikelet.Each spikelet consists of one small flower,one pair of empty glumes and one pair of rudimentary glumes.A normal flower of rice consists of one lemma,one palea,two lodicules,six stamens and one pistil.At present,people have already understood and mastered some of the regulatory mechanisms of plant flower development,such as the ABC model.The study on the floral organs of monocots is slightly behind that of dicots,and there are significant differences in the development and morphogenesis of floral organs between dicots and monocots.People have had a preliminary understanding of the development of rice flower organs,and also established an ABCDE model which is suitable for rice flower organ development.However,a thorough understanding of the mechanism still requires a lot of in-depth researches,especially in the origin of rice lemma and palea is controversial.In this study,the materials we used include japonica rice variety 9311,short palea 1(spl)and the genetically isolated population.spl is the mutant of 9311 treated by Co60(30KR)and its palea is not normal.The cloning of the mutated gene was carried out.The main findings obtained are as follows.1.Under the same environment and the same period of sowing conditions,comparing with wild type 9311,the mutant spl showed a slightly earlier heading date and reached a significant difference;At the same time,there were extremely significant differences between wild type 9311 and the mutant spl in the plant height,the number of primary branches,the number of spikelets of per panicle and the seed setting rate.In general,the mutant sp1 has a shorter palea,a shorter plant height,and a smaller number of primary branches and a lower seed setting rate.The short palea rate of the mutant spl was 95.57%,and the palea of sp1 could not be hooked with the lemma.The second round of the structure-lodiclue,the third round of the structure-stamen and the fourth round structure-pistil were exposured to the air.2.The panicles of wild type 9311 and mutant sp1 plants from Sp4 to the 4th stage of young panicle differentiation were excised and observed,and compared under the scanning electron microscope.The results showed that the development of sp1 palea were slower than 9311 palea in the Sp4-Sp7.In the 4th stage of young panicle differentiation,the development of the spikelet was completed,and the sp1 palea was short and could not form a complete closed structure with lemma.The results showed that the characteristics of mutant spl palea showed abnormal changes in the early stage of spikelet development.3.The paleas of F1 of 9311/sp1 and spl/9311 performed normally,which showed that short palea was a recessive trait.Statistical analysis of phenotypic separation of two F2 was carried out.After chi-square detection,the 3:1 isolation genetic law of a pair of alleles was observed(χ2(1,0.05)<3.84),indicating that the sp1 phenotype is controlled by the recessive homozygous gene at the single site.4.The target gene was mapped to the 127.7 kb interval on the long arm of chromosome 1 using 1170 recessive extremities isolated from the sp1/Nippon F2 population.There are 24 open reading frames in the area.By sequencing 9311 and spl,it was found that only ORF14 had a difference site between the mutant and the wild type,and the remaining 23 ORFs did not differ between the two.spl has a single base G deletion at position 447 of ORF14,resulting in frameshift and early termination of translation.The test of transgenic complementation confirmed that the gene SP1 is the target gene.5.Real Time-PCR and GUS tissue staining showed that SP1 gene was only expressed in the internodes,young panicles(0-5cm)and the young spikelets and stamens,while in other tissues including roots,stems and leaves,the SP1 did not expressed.The result showed that SP1 gene is specifically expressed in rice tissues.Subcellular localization result showed that SP1 is localized in the nucleus.