| Rice（Oryza sativa）is one of the main food crops for the world population.Heading date,one of the most important agronomic traits,determining the rice adaptability in different regions,and affecting rice yield and quality.Moreover,rice is a model plant for monocotyledonous crops.Researchs on the cloning and function of rice heading date genes have considerable theoretical and practical significance for revealing the molecular regulation mechanism of rice flowering pathway and improving the yield of agricultural crops.In this study,a late heading date mutant,designated as late heading date 3（lhd3）,was screened from Zhonghua 11（Oryza sativa L.subsp.japonica）mutant library inducd by 60Co-γirradiation.The phenotypic of lhd3 was identified under natural LD condition.The genetic analysis,map-based cloning and high throughput resequencing analysis were performed to isolated LHD3 gene.The subcellular localization of LHD3and expression pattern of LHD3 gene were perfomed.And LHD3 gene involved photoperiodic flowering pathway were also investigated.The main results and conclusions are as follows:1.The heading date of lhd3 mutant was delayed about one week compared with the WT under both natural SD and LD conditions.In addition,the agronomic traits of lhd3 were also changed,such as increased plant height,changed grain shape,and significantly decreased 1000-grain weight.2.Genetic analysis showed that lhd3 was controlled by a recessive single gene.Bulked Segregant Analysis（BSA）was adopted to primary map the LHD3 gene.Using a bulk DNA pool from lhd3/Nipporbare F2 individuals with the mutant phenotype,LHD3 was located between the ZNC-6 and ZNC-9 markers on rice chromosome 3,at a physical distance of 7.3 Mb.To further fine map lhd3,six Indel molecular marker was designed based on the sequence difference between Zhonghua11 and Nipponbare.The polymorphism primers were subsequently used to screen 148individuals,which finally mapped the LHD3 locus between Chr3-14.9M and Chr3-16.7M markers,within a physical distance of 1.8 Mb.Fifty F2 recessive single plants obtained from lhd3/ZH11 F2 generation and the same amount of DNA was mixed for the re-sequencing.The wild-type ZH11 was also sequenced as a control.The mutated SNPs and Indels within previous mapped interval were detected and analyzed.We found a 1-bp deletion in the coding region of LOC＿Os03g28310 gene,which encodes a DNA＿J family protein,which result in the premature termination of the encoded amino acid.3.To verified the LHD3 candidate gene,genetic complementation test was conducted.A 5,898-bp genomic DNA fragment containing the entire LHD3 coding region,2.5 kb upstream and 1.2kb downstream sequences was inserted into the binary vector p CAMBIA1300 to generate the transformation vector p LHD3:LHD3 and transformed into the lhd3 mutant.The heading date of lhd3 was restored to normal in6 p LHD3:LHD3 transgenic lines,wheras all 8 of the p CAMBIA1300 control transgenic lines failed to recover the wild type phenotype.The results demonstrated that the mutant of LHD3 responsible the lhd3 penotypes.4.To indentified the subcellular localization of LHD3,a transient expression experiment was conducted to analyze LHD3 expression in tobacco epidermal cells.The N terminal of full-length LHD3 ORF was fused to GFP under the control of the cauliflower mosaic virus（Ca MV）35S promoter,and obtained the p35S:GFP-LHD3construction,Then the p35S:LHD3-GFP vector and H2B-mcherry（Nuclear localized marker）were injected into tobacco leaves together and observed by laser confocal microscope.The results showed that he GFP fluorescence signal of LHD3-GFP was co-expressed with m Cherry signal of H2B-mcherry in nucleus,which showed that the LHD3 localized to nucleus,To identified the expression pattern of LHD3,the expression levels of LHD3 was detected by quantitative real time-PCR（q RT-PCR）under NLD in WT.The results showed that LHD3 expression had obvious circadian rhythm patten,and the expression levels in different tissues were significantly different,with the highest expression in stems and the lowest expression in young panicles.5.To investigated LHD3 gene involved the pathway of photoperiodic flowering,the expression levels of flowering-related genes were analyzed by q RT-PCR.The results showed that the expression levels of Ehd1,Hd3a and RFT were dramatically decreased in lhd3 mutant both SD and LD conditions,while the expression levels of Hd1 were not significantly difference between lhd3 and WT.The expression level of Hd1 gene was slightly increased in lhd3 mutant within 16h after pening the light in SD conditions,while the expression levels not significantly difference in LD conditions.Under LD condition,the expression levels of Ghd7 and MADS50 slightly decreased in lhd3 mutant Moveover,the expression levels of other flowering-related genes upstream of Hd3a/RFT,were not significantly different between lhd3 and WT under both growth conditions.It is speculated that LHD3 may promote rice heading date mainly through Ehd1-Hd3a/RFT1 pathway.6.To further confirm the pathway of LHD3 involed in photoperiodic flowering,the overexpression vectors of Hd3a-ox,RFT-ox and Ehd1-ox drived by Ca MV35S promoter were constructed and transformed into lhd3 mutant.The heading date of Hd3a-ox and RFT-ox transgenic plants was significantly earlier than that of control plants.Due to the Ehd1-ox transgenic plants have just obtanted,the phenotype identification is being carried out.The results preliminarily showed that LHD3 was involved in the flowering pathway of Hd3a/RFT.In conclusions,LHD3 is a new gene locus involed in the photoperiod flowering pathway in rice.The study on the functions of LHD3 and its participation in the photoperiod flowering pathway will enrich the molecular network of rice flowering and will provide the germplasm resources and theoretical support for molecular design and breeding. |
