Clonging And Functional Analysis Of LoMYB80 And LoAMS Gene Related To Anther Development In Lily(Lilium Oriental Hybrids)

As one of the most popular cut flowers,lily holds on an important role in the global market.However,the lily flower has huge anthers with large amounts of pollen grains which could pollute the petals and surrounding surfaces if they are not removed timely.This phenomenon brings many troubles for commercial sales.So,controlling the anther’s development and reducing pollen production through genetic engineering is an effective way to solve the problem.The study of the genes related to anther development could provide effective genes for genetic engineering breeding and useful theory for molecular breeding.In this study,LoMYB80 and LoAMS were isolated from anthers of Lilium Oriental Hybrids‘Siberia’.Further more,their biochemical characteristics and the functions during anther development were also studied.The main results are as follows.(1)The LoMYB80 gene was isolated from anther of 4 cm lily flower bud which ORF was 909 bp encoding a protein of 302 amino acids.Multiple alignlent and protein structure analysis showed that L0MYB80 belonged to R2R3 MYB family and shared a 44 amino acid sequence with other plant MYB80s.Phylogenetic tree analysis showed LoMYB80 had a closest genetic relationship with OsMYB80(Oryza sativa)and TaMYB80(Triticum aestivum)which were all monocotyledonous plants.Transient expression of LoMYB80-GFP revealed LoMYB80 localized in nucleus.The transcriptional activation experiment showed LoMYB80 was a transcription activation factor and the transactivation activity depended on the last 70 amino acids which were close to the C-terminal region.(2)Real-time quantitative PCR analysis revealed the highest relative expression of gene was in the anther of 10 cm and then was in 4 cm during 1-10 cm flower buds.The relative expression in microspore/pollen was much higher than the whole anther in 10 cm bud,and was much higher than the microspore/pollen of 8 and 9 cm buds;It is indicated that LoMYB80 might play an important role in microspore releasing and pollen coat formation.The LoMYB80 gene expression was significantly higher in anther than other organs in the period of most flower buds were in 4 cm.The 989 bp sequence upstream from the start ATG of LoMYB80 was cloned using the hiTAIL-PCR method,and the LoMYB80 gene was detected mainly expressed in the young anthers which were in the stages from 6 to 10 by analyzing the histochemical localization of GUS expression.According complementation experiment,the transgenic ms188 mutant lines could produce normal pollen grains,and could restore fertility partially.However,the number of the pollen grains was far smaller than wild type.(3)The LoAMS gene was isolated from anther of 3 cm lily flower bud which ORF was 1626 bp encoding a protein of 541 amino acids.Multiple alignment and protein structure analysis showed that LoAMS belonged to MYC family and contained a typical bHLH domain.Phylogenetic tree analysis showed LoAMS had a closest genetic relationship with EgAMS(Elaeis guineensis)and PdAMS(Phoenix dactylifera)which were all monocotyledonous plants.Transient expression of LoAMS-GFP revealed LoAMS localized in nucleus.The transcriptional activation experiment showed LoAMS was a transcription activation factor and the transactivation activity depended on 204 amino acids which were close to the C-terminal region.(4)Real-time quantitative PCR analysis revealed the highest relative LoAMS gene expression was in the anther of 10 cm and then was 3 cm during 1-10 cm flower buds.The relative expression in microspore/pollen was much higher than the whole anther in 10 cm bud,and was much higher than the microspore/pollen of 8 and 9 cm buds;It is indicated that LoAMS might play an important role in the period of microsporocyte meiosis and pollen coat formation.The LoAMS gene expression was significantly higher in anther than other organs in the period of most flower buds were in 3 cm.The 389 bp sequence upstream from the start ATG of LoAMS was cloned using the hiTAIL-PCR method,and the promoter contains 6 pollen-specific elements.These results suggest that both LoMYB80 and LoAMS are involved in regulation of lily anther development,and could be the candidate genes for genetic engineering breeding.Our study provides theoretical basis for breeding new lily varieties with little or no pollen.