| Chrysanthemum(Chrysanthemum morifolium Ramat.),as one of four popular cut flowers in the world and one of the ten most famous flowers in China,has great ornamental and economic value.Aphids,the main pests in chrysanthemum,affect the yield and quality of plants seriously.Currently,the chemical methods of controling aphids not only cause environmental pollution but also lead to aphids population resistance.The prevention of aphids has been a major problem in chrysanthemum production.MYB transcription factors have many functions involved in varieties of plants life activities,and its regulation in aphid resistance has been reported.However,in the chrysanthemum,there is little information about MYB genes.In the present study,we isolated two MYB genes CmMYB58.1 and CmMYB58.2 in chrysanthemum.Their expression feature,transcriptional activity and subcellular localization were analyzed.The CmMYB overexpression chrysanthemum lines were generated through Agrobacterium tumefaciens mediated leaf disc transformation,and the aphid resistance of these transgenic lines were assessed as well.Then the mechanism of aphid resistance caused by CmMYB58.1 and CmMYB58.2 was explored in terms of their roles in regulating lignin biosythesis.The main conclusions are as follows:1.Two MYB genes CmMYB58.1 and CmMYB58.2 were cloned from chrysanthemum with the method of RT-PCR and RACE-PCR.The ORF of CmMYB58.1(KT763374)and CmMYB58.2(KT763375)was 753bp and 600bp,respectively.The amino acid sequence was aligned and the result displayed that CmMYB58.1 and CmMYB58.2 belong to typical R2R3-MYB subfamily.2.The relative expression of CmMYB58.1 and CmMYB58.2 in chrysanthemum after aphid feeding were done by qRT-PCR.The results indicated that CmMYB58.1 and CmMYB58.2 both can be induced by aphid stress specifically,different from mock puncture treatment.And the relative expression of CmMYB58.1 and CmYB58.2 in different tissues of chrysanthemum were also detected,the results showed that CmMYB58.1 and CmMYB58.2 showed the highest expression level in leaves,higher in stems and the least in roots,suggesting the tissue specific expressions of CmMYB58.1 and CmMYB58.2.3.Recombinant vectors pGBKT7-CmMYB58.1 and pGBKT7-CmMYB58.2 were constructed and transformed into the yeast strains Y2H.The yeast one hybrid assay demonstrated that CmMYB58.1 functioned as a transactivator,and the Y2H strain containing pGBKT7-CmMYB58.1 could become blue on SD/-His-Ade medium with X-a-gal,while no obvious transcriptional activity was detected in CmMYB58.2.4.The green fluorescent protein expression vectors pMDC43-GFP-CmMYB58.1 and pMDC43-GFP-CmMYB58.2 were structured and transformed into the onion epidermal cells.The subcellular localization assay showed that CmMYB58.1 and CmMYB58.2 were localized to the nucleus in vivo.5.Through Agrobacterium tumefaciens-mediated leaf disc transformation,CmMYB58.1 and CmMYB58.2 were transformed into 'Jinba' which is an aphid sensitive variety of chrysanthemum.The CmMYB58.1 and CmMYB58.2 overexpression chrysanthemum lines both increased the ability of aphid resistance.Lignin contents were increased in overexpression chrysanthemum lines.And the expression levels of Cm4CLl(4-hydroxycinnamateCoAligase1),CmCCR1,CmF5Hl(Ferulate 5-hydroxylase)and CmCAD6(Cinnamylalcoholdehydrogenase6)were increased,while CmC4H and CmCOMT(Caffeicacid-ferulicacidO-methytransferase)were decreased in CmMYB58.1 overexpression chrysanthemum lines,compared with controls;the expression levels of CmPALl(Phenylalanine ammonia-lyase 1),CmC4H(Cinnamate4-hydroxylase),Cm4CLl(4-hydroxy cinnamoyl CoA ligase 1),CmHCT(Hydroxycinnamoyl CoA-Shikimate/Quinate-hydroxycinnamoyl transferase),CmC3Hl(Coumarate3-hydroxylase1),CmCCoAOMT1(Caffeoyl CoA O-methyltransferase 1)and CmCCRl(Cinnamyl CoA reductase 1)were increased in CmMYB58.2 overexpression chrysanthemum lines,while the other genes have no obvious change,compared with controls.The data indicated that CmMYB58.1 and CmMYB58.2 improved the ability of plants resistance to aphid via promoting the accumulation of lignin. |
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