Functional Analysis of VvARF18 Gene in Red Globe Grape
[Objective]Auxin response factor(ARF)is a significant regulatory factor in the auxin signaling pathway and plays an important role in plant growth and development as well as various physiological processes.Analysis of the Red Globe grape VvARF18 promoter,heterologous expression,endogenous hormone content and its expression in response to hormones was made in order to explore the mechanism of VvARF18 gene in the auxin(IAA)signaling pathway and flower bud differentiation process in Red Globe grapes.[Method]The VvARF18 gene sequence was obtained by homologous cloning by using facility Red Globe grape flower buds as experimental materials.The cis-acting elements of the promoter were analyzed using the online database PLACE.The plant overexpression vector pC2300-VvARF18 was constructed based on the pCAMBIAI2300 plant expression vector by double enzyme digestion and homologous recombination method.The recombinant vector pC2300-VvARF18 was transformed into Agrobacterium tumefaciens strain GV3101 by using electrical shock method.The tobacco leaves were used as explants and transferred into tobacco by Agrobacterium-mediated callus transformation method,and positive transgenic seedlings were obtained by PCR.The quantitative real-time PCR(qRT-PCR)was used to analyze the expression level of VvARF18 transgenic tobacco lines,and the transgenic lines with high expression level were screened and cultured to T3 generation,and treated with IAA and GA3 to analyze the expression level of VvARF18.The content of IAA,GA,ABA and CTK in flower buds and leaves of transgenic tobacco were determined by enzyme-linked immunosorbent assay.[Result]VvARF18 of Red Globe grape located on chromosome 13,and contained 3 exons and 2 introns.There are multiple cis-acting elements in the VvARF18 promoter region that respond to light,plant hormones,and stress.The phenotypic analysis found that the process of flower bud differentiation was faster in transgenic tobacco than in wild-type tobacco.The qRT-PCR results showed that the expression of VvARF18 showed an increasing and then decreasing trend during the four periods of flower bud development in transgenic tobacco,and the highest expression level was reached in the S3 stage.The results of IAA,CTK,GA and ABA determination in flower buds and leaves of transgenic tobacco plants showed that the content of four plant hormones in flower buds and leaves of transgenic tobacco plants were higher than those of wild-type plants.The change trend of GA/IAA during the four periods of transgenic tobacco flower bud development were consistent with the expression trend of VvARF18.The expression level of VvARF18 in transgenic tobacco plants treated with IAA and GA3 decreased with the increase of IAA treatment concentration and also decreased with the extension of GA3 treatment time.[Conclusion]Grape VvARF18 negatively regulated auxin to participate in the process of plant flower bud differentiation,which could interact with key factors in the gibberellin signaling pathway to synergistically regulate hormone levels in plant flower buds and had a facilitative effect on plant flower bud differentiation.
Red Globe grapeVvARF18transgenic plantsflower bud differentiationphytohormone
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