Regulatory Function Of EARLI1Subfamily Gene AT4G12490on Flowering Transition Of Arabidopsis Thaliana And Antimicrobial Activities Of Its Encoding Protein

Angiosperms accomplish the transition from vegetative growth to reproductive growth by flowering. The timing of floral induction is controlled accurately by environmental factors and development status of plants. The sophisticated regulatory networks of plants can monitor changes in the environment. To maximize seed production, plants must undergo a sufficient vegetative growth before entering the reproductive stage. In Arabidopsis, the flowering time is considered to be controlled by six major pathways, including photoperiod, gibberellin, age, autonomous, vernalization and ambient temperature pathways, which are implicated in flowering transition under different conditions.The biological function of EARLI1subfamily gene AT4G12490in floral induction of Arabidopsis was studied in the present work. The proteins of EARLI1subfamily are constituted by a signal peptide, a proline-rich domain (PRD) and an eight cysteine motif (8CM), and the8CM coding sequences of them are very conservative. Phenotypic observation indicated that the8CM RNA interference lines of EARLI1subfamily in the genetic background of Col-FRI-Sf2initiated flowering transition earlier than the wild-type plants. It suggested that EARLI1subfamily genes functioned in maintenance of vegetative growth and prevention of premature reproductive growth.In order to comprehensively understand the mechanism of EARLI1subfamily gene AT4G12490in regulation of Arabidopsis flowering, two RNA interference vectors, AN-90-PRD and AN-90-8CM, targeted to the coding sequences of PRD and8CM of AT4G12490, respectively, and an overexpression vector, AN-90, containing the intact open reading frame of AT4G12490, were constructed in this work. RNA interference lines and overexpressing lines of AT4G12490were prepared by Agrobacterium-mediated transformation of Col-0wild-type Arabidopsis plants using floral dip method in combination with vacuum treatment. At the same time, homozygous mutant has been screened from the progeny of heterozygous T-DNA insertion lines of AT4G12490. Comparison to the phenotypic characteristics of wild-type, overexpressing and RNA interference plants demonstrated that downregulation of AT4G12490could lead to earlier flowering, indicating that efficient expression of AT4G12490could maintain the vegetative growth and delay the flowering time.Subsequently, the influences of upregulation, downregulation and knockout of AT4G12490on the expression of the important genes involved in different pathways of floral induction were analyzed using the materials above-mentioned. Under long-day photoperiods, AT4G12490mainly expressed in seedlings, mature rosette leaves and stems, the most similar to FLK of autonomous pathway and late-flowering gene FRI of vernalization pathway. The expression of AT4G12490varied in different stages of development, the transcription level of it was higher in early stage of vegetative growth, also in early stage of reproductive development. Under long-day photoperiods, EARLI1subfamily genes, such as AT4G12490> EARLI1and AZI1, showed daily rhythmicity in expression. Either under long-day photoperiods or under short-day photoperiods, downregulation of AT4G12490resulted in increase of mRNA abundance of florigen gene FT and flowering integrator gene SOC1, and this effect was more strong under long-day photoperiods. Simultaneously, the circadian rhythm expression pattern of the genes involved in promotion of flowering transition was more obvious. In contrast, downregulation of AT4G12490had no effect on expression and oscillation of the circadian clock genes, including CCA1、LHY、G1. Besides, along with the decrease of AT4G12490expression, the transcription of FLC, the potent repressor of flowering in vernalization and autonomous pathways, and RGA, a repressor of GA signal transduction, were inhibited, while VIN3involved in vernalization pathway and the genes related with GA biosynthesis were activated. The results of high performance liquid chromatography analysis showed that interference of AT4G12490expression could increase the content of endogenous GA, it is consistent with the phenotype of RNA interference lines, including slender, tall and early flowering. All these results indicated that AT4G12490could influence different flowering pathways.In order to determine the subcellular localizaion of AT4G12490protein, the coding sequence of it without the stop codon were amplified and inserted into pCAMBIA1302to produce a fusion expression vector, and transgenic tobacco plants harbouring35S::AT4G12490-GFP construct were regenerated by Agrobacterium-mediated leaf disc method. Observation under laser scanning confocal microscope indicated that AT4G12490protein expressed in tobacco was localized to cell surface, suggesting this protein might be related with sensing of environmental stimuli and signal transduction. Moreover, constitutive expression of AT4G12490in tobacco plants also could promote the vegetative growth and delay the flowering process.Furthermore, recombinant protein of AT4G12490lacking the signal peptide was expressed in Escherichia coli. At first, the coding sequence of PRD-8CM was amplified from genomic DNA of Col-0wild-type Arabidopsis plants and ligated into pPROEX-HTb. Then the recombinant plasmid was introduced into BL21(DE3) strain of E. coli. SDS-PAGE and Western blotting analyses indicated that the recombinant protein of AT4G12490expressed in E. coli after induction with IPTG mainly existed in inclusion bodies. Because the fusion protein expressed in E. coli has6×histidine tag, it was purified with Ni-NTA affinity column after lysis of inclusion bodies with urea. The result of antimicrobial test showed that the growth of Botrytis cinerea and Saccharomyces cerevisiae could be inhibited effectively by recombinant AT4G12490. At the same time, inducible expression of AT4G12490with galactose in yeast cells also could repress the propagation of Saccharomyces cerevisiae.Function identification of AT4G12490in regulatory network of flowering time in Arabidopsis can provide theoretical supports to prolongation of the vegetative growth stage and increase of the biomass accumulation of plants by genetic engineering, it is significant in development of biomass energy. Study on the antimicrobial resistance of AT4G12490has potential applications in crop improvement.