| Pseudomonas syringae is a common pathogenic bacterium in variety of plants which could cause the hypersensitive response or freezing lesions against host plants. The ice nucleation protein (INP) which could lead the water within the plant tissue and cell form the crystals was a induced secreted protein after excitation by signal molecules. Therefore, study of the transcription activity of this gene and regulatory factors for in-depth understanding of ice nucleation activity and the incidence of pathogenic mechanisms, and explore the biological control mechanism was important. P.syringae MB03 was a high ice nucleation activity strain, the ice nucleation activity gene inaQ had been cloned by in situ hybridization (GenBank: EU360731). In this study, the ice nucleation gene promoter as the research object, green fluorescence protein as a reporter protein, studied the transcription and expression activity of this promoter under the different medium, different temperatures and different exogenous chemical inducer in the P. syringae, Escherichia coli (E. coli) and P. putida, respectively. Determine that promoter as a low-temperature-dependent promoter, and the activity significantly induced by 3,5-dinitrosalicylic acid.Ice nucleation gene inaQ contains upstream 522 bp sequence and the coding structure gene of 1200 aa sequence. Through the bioinformatics analysis, inaQ promoter sequence present in several reported ice nucleation genes upstream and a P.syringae pv syringae B728a ice nucleation protein octamer regulatory region. To predict the promoter region, the result show two regions which simila to the E. coli conservative-35 region and-10 regions of promoter, the sequence was TTGCTG and GGTTAAATT, respectively.RT-qPCR was used to analysis the inaQ gene relative quantitative of the transcription activity of e in 16℃and 28℃. The result showed that the transcription level of this gene in 16℃is significantly higher than 28℃, and in the late of logarithmic phase the transcription activity is about 2 times, and in the stationary is nearly 6 times, in addition the 16℃it benefit for the gene continue transcription.An ice nucleation gene upstream promoter fragment and green fluorescent protein (GFP) fragment was amplified by PCR and fusioned by SOE-PCR, and insert into the PstⅠ/EcoR I sites of plasmid pUCP 18 to generate a E. coli-P. putida recombinant plasmid pInaQ522GFP. Expression determination by GFP as the reporter, showed that GFP can be expressed either in recombinant E. coli either in P. putida through the optical fluorescent microscopic examination and SDS-PAGE analysis. According to the predict obtained two different length promoters-297 bp and 111 bp, respectively-and used the same method to construction recombinants vector, named pMB345 and pMB346. The GFP fluorescence intensity of the E. coli recombinants strains MB346 and MB348 were 356.7 and 352.5, respectively and MB350 had not GFP expression, it suggest that the length of the promoter is not important for its activity and the-10 region and-35 region is necessary.OprL gene was widely spread in gram-negative bacteria such as E. coli and P. putida. In this study the promoter of this gene was a control to compare the activity of the ice nucleation gene promoter. In E. coli harboring ice nucleation gene promoter the maximum fluorescence intensity was 456.7 and the average fluorescence intensity was 330.1, on the other hand the recombinant harboring oprL promoter was 323.7 and 188.9, so that the ice nucleation promoter had stronger activity. And in the P. putida the two promoters had similar fluorescence intensity was 99.2 and 130.9, the oprL promoter slighter better.Select different medium, different temperatures and different chemical inducer to induced promoter in order to explore the expression condition of this promoter. The fluorescence intensity increased by 32.5% and 27.3% when using the KB and NAD medium than the LB medium culture in the same condition. The fluorescence intensity of the E. coli and P. putida improved 17.9% and 30.1% when cultured at 16℃, indicating that this gene has relatively high expression activity at low temperature. Different inducer had up-regulation effect or down-regulation effect, sodium sulfate, sodium nitrate,3,5-dinitrosalicylic acid, hydroquinone, 4-hydroxy-3-nitrophenyl acetic acid had up-regulation in which the 3,5 dinitrosalicylic acid had the most significant effect. And the hot water extract of mustard seeds and some chemical substances had the same effect. The chemical component of inducer substance was analyzed by HPLC. |
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