Cloning And Functional Analysis Of The Circadian Gene LHY Promoter In Fraxinus Mandshurica

In this experiment, the gene LHY promoter sequences was isolated from Fraxinus mandshurica by Site Finding-PCR and the cis-acting elements of gene LHY promoter was analyzed. The plant transient expression vector pPXGFP-P-LHY and plant expression vector pPCXGUS-P-LHY were constructed. The activity of LHY promoter was researched by GFP(green fluorescent protein) and GUS organization staining. The response of LHY promoter to abiotic stress was analyzed by GFP, GUS enzyme activity and quantitative expression of the gene GUS. In addition, in order to explore the relationship between DNA methylation and drought stress,the changes in DNA methylation patterns of parents Fraxinus mandshurica and F1 hybrids in drought stress was studied. The main results are as follows:1、Clone and activity analysis of gene LHY promoter in Fraxinus mandshuricaIn this study, the promoter sequence of gene LHY of 1360bp was isolated. Bioinformatics analysis showed that the LHY promoter contains important transcription essential elements(TATA box, CAAT box), some adversity regulatory elements(such as drought-responsive elements), hormone-responsive elements(such as gibberellin, jasmonic acid and other regulatory elements), light-responsive elements(AE-box, BOXI, GAG-motif, LAMP-element, et al.) and Circadian rhythms associated components. The activity of LHY promoter was proved by GFP and GUS staining after Nicotiana tabacum was infected by transiently.2、The variation of activity of LHY promoter in response to abiotic stressThe materials of suspension cells of Betula platyphylla which were infected by pPXGFP-P-LHY was treated with NaCl. The GFP results showed that the promoter of LHY in the expression vectors pPXGFP-P-LHY could express. Compared to the control, the NaCl stress enhanced the expression of the promoter by the brightness of the fluorescence.Additionally, the Nicotiana tabacums infected by pPCXGUS-P-LHY were also treated with NaCl and PEG. The results showed that both the GUS enzyme activity and the expression of GUS increased first and then reduced as drought and salt stress increased.Compared to the control, at 3 h of drought stress and 6 h of salt stress, both the GUS enzyme activity and the expression of GUS reached the maximum. It indicated that LHY promoter could still express under abiotic stress and adversity regulatory elements of LHY promoter were regulated by NaCl and PEG.3、Methylation analysis of promoter region and coding region of circadian gene LHYIn this study, the material of female parent Fraxinus mandshurica, male Fraxinus Americana and their hybrids F1 were treated with drought. The bisulfite sequencing method was used for gene LHY promoter and coding region methylation analysis, the results showed that, compared to the treat control and parent control, the methylation level of the F1 subjected to drought decreased because the original sites of methylation occurred demethylation. On the basis of the studies above, the quantitative expression of LHY gene was analyzed by fluorescence quantitative PCR technology, the results showed that the expression of LHY gene of F1 was higher than that of parents under drought stress.