Changes Of DNA Methylation When Exogenous5-aminolevulinic And P-hydroxybenzoic Acids Mitigate Heat Stress Of Cucumis Sativus And Gene Fhy3Is Transformed To Arabidopsis Thaliana

As the influence of the greenhouse, high temperature occurs more frequently. Heat is thecommon adverse factor in vegetable cultivation, which cause the reduction of productivity.Cucumber is one of main cultivated vegetables in our country. It is a kind of thermophilic andnot high temperature resistant vegetables. Therefore, it is very important to increase the heattolerance of cucumber. In the study, we elucidate the mechanism of heat stress mitigated byexogenous ALA and PHBA. We investigated the effect of exogenous ALA or PHBA on theplant growth parameters, antioxidant enzymes activities, the contents of proline and solublesugar, and the changes of DNA methylation under heat stressed of cucumber seedlings. Wepicked out the differential fragments to clone the methylation polymorphic fragments andanalyzed their sequence by using bioinformatics. Our work might help to elucidate themechanism of heat stress mitigated by exogenous ALA and PHBA on the sides of antioxidantenzymes and DNA methylation. It also could lay the foundations for applying methylationpolymorphic genes and give references to studying the mechanism of stress mitigated byother substances.In recent years, growing attention has been focused on the safety of the geneticallymodified food. In transgenic plants, endogenous and environmental factors will influence themethylation of transformed exogenous genes and thus affect the phenotype of the transgenes.On the other hand, the transformed exogenous genes can cause the silencing of endogenousgenes if the two genes are sufficiently homologous in transgenic plants. Transgenes will takesome unintended consequences in many transgenic plants. The unintended consequences oftransgenes would be detected in view of DNA methylation after we compared the transgenicseedlings with the wild type plantlets or analyzed the differences between the recipient andtransgenic Arabidopsis. When some MSAP fragments were found to have similarities withArabidopsis endogenous genes, the transcript levels of three candidate genes were studied indifferent organs. Meanwhile, the methylation levels of the three genes was studied using thebisulfite sequencing PCR (BSP) technique to confirm the cytosine methylation alterationsobtained by MSAP method. Our aims were to investigate whether transgenes can influencethe methylation variations of genomic DNA sequences including some endogenous genes thatare not homologous with the transformed exogenous gene in transgenic plants. Our work may also lay a foundation for understanding the unintended consequences of the transformedexogenous genes.At the two-leaf stage, cucumber seedlings were selected and pretreated with3μM ALAor0.5mM PHBA at normal temperature (25/18°C) for2d. Then the cucumber seedlings wereexposed to heat (42/38°C). Samples of the second leaf were used to determine the activitiesand contents of the antioxidants. After extracting genomic DNA from the second leaves, DNAmethylation was mensurated by using the methylation-sensitive amplified polymorphism(MSAP) method. Then DNA methylation polymorphic fragments were isolated, cloned andsequenced. The transcript levels of genes in different tissues of cucumber seedlings weredetermined by real-time quantitative PCR. We also measured the veracity of the MSAP bybisulfite sequencing PCR.The fhy3mutant (fhy3) and wild type (WT) are of Arabidopsis thaliana Nossen ecotype,and35S:GUS-FHY3(GUS-FHY3) is a transgenic line in the fhy3mutant background. Seedswere germinated and grown at22°C, and the resotte leaves, roots, flowers and internodes ofthe three kinds of plantlets were collected and then were respectively ground with liquidnitrogen for extraction of genomic DNA and total RNA. DNA methylation was mensuratedby using the method of MSAP. Then DNA methylation polymorphic fragments were isolated,cloned and sequenced. The transcript levels of genes in different tissues of Arabidopsisthaliana were determined by real-time quantitative PCR. We also measured the veracity of theMSAP by bisulfite sequencing PCR. The main results of this study were listed as thefollowing:(1) Effects of exogenous ALA and PHBA on the growth of cucumber seedlings underheat stressIn the experiments, heat significantly decreased the growth of cucumber seedlings, andthe edges of the second leaves to be dried up. However, the growth parameters weresignificantly elevated in the ALA/PHBA+heat treatment, the withering rate of the secondleaves decreased. The growth of cucumber seedlings was inhibited observably by heat stress.ALA and PHBA pretreatment alleviated the inhibition of heat stress on the growth ofcucumber seedlings.(2) Effects of exogenous ALA and PHBA on antioxidant enzymes and other relatedindexes of cucumber leaves under heat stress In the ALA or PHBA pretreatment experiment, heat increased the levels of MDA andthe contents of ROS. The combination of ALA or PHBA pretreatment and heat stressincreased the activities of antioxidant enzymes such as SOD, CAT, GPX, GSH-Px, APX,MDHAR, DHAR and GR and thereby decreased the level of MDA and ROS. We proposethat exogenous ALA or PHBA increases antioxidant enzymes activities in cucumber leavesand thereby decreases the level of ROS, thus protecting the growth of cucumbers under heatstress.(3) Effects of exogenous ALA and PHBA on DNA methylation of cucumber leaves underheat stressThe changes of DNA methylation in cucumber leaves were measured by MSAP.According to the results of MSAP, the levels of DNA methylation in cucumber plants were19%-21%. Heat and ALA or PHBA pretreatment made the DNA methylation changed incucumber genomic. All the fragments showed the significant similarities to cucumbergenomic sequences. The levels of related genes in different tissues of cucumber seedlingswere determined by real-time quantitative PCR. The veracity of the MSAP was proved bybisulfite sequencing PCR.(4) The changes of DNA methylation in recipient and transgenic Arabidopsis leavesThe changes of DNA methylation in Arabidopsis thaliana leaves were measured byMSAP. According to the results of MSAP, the levels of DNA methylation in Arabidopsisthaliana were10%-12%. The recipient or transgenic gene fhy3made the DNA methylationchanged in Arabidopsis thaliana genomic. All the fragments showed the significantsimilarities to Arabidopsis thaliana genomic sequences. The levels of related genes indifferent tissues of Arabidopsis thaliana were determined by real-time quantitative PCR. Theveracity of the MSAP was proved by bisulfite sequencing PCR.