Cloning And Expression Analysis Of Tomato DNA Methyltransferase SlDRM2L Gene

As be fixed,plants are affected by many abiotic stress factors during growth and development.With the developments of industrialization,large amounts of greenhouse gases emitted have caused global warming,and the ambient temperature is constantly increased.These effects have prompted a large number of plants to improve their tolerance to harsh environments and increase survival rates by changing their genetic mechanisms.The main way for plants to improve their tolerance is to use epigenetics such as DNA methylation to adapt the harsh environment.DNA methylation mainly modifies genomic DNA under the catalysis of methyltransferase in response to the stress of the external environment.In plants,the main DNA methyltransferase which involved in the initiation of DNA methylation is DRM2.It is more comprehensive in the study of plant tolerance.For example,in the salt stress tolerance studies of Arabidopsis DRM2 have shown that DNA methyltransferase may indirectly promote the expression of cellulose synthase to regulate the level of cellulose synthesis conferring tolerance in Arabidopsis.There is also a corresponding study on the tea plants,they can regulate the changes of genomic methylation by changing the expression level of Cs DRM2 gene in order to participate in the response to the stress of the low temperature.In this study,the NCBI database was used to search for a sequence with high homology to Arabidopsis cytosine methyltransferase DRM2 in tomato,which is named as SlDRM2L.The full-length c DNA sequence of tomato DNA methyltransferase SlDRM2L gene was obtained through Gen Bank(accession number NM?001246974).We used biological information technology to analyze the tomato SlDRM2L protein domain,physical and chemical informations and completed the prediction of the three-dimensional structure.At the same time,the protein sequences encoded by the tomato SlDRM2L gene were searched for homologous sequences in other species to analyze their protein conserved domains and relatedness.Through these biological information,we learned that SlDRM2L,as a hydrophilic protein composed of 603 amino acids,has the closest relationship with tobacco.Using wild-type tomatoes as model plants for molecular level research,and the expression of this gene in tomato tissue was analyzed by fluorescence quantitative PCR,the results showed that the SlDRM2L gene was expressed in all tissues.And the highestexpression levels are in leaves and flowers.And the constructed green fluorescent transient expression vector p BI121-SlDRM2L-GFP was used for subcellular localization experiments through in tobacco transient expression system.The observation of the laser confocal microscope revealed that the SlDRM2L protein was localized in the nucleus.We constructed the gene silencing expression vector and obtained the down-regulated transgenic plants by genetic transformation and plant tissue culture.The size and weight of the down-regulated transgenic and wild-type tomato fruits were recorded and compared in details: after the measurements of vernier caliper and analytical balance the transgenic fruits were found to be smaller and lighter than the wild-type tomatoes.In analyzing the methylation level of MuDR transposon in wild-type and transgenic tomato materials,we found that the methylation level of MuDR transposon in transgenic plants with down-regulated SlDRM2L was significantly lower than that of wild-type tomato plants.It shows that the down-regulation of SlDRM2L will affect the methylation levels of plants,and from this we can further speculate that SlDRM2L is a DNA methyltransferase involved in DNA methylation.The influence of high temperature is a stress factor on plant growth and development has also received attention.It has been reported that under high temperature environment,the level of plant DNA methylation and even the regulation of plant fertility will be induced by high temperature stress.Therefore,in the study,we also analyzed whether high temperature treatment affects the transcription level of tomato DNA methyl transfer gene SlDRM2L.Under the high temperature(39 ?,4 h),the gene transcription level of tomato SlDRM2L was significantly increased,which indicated that DNA methylation mediated by SlDRM2L may be induced by high temperature.At the same time,the transcriptional levels of HSFA2,HSP90,HSP21 related genes of high temperature response pathway in transgenic plants were also analyzed by semi-quantitative PCR experiments.The results showed that the expression of HSP21 in these three genes was up-regulated,suggesting that SlDRM2L may be a DNA methyltransferase that mediates DNA methylation modification of HSP21.This indicates that SlDRM2L in tomato may participate in the response to high temperature stress by modifying and regulating the HSP21 gene.These molecular biology experiments are of great significance for in-depth understanding and studying of the molecular mechanism and biological functions of SlDRM2L.It provides relevant scientific basis for discovering the roles of SlDRM2L inplant growth and development.It has laid a theoretical foundation for further study on the function of SlDRM2L.