Construction Of S1DNMT2 Gene's RNAi Expression Vector And Functional Analysis

During the growth and development of plants,they may be subjected to certain biological and abiotic stresses.In response to abiotic stresses,plants undergo a series of physiological and biochemical reactions to adapt to different environmental changes.DNMTs are methyltransferases in DNA methylation.DNMT2 has a highly conserved domain in plants,but no catalytic activity.Nowadays,there are few experimental studies on the mechanism and functional analysis of DNMT2 of DNA methylation in plants.Therefore,studying the function and role of DNMT2 in the process of growth and development or stress resistance of plants is important and meaningful to improve crops?tomato?resistance and quality.In this paper,tomatoes,the model plant,are used as the material.And the amino acid sequences of DNMT2 protein in 11 species were obtained through NCBI database comparison.The analysis of the conserved protein domains by DNAMAN software revealed that these amino acid sequences had relatively conserved protein domains.The evolutionary tree analysis and DNA methylation domain analysis of the amino acid sequences of these 11 proteins show that the relationship between SlDNMT2 protein and St DNMT2 protein was close,and the homology was as high as 95.54%.Simultaneously,we construct multiple plant expression vectors of SlDNMT2 for a series of physiological and biochemical analysis.First,the fusion expression vector p ART27-SlDNMT2-e GFP was constructed to observe the subcellular localization of SlDNMT2.The results showed that the SlDNMT2 protein was localized in the nucleus.Using plant genetic engineering technology and RNAi interference technology,we construct the transgenic RNAi expression vector p BI121-SlDNMT2-si RNA,and infect the tomato through Agrobacterium-mediated infection method,and then through plant tissue culture technology and molecular biology technology,we successfully obtain the transgenic positive plants.Next,Real-time PCR technology was used to confirm that the SlDNMT2 gene had been down-regulated in the transgenic RNAi plants.High salt,heavy metal pollution and drought are important environmental factors during growth and development of plants.For this reason,we analyzed the growth and development status of transgenic plants under high salt,heavy metal and drought conditions.First,we treated wild-type tomatoes?AC?and transgenic tomato plants with200 mmol/L Na Cl and 300 mmol/L D-Mannitol,and the results showed that under 200mmol/L salt stress and 300 mmol/L D-Mannitol treatment,the growth and development status of wild type is better than that of transgenic plants,and there is a significant difference in their fresh weight,which indicates that SlDNMT2 RNAi transgenic plants are more sensitive to salt and D-Mannitol stress.Secondly,we also analyzed the growth and development of SlDNMT2 RNAi transgenic plants under different divalent heavy metal ion stress,and the results illustrated that under Zn²⁺stress conditions,the growth status of transgenic RNAi plants was poor compared with wild type.This result demonstrated that the transgenic RNAi plants were more resistant to Zn²⁺.However,under Fe²⁺stress,the roots of transgenic RNAi plants are more developed compared with wild type?AC?,and the total number of roots of transgenic RNAi plants is more than that of wild type?AC?.This result indicates that the roots of transgenic RNAi plants have a higher resistant to Fe²⁺stress.Finally,we treated the wild-type tomato and SlDNMT2 RNAi transgenic plants grown in soil culture for drought treatment for 10days,and the results showed that the growth status of the transgenic plants was worse and the leaves are more curled.These experimental results further verified,transgenic RNAi plants are less tolerant to drought stress than wild type.Observing the number of stoma in the leaves of transgenic RNAi plants through a microscope,it was found that the distribution of stoma in the leaves of transgenic plants was more densely compared with wild type.According to reports,the TINY1 and SAG1 genes may be involved in response to drought stress in Arabidopsis.We used semi-quantitative techniques to analyze the expression of their homologous genes named Sl TINY1 and Sl SAG1 in tomato.The results demonstrated that,compared with AC,there was no significant change about the content of Sl SAG1 gene in transgenic plants.However,the content of Sl TINY1 gene was down-regulated in transgenic plants.In summary,thanks to the above results and analysis of related data,it is found that the SlDNMT2 gene is likely to involve in the response pathway to deal with stress in plants,but the specific regulatory mechanism needs to be further investigated.This article lays a good scientific foundation for further analysis of the functional role of the SlDNMT2 gene in the process of DNA methylation,and is of great significance for further exploring the molecular mechanism of DNMT2 in plants.Meanwhile,it also provides a scientific and experimental basis for further studying genetic breeding and resistance of crop to stress.