Transcriptional Regulation Of Cadmium Tolerance Protein MTP-like Genes In Taxus Chinensis

The southern yew(T.chinensis)is one of our most important wild yew trees.Taxus chinensis is also known as cypress pine,yew,yew pine,and torreya.The paclitaxel contained in it has extremely high economic value and is the first-selected specific drug for cancer treatment.However,Taxus chinensis has a small distribution range in the wild,slow growth,poor environmental adaptability,and indiscriminate deforestation,and the wild plants are almost extinct.Strengthening the research on Taxus chinensis,improving its environmental adaptability and protecting the ecological environment of its population has become an effective way for the comprehensive development and utilization of Taxus chinensis.Taxus chinensis is a rare medicinal plant,which will affect production safety in a cadmium-contaminated environment.It is necessary to study the response of Taxus chinensis to cadmium.In this thesis,Hoagland nutrient solution was used for hydroponics of Taxus chinensis,with heavy metal cadmium as the stress factor,and leaves as materials.First,the effect of different cadmium treatment time on the leaves of Taxus chinensis was studied,and the most obvious response to cadmium stress was determined.It was determined that the most obvious treatment time in response to cadmium stress was 6days of cadmium treatment;secondly,within the optimal response time,the leaves of Taxus chinensis were treated with different concentration gradients of cadmium(0,100,200,300 μmol/L),and the optimal treatment concentration was found to be 200μmol/L.Then we treated Taxus chinensis under the conditions of cadmium treatment for 6 days and cadmium treatment concentration of 200 μmol/L.The internal physiological and biochemical changes of the plant were significant,and several indicators reached their peaks,indicating that Taxus chinensis mobilized its internal response system.In order to analyze the response mechanism of Taxus chinensis to heavy metal cadmium,we chose cadmium treatment for 6 days and cadmium treatment concentration of 200 μmol/L as the experimental conditions for the next step of transcriptome sequencing.By screening the annotated gene library of the transcriptome,we screened two heavy metal transporter-encoding genes,MTP1 and MTP11.q RT-PCR analysis showed that both MTP1 and MTP11 genes were expressed in the rhizomes and leaves of Taxus chinensis;among them,the expression level of MTP11 gene in leaves was significantly higher than that in rhizomes.Subcellular localization experiments demonstrated that the expression of Tm MTP1 and Tm MTP11 is concentrated in the cytoplasmic membrane,which is consistent with MTP having a transmembrane domain.We speculate that Tm MTP1 and Tm MTP11 enhance cadmium tolerance by transporting cadmium to sequestered storage in the cytoplasmic membrane.By means of chromosome walking,the mobile subsequences of Tm MTP1 and Tm MTP11 were successfully cloned,and the MYB family factors induced by cadmium stress were screened by bioinformatics,and the potential upstream regulators of Tm MTP1 and Tm MTP11 were screened out.The experimental results showed that the expression of transcription factors Tm MYB4 and Tm MYB34 was induced firstly when Taxus chinensis received heavy metal cadmium stress.Inducible expression of Tm MYB4 and Tm MYB34 binds to the promoter of MTP gene and regulates their expression levels,resulting in tolerance to cadmium stress.