Cloning And Functional Identification Of NRAMP In Taraxacum Antungense Kitag

Dandelion is a perennial herb of Compositae,some researchers and scholars suggested that dandelion has the potential advantage in improving soil in the 1960s(Mossop et al,2000).In order to investigate the reason why dandelion can absorb heavy metals in large quantities and the related molecular mechanisms in the adsorption process,based on the sequencing of the dandelion transcriptome in Dandong,a NRAMP gene sequence are screened and Ta NRAMP is obtained by cloning and analyzed by software.And real-time fluorescence quantitative PCR is used to determine the expression changes of Ta NRAMP gene under Cd stress.Then the PRI101-Ta NRAMP overexpression vector is constructed to verify the function of NRAMP gene by Agrobacterium mediated in dandelion.The results of the experiment are as follows:1.The NRAMP gene has been cloned,with a length of 1647 bp,encoding 548 amino acids.The conservative structure domain of the Ta NRAMP amino acid sequence is the same as the Arabidopsis At NRAMP protein,the results of phylogenetic tree show that the most close relation with the Ta NRAMP is the sunflower of the Compositae plant;the results of the two stage structure prediction indicates that the protein consists of three kinds of modules,which are 39.42% a-spiral,19.16% beta extension and 41.42% irregular curling modules.The prediction results show that Ta NRAMP has 10 transmembrane domains,which are 124~146,159~181,196~218,225~247,267~286,307~329,366~388,408~425,476~498,503~525 amino acids in the protein sequences.2.The expression of Ta NRAMP gene in four tissues of root,petiole,leaf and flower of Taraxacum is analyzed by q RT-PCR.The results shows that the quantity of Ta NRAMP gene expression in four organs is differential expression.The relative expression of Ta NRAMP gene in root was about 0.5 times higher than that of flower,3 times higher than leaf and 31 times higher than petiole,presenting the state of root > flower > leaf > petiole,showing significant difference.3.Under the treatment of Cd,the expression of Ta NRAMP gene showed a great change with time.Between 0-2 hours after Cd treatment,the relative expression of Ta NRAMP gene was gradually and upwardly increased,showing a significant difference.After 2 hours of treatment,the relative expression level of Ta NRAMP gene began to drop sharply,and the down-regulation range was as high as 20-fold,and it gradually began to maintain a stable low-level expression after a violent decline.At different Cd treatment times,the relative expression of genes in leaves and roots showed significant differences.At 24 h and 48 h,the genes in the roots were barely expressed.4.The PRI101-Ta NRAMP overexpression vector is successfully constructed and transferred to dandelion,and then obtain two transgenic plants.Then the two strains are treated with 0.5μm Cd,compared with the wild type dandelion,the experiment result shows that the content of heavy metal adsorbed on the aboveground part of transgenic plants is 2-3 times compaired with the wild type,and it proves that the NRAMP gene has a direct effect on the adsorption of Cd in the taraxacum.