Cloning And Functional Analysis Of SaNramp6 Gene From Sedum Alfredii Hance

Hyperaccumulating ecotype of Sedum alfredii is a new identified Cd/Zn/Pb hyperaccumulator which was found in an ancient Cd/Pb/Zn mined area in Quzhou city of Zhejiang province. Here, we obtain S. alfredii Hance natural resistance-associated macrophage protein(Nramp) which has been found to play an important role in tolerance of heavy metal stress and report the characterization, expression, preliminary bioinformatics analysis and functional validation of it. The primary findings are depicted as follows:1. Through local alignment among the transcriptome data of S. alfredii, we obtained a sequence with high homology to AtNramp6 gene and designated as SaNramp6. Using 5’ RACE and 3’ RACE amplification, the full-length cDNA was cloned, which was 2,055 bp with an open reading frame(ORF) of 1,638 bp, a 5′-untranslated region(UTR) of 95 bp and a 3′-UTR of 322 bp. The ORF of SaNramp6 encoded a polypeptide of 545 amino acids with a calculated molecular weight of about 58.44 kDa. The BLASTp searching at NCBI shows that SaNramp6 was characterized structurally by the presence of Nramp domain with eleven transmembrane structures. Furthermore, genomic sequence of SaNramp6 was also cloned and sequence analysis showed this gene contains 10 introns.2. Total RNAs of root, stem and leaf of S. alfredii after Cd treatment was extracted and reverse transcribed for RT-qPCR which was used to analyze tissue specificity and spatial expression pattern of SaNramp6. The results showed that, without treatment, the expressions of SaNramp6 in leaf and root are higher. After treatment, the expression in the root tissue was enhanced in a small extent during early time points while remarkable elevation was detected at the time points of 96 h and 1 week. However, the expression of SaNramp6 in leaf declined. It indicated that this gene functions in root and it may participate in the Cd absorption and transport.3. SaNramp6 fused to green fluorescent protein was located to the plasma membrane in both onion(Allium cepa) epidermal cells and Nicotiana benthamiana lower leaf epidermal cells.4. We analyzed the function of SaNramp6 in yeast cell using yeast functional complementation study. The results of growth conditions, growth curves and Cd content revealed that, the transformants of SaNramp6 could accumulate more Cd, and the growth of the control stain with empty vector was better than the transformants of SaNramp6. It indicated that SaNramp6 could enhance the Cd content and the sensitivity of ycf1 yeast mutant under Cd stress condition.5. Positive transgenic Arabidopsis thaliana was obtained after transformed into A. thaliana by floral dip. After Cd treatment, the results of phenotype and physiological indexes indicated when overexpressed in Arabidopsis, SaNramp6 confers increased sensitivity to Cd. The results of NMT and determination of Cd content indicated the transgenic Arabidopsis has higher Cd absorption rate and Cd content. Also using Arabidopsis functional complemention studies, we found SaNramp6 leaded to enhance Cd content and make more toxicity.