Cloning And Functional Research Of StNRAMP2 Cadmium Transporter In Potato(Solanum Tuberosum L.)

Potato {Solanum tuberosum L.)is the fourth largest food crop in the world.Heavy metal pollution in soil have attracted great attention.The study of the molecular mechanism of heavy metal accumulation in potato is of great significance to reduce the accumulation of heavy metals in edible parts of potato.Natural Resistance Associated Macrophage Protein is a kind of membrane proteins that transport Cd.This gene family has been studied in Oryza sativa and Arabidopsis thaliana,but the function of NRAMP gene in potato is rarely reported.Therefore,in this study,NRAMP family genes were identified from the potato genome,and bioinformatics analysis and gene expression analysis under different heavy metal stress were carried out to screen out the gene StNRAMP2 in response to cadmium stress.The cadmium tolerant potato variety’Yunshu505’ was used as the experimental material.The StNRAMP2 Gene was silenced by virus-induced Gene Silencing(VIGS),and heterologous expression of StNRAMP2 in tomato was detected after heavy metal stress.In order to provide a theoretical basis for studying the molecular mechanism of cadmium accumulation in potatoes,the relative expression of StNRAMP2 gene,the content of heavy metals and the activity of antioxidant enzymes were investigated.The main research results are as follows:(1)A total of five NRAMP genes were retrieved from potato genome,and the NRAMP genes were divided into four subfamilies by evolutionary analysis.The length of RNAMP genes ranged from 3490 to 8383 bp,encoding 481-1286 amino acids.The molecular weight is 53.33 kDa-139.32 kDa.The cis-acting elements in StNRAMP gene family members are different.StNRAMP3 only contains MYB cis-acting elements,while StNRAMP1 and StNRAMP4 contain abscisic acid responding elements.StNRAMP5 contains many plant hormone response elements,including gibberellin response element TATC-box,salicylic acid response element TCA-element,and plant auxin response element TGA-box,suggesting that the gene expression is co-regulated by a variety of plant hormones.(2)Real-time quantitative PCR analysis showed that the expression of potato NRAMP1 gene family members differed in response to different heavy metal stress.StNNRAMP1 hardly responded to heavy metal stress.Compared with 0 h stress,StNRAMP1 significantly responded to Cd stress,and the expression level reached the highest at 6 h stress in leaf tissues.These results suggest that StNRAMP2 may be involved in response to Cd stress.(3)Under 100 mg/kg Cd stress,the content of Cd in non-silenced StNRAMP2 potato plants was as high as that of root>old leaf>mature stem>young stem>young leaves>tubers.cadmium content in silenced StNRAMP2 was root>old leaf>Mature stem>Young stem>Young leaves>Tubers,compared with unsilenced StNRAMP2 gene plants,cadmium content in tubers was slightly increased in silenced plants,while cadmium content in other tissues was significantly decreased in silenced plants,which was reduced by 0.156 mg/kg and 66.30%in mature stems.In the old leaves,cadmium concentration was decreased by 0.164 mg/kg and 63.48%,indicating that potato StNRAMP2 gene is mainly involved in the transport of cadmium to older tissues,and silencing NRAMP2 gene is helpful to reduce the content of cadmium in potatoes and promote the transport of cadmium from stem to leaf.(4)Under 100 mg/kg Cd stress,StNRAMP2 overexpression in tomato showed no significant difference in Cd in stem of transgenic tomato compared with wild-type plants,while Cd concentration in root and leaf of transgenic tomato was significantly higher than that of non-transgenic plants,and the difference was most significant in root After 15 d of cadmium stress,the cadmium content of transgenic plants was 3.5-fold higher than that of wild type plants,while the cadmium content of transgenic plants was 3-fold higher than that of wild type plants under 30 d of cadmium stress.In leaves,StNRAMP2 gene was increased by 1.5-fold after 15 d stress and 1.6-fold after 30 d stress.Overexpression of StNRAMP2 gene increased the content of heavy metals in roots and leaves of tomato,suggesting that the function of StNRAMP2 gene was related to cadmium accumulation.