Function Analysis Of BrpHMA1 And BrpHMA2-2

Heavy metal transporting ATPase(HMA)is a transmembrane transporter protein,which generally contains six to eight transmembrane fragments.It belongs to the P-type ATPase family and provides energy for metal transport mainly through hydrolysis of ATP.HMA proteins participate in the absorption and transport of metal elements during plant growth and development,and play an important role in plant metal transport.Chinese flowering cabbage(Brassica parachinensis L.)is a special vegetable which is very popular in South China.Recent years,the rapid economic development and the use of various chemical products have brought heavy metal pollution to soil more seriously,and the vegetables we eat ordinary are polluted inevitably.In this paper,c DNA fragments of BrpHMA genes of the Chinese flowering cabbage were cloned based on the previous transcriptome data analysis on the Cd stressed-Chinese flowering cabbage and the whole genome sequence information of Chinese cabbage.The bioinformatics analysis were further performed on BrpHMA proteins together with the other species HMA proteins.The expression pattern of the BrpHMAs were analyzed.The function of BrpHMA1 and BrpHMA2-2 were analyzed through heterologous expression in yeast and Arabidopsis.We obtained the major results as follows:1.By analyzing the previous transcriptome data of the Cd stressed-Chinese flowering cabbage and the whole genome sequence information of Chinese cabbage,the primers were designed for cloning of BrpHMAs,and seven BrpHMA genes were cloned.By constructing a phylogenetic tree together with HMAs proteins of Arabidopsis and rice,BrpHMA proteins were classed into three major categories,namely P_1B-1,P_1B-2and P_1B-4ATPase subfamily.BrpHMA1 belongs to the P_1B-4subfamily,BrpHMA2,BrpHMA2-1,BrpHMA2-2,BrpHMA3 and BrpHMA4 belong to the P_1B-2subfamily,and BrpHMA5 belongs to the P_1B-1subfamily.2.Seedlings or elder plants of Chinese flowering cabbage was treated with different cadmium concentrations,and RNA was extracted from different parts of plants for RT-q PCR to analyze the expression of BrpHMAs.Results show that the all the analyzed BrpHMA genes are responsive to cadmium stress:BrpHMA1,BrpHMA2-2 were obviously induced by cadmium,and the expressions of BrpHMA2-1,BrpHMA3,BrpHMA4,BrpHMA5 up-regulated or down-regulated under different cadmium concentration or after different time of treatment.3.Gene expression analysis by uisng GUS driven by gene promoter showed that both BrpHMA1 and BrpHMA2-2 present tissue-specific expression.GUS driven by the BrpHMA1 promoter is only expressed in anthers.GUS driven by the BrpHMA2-2promoter is expressed majorly in the vascular tissues,and also in filigree,anthers and stigmas.4.Subcellular localization analysis of 35S::eGFP-BrpHMA1 and35S::eGFP-BrpHMA2-2 proteins using the Arabidopsis protoplast transient expression system revealed that BrpHMA2-2 protein is located on the plasma membrane.No fluorescence was observed in the eGFP-BrpHMA1-transfected protoplasts,and the cellular location of BrpHMA1 needs to be further studied.5.BrpHMA1 and BrpHMA2-2 genes were heterologous expressed in yeast cells.The growth of the yeast cells transformed with BrpHMA1 gene or with empty vectors had no significant difference under cadmium stress.While yeast cells transformed with BrpHMA2-2 grew worse and were more sensitive to cadmium stress than the cells transformed with empty vectors.6.Homozygous transgenic Arabidopsis thaliana were obtained by transforming with BrpHMA1 or BrpHMA2-2 mediated agrobacterium.No significant difference were obversed in the growth of the transgenic and wild-type plants under cadmium stress,but the contents of chlorophyll in the transgenic plants was obviously lower than that in wild-type plants.It showed that the overexpression of BrpHMA1 or BrpHMA2-2 decreased the Cd tolerance of plants.