Cloning and Expression Analysis of CAX3 and CCS Genes of Impatiens uliginosa
The CAX(Ca2+/H+exchanger)and CCS(copper chaperone for Cu/Zn-SOD)proteins in the plants are important tonoplast membrane transporters and copper chaperones,which play important roles in protec-ting plants from heavy metal threats through different mechanisms.In the previous research,it was found that Impa-tiens uliginosa had good tolerance to copper stress,and the genes contributing to copper resistance were screened out by transcriptome sequencing.Based on these,the copper resistance-related genes,CAX3 and CCS,were cloned by RT-PCR technology using I.uliginosa as material,and were named IuCAX3 and IuCCS respectively in this stud-y.Their cDNA sequence lengths were 1 320 and 975 bp,encoding 439 and 324 amino acids,respectively.The bioinformatics analysis showed that IuCAX3 was an unstable protein with a conserved domain of the caca2 superfam-ily located on plasma membrane and vacuole.IuCCS was a stable protein with a conserved domain of the PLN02957 superfamily located on chloroplast.The phylogenetic analysis based on amino acid sequences of IuCAX3 and IuCCS showed that they were both clustered with those of I.glandulifera with the similarity as 98.18%and 89.81%respec-tively.Different copper concentrations(0,5,10,15,20 and 25 mg·L-1)were set to study the expression level of IuCAX3 and IuCCS in I.uliginosa leaves.qRT-PCR analysis showed that the expression level of IuCAX3 and IuCCS genes were both up-regulated obviously under copper treatments,and the range of up-regulation was the largest when copper concentration was 15 mg·L-1 with the largest expression level on the 12th day of treat-ment.With the expansion of treat time,the expression level of IuCAX3 gene increased first and then de-creased,and that of IuCCS changed different under different copper concentrations,which showing a rising-falling-rising trend when copper concentration not higher than 15 mg·L-1 and a rising-falling trend when cop-per concentration higher than 15 mg·L-1.The expression level of IuCCS gene was generally higher than that of IuCAX3 gene.It was speculated that IuCAX3 might mainly act at early stage of copper treatment,while the effect of IuCCS became more significant at the late stage.These results could provide some references for fur-ther research on the molecular mechanism of copper resistance of I.uliginosa.
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