滇水金凤耐铜相关基因CAX3和CCS的克隆及表达分析

Cloning and Expression Analysis of CAX3 and CCS Genes of Impatiens uliginosa

植物CAX(Ca2+/H+反向转运蛋白)和CCS(铜锌超氧化物歧化酶铜伴侣蛋白)分别是重要的液泡膜转运蛋白和铜伴侣蛋白,通过不同机制在缓解重金属胁迫中发挥着重要作用。本课题组前期研究发现滇水金凤(Impatiens uliginosa)对铜胁迫具有很好的耐受性,并通过转录组测序筛选出耐铜相关基因。在此基础上,本研究以野生滇水金凤为材料,通过RT-PCR技术克隆获得耐铜相关基因CAX3和CCS,分别命名为IuCAX3和IuCCS,其cDNA序列长度分别为1320 bp和975 bp,分别编码439个和324个氨基酸。生物信息学分析结果表明,IuCAX3为不稳定蛋白,具有caca2超家族的保守结构域,定位在质膜、液泡上;IuCCS为稳定蛋白,具有PLN02957超家族的保守结构域,定位在叶绿体上。系统进化分析结果显示,IuCAX3和IuCCS均与喜马拉雅凤仙花的相应基因聚为一支,相似性分别为98.18%和89.81%。通过设置不同的铜处理浓度(0、5、10、15、20、25 mg·L^(-1))研究了IuCAX3和IuCCS基因在滇水金凤叶片中的表达情况,qRT-PCR分析结果表明,铜处理能诱导两个基因大幅上调表达,且铜浓度为15 mg·L^(-1)时的上调幅度最大,并均在处理12 d达到最高水平;随处理时间延长,IuCAX3的表达量先升高后降低,IuCCS的表达量在铜浓度不高于15 mg·L^(-1)时呈先升后降再升的趋势,但当铜浓度高于15 mg·L^(-1)后则呈先升后降的趋势,而且铜处理下IuCCS的表达水平明显高于IuCAX3,因此推测IuCAX3基因主要在铜胁迫前期起作用,而IuCCS基因在铜胁迫后期仍发挥重要作用。本研究结果可为深入研究滇水金凤耐铜分子机制提供一定的参考依据。

The CAX(Ca^(2+)/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 protecting plants from heavy metal threats through different mechanisms.In the previous research,it was found that Impatiens 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 study.Their cDNA sequence lengths were 1320 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 superfamily 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%respectively.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 treatment.With the expansion of treat time,the expression level of IuCAX3 gene increased first and then decreased,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 copper 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 further research on the molecular mechanism of copper resistance of I.uliginosa.