大豆GmDnaJ1蛋白对几种重金属胁迫的响应研究

Expression characterization of soybean GmDnaJ1 in response to heavy metal stresses

关键词:  大豆  DnaJ-like蛋白  重金属胁迫  重金属响应  分子伴侣

Key words:  soybean  DnaJ-like protein  heavy metal stress  heavy metal response  molecular chaperone

摘要:

通过对大豆铝胁迫下的转录组测序分析,发现一个差异表达的基因,其编码一个具有101个氨基酸残基的DnaJ-like分子伴侣蛋白-GmDnaJ1(Glycine max DnaJ1),等电点为8.97; 序列分析表明该蛋白具有典型的高度保守的J domain功能域,是一种类型III的J蛋白; 通过对其序列的同源性及进化关系分析,推测该蛋白可能响应重金属胁迫。为进一步探究GmDnaJ1是否能够对重金属胁迫产生应答反应,试验分别以0或100 μmol·L^-1 Cu²⁺、Pb²⁺和Cd²⁺溶液胁迫处理的不同时间(0、12、24、48和72 h)大豆根尖RNA为材料,通过实时定量PCR研究了该蛋白基因的表达特征。结果表明:与对照相比,GmDnaJ1受Cu、Pb和Cd等重金属的诱导而强烈表达,呈现先升高后降低的趋势,其中Pb、Cd处理24 h后表达水平达到峰值,而Cu处理48 h后达到峰值; 此外,GmDnaJ1对Cu、Pb和Cd胁迫的响应程度也不同,表明该基因对这三种重金属的响应模式存在差异。根据以上研究结果,推测大豆GmDnaJ1蛋白不仅响应铝毒胁迫,而且可能在响应重金属胁迫方面具有重要的作用,参与了大豆对重金属毒害的抵抗。该结果为深入研究GmDnaJ1在重金属胁迫响应中的功能及其分子机制提供了一定的依据。

Abstract:

Although necessarily contributing to regulating the activities of the proteins that involved in maintaining growth and development of living organisms,heavy metal elements,such as Cu,Pb and Cd,will become detrimental to plants at excess concentrations in the environment. However,plants can respond to and survive in heavy metal contaminated conditions through different strategies,among which arousing multiple heavy metal-related genes is very essential. Therefore, identification and functional analysis of crucial genes that associated with heavy metal response is of great importance in elucidating the plant tolerance mechanism and finally improving the resistance to heavy metal stress. DnaJ proteins,belonging to molecular chaperone family,were widely found to be very important in response to biotic and abiotic stresses. In the present study,we first identified an aluminum responsive gene, GmDnaJ1,(Glycine max DnaJ1)from soybean transcriptome sequencing data. Then we analyzed the sequence of this gene and found that it encoded a DnaJ-like protein,containing 101 amino acids with the isoelectric point of 8.97. By means of homogeneous analysis,we further revealed that GmDnaJ1 had the highly conserved J domain structure,which was the typical characteristics of type III DnaJ proteins. Phylogenetic analysis of GmDnaJ1 with other J proteins from different plant species implied that the protein probably exerted its role in response to heavy metal stress. In order to investigate the possible function of GmDnaJ1,we used pre-cultured and uniformly grown soybean seedlings and then treated with 0 or 100 μmol·L^-1 Cu²⁺,Pb²⁺ and Cd²⁺ solutions. The root tips of each sample were subsequently harvested at 0,12,24,48 and 72 h,respectively. RNA were extracted and subjected to reverse transcription. After that,real time quantitative PCR(RT-qPCR)was performed to reveal the expression patterns of GmDnaJ1 under different heavy metal stresses for different times. The results indicated that comparing with the controls,GmDnaJ1 could be dramatically up-regulated by Cu,Pb and Cd stresses over time. The expression level of GmDnaJ1 increased at first and then decreased. We noticed that the peak of GmDnaJ1 expression appeared at 24 h after being treated with Pb or Cd solution,whereas the peak of the gene occurred at 48 h when the soybean suffered from Cu stress; Furthermore,we observed that the extent of GmDnaJ1 expression varied in response to different heavy metal elements,implying a possibility that GmDnaJ1 exhibited different response models when the plants suffered from different heavy metal stresses. The above results suggested that the soybean GmDnaJ1 not only involved in aluminum response,but also played an important role in response to heavy metal stresses,such as Cu,Pb and Cd,and probably participated in the resistance to heavy metal toxicity. The present findings will provide some experimental basis for the functional analysis of GmDnaJ1 and its molecular mechanism in response to heavy metal stress.