| There are many microorganisms that can grow normally under heavy metal stress in natural environment, such as yeast, algae, bacteria and so on. In the process of removing heavy metals and adapting to the environment, these microorganisms have to have a series of complicated physiological and biochemical reactions in their microbial cells. The Antarctic yeast Rhodotorula mucilaginosa AN5 was taken as the research object to analyze the transcriptomic changes under the stress of Cu2+ by Illumina sequencing analysis, the adaptation mechanism to the heavy metals of Antarctic yeast AN5 from m RNA level. Two genes which had significant differences were carried out for further research, including the cloning analysis, purification and expression of WD repeat protein(WDR); the cloning and sequence analysis of glutathione S-transferase(GST).(1) The RNA-seq sequencing of the Antarctic yeast AN5 was detected. The control group got 41 738 378 read sequences and the experimental group received a total of 51 247 104 sequences. 407 differentially expressed(more than 2 fold) genes were confirmed, including 305 up-regulated genes and 102 down regulated genes. These genes were classified through the GO annotation, and most unigenes were attributed to biological processes. 6 414 unigenes were analyzed by the egg NOG to understand the distribution of gene function. A total of 3 282 isotig got the KEGG annotation, whose proportion of metabolic pathways was 32.3%. Through these analysis, the differentially expressed genes caused by environmental stress were confirmed.(2) Three genes from differentially expressed genes were selected to do the fluorescence quantitative PCR, including the WD repeat protein gene, glutathione S-transfer and heat shock protein 90(Hsp90). The results showed that the expressions of GST and WDR were up-regulated by the heavy metal stress, the expression of HSP90 was increasing at 4 h and 12 h while the expression at 48 h had a declining tendency. GST gene expression significantly increased at 48 h, which was about 5 times higher than control group. The expression of HSP90 gene at 12 h was significantly higher than control group.(3) WD-repeat proteins are involved in the process of transcription, signal transduction and cell proliferation. In this experiment, an important differential gene WD-repeat from the transcription group of the Antarctic yeast AN5 was cloned. The length of the obtained c DNA sequence was 1 079 bp, which encode 361 amino acids. The results of bioinformatics analysis showed that the molecular weight of WDR was 40.91 k Da and the isoelectric point was 5.25, which was mainly hydrophilic. No signal peptide sequence was detected in the transmembrane region. And Tag His was added into the downstream. The recombinant plasmid was successfully constructed on the p ET-28 a vector. And IPTG was used to induce the expression and the target protein was purified by affinity chromatography.(4) Glutathione S-transferase plays a key role in the metabolism of endogenous and exogenous toxic substances in the organism. GST can be combined with some toxins in the cell to prevent toxic accumulation of toxic substances caused by excessive accumulation of cells. In addition, it can also be directly coupled to some toxic substances in the environment. After the Antarctic yeast AN5 GST gene cloning and sequence analysis the fact was found that the c DNA length of Antarctic yeast AN5 GST genes was 969 bp. The number of amino acids was 284 and protein molecular weight was 32.58 k Da, theorical p I was 7.74, hydrophilic was strong, and it not only did not exist obvious transmembrane region but also was not a signal peptide. The two stage structure and the three stage structure prediction showed that the protein had a lot of helices and coil.The above research is to provide some reference for understanding the mechanism of the heavy metal in the Antarctic yeast. |
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