Study On Cold Tolerance Mechanisms Of Rhodotorula Mucilaginosa CTD02

Because of extreme conditions under deep sea,such as high pressure,low temperature(except for the hydrothermal vents)and darkness etc.,microorganisms live there rely on their special physiological metabolic pathways to adapt to such extreme environment.But until now,people's understanding of the physiological mechanism of these microorganisms living in the deep sea,especially fungi to adapt to the extreme environment is still very limited.In this paper,Rhodotorula mucilaginosa CTD02,isolated from 5000 m depth of Yapu trench,was used as a model to study its cold tolerance mechanisms with two aspects--growth characteristics and transcriptome expression.Temperature growth experiment showed that CTD02 could grow at 4 ? and 25 ?,but it grew slowly at 4 ?,suggesting that the strain had cold adaptability.At the same time,the unsaturation of fatty acids in CTD02 cell membrane at 4 ? was significantly higher than that at 25 ?,suggesting that the unsaturation of fatty acids in CTD02 cell membrane was closely related to its low temperature adaptability.Further studies were carried out to understand the low temperature adaptation mechanism of CTD02 by genomics and transcriptome analysis.The whole genome sequencing analysis showed that the genome size of the strain was 20.24 Mb,and the GC content was 60.49%.The results of transcriptome analysis showed that the expression of 3107 genes was significantly different between 4 ? and 25 ?,of which 2112 genes were significantly up-regulated and 995 genes were significantly down regulated.Low temperature inhibited the gene transcription of CTD02 in cell metabolism,cell division,protein translation,substance transport,extracellular protein secretion,cell movement and DNA damage,which were the main reasons for the slow growth of CTD02 at low temperature.In addition,the transcription level of genes in the pathway of unsaturated fatty acid metabolism and synthesis of CTD02 changed significantly under low temperature.Therefore,we selected seven representative genes of fatty acid synthesis pathway by using real-time fluorescent quantitative PCR(RTq PCR),and the validation results were consistent with the transcriptome data analysis.We further selected two genes with the largest difference in gene expression in fatty acid synthesis pathway.One is the long-chain fatty acid Co A ligase 2(ACSBG2)gene and the other one is acetylcoa acyltransferase 1(ACAA1)gene for gene silencing.After we silenced the two genes,we measured the difference of gene expression level in fatty acid synthesis pathway by RT-q PCR and found that the expression level of downstream genes of the two genes was significantly down regulated.At the same time,the growth rate of the recombinant strain after gene silencing was higher than that of the wild type strains at low temperature.Based on the above data,we believe that ACSBG2 gene and ACAA1 gene are two key genes in fatty acid synthesis pathway of CTD02,which are closely related to their low temperature adaptability.ACSBG2 gene shortens the length of fatty acid chain of cell membrane by reducing the activation ability of acetyl group at the end of fatty acid,so as to improve the fluidity of cell membrane at low temperature.ACAA1 gene revealed that eukaryotic microorganisms could inhibit cholesterol synthesis by regulating the metabolism direction of acetylcoa at low temperature,thus increasing the synthesis of intracellular fatty acids and ensuring the fatty acid supply of cell membrane at low temperature.After gene silencing at room temperature and low temperature,the content of fatty acids in the cell membrane of the recombinant strain also confirmed our hypothesis.Adjusting the ratio of the content of unsaturated fatty acids in the cell to the short chain unsaturated fatty acids are a main way for CTD02 to cope with the cold environment.