The Relationship Between Rkhogl Gene And Cold Adaption Of Rhodosporidium Kratochvilovae

High osmolarity glycerol mitogen-activted protein kinase signaling transduction pathway(HOG-MAPK pathway)is the most prominent pathway that allows yeast cells to perceive and quickly respond to altered omolarity.In eukayotes,this pathway is proved to be vital in stress responses,including oxygen stress,citric acid stress,thermal shock and clod stress.However,the relationship between the HOG-MAPK pathway and the biosynthesis of polyunsaturated fatty acid(PUFA)at low temperature has not been reported in oleaginous fungi.Our previous studies found that the contents of PUFA in cell membrane increased significantly as the culture temperature decreased in Rhodosporidium kratochvilovae YM25235 strain,and the inhibition of PUFA synthetic could affect the cell growth at low temperature.Those results indicated that PUFA may associated with the cold adaption of YM25235.In this study,we intend to clone and analysis the function of Rkhogl,one of the vital genes in HOG-MAPK pathway,and further explored the roles of Rkhogl and the HOG-MAPK pathway in the temperature adaptability and PUFA biosynthesis at low temperature in R kratochvilovae YM25235.Firstly,the mRNA level and the phosphorylation level of Rkhogl in YM25235 at low temperature,and also the the content of glycerol were analyzed.And the results shown that the mRNA expression level,phosphorylation level of Rkhogl gene and the content of glycerol in YM25235at 15? within 1 h was higher than that of 30?.Therefore we inferred that Rkhogl gene might be involved with the cold adaption of YM25235.Secondly,cloning and function analysis of Rkhog1gene.A pair of gene-specific primers was designed based on the RNA-seq data of YM25235.A full-length cDNA fragment of 1080 bp,designated RKHogl,was obtained from YM25235 using cDNA as template by PCR amplification,which encodes 365 amino acids with an estimated molecular mass of 40.8 kDwith the isoelectric point of 5.33.Further sequence analysis revealed that the deduced amino acid sequence of Rkhogl contained the conserved ATP-binding signature of protein kinases,the active site of Ser/Thr protein kinase,a C-terminal common docking motif and a TGY motif.Multiple sequence alignment of Rkhogl show that the deduced amino acid sequence shared the highest identity of 97.8%to Rhodotorula graminis Hogl,64.8%to Saccharomyces cerevisiae Hogl and 71.1%to Candida albicans Hogl.Those results indicated that the cloned cDNA fragment was a novel hog1 gene.To elucidate the function of the isolated novel putative Rkhogl gene,Rkhogl was subcloned into pWXY3.0,and the resultant recombinant plasmid pW3Rkhogl was further transformed into hogl null mutant Saccharomyces cerevisiae strain to analyze the relationship between Rkhogl gene and the salt stress,osmotic stress and low temperature stress.This Rkhogl gene from YM25235 could rescued the function of Schogl in S.cerevisiae hogl null mutant stainand the Rkhog1rescued strain showed smilar phenotypic types with the wild S.cerevisiae strain in the glycerol production and the salt-tolerance,osmotic-tolerance andcold-tolerancelt indicated that the Rkhog1 gene cloned from YM25235 was a functional hogl gene.Finally,analyzing the relationship between Rkhog1 gene and the PUFA biosynthesis at low temperature.Rkhogl was subcloned into pRH2304,and the resultant recombinant plasmid pW3Rkhogl was further transformed into YM25235.The stress resistance analysis shown that overexpression of Rkhogl gene could increase the biosynthesis of glycerol and enhance the salt-tolerance,osmotic-tolerance,cold-tolerance of YM25235.Fatty acid analysis shown that the overexpression of Rkhogl gene in YM25235 could increase the content of linoleic acid and a-linolenic acid from 28.16%and 8.87%to 36.76%and 21.11%increased by 1.30 times and 2.38 times in comparison with control under the condition of 15?,and the content of oleic acid was significantly decreased.Further,the mRNA expression level of?12-fatty acid desaturase gene RKD12,which is responsible for the sequential conversion of oleic acid to linoleic acid and a-linolenic acid analysis,also shown that it increased by 3.5 times at 15?,but unchanged at 30?.It indicated that under low temperature condition Rkhogl gene can specifically increase the mRNA expression level of RKD12 gene,resulting in the increase of the content of PUFA and improve the cold adaption of YM25235.In addtion,the mRNA expression level of the transcription factor Msn4 in YM25235 was increaed at 15?,and overexpression of Rkhogl gene could make this increase more significant,and at 30? there have no significant change.Those results indicated that the expression of Rkhogl gene could increase the transcription of Msn4 gene,and the transcription factor Msn4 might associated with the biosynthesis of PUFA at low temperature in YM25235.In conclusion,a new hogl gene named Rkhogl in HOG-MAPK pathway was identified in this study,which not only correlated with the stress resistance of low temperature,hyperosmotic and salt in R.kratochvilovae YM25235,but also correlated with the mRNA expression level of RKD12 gene,Msn4 gene and PUFA biosynthesis at low temperature.This study will be helpful to illuminate the signal transduction pathway and regulatory mechanism for the biosythesis of PUFA at low-temperature in R.kratochvilovae YM25235,help to reveal the mechanism of cold adaptability in oleaginous micro-organisms,improve the production of unsaturated fatty acid linoleic acid and a-linolenic acid through the genetic engineering means and further lay the foundation for large-scale production of PUFA.