The Relationship Between Histidine Kinase HisK2301 And Cold Adaption Of Rhodosporidium Kratochvilovae

Histidine kinases(HKs)are transmembrane transferase proteins,they function as cell receptors of signaling molecul.es during the signal transduction process.Prokaryotic HKs generally contain two highly conserved domains,histidine kinase-like ATPase catalytic(HATPase)domain and the histidine kinase(HisKA)domain.As signal transduction,the HKs act as sensor to sense the changes of external environment conditions,the HATPase domain of histidine kinase binds ATP and autophosphorylates a conserved histidine residues of HATPase domain.HKs generally act as components of two-component signal transduction systems(TCSs)for signal transduction.TCSs are widely distributed in prokaryotes and eukaryotes,which generally contain a sensor HK and a cognate response regulator(RR).Two-component signal transduction pathway includes a signal input,histidine kinase autophosphorylation,response regulator phosphorylation and signal output.HKs autophosphorylate a conserved histidine(H)residue in response to an environmental signal.The phosphate group is then transferred to a conserved aspartic acid(D)residue in a response regulator(RR)protein.The activated RR carries the signal downstream to directly control the transcription of target genes.A oleaginous and cold-adaptive yeast strain Rhodosporidium kratochvilovae YM25235 is used in the study.Transcriptome sequencing was first applied to analyze the underlying cold adaption mechanims for this strain cultured at 15 ? and 30 ?,respectively.The result showed that cold adaption of YM25235 was significantly associated with the MAPK cascade signaling pathway.The mRNA expression levels of 11 genes of the total detected 73 genes in the MAPK cascade signaling pathway analysis showed more than 2 time changes.Among them,9 were significantly down-regulated,especially the genes that associate with the signal transduction pathway of osmotic stress almost showed down-regulated at mRNA expression levels.In addition,we found that 5 potential histidine kinase gene sequences were obtained by transcriptome sequencing,but only the histidine kinase gene HisK2301 was up-regulated.Analysis of the mRNA expression level of histidine kinase gene HisK2301at 15 ? showed 2.6 times higher than that at 30 ?.Therefore,we inferred that the two-component signal transduction system including.histidine kinase HisK2301 might be involved with the cold adaption of the Rhodosporidium kratochvilovae YM25235.According to the sequence information obtained from transcriptome sequencing,gene-specific primers were designed and used ampified the HisK2301 gene by PCR amplification.A full-length 3888bp cDNA sequence was obtained.Analysis result indicated that this sequence contained a complete open reading frame that encods 1295 amino acids.Analysis also showed that the encoded protein histidine kinase HisK2301 contained three conserved HisKA,ATPase and REC domains Besides,LCR domain and 6 HAMP domains locating at the N terminal of histidine kinase HisK2301,as well as two potential transmembrane regions.A GFP-tagged fusion proteins of HisK2301 expressed in YM25235 to domenstrated that HisK2301 is membrane-bounded protein.All results preliminary indicated the HisK2301 sequence is a novel and potential histidine kinase gene,and the encoded histidine kianse HisK2301 belongs to group III fungal hybrid-type histidine kinase,but unlike the reported other fungal type III histidine that generally are soluble protein and located in cytosol,HisK2301 obviously is a tranmembrane protein.In order to verify the function of the obtained sequence HisK2301 and its correlation with cold adaptation,the gene was inserted into the plasmid pYES3/CT and pRH2304 to generate recombinant plasmids pY3HisK2301 and pRHHisK2301,respectively.After verification,the recombinant plasmids were transformed into Saccharomyces cerevisiae INVScl and YM25235 for expression,respectively,to analyze its effects on the resistance of transgenic cells to low temperature,hyperosmotic,salt,oxidative and metal ion stresses.The results showed that HisK2301 gene can imporve the resistance to low temperature,high osmotic pressure and salt stresses of INVScI and YM25235 cells transformed with HisK2301 gene.The N-terminal transmembrane region of HisK2301 was further deleted and the function of this mutant was analyzed in INVScl with the same method.The results showed that deletion mutant could not improve the stress resistance of the transgenic yeast,which might result from the deletetion of the transmembrane region of histidine kinase HisK2301 and the deletion mutant could not target to the cell membrane and lost the ability to perceive environmental stress signals.Fatty acid analysis of YM25235 transformed with HisK2301 gene by gas chromatography showed that the content of linoleic acid and a-linolenic acid increased by 5.06%and 1.56%in comparison with YM25235 transformed with control plasmid pRH2304,and the content oleic acid was decreased by 3.6%accordingly.Analysis of the mRNV expression levels of ?12-fatty acid desaturase gene which is responsible for the sequential conversion of oleic acid to linoleic acid and a-linolenic acid also showed significant increase at 15 ?,but unchanged at 30 ?.These indicated that HisK2301 gene can specifically increase the mRNA expression level of A1-fatty acid desaturase gene,resulting in the increase of the content of linoleic acid and a-linolenic acid and improve the cold adaption of YM25235.In conclusion,a new histidine kinase gene HisK2301 is identified in the study,which not only correlated with the stress resistance to low temperature,hyperosmotic and salt for R.kratochvilovae YM25235 but also with the mRNA expression level of?12-fatty acid desaturase gene and polyunsaturated fatty acids biosynthesis at low temperature.This study will be helpful to reveal the signal transduction pathway for the biosythesis of linoleic acid and a-linolenic acids at low-temperature in R.kratochvilovae YM25235,uncovering the regulatory mechanism of linoleic acid and a-linolenic acids biosynthesis at low-temperature in YM25235.In addition,this study will further contribute to uncovering the regulation mechanism of PUFA biosynthesis in fungi and providing a reference for the elucidation of fungal cold adaption mechanisms.Exploring the regulation mechanisms of polyunsaturated fatty acid biosynthesis at low temperature will also be helpful for future ressarch and applications in PUFA production by using regulation mechanisms.