Studies On The Association Of Zinc Finger Protein Gene RkMsn4 With The Cold Adaptation Of Rhodosporidium Kratochvilovae

Transcription factor Msn4 is a typical C2H2 type zinc finger protein,which is the main transcription factor regulating the response of external stress in eukaryotic cells.It also plays an important role in the stress response regulation of eukaryotic cells,such as starvation stress,salt stress,oxidative stress,hypertonic pressure stress and low temperature stress.However,there have been no reports to study the relationship between the transcription factor Msn4 and the biosynthesis of polyunsaturated fatty acids（PUFAs）in fungi at low temperatures.Our previous studies have showed that the contents of PUFA in cell membrane was increased significantly as the culture temperature decreased in the oleaginous yeast—Rhodosporidium kratochvilovae strain YM25235,which promoted the cold adaption of the strain at low temperature,but the specific underlying regulation mechanism is still unclear.In this study,the Msn4homologous gene RkMsn4 of YM25235 was used;to analyze its effects on the cold adaptation of the YM25235 and the biosynthesis of PUFA in YM25235 at low temperature.The mRNA transcription level of RkMsn4 gene and the glycerol content in YM25235 cultured at low temperature were first analyzed.The results showed that the increase of mRNA transcription level of RkMsn4 gene was observed after cultivation at15°C for the first 1h.When the YM25235 was cultured at 15°C for 8 h,the mRNA transcription level of RkMsn4 gene was increased by 1.41 times.The content of glycerol in YM25235 cultured at 15°C also increased accordingly.This indicated that the RkMsn4 gene might be involved in the cold adaptation of YM25235.The RkMsn4 gene was cloned and its sequence was analyze.The results showed that the amplified Rk Msn4 gene contained a complete open reading frame of 2625 bp,potentially encoding a protein of 874 amino acids with a estimated molecular weight of94.5kD.Sequence analysis showed that the encoded amino acid sequence of the RkMsn4 gene had a highly conserved Zn F-C2H2 domain,protein kinase binding region,Fungal_trans domain and CHAD domain,which shared 99%identity to that of Rhodotorula toruloides Msn4 gene and 70%identity to that of Leucosporidium creatinivorum Msn4 gene.Phylogenetic analysis also indicated RkMsn4 encoded protein shared closer relationship with Msn4 proteins from red yeasts.These results indicated that the RkMsn4 gene cloned from YM25235 is a potential Msn4 gene.To uncover the association of RkMsn4 gene with the cold adaptation of YM25235and the PUFA biosynthesis in YM25235 at low temperature,the recombinant plasmid pRHRkMsn4 was constructed and further transferred into the YM25235 strain for overexpression.Enviromental stress resistance analysis showed that overexpression of RkMsn4 gene can significantly increase the content of glycerol in YM25235 strain,and enhance the tolerance of transgenic YM25235 to high osmotic pressure,salt stress and low temperature stress.GC analysis of fatty acids in the RkMsn4 gene-overexpressed YM25235 cultured at 15°C showed that the contents of linoleic acid（LA）andα-linolenic acid（ALA）were increased from 16.81%and 6.14%to 31.56%and 17.32%respectively,in comparison with that of YM25235 transformed with pRH2304 and cultured at 15°C.The content of oleic acid（OA）in the RkMsn4 gene-overexpressed YM25235 was also decreased accordingly.Further analysis also showed that overexpression of RkMsn4 gene caused a increase of mRNA transcription level of theΔ^12/15-desaturase gene RKD12 by 2.54 times at 15℃.These results indicated that overexpression of RkMsn4 gene increased the mRNA transcriptional level of RKD12and the PUFA biosynthesis in the YM25235 at low temperature,subsequently improving the cold adaptation of YM25235 at low temperature.The interaction between RkMsn4 protein and the RKD12 gene promoter RKD12P was analyzed by yeast one-hybrid assay,with the aim to reveal the relationship between transcription factor RkMsn4 and the increase of mRNA transcriptional level of RKD12at low temperature.3617bp upstream sequence of the RKD12 coding sequence,designated as RKD12P,was first cloned.Sequence analysis showed that this sequence contained typical eukaryotic promoter elements such as TATA-box,BRE,CAAT-box,GC-box and DPE,as well as environmental stress response related stress response element（STRE）,Low-temperature-responsive element（LTRE）and metal-responsive element（MRE）.For functional identification of the RKD12P,this fragment was inserted into vector pRH2304 by replacing the promoter PRtGPD1 upstream of the RtGFP gene to construct a new recombinant plasmid pRHRKD12PGFP,which was then transformed into the YM25235.Both fluorescence microscopic observation and western blotting analysis indicated that RKD12P was a functional promoter.The promoter RKD12P and RkMsn4 gene were further inserted respectively plasmids pAbAi and pGADT7-Rec2 of yeast one-hybrid system to generate recombinant plasmids pAbAiRKD12P and pGADT7Rec2RkMsn4,which were then cotransformed into Saccharomyces cerevisiae Y1H yeast.Antibiotic resistance test indicated that the transcription factor RkMsn4 encoded by interact with the promoter RKD12P.These results showed that the gene RkMsn4-encoded protein might functioned as a transcription factor to interact with RKD12P at low temperature and improve the transcription of RKD12 gene.In order to reveal other regulatory proteins involved in the increase of RKD12gene transcription at low temperatures and Chromatin immunoprecipitation（Ch IP）method was used to isolate the proteins interacted with the promoter RKD12P at low temperature.The results showed that a total of 14 nuclear-localized proteins were obtained.Among them,6 kinds proteins were found in both samples cultured at 15℃and 30℃.2 proteins were only present in the sample cultured at 30℃.The rest 6proteins were only present in the sample cultured at 15℃,and some of them have been reported to be involved with environmental stress response.Studies of the effects of these proteins on the transcriptional regulation of RkD12 gene at low-temperature will be carried out in our furure work.Our previous data have showed YM2535 is an oleagnious yeast,and it also produces carotenoids such asβ-carotene,torularhodin and torulene.Therefore,the effects of RkMsn4 gene overexpression on the biosynthesis of carotenoids and lipid in YM25235 were simultaneously analyzed.Total carotenoids and lipid were extracted from the RkMsn4 gene-overexpressed YM25235 cultured at 30°C,and the mRNA transcription level of some genes related to the carotenoids and lipid biosynthesis was analyzed.The results showed that overexpression of RkMsn4 gene increased the mRNA transcription level of carotenoid biosynthesis-related genes the the transgenic YM25235,resulting in a significant increase of the carotenoids content.However,the lipid content in the the transgenic YM25235 was slightly lower than control,although increased mRNA transcription level of lipid biosynthesis-related genes was observed.It is speculated that more intracellular acetyl-CoA molecules were used as substrate of the biosynthesis of carotenoids caused by the overexpression of RkMsn4 gene in YM25235.In summary,overexpression of the RkMsn4 gene improved the resistance of the YM25235 to high osmotic stress,salt stress,and low temperature stress.It also increased the transcription level of RKD12 gene and the biosynthesis of PUFA in YM25235 at low temperature,improving the cold adaptation of YM25235.The increase of the RkMsn4 transcription level was the result of the interaction of RkMsn4protein with RKD12 gene promoter at low temperature.Furthermore,overexpression of RkMsn4 gene promoted the biosynthesis of carotenoids and decreased the the biosynthesis of lipid in YM25235.This study will contribute to uncover the cold adaptation mechanisms,signal pathway of PUFA biosynthesis and its regulation at low temperature,the associtation of the RkMsn4 gene with the biosynthesis carotenoids and lipid in YM25235,providing a basis for the further research of related theories and applications.