Characterization And Functional Analysis Of Dm-SEPP CDNA And Its Role In Cold Response

Freezing avoidance is an essential trait for Antarctic Notothenioids to survive insubzero temperature.The reason why Dissostichus mawsoni,the Antarctic Notothenioids,as the main specie of cold icefish that inhabit the Southern Ocean in subzero degree water of-1.9°C deserves explored.Our previous study using transcriptome and comparative genome analysis indicated a significant gene copy number gain of Dissostichus mawsoni selenoprotein P(Dm-SEPP)during the evolutionary radiation of Antarctic Notothenioids,suggesting that Dm-SEPP contributes to this process,but the detailed structure and function of this gene product remain unclear.Here,we identified the Dm-SEPP c DNA characterization and functional analysis of Dissostichus mawsoni and made a preliminary study of its responsive mechanisim at low temperature.The detailed results are summarized as follows:1)We predicted the SECIS elements of Dm-SEPP through SECISearch3 and calculated its theoretical isoelectric point(p I)and molecular weight(MW).The results showed that Dm-SEPP c DNA contains two SECIS elements in the 3’-UTR,separately 1427-1502 bp and 2001-2074 bp;PI is 5.68,MW is 40.96 k Da;its ORF contains 17 selenocysteines(U).The configuration patterns of the selenocysteine codons are either UXU or UXUXU,where U is selenocysteine and X could be any amino acid.The SECIS elements of SEPP in the three species are conserved and the m RNA organization has the common pattern: AUGA_AA_GA.2)Amino acid sequence comparison of SEPP between D.mawsoni and other selected organisms showed that the highest amino acid identity was determined to be 63%,whether with Fugu or Nile tilapia.And most Sec residues(U)are conserved.The phylogenetic tree of the Dm-SEPP gene showed that the Dm-SEPP gene is closely related to the Fugu SEPP gene.Among the model organisms,the zebrafish Em-SEPP gene is the most closely related to the Dm-SEPP gene.3)Then the c DNA encoding Dm-SEPP(containing ORF and 3’-UTR)was cloned into pc DNA3.1(-)vector via the unique Xho I site and the neighboring Eco RI site.For convenient detection,a FLAG-tag encoding sequence was inserted before the stop codon(TAG)to express Dm-SEPP-FLAG.For the expression of the Dm-SEPP-FLAG,codon optimization was performed by changing the codon-adaptation index from 0.22 to 1.0 without altering the amino acid sequence.Dm-SEPP-FLAG was transfected into HEK-293 T cells,and then the cells were exposed to a temperature of 28°C as low temperature treatment.RT-q PCR and Western Blot showed that Dm-SEPP-FLAG was highly expressed in HEK-293 T cells both at 37°C and 28°C.It was also observed that over-expression of Dm-SEPP-FLAG could decrease ROS levels and increase cell viability at low temperature.The present data suggested that D.mawsoni Dm-SEPP protects HEK-293 T cells atlow temperature,probably via inhibition of ROS-related damage.4)To study the role of SEPP in cold adaptation of fish,we examined the expression of Em-SEPP in zebrafish ZF4 cells exposed to a lower temperature of 18°C for different times(3,5,30 days).Western Blot showed that the protein level of Em-SEPP obviously increased briefly then decreased gradually and maintained a higher level at low temperature throughout the experiment,with the highest level on the 3rd day.RT-q PCR showed that the m RNA levels of Em-SEPP were also up-regulated at low temperature,especially on the 3rd day.Em-SEPP-targeting sh RNA-expressing lentivirus was used to knockdown and low its expression.The m RNA and protein levels of Em-SEPP significantly decreased after lentivirus transduction.And Em-SEPP-sh RNA expressing ZF4 cells showed increased cell death both at 28°C and 18°C,suggesting a protective role of Em-SEPP at low temperature and normal culture condition.And knockdown of Em-SEPP led to increased ROS levels in ZF4 cells both at 28°C and 18°C.In conclusion,we studied the Dm-SEPP’s protective function in cold adaption of D.mawsoni through over-expression of Dm-SEPP in 293 T cells;and further study discovered ZF4 intrinsic Em-SEPP was induced at low temperature and knockdown of Em-SEPP showed a similar result.Above data indicated that SEPP might play an important protective role on fish survival in cold environment.