CbCOR15, A Cold-regulated Gene From Alpine Chorispora Bungeana, Confers Cold Tolerance In Transgenic Tobacco

We isolated a novel cold-responsive gene,CbCOR15,from Chorispora bungeana, a rare alpine subnival plant and a valuable species for research targeting the mechanism of cold-hardiness and stress-responsive genes.We investigated the patterns of tissue-specific expression and stress-related expression of CbCOR15 by Semi-quantitative RT-PCR.The function of CbCOR15 in stress protection was investigated by transgenic approach.We got results as follows:1.RACE(Rapid Amplification of cDNA Ends) was performed to isolate cold-regulated CbCOR15(EF208112) from alpine Chorispora bungeana.The full-length CbCOR15 cDNA of alpine Chorispora bungeana comprises 612 bp -- 423 bp in the open reading frame(ORF),83 bp in the 5'- untranslated region(UTR),and 106 bp in the 3'-UTR with a poly(A) tail.The ORF encodes a polypeptide of 140 amino acids,for which the calculated isoelectric point and molecular mass are predicted to be 5.20 and 14.9 kDa,respectively.The deduced protein CbCor15 has an amino acid sequence that is highly conserved with that of ApCOR15a,AtCOR15a, AtCOR15b,BN115,and CBCOR15.This similarity indicates that CbCOR15 belongs to the COR15 gene family,members of which are frequently expressed in vegetative tissues in response to low-temperature environmental stresses.2.Sequence analyses showed that the CbCor15 protein is highly hydrophilic, especially the C-terminal two-thirds of the polypeptide.CbCor15 amino acid sequence analysis indicated that the N-terminal has several features in common with transit peptides that target proteins to the stromal compartment of chloroplasts.3.CbCOR15 expression was detected only in the leaves after 1 d of treatment at 2℃,thereby demonstrating that this gene is leaf tissue-specific.Expression was induced at both 4℃,0℃and -4℃and was maintained at high levels for at least 7 d. Expression was also induced by ABA,PEG,but not by H₂O₂.4.We introduced CbCOR15,CbCOR15m with the CaMV 35S promoter,into tobacco.The transgenic tobacco plants were confirmed by PCR,and real time RT-PCR and western blot analysis were used to detected expression of CbCOR15, CbCOR15m.The electrolyte leakage,chlorophyll content,and RWC did not differ between transgenic lines -- high versus weak expression -- indicating that the extent of tolerance is not directly correlated with CbCOR15 accumulations.This suggests that freezing tolerance is a multi-genic trait,resulting from several factors.Therefore, the effective threshold for such tolerance is apparently attained only through the collective action and cumulative effect of individual COR/LEA proteins,each with its own limited influence.5.All transgenic lines of CbCOR15 showed greater chilling/freezing tolerance than the non-transgenics and the transgenic lines of CbCOR15m.When chilled,the transgenic T₁ plants of CbCOR15 had less electrolyte leakage and MDA content, higher chlorophyll content,and greater relative water content.This suggests that the CbCOR15 gene product functions in plasma membrane protection,prevents water loss and crystallization of cellular components,and increases chilling tolerance.Our results also demonstrated that freezing tolerance is significantly increased in CbCOR15 transgenic plants.We conclude CbCOR15 plays an important role in conferring cold/freezing tolerance in transgenic tobacco plants,similar to the functioning of AtCOR15a and Wcor15.In conclusion,CbCOR15 expression was detected by low temperature,ABA, PEG,but not by H₂O₂.Chilling and freezing tolerance is significantly increased in CbCOR15 transgenic plants.CbCOR15 gene product functions in plasma membrane protection,prevents water loss and crystallization of cellular components.