| Metallothionein(MTs)is a group of low molecular weight proteins with character of high cysteine(Cys)residue content and metal-binding ability.Five cDNA sequences of metallothionein(MT)genes were isolated from sugarcane variety of ROC22(Saccharum spp.hybrid cv.ROC22)and the other one was obtained from the mature seeds originating from ROC22 selfing.Recombinant expression plasmid containing target ScMTs were constructed and then transferred into yeast(Pichia pastoris)successfully.Spot assay was performed to ascertain the response of yeast cells transformed with recombinant plasmid(pPICZ-ScMTs)or vector alone(pPICZ)to Cd2+ and Cu2+ stresses.Furthermore,tissue-specific expression pattern of ScMTs genes and their expression profiles under different stress conditions in sugarcane were investigated by RT-qPCR.Main results are as follows:1.Cloning and sequence analysis of ScMTs genes from sugarcaneFive cDNA of metallothionein(MT)genes were isolated from sugarcane plantlets of ROC22.The full-length of cDNA sequences obtained were 456 bp for ScMT1-1(GenBank Acc.:KU950383),448 bp for ScMT2-1-5(GenBank Acc.:KU950385),419 bp for ScMT3-1(GenBank Acc.:KU950384),318 bp for ScMT7(GenBarik Acc.:KU950387)and 373 bp for ScMT10(GenBank Acc.:KU950388),respectively,and their open reading frames(ORFs)were 228 bp,252 bp,198 bp,237-bp and 237 bp,correspondingly.In addition,another cDNA of MT gene,named as ScMT4(GenBank Acc.:KU950386),was isolated from the mature seeds of ROC22 selfing.The cDNA of ScMT4 was 251 bp with a 159 bp ORF.According to the arrangement of Cys-containing motifs,the deduced MT proteins of ScMT1-1,ScMT2-1-5,ScMT3 and ScMT4 were categorized to plant typical Type 1,Type 2,Type 3 and Type 4,respectively,while the Cys-rich domains pattern of ScMT7 and ScMT10 were different from all the mentioned four types MTs,and might be two new types of plant MT.2.Effects of heavy metal stresses on recombinant yeast growthScMTs were successfully constructed into expression plasmids pPICZ and then transferred to yeast(P.pastoris).Serial dilutions of each recombinant yeast liquid culture were spotting in solid medium containing 200 μmol/L,400 μmol/L,600μnol/L Cd2+ or 50 μmol/L,100 μmol/L,150 μmol/L Cu2+,respectively.When challenged with Cd2+ or Cu2+ stress,there were no differences between the recombinant yeast cells transformed with pPICZ-ScMT1-1 or pPICZ-ScMT2-1-5 and vector alone(pPICZ)in the growth status.It revealed that neither ScMT1-1 nor ScMT2-1-5 played a positive role in yeast tolerance to Cd2+ and Cu2+.The growth status of the recombinant yeast cells transformed with pPICZ-ScMT3-1 were significantly better than control,suggesting its positive effect of ScMT3-1 in improving Cd2+ and Cu2+ tolerance in yeast.The growth status of the recombinant yeast cells transformed with pPICZ-ScMT4 was similar to that of the control,revealing it without playing a positive role in yeast tolerance to Cd2+ and Cu2+.Therecombinant yeast cells transformed with pPICZ-ScMT7 did not grow better than that of the control under Cd2+ stress,while it showed better growth status under Cu2+ stress,suggesting its positive regulation of Cu2+ tolerance in cells.3.Expression parterns of the ScMT Genes in sugarcaneThe tissue-specifie expression analysis of ScMTs in mature sugarcane showed that ScMT1-1 was maiuly expressed in leaf where the expression level was 27.6 times higher than that in the pith.ScMT2-1-5 was mainly expressed in the meristem of stem with 5.17 times higher than that in the pith.ScMT3-1 was mainly expressed in the leaf with 3.37 times higher than that in the pith.The expression of ScMT4 was only detected in mature seeds.ScMT7 and ScMT10 were mainly expressed in the bud with 47.04 and 23.36 times respectively higher than those in the pith.Different response of ScMTs to various exogenous stresses was found in different sugarcane genotypes,suggesting the functional differentiation in sugarcane metallothionein gene family.More specifically,ScMT1-1 was positively regulated by salicylic acid(SA)and HZOZ,while no significant up-regulation was detected in response to Cd2+ or Cu2+ stress.Moreover,it was mainly involved in the response to polyethylene glycol(PEG)osmotic stress in the overground parts of sugarcane.ScMT2-1-5 was negatively regulated by abscisic acid(ABA)and methyl jasmonate(MeJA),while it was positively regulated by SA.ScMT2-1-5 was positively response to PEG osmotic stress,and its up-regultation trend was more stable in sugarcane underground parts than that in overground parts.Moreover,it mainly responded to Cu2+ stress in underground parts of sugarcane.Besides the expression level of ScMT2-1-5 existed specificity to treated concentration in the response to H2O2 or NaCl stress.ScMT3-1 was positively regulated by ABA,MeJA and SA,and the above induction showed to be a dose-responsive effect.ScMT3-1 was induced by H2O2 stress and was inhibited by NaCl stress.ScMT3-1 might play a positive role in responding to PEG stress in underground parts with 14.55 times higher than that in the control under 25%PEG treatment.Besides,the expression scenarios of ScMT3-1.showed a slight up-regulation in response to Cu2+ stress,and the up-regulation trend was more obvious in underground parts than that in overground parts.Unlike Cu2+ stress,ScMT3-1 mainly responded to Cd2+ stress in overground parts.ScMT7 was up-regulated by MeJA,SA and H2O2 stresses,while it was inhibited by NaCl stress.ScMT7 might play a positive role in responding to PEG stress in underground parts of sugarcane,in which its expression level reached to 325.83 times higher than that of the control when treated with 10%.PEG.It mainly involved in positive response to Cd2+ stress in overground parts of sugarcane.Meanwhile,it involved in positive response to Cu2+ stress in both overground and underground parts,and its expression level was about 41.44 times higher than that of the control.ScMT10 was negatively regulated by ABA and MeJA,and its expression level existed specificity to treated concentration in response to SA.The expression of was inhibited by both H2O2 and NaCl stresses.Moreover,the ScMT10 was involved in the positive response to PEG osmotic stress in both parts and inhibited by heavy metals of Cu2+ or Cd2+. |
