Cloning,Expression Analysis And Functional Characterization Of CsSPMS Associated With Cold Tolerance In Tea Plant [Camellia Sinensis(L.)O.Kuntze]

Low temperature is one of the major environmental factors,limiting geographical distribution,which also effects on crops yields and qualities.A great loss occurred on the world because of the low temperature each year.In recent years,the mechanism of chilling tolerance in plant had been a hot spot in the plant biology researches.Since low temperature is one of the major factors affecting tea quality and production,underlying the mechanism of resisting to low temperature in tea plant(Camellia sinensis L.)warrants serious attention.Spermine,a physiologically active substance in plants,is closely related to resistance.The full-length cDNA sequence of SPMS gene was cloned using reverse transcription-PCR(RT-PCR)combined with RACE techniques from tea plant cultivar'Yingshuang',following its subcellular localization,gene expressions were investigated.The results are of critical importance to understand the cold stress related spermine in tea plant as well as expression and genes regulation,and provide the academic evidence for illuminating spermine and SPMS involve in cold resistance at a molecular level in tea plant.The main results were as follows:1.The full-length cDNA sequence of SPMS gene was cloned using reverse transcription-PCR(RT-PCR)combined with RACE techniques from tea plant cultivar'Yingshuang'.This gene was named CsSPMS with GenBank accession number KJ580429.It was 1 374 bp in length,containing a 1 113 bp open reading frame(ORF)which encoded 371 amino acid residues of 41.28 kD molecular weight.2.Amino acids sequence alignment shown that CsSPMS had a high level of similarity with Oryza sativa,Arabidopsis thaliana and Populus tomentosa.Deduced amino acids sequence of 149-260 contained 6 typical conservative domains,which were S-adenosylmethionine and decarboxylation adenosine methionine(spermidine,spermine precursor)special domain,and N-terminus as well as C-terminus were highly conserved.Phylogenetic tree shown that CsSPMS was most closely to that of Arabidopsis thaliana,which belonged to the same branch.Hydrophilic/hydrophobic analysis indicated that the 100 glycine(Gly)was most hydrophobic,the 300-glutamine(Gln)was hydrophilic strongest,most of amino acids were hydrophobic.Protein secondary structure analysis revealed that the protein contains 125 a-helix,accounting for 33.69%;86 extension chain,accounting for 23.18%;25 ?-fold,accounting for 6.74%;135 no rules twist,accounting for 36.39%.3.Online subcellular localization prediction result said that CsSPMS protein most likely localized in the nucleus.Furthermore,the localization assays indicated that CsSPMS protein localized in the cell nucleus,after observing its subcellular localization by the fusion expression vector pJIT166-GFP/SPMS.4.The CsSPMS gene expression levels in tea plant were analyzed by quantitative real-time PCR qRT-PCR.CsSPMS gene expressed in all the tissues,including root,stem,leave,flower and fruit detected by qRT-PCR,the highest expression level were in root and stem.The qRT-PCR analysis also indicated that the expression level of CsSPMS was up-regulated in roots,stems and leaves by cold treatment.The top expression level of CsSPMS in leaves was after 2 h treatment,while after 12 h treatment in roots.5.The relationship between CsSPMS expression and cold tolerance were performed under cold stress using four tea plant cultivars 'Longjing 43','Zhongcha 108','Yingshuang,and 'Baicha 1'.Among four cultivars,cold-resistant capability of 'Longjing 43' and 'Zhongcha 108' are higher than 'Yingshuang' and 'Baicha 1'.The results shown that the CsSPMS expression level under non-cold condition in 'Longjing 43' was highest,followed by 'Yingshuang' and 'Baicha 1',and 'Zhongcha 108' at the lowest level.Expression levels of CsSPMS were up-regulated by cold stress in all cultivars,especially in'Zhongcha 108',whose value reached to 8.8-fold.Expression levels of CsSPMS in'Longjing 43','Zhongcha 108' were significantly higher than 'Yingshuang','Baicha 1' by cold treatment.All results revealed that CsSPMS expression levels were closely related to cold tolerance capability in tea plant cultivars.6.The cold-resistance ability of exogenous Spm supplying was analyzed by measuring physiological parameters under cold stress.Proline contents,as well as MDA contents in tea plants after Spm treatment were higher than control ones at 1d,4d,10d timecourse;The SOD,CAT,POD activity of exogenous Spm supplying samples were significantly increased at Id,4d,10d,14d of the four time periods under cold treatment.The results show that exogenous Spm supplying samples contain more cold resistant compositions than the control group,so that exogenous Spm supplying tea plant has higher cold-tolerance.