Studies On Molecular Ecological Adaptation Mechanism Of Diversied Physiological Responses To Low-Temperature Among Invasiva Plant Croftonweed (Eupatorium Adenophorum) Populations

Croftonweed（Eupatorium adenophorum Spreng.） is a perennial rosette sub-shrub herbaceous plant belonging to Compositae family.In China,this weed has become one of the most important invasive species,which has distributed in Yunnan,Guizhou,Sichuan, Guangxi,Tibet Provinces and Autonomous regions of southwest China.Currently,it has expanded into the middle valley of Yangtze River and colonized in Chongqing and Hubei, still continuing to spread eastward and northward（Qiang 1998,2005）.The whole Yangtze River areas may be potentially invaded based on the ecological niche model of Papes and Peterson（2003）.Investigation of Croftonweed ecological adaptation mechanism will be helpful to reveal its invasion mechanism and understand its invasion ability.Considering Croftonweed has chronically adapted to tropic and south subtropic climate conditions in its native areas,whether or not it could exceed the low-temperature limitation in the procession of diffusion northward will become the critical issue for new invasion.In this paper,Croftonweed was used as plant material for study.Firstly,mass plant material were produced by tissue culture methods,then physiology and biochemistry methods were taken to compare the physiological responses of different geographical population under low-temperature stress.The result showed that this specie had naturally inovlved low-temperature populations through physiological adaptability differentiation during its invasion process in China,which could allow it to invade northward and lower extreme temperature regions.In order to explore Croftonweed low-temperature physiological differentiation reason（whether genetic structure and molecular variation leading to diversity manifestations or just limited environment response difference）,the conservative low-temperature key transcription factor CBF/DREB1 of higher plants was used as research starting point.On the basis of cloning EaCBF1 and its downstream EaCOR gene, their expression models of different geographical populations were compared and expression difference reasons were explored.In addition,EaCBF1 gene function was identified through transgenic method.Mechanism of molecular ecology adaptation to low-temperature in Croftonweed would be elucidated according to above results in order to provide the evidence of its further invasion to northeastern part of China.The main study results are given as followings:1 Croftonweed tissue culture1.1 Croftonweed asexual propagation system establishmentAn asexual propagation system of Croftonweed was estabolished to provide plant material for related experiments.The results showed that the optimum media for cluster bud multiplication and root induction are MS+2.0 mg·L^-1 6-BA+0.05 mg·L^-1 NAA and 1/2 MS（with half of macro-elements）+0.1 mg·L^-1 IAA,respectively.1.2 Croftonweed in vitro regeneration system establishmentTo provide plant material and technical foundation for research of different gene function from Croftonweed in vivo,callogenesis and redifferentiation conditions from Croftonweed leaf were investigated.The results showed that the optimum media for leaf callogenesis and redifferentiation were MS+0.1 mg·L^-1 6-BA+0.1 mg·L^-1 NAA and MS +2.0 mg·L^-1 6-BA+0.2 mg·L^-1 NAA,respectively.Then 0.7%agar:3%sugar is the best ratio of support and carbon resource for leaf callus formation.1.3 Different Croftonweed populations’ tissue culture characteristicsTissue culture characteristics of 9 Croftonweed geographical populations are compared.The results showed that there were significant differences between their rates of proliferation per month（from 3.65 to 7.60）.Huangguoshu Guizhou（HGG） population had the highest leaf-callus induction rate.Callus redifferentiation ability of each population was in consistance with that of single mass callus formation,but 9 different populations of the rooting capacity of 9 populations was not significantly different.2 Croftonweed low-temperature physiological responses2.1 Different Croftonweed populations’ low-temperature tolerance abilitiesA freezing injury classification standard was proposed according to injury symptoms of Croftonweed under -5℃.Low-temperature tolerance abilities were compared among 9 geographical populations through calculating freezing injury index and semi-lethal low-temperature.The results showed that the semi-lethal low temperature was positive correlation with freezing injury index under -5℃treatment after 4 days.Baise Guangxi （BSG） population had the highest semi-lethal low-temperature and freezing injury index, which was belong to the cold sensitive population.Meanwhile,these two parameters of HGG population were lowest,which was the cold tolerance population.2.2 Different Croftonweed populations’ low-temperature physiological responsesPhysiological responses to freezing temperature were determined to elucidate mechanisms of freezing tolerance.Under freezing stress,physiological changes,including increases in malondialdehyde（MDA） and total soluble protein contents;reductions in total soluble sugar,chlorophyll contents,and ratios of variable chlorophyll fluorescence to maximum chlorophyll fluorescence（Fv:Fm）;and fluctuation of superoxide dismutase （SOD） activity,were observed among all nine populations.However,different degrees of physiological responses were found among populations with diverse low-temperature sensitivities.Treatment 4 d at -5℃,MDA content increased 24.94-fold in leaves of the sensitive BSG population compared with untreated leaves.Total soluble protein content in leaves of the tolerant HGG population increased to the highest value among nine populations.SOD activity of the freezing-sensitive BSG population decreased 35.68%of the control,whereas the tolerant HGG population reduced to 69.60%.Moreover,soluble sugar of the tolerant HGG population decreased 29%,less than the sensitive BSG population（87%）.There were fewer declines in the percentages of chlorophyll content and Fv:Fm value in HGG than in BSG（less 44%and 32%）.Freezing injury index had significant negative correlations with Fv:Fm values and chlorophyll contents（-0.619 and -0.622,respectively）.3 Croftonweed low-temperature molecular ecology adaptation mechanisms3.1 cDNA full-length sequences clone of Croftonweed transcription factor EaCBF1 gene,its downstream EaCOR gene and internal marker EaACTIN geneThree full length of cDNA were cloned using RACE from Croftonweed leaf.The amino acid sequence coded by EaCBF1 possessed three characteristic structure domains: alkaline nuclear location region,typical AP2 domain and acid activation region,which was belonged to A1 subfamily of DREB and 63%homology with LeCBF3 protein.Its molecular weight and isoelectrie point were 24.56 KDa and 4.87,respectively.EaCOR coded an high hydrophilicity polypeptide containing one dehydration feature structure, which molecular weight and isoelectric point were 28.72 KDa and 4.66,respectively.This protein had relatively close evolution relationship with Brassica napus COR25 and Capsella bursa-pastoris COR29,and had 61%homology with coffee CcDH3 protein. EaACTIN coded a 377 amino acidsβ-actin protein.Its molecular weight and isoelectric point were 41.75 KDa and 5.16,respectively,which had the closely evolution relationship with Stevia rebaudiana SrActin and their amino acid sequences were same.3.2 DNA sequences clone of Croftonweed transcription factor EaCBF1 gene and its downstream EaCOR geneLong PCR and Tail-PCR methods were used to clone EaCBF1 and EaCOR DNA sequences or EaCBF1 flanking sequence from Croftonweed leaf,respectively.The results showed that EaCBF1 gene had no intron and EaCOR gene was consisted of two extrons and one intron.EaCBF1 5’ flanking sequence（1 020 bp） and 3’ flanking sequence（706 bp） were got by three rounds of independent Tail-PCR experiments.Its 5’ flanking sequence contains TATA box（-69～-66 bp）,G-box（-116～-110 bp）,two MYC recognition site （-135～-129 bp and -108～-103 bp） and two MYB recognition site（-259～- 255 bp and -282～-287 bp）.3.3 Different Croftonweed populations’ EaCBF1 expressions under low-temperatureSemi-quantitative RT-PCR and Northern blot were used to study EaCBF1 gene expression in vegetative organs of 4 Croftonweed populations under 4℃.The results showed that EaCBF1 did not express in Croftonweed leaf,stem and root before treatment. After 4℃treatment 0.5 h,this gene began to expression.Its expression level continuously increased following treatment time prolonged.The above phenomenon means that EaCBF1 belongs to low-temperature induction gene.EaCBF1 gene expression abundance is closely related to Croftonweed low-temperature tolerance capacity,its expression levels in nutritional organs of low temperature-resistant populations are higher than that of low-temperature-sensitive populations during 4℃treatment.EaCBF1 mRNA transcription level of the cold tolerance HGG population and that of the cold sensitive BSG population were the highest and lowest among four populations at the same time, respectively.Its EaCBF1 mRNA transcription level in leaf,stem and root was 56%,63.7 %and 90.3%of the HGG after treatment 24 h.EaCBF1 gene expression had organ specificity,and their expression levels were in oder:leaf＞stem＞root.EaCBF1 gene was single copy gene,but its southern blot hybridization fragment position was different among four populations which was maybe the reason that caused EaCBF1 differentially expression levels.3.4 Different Croftonweed populations’ EaCOR expressions under low-temperature Semi-quantitative RT-PCR and Northern blot were used to study EaCOR gene expression in vegetative organs of 4 Croftonweed populations under 4℃.The results showed that EaCOR did not express in Croftonweed leaf,stem and root before treatment.After 4℃treatment 1h,this gene began to expression.Its expression level continuously increased following treatment time prolonged too.The above phenomenon means that EaCOR belongs to low-temperature induction gene as well.EaCOR gene expression abundance is closely related to Croftonweed low-temperature tolerance capacity,its expression levels in nutritional organs of low temperature-resistant populations are higher than that of low-temperature-sensitive populations during 4℃treatment.EaCOR mRNA transcription level of the cold tolerance HGG population and that of the cold sensitive BSG population were the highest and lowest among four populations at the same time,respectively. EaCOR mRNA transcription level of the cold tolerance HGG population was the highest among four populations during 4℃treatment.The cold sensitive BSG population had the lowest transcription level at the same time.Its EaCOR mRNA transcription level in leaf, stem and root was 66%,56.4%and 89%of the Huangguoshu Guizhou after treatment 24 h.EaCOR gene expression had organ specificity,their expression levels were in oder: leaf＞stem＞root.EaCOR gene expressions in leaf,stem and root were all lagged behind the EaCBF1 gene through comparison of their expression patterns,which showed that EaCOR was the downstream gene of EaCBF1.Both EaCBF1 and EaCOR had the same expression change trend among four populations,which indicated that differentiation of different physiological response to low-temperature was determined by EaCOR differentially expression levels through regulation of EaCBF1 expression among four Croftonweed populations.3.5 Croftonweed EaCBF1 gene function analysisEaCBF1 gene was connected into eukaryotic expression vector pBI121 after introducing BamHⅠrestriction site on its sequence terminal.Then Agrobacterium tumefaciens containing EaCBF1 gene was obtained through transformed pBI121-CBF plasmid into Agrobacterium tumefaciens by freeze-thaw method.Transformation conditions for Agrobacterium tumefaciens of Croftonweed were investigated,the results showed that Kan 50 mg·L^-1 used as antibiotics selection pressure,3 days pre-culture with Agrobacterium tumefaciens suspension infection 5 min was optimum transgenic condition, Croftonweed exogenous EaCBF1 gene transformation rate was 4.4%using this treatment. Among GUS staining positive 23 resistant shoots,2 shoots’（TG-1 and TG-2） cold tolerance abilities were enhanced.Southern bolt showed that both TG-1 and TG-2 had accepted one copy of exogenous EaCBF1 gene,Northern blot showed that EaCBF1 were expressions in two transgenic shoots under room temperature,their expressions were more strongly than the non-transgenic shoots（BSG） under 4℃.