ORIGINAL ARTICLE
Ectopic expression of a novel peach (Prunus persica )CBF transcription factor in apple (Malus 3domestica )results in short-day induced dormancy and increased cold hardiness
Michael Wisniewski ?John Norelli ?
Carole Bassett ?Timothy Artlip ?Dumitru Macarisin
Received:27September 2010/Accepted:10January 2011/Published online:28January 2011óSpringer-Verlag (outside the USA)2011
Abstract Low,non-freezing temperatures and/or short daylength (SD)regulates cold acclimation and dormancy in fruit trees.Regarding cold acclimation,C-repeat binding factor (CBF /DREB )transcriptional activator genes have the well-documented ability to induce the expression of a suite of genes associated with increased cold tolerance.We isolated a full-length cDNA of a peach CBF gene,desig-nated PpCBF1(GenBank Accession HM992943),and constitutively expressed it using an enhanced 35S promoter in apple.Unexpectedly,constitutive overexpression of the PpCBF1in apple resulted in strong sensitivity to short daylength.Growth cessation and leaf senescence were induced in transgenic lines exposed to SD and optimal growth temperatures of 25°C over a 4-week period.Fol-lowing 1–4weeks of SD and 25°C trees were returned to LD and 25°C in the greenhouse.Control (untransformed)plants continued to grow while transgenic lines receiving two or more weeks of SD remained dormant and began to drop leaves.Constitutive overexpression of the PpCBF1in apple resulted in a 4–6°C increase in freezing tolerance in both the non-acclimated and acclimated states,respec-tively,compared with untransformed M.26trees.This is the ?rst instance that constitutive overexpression of a CBF gene has resulted in SD-induction of dormancy and to our knowledge the ?rst time apple has been shown to strongly respond to short daylength as a result of the insertion of a transgene.
Keywords Cold acclimation áLeaf senescence áAP2/ERF áDormancy áTransgenic apple Abbreviations AP2
Apetala2
CBF or CBF /DREB C-repeat binding factor ERF
Ethylene response factor
DRE /CRT /LTRE
Dehydration responsive element/C-repeat/low temperature responsive element COR
Cold-regulated genes
RT-qPCR Reverse transcription,quantitative real-time PCR SD Short daylength LD Long daylength LT Low temperature HT
High temperature
Introduction
The ability to cold acclimate and undergo a period of dor-mancy is essential to temperate woody plants in order to survive freezing winter temperatures.Seasonal regulation of growth and freezing tolerance is marked by phenological events such as growth cessation,bud set,the onset of dor-mancy,cold acclimation,deacclimation,and bud burst (I-ban
?ez et al.2010;Kalberer et al.2006).Cold acclimation results in and is governed by a distinct set of changes in gene expression (Chinnusamy et al.2006;Welling and Palva 2006;Thomashow et al.2001).The same is true for dormancy (Rohde and Bhalerao 2007).In woody plants,the processes of dormancy and cold acclimation overlap and both can be induced by low,non-freezing temperatures
M.Wisniewski (&)áJ.Norelli áC.Bassett áT.Artlip áD.Macarisin
U.S.Department of Agriculture,Agricultural Research Service (USDA-ARS),The Appalachian Fruit Research Station,Kearneysville,WV 25430,USA
e-mail:michael.wisniewski@https://www.doczj.com/doc/ec18955967.html,
Planta (2011)233:971–983
DOI 10.1007/s00425-011-1358-3
(LT)and short daylength(SD)(Welling and Palva2006; Kalberer et al.2006;Wisniewski et al.2003;Weiser1970; Sakai and Larcher1987).The extent to which each process is regulated by either LT or SD,however,is species speci?c (Heide2008).Interestingly,a distinct contrast exists within the Rosaceae regarding environmental control of growth cessation and dormancy.In apple(Malus9domestica)and pear(Pyrus communis),both in the tribe Pyreae,growth cessation and the onset of dormancy are relatively unre-sponsive to SD and instead require LT(Heide and Prestrud 2005)while in peach(Prunus persica)and other Prunus species of the tribe Amygdaleae,growth cessation and dormancy are induced mainly by SD and enhanced by LT (Heide2008).
Plant phenology is strongly controlled by climate and as such has become a strong bioindicator of ongoing climate change(Gordo and Sanz2010).Despite the recognized role of temperature in the onset and release of dormancy,and in cold acclimation and deacclimation,the impact that climate change will have on the phenology of fruit trees is not known.Increasing episodes of devastating spring frosts, however,may be more common(Gu et al.2008)and in a review of several studies,Ball and Hill(2009)indicated that elevated atmospheric CO2concentrations can have a negative impact on acclimation and enhance vulnerability to frost damage.Therefore,there is a need to better understand the molecular control of dormancy and cold acclimation in trees(Welling and Palva2006).
CBF proteins belong to the CBF/DRE binding(DREB) sub-family of the Apetala2-ethylene responsive factor (AP2/ERF)super family of transcription factors(Nakano et al.2006)that bind to a cis-element(DRE/CRT/LTRE) containing a conserved CCGA core sequence(Baker et al. 1994;Yamaguchi-Shinozaki and Shinozaki1994).In Arabidopsis,LT-inducible CBF genes have been demon-strated to regulate a large number of cold-regulated(COR) genes,whose products are thought to contribute to freezing tolerance.The role of CBF in cold response and acclima-tion has been well documented in both herbaceous (Thomashow et al.2001)and woody plants(Welling and Palva2006).In relation to woody plants,CBF transcription factors have been isolated and shown to be cold-responsive in birch(Betula pendula)(Welling and Palva2008),poplar (Benedict et al.2006),eucalyptus(Navarro et al.2009;El Kayal et al.2006),grape(Xiao et al.2006),sweet cherry (Prunus avium)(Kitashiba et al.2004),citrus(Champ et al. 2007),blueberry(Vaccinium corymbosum)(Polashock et al.2010),and dwarf apple(Malus baccata)(Yang et al. 2010).
In comparison with herbaceous plants the regulation of CBF in woody plants appears to be more complex.The expression pattern of speci?c CBF genes within a species can be different in annual versus perennial tissues (Benedict et al.2006)and the timing of CBF expression can be different in plants prior to and after the onset of dormancy(Welling and Palva2008).In birch under LD, Welling and Palva(2008)demonstrated CBF induction in response to low,non-freezing temperatures within15min, similar to CBF in Arabidopsis.However,under SD con-ditions CBF induction was delayed and was upregulated for a longer period of time.In dormant birch trees exposed to freezing temperatures(-10°C),CBF expression and subsequent induction of COR genes did not occur until thawing.
To better understand how various phenological parame-ters(timing of acclimation and deacclimation,?owering, fruit development,etc.)of fruit trees may be impacted by climate change we have begun to isolate peach and apple CBF genes and characterize their expression and function.In the present study,we examined the functional role of a CBF gene obtained from peach,designated PpCBF1,by over-expressing it in a rootstock variety(M.26)of apple.We examined its level of expression in several transgenic apple lines and the impact of its constitutive overexpression on cold hardiness,growth and other aspects of plant morphol-ogy.Most interestingly,constitutive overexpression of PpCBF1in apple had a dramatic impact on the photoperiodic sensitivity of apple.In comparison with untransformed trees, transgenic apple lines carrying PpCBF1exhibited growth cessation and leaf senescence in response to SD treatments alone.This is the?rst report of constitutive overexpression of a CBF gene resulting in SD-induction of dormancy and the ?rst time that apple has been made sensitive to daylength through the introduction of a transgene.
Materials and methods
Cloning of PpCBF1and construction
of PpCBF1-overexpression vector
A full-length coding sequence for a CBF transcription factor was obtained as an expressed sequence tag(EST)from a cDNA library derived from peach bark tissue collected in December.The clone,Prunus persica CBF1(PpCBF1) (GenBank accession HM992943),was sequence veri?ed, then mobilized into pRTL2under the control of an enhanced 35S promoter(Payton et al.2001)to form35S::PpCBF1. After digestion with Hin dIII,the35S::PpCBF1fragment was ligated into an appropriately digested pBINPLUS/ARS vector(Belknap et al.2008).Agrobacterium tumefaciens strain EHA105(pCH32)was transformed with the pBIN-PLUS/ARS vector containing the enhanced35S-PpCBF1 fragment via electroporation and veri?ed via PCR using PpCBF1-speci?c primers,forward:50-agggcttcttctttctccac-30, reverse:50-aaatctttatgttcgactcactca-30.
Analysis of peach and apple CBF derived polypeptide and promoters
Alignment of the peach and apple CBF/DREB family polypeptides was done with COBALT(Papadopoulos and Agarwala2007).Domain features were identi?ed accord-ing to Nakano et al.(2006).For promoter analysis,the?rst 500bp upstream of the translation start site were identi?ed from the peach and apple genome sites using the nucleotide sequences of PpCBF1and MdCBF1and2to locate the respective genes.The resulting sequences were analyzed with the PLACE program(Higo et al.1999)to identify cis-elements involved in regulating key processes.In addition, some elements were identi?ed based on analyses con-ducted by Michael et al.(2008)on the nuclear transcrip-tome of Arabidopsis under different light and temperature cycles.
Transformation of apple
Clonally propagated apple(Malus9domestica)‘M.26’leaves underwent Agrobacterium-mediated transformation as described by Borejsza-Wysocka et al.(1999).Trans-formation of the resulting regenerants was veri?ed via PCR using primers for the following genes:elongation factor1 (EF1a),used as a positive control for PCR and VirG,used to assess whether there was residual,endophytic Agro-bacterium contamination.A combination of35S promoter (TEV50)-and PpCBF1(CBF557rev)-speci?c primers were used to select for transformants.Primers for EF1a were, forward:50-gacattgccctgtggaagtt-30,reverse:50-ggtctgac-catccttggaaa-30.For VirG the primers were:forward: 50-gccggggcgagaccatagg-30,revers:50-cgcacgcgcaaggca acc-30.The TEV50–CBF557rev primers were:forward: 50-cgaatctcaagcaatcaacc-30,reverse:50-agctaagcattggggtgg agaaag-30.
Plant material
Explants were propagated in tissue culture as described by Norelli et al.(1988)and Ko et al.(2002),with root induction as described by Bolar et al.(1998).Young trees were transferred to soil and grown in a greenhouse with Na vapor supplemental lighting to maintain the daylength at 16h,and a maximum–minimum temperature range of 35/20°C.These growth conditions were de?ned as long day (LD)and high temperature(HT).Trees were watered daily and fertilized regularly.
RT-qPCR and semi-quantitative PCR
Transcript abundance of PpCBF1transgene or native apple CBF genes to low temperature was evaluated using reverse transcription-quantitative real-time PCR analysis (RT-qPCR).Gene-speci?c primers were designed for each CBF gene as follows:for PpCBF1,forward:50-aggg cttcttctttctccac-30,reverse:50-aaatctttatgttcgactcactca-30; for MdCBF1,forward:50-gctggcagatatggcggaaggaat-30, reverse:50-cataagggcacgtcagcacaaaca-30;for MdCBF2, forward:50-atccgacggccgagatggca-30,reverse:50-ccaaactc cgctggccggaa-30.EF1a was used as an endogenous con-trol using the primer combination described above.Total RNA was isolated from leaf tissue using Concert Plant RNA Reagent(Invitrogen,Carlsbad,CA,USA)and DNase treated(Turbo DNA-free Kit;Ambion,Austin, TX,USA).RT-qPCR analysis was performed using50ng of total RNA,SuperScript III Platinum SYBR Green One-Step RT-qPCR Kit with ROX(Invitrogen),and20pmol of each primer per reaction.The ABI7900(Applied Biosystems,Foster City,CA,USA)was set to cycle as follows:cDNA synthesis at48.0°C for30min;95.0°C denaturation for5min;40cycles of95.0°C for15s followed by55.0–57.0°C(depending on primers used)for 1min;40.0°C for1min;dissociation step.There were three technical repetitions per biological replicate(plant or tree)for both test gene and reference.The standard curve method was used to calculate transcript abundance relative to EF1a reference gene(user bulletin no.2; Applied Biosystems;Nicot et al.2005).Differences between treatments were evaluated using a generalized linear model(SAS Institute Inc.)in which technical rep-licates were nested within biological replicates in order to weight the importance of biological variation over tech-nical variation.
Semi-quantitative PCR was conducted to analyze the expression of a cold-inducible dehydrin(MdDhn1)gene (Wisniewski et al.2008)and two native apple genes (MdCBF1and MdCBF2).Total RNA was extracted as described above.Thermal cycling was performed using a GeneAmp PCR System9700(Applied Biosystems)with 48.0°C for30min(cDNA synthesis);95.0°C for5min (denaturation);and40cycles of95.0°C for15s,55.0°C for30s,68.0°C for30s(PCR ampli?cation).Initial RT-PCR reaction volumes were50l L,with10l L ali-quots being removed at20,25,30,35,and40cycles in order to determine the optimum number of cycles;sub-sequent reactions were run in10l L reaction for30 cycles.EF1a was used as an internal standard.CBF pri-mer pairs were as described above.MdDhn1primer pair was forward:50-cgccatggacaagatcaaagaaaa-30,reverse: 50-cactcgcgcacgtaagaaagaaat-30.For EF1a,as described above.PCR reaction products were separated by agarose gel electrophoresis stained with ethidium bromide (Sigma-Aldrich,St.Louis,MO,USA)and scanned using a Typhoon Trio Variable Mode Imager(GE Healthcare, Fair?eld,CT,USA).
Ion leakage assays
Ion leakage assays were performed essentially as described by Arora et al.(1992)for transgenic and control (untransformed)plants.In brief,0.5cm leaf disks were placed in209150mm tubes with70l L water,and the tubes in turn placed in a controlled temperature bath.After an initial equilibration at0°C,the temperature was decreased in1.5°C per0.5h decrements.Upon removal from the bath,the tubes were placed on ice for24h,and then allowed to thaw.After the disks were allowed to thaw, 15mL of water was added,the disks vacuum in?ltrated, the tubes shaken at250rpm for3h,and conductance measurements were recorded with a conductivity meter (YSI model35,Yellow Spring Instruments,Yellow Springs,OH).The tubes were then autoclaved,and the conductivity re-measured.The LT50(lethal temperature at which50%of the tissue is killed)was then derived from these data according to the method of Zhang and Willison (1987).Percent injury was determined using the formula:?%injury?%LetTà%LecT=100à%LecT ?100; where%L(t)and%L(c)are the measurements of percent-age of ion leakage for the respective freeze-treatment temperature and unfrozen control,respectively.This adjusts the data,taking into account background leakage from unfrozen controls.The data were then adjusted to set maximum leakage at100%using the following equation: (T max-T actual/T max)9100,where T max represents maxi-mum leakage obtained from autoclaved tissue and T actual represents leakage obtained from samples prior to auto-claving.This approach normalizes the data over a range from0to100%.All measurements were replicated three times where a replicate represents an individual tree. Daylength and temperature treatments in freezing tolerance studies
Non-acclimated leaves were harvested from trees main-tained under the greenhouse conditions described above. Acclimated leaves were harvested from trees kept at4°C for2weeks under either constant light(100l mol pho-tons m-2s-1)(LD,LT)or a combination of4°C and short daylength(8h light and16h dark)(SD,LT). Daylength and temperature treatments
in dormancy studies
LT treatment
Six-month old,greenhouse-grown T166(n=20)and untransformed M.26(n=15)were placed in an environ-mental chamber at4°C with low(100l mol photons m-2s-1)constant light(LD,LT).Trees were returned to a greenhouse after1,2,3,or4weeks of chilling exposure.Greenhouse conditions included supplemental light to provide16h of light, along with temperatures above20°C(LD,HT).LT and SD treatments(described in the next section)were done simulta-neously on different sets of trees.Therefore,both sets of trees were exposed to identical greenhouse conditions prior to treatment.
SD treatment
Six-month old,greenhouse-grown T166(n=20)and untransformed M.26(n=15)were placed in a Conviron PGV36growth chamber(Conviron,Winnipeg,MB, Canada)with8h light(300l mol photons m-2s-1), 25°C/16h dark,20°C(SD,HT).Trees were returned to a greenhouse after1,2,3,or4weeks of SD exposure. Greenhouse conditions included supplemental light to provide16h of light,along with temperatures above20°C (LD,HT).
Results
PpCBF1and apple CBF sequence analysis
There are four complete CBF genes in Arabidopsis(Haake et al.2002;Medina et al.1999)and peach(data not shown), and?ve full-length CBF genes in apple(Table1).The coding region of the PpCBF1gene is720nt in length, encoding a protein with a calculated molecular mass and p I of26.5kDa and6.76,respectively.The predicted poly-peptide has sequence motifs consistent with the CBF/ DREB1family as de?ned by Nakano et al.(2006)(Fig.1). The coding regions of MdCBF1(GenBank Accession HM992942)and MdCBF2(GenBank Accession HM992941) are830and555nt,respectively.MdCBF1encodes a pre-dicted protein of23.9kDa,with a p I of5.21.Both apple predicted polypeptides also contain motifs consistent with the CBF/DREB1sub-family of the AP2/ERF gene super-family(Fig.1).MdCBF1is identical to a previously Table1Similarity of apple full-length CBF polypeptides to PpCBF1
Gene ID Total score Query coverage(%)E value
MdCBF2295993e-85 MdCBF3292994e-84 MdCBF4285994e-82 MdCBF5199894e-56 MdCBF1199894e-56
Similarity was determined using BLASTp.Query sequence was PpCBF1
reported but uncharacterized M.9domestica gene also termed MdCBF1(GenBank accession DQ074478;Hellens et al.2005).Comparison of PpCBF1with apple CBF poly-peptides identi?ed from the apple genome sequence (Velasco et al.2010)indicated closest homology to MdCBF2(Table1).Amino acid identity between PpCBF1 and MdCBF2was67%compared with60%amino acid identity between MdCBF2and MdCBF1.Alignment of the peach and apple sequences with each other indicated a high degree of conservation,particularly around the AP2DNA binding domain(Fig.1).Identity between the DNA binding domains of PpCBF1and MdCBF2was92%,whereas identity between MdCBF1and2was only82%.
An analysis of the upstream nucleotide sequence of the peach and apple CBF genes predicted a number of regu-latory elements(Fig.2).Several MYB or MYC elements were present in the promoter regions of all three CBF (MdCBF2,MdCBF1,PpCBF1)genes analyzed(data not shown).Interestingly,a consensus C-repeat/DREB core element(CCGAC)was found in the MdCBF2promoter region nearly100bp downstream of the TATA box, whereas the MdCBF1and PpCBF1promoters contained a C-repeat-like element found in monocots(CCGAA).The MdCBF2promoter also contained an Inducer of CBF Element(ICEr2)-like sequence.No such element was found in the promoters of PpCBF1or MdCBF1. Transgenic apple lines overexpressing PpCBF1 Transformation of M.9domestica‘M.26’with PpCBF1 resulted in several transgenic lines that were con?rmed in tissue culture plants by PCR using a combination of35S promoter-and PpCBF1-speci?c primers,as well as Agro-bacterium VirG primers to con?rm that PCR ampli?cation resulting from the35S-PpCBF1primers was not due to Agrobacterium contamination(data not shown).As expected,leaves of greenhouse-grown plants of T126, T164,and T166had signi?cantly(P\0.0001)greater levels of PpCBF1transcripts than leaves of M.26or pBinPlus.ARS(empty vector)transgenic T5plants as measured by RT-qPCR(Fig.3).Of the three PpCBF1 lines,T166plants exhibited signi?cantly(Tukey multiple comparison P=0.05)greater PpCBF1transcript abun-dance than T126and T164plants.As found in other systems(Welling and Palva2008),constitutive over-expression of the PpCBF gene in apple initially resulted in reduced growth during acclimation from tissue culture to greenhouse growing conditions;however,with time,
differences in plant stature between wild-type and trans-genic plants disappeared(data not shown).Visual exami-nation indicated that leaves of transgenic lines tended to be somewhat smaller and both major and minor veins accu-mulated higher amounts of anthocyanins when plants were cold acclimated(Fig.4).The effect of apple line(M.26, T5,166,164,126)on leaf dry weight was highly signi?cant (P B0.001).Leaves of all transgenic lines exhibited a signi?cantly higher dry weight(P B0.05)per unit leaf area compared with M.26(wild-type)and T5(vector only)
lines(Fig.5).The highest leaf dry weights were in the T166line.
Effect of constitutive overexpression of PpCBF1
on freezing tolerance
Freezing tolerance was initially examined in non-accli-mated plants of all three lines(T126,T164,and T166)and compared with untransformed M.26plants,as well as plants transformed with the vector alone(T5).In non-acclimated plants,the LT50of T126and T164trees was improved by approximately2°C compared with untrans-formed M.26and T5(vector alone)trees(data not shown). In contrast,the T166trees exhibited a marked increase in freezing tolerance in both the non-acclimated and accli-mated(2weeks at4°C under SD)states(Fig.6a and b).
Fig.3Abundance of PpCBF1transcripts in leaves of greenhouse-grown trees of?ve
apple lines as assessed by RT-qPCR and expressed
as percent of relative abundance of line T164,set at100%.
M.26=untransformed parental line;T5=pBINPLUS/ARS(empty
vector)transgenic line;T126,T164,and T166=transgenic lines
containing PpCBF1
Fig.4Anthocyanin accumulation in cold acclimated leaves of
untransformed M.26parental line and T166PpCBF1transgenic.
a Whole leaves of M.26were slightly larger than those of T166which
had enhanced anthocyanin accumulation in veins.b Close-up view of
the abaxial leaf surface of M.26.c Close-up view of the abaxial leaf
surface of transgenic T166
The LT 50of T166trees was -9.5and -13.0°C in non-acclimated and acclimated trees,respectively,representing a 4–6°C increase in freezing tolerance,compared with untransformed M.26trees.Therefore,further character-ization focused on the T166line which also had the highest level of constitutive overexpression of PpCBF1.
Constitutive overexpression of PpCBF1resulted in high levels of expression of a native cold-inducible dehydrin gene (Fig.7a)in non-acclimated plants compared with untrans-formed,non-acclimated M.26plants and slightly higher levels of dehydrin gene expression in acclimated plants compared with untransformed M.26plants.In contrast,2weeks of SD exposure did not increase levels of dehydrin gene expression in either leaf or bark tissue (Fig.7b).Response of native apple CBF genes to SD-treatment,LT and overexpression of PpCBF1
Two weeks of SD-treatment did not appear to elevate the level of apple CBF genes analyzed in this study in either leaf or bark tissues (Fig.7c).The effect of constitutive overexpression of PpCBF1on the native MdCBF1and MdCBF2genes during exposure of plants to 5°C was assessed by RT-qPCR (Fig.8).Constitutive expression of PpCBF1in apple did not appear to have a signi?cant effect on the relative abundance or timing of the expression of either MdCBF1(Fig.8a)or MdCBF2(Fig.8b)in T166plants compared to untransformed M.26(P =0.29and P =0.50for MdCBF1and MdCBF2,respectively).
PpCBF1overexpression in apple results in short daylength-induced dormancy and leaf senescence In the initial examination of the effect of PpCBF1over-expression on the freezing tolerance of acclimated plants,trees were subjected to low temperature (4°C)and a short daylength (8h day and 16h night)for 14days.After the 14-day period and the assessment of freezing tolerance,plants were returned to normal greenhouse conditions.It was observed that while untransformed plants continued to grow,T164and T166plants had stopped growing and set a terminal bud (Fig.9a and b).This was
followed
Fig.5Effect of PpCBF1transgene expression on mean dry weight ±SE of apple leaf disks.M.26=untransformed M.26parental line;T166,T126,and T164=transgenic apple lines expressing PpCBF1;T5=pBinPlus.ARS (empty vector)transgenic.n =6(3different leaves from 2different plants of each line).The effect of line on leaf dry weight was signi?cant at P B 0.001using a one-way ANOVA.Different letters indicate signi?cantly different (P B 0.05)means as determined using a Duncan’s mean
separation
Fig.6Freezing tolerance of T166transgenic PpCBF1trees of apple expressed as percent injury of leaves assessed by electrolyte leakage.The temperature representing 50%injury (LT 50)values are indicated in each graph.M.26=untransformed M.26parental line,T166=transgenic apple lines overexpressing PpCBF1.a Non-acclimated trees and b trees acclimated (LT and SD)for 2weeks.Data are means ±SD,n =9(3plants and 3leaf disks from each plant at each temperature)
by the onset of a basipetal pattern of leaf senescence (Fig.10).
Further experiments were then conducted on T166and untransformed M.26to determine if growth termination and leaf senescence were due to a hypersensitivity to low temperature or if the trees were now sensitive to SD.These results are summarized in Fig.11.Both T166and M.26trees responded to being kept at 4°C under constant light for 1–4weeks by reducing growth and setting a terminal bud.After 4-week exposure to LT and LD the majority of trees returned to the greenhouse no longer exhibited ter-minal growth and had set a terminal bud (Fig.11).In comparison with untransformed M.26trees,T166trees exhibited a greater amount of leaf senescence compared with untransformed plants shortly after trees were returned to the greenhouse.In contrast to low-temperature treatments under constant light,there was a differential effect of short daylength (8h day/16h night)at warm temperatures (25°C)on dormancy induction in PpCBF1transgenic T166versus untrans-formed M.26trees that was quite dramatic.In untrans-formed M.26trees,SD-exposure for 1–4weeks had no impact on growth either during the treatment or after the plants had been returned to the greenhouse under LD conditions.In PpCBF transgenic T166trees,however,1–4weeks of SD-exposure progressively stimulated leaf senescence and terminal bud set,or in other words,dor-mancy.While a quantitative assessment of dormancy was not conducted due to the low number of transgenic plants available,it was observed that in T166trees that two or more weeks of SD-exposure resulted in 100%terminal bud set and a basipetal pattern of leaf
senescence.
Fig.7Semi-quantitative PCR analysis of a cold-inducible dehydrin or CBF (MdCBF1,MdCBF2)genes in apple.a Dehydrin gene expression in non-acclimated (NA)and cold-acclimated (ACC)leaf tissues of untransformed (M.26)and transgenic (T166)apple.The constitutively expressed elongation factor gene was used as a control.b Ehydrin gene expression in leaf and bark tissues of M.26apple at Time 0(T 0)and after 2weeks of SD exposure.c Apple CBF (MdCBF1,MdCBF2)in leaf and bark tissues of M.26at Time 0(T 0)and after 2weeks of SD exposure.Elongation factor served as a
control
Fig.8Effect of PpCBF1on relative transcript abundance of native MdCBF genes assessed by RT-qPCR after transfer to low temperature (5°C).a MdCBF1.b MdCBF2.M.26=untransformed parental line;T166=transgenic line containing PpCBF1
Discussion
Translation of the cDNA encoding the peach CBF1gene (PpCBF1)yielded a typical ERF family transcription factor containing a single AP2-domain (Fig.1).Motifs speci?c to CBF/DREB proteins are present,and contact amino acids involved in DNA binding at the GCC-box regulated by A.thaliana ERFs (Allen et al.1998)are conserved in the peach polypeptide,except for replacement of the arginine at position 77in AtCBF4with glycine at position 81in PpCBF1.According to BLASTn analysis,the
peach
Fig.9Effect of PpCBF1on relative shoot growth (%)of apple after being returned to the greenhouse following acclimation for 2weeks at 4°C (LT)and SD.a Stem shoot growth during 11days of deacclimation in the greenhouse as a percent of total shoot growth (mean ±SD,n =5).Percent of total shoot growth is de?ned as the growth that occurred after returning the trees to greenhouse condi-tions for 11days divided by the total shoot length prior to the 11-day growth period 9100.b Representative samples of M.26and T166plants.M.26=untransformed parental line;T5=pBINPLUS/ARS (empty vector)transgenic;T166,T164and T126=transgenic apple lines overexpressing
PpCBF1
Fig.10Basipetal pattern of leaf senescence in PpCBF1transgenic T166and untransformed M.26plants after trees were returned to greenhouse conditions following 2-week exposure to 4°C and
SD
Fig.11Effect of either 4weeks of SD exposure or 4weeks of LT (4°C)exposure on dormancy of untransformed M.26apple trees or transgenic (T166)apple overexpressing a peach CBF (PpCBF1)gene.M.26Con are control trees that remained under greenhouse condi-tions and LD.Trees shown are representative of groups of trees used in the experiment.Whereas the growth of untransformed trees (M.26)was unaffected by 4weeks of SD exposure,T166trees exposed to SD stopped growing,set a terminal bud and underwent a basipetal pattern of leaf senescence.In contrast,4weeks of LT exposure caused both M.26and T166trees to stop growing and set a terminal bud.T166plants also exhibited rapid leaf senescence
genome(https://www.doczj.com/doc/ec18955967.html,/peach/genome),appears to contain four CBF genes arranged in tandem on linkage group 5.This tandem arrangement is similar to the arrangement of three of the four CBF genes present in the genome of Arabidopsis(Thomashow et al.2001).
Previous analysis of stress gene expression in peach (Bassett et al.2009)and apple(Wisniewski et al.2008) demonstrated that homologs of small gene families were not responsive to the same stress treatment,possibly re?ecting differences in promoter function.We therefore compared the promoters of the peach and apple CBF s (Fig.2).Several cis-regulatory elements were predicted in the500bp upstream of the translation start sites of each gene.A number of MYC and MYB transcription factor binding sites were found in all three promoters,but their locations were not the same for each gene(data not shown).MdCBF2contains a consensus CRT/DREB ele-ment,suggesting that this gene might be self-regulated. Both MdCBF1and PpCBF1contain elements associated with cold regulation in monocots(Dunn et al.1998),but it is not clear whether these elements also function in dicots. MdCBF2also contains an ICEr2-like element which pre-dicts its expression could be regulated in a fashion similar to that of the At CBF3gene(Chinnusamy et al.2003).Only MdCBF2had a consensus Gbox element in its promoter. Gbox elements are associated with light regulation and work in combination with other cis-elements to form a circadian network regulating gene expression throughout the day(Michael et al.2008).Based on our analysis of cis-elements,including variation in sequence and location within the promoter,we conclude that regulation of these three CBF s is likely to be substantially different in response to the same treatment,e.g.,cold or light.
The predicted polypeptides of MdCBF1and MdCBF2 are similar to the predicted PpCBF1polypeptide(Fig.1) and are also typical of the CBF/DREB1sub-family of the AP2/ERF transcription factor family as described by Nakano et al.(2006).We have identi?ed a small family of apple CBF genes(data not shown)consisting of at least ?ve complete members(https://www.doczj.com/doc/ec18955967.html,/apple/ genome)listed in the homology analysis presented in Table1.Based on this analysis we report that MdCBF2is the most likely homolog of PpCBF1and that of the apple genes examined,MdCBF1is the most highly diverged from the peach gene.
The transgenic lines overexpressing PpCBF1all initially displayed a slow growth and lagged behind non-transgenic M.26plants propagated from tissue culture.Overexpres-sion of native or heterologous CBF/DREB1genes has been observed to cause dwar?ng in other plant systems.For example,overexpression of native A.thaliana CBF/ DREB1genes in Arabidopsis(Liu et al.1998;Kasuga et al. 1999;Gilmour et al.2000)has all shown varying degrees of dwar?ng in addition to other phenotypic alterations. Heterologous overexpression systems displaying pheno-typical abnormalities or changes in leaf morphology include transgenic tomato and transgenic potato over-expressing an Arabidopsis CBF(Hsieh et al.2002;Pino et al.2008),as well as transgenic Arabidopsis over-expressing cherry(Prunus avium L.)CBF(Kitashiba et al. 2004)or birch(Betula pendula Roth)CBF s(Welling and Palva2008).
There was an association between the level of PpCBF1 transcript abundance(Fig.3)and the level of phenotypic effects observed in transgenic apple lines.Correlations between expression levels of CBF/DREB1genes that are overexpressed and growth habit have been noted by Liu et al.(1998),Kasuga et al.(1999),and Kitashiba et al. (2004).The high levels of anthocyanin present in leaves of the T166(Fig.4)and their higher dry weights(Fig.5)have not been speci?cally noted in other CBF overexpression studies but are changes that have been associated with the process of cold acclimation(Hannah et al.2005).Pino et al.(2008)observed that compared with the wild type,the leaves of transgenic potato overexpressing an AtCBF were darker green,had higher chlorophyll,and lower anthocy-anin levels,with a greater number of stomates and dis-played greater photosynthetic capacity.The common and divergent phenotypic characteristics observed in apple versus potato associated with CBF1overexpression may re?ect species-speci?c components of the CBF regulon.It is possible that a good deal of variability may have evolved in the assemblage of genes that are regulated by CBF in any particular species since response to cold initiates a global change in Arabidopsis(*306genes)of which only 12%are certain members of the CBF regulon(Fowler and Thomashow2002).Benedict et al.(2006)have suggested that the winter-driven evolution of dormancy in perennial plants may have given rise to speci?c roles for these ‘master switches’in the different annual and perennial tissues of woody species.
Overexpression of Arabidopsis CBF genes in other species such as poplar(Benedict et al.2006)and potato (Pino et al.2008),as well as the ectopic expression of CBF genes from other species in Arabidopsis(Welling and Palva2008;Polashock et al.2010;Kitashiba et al.2004) has resulted in increased levels of freezing tolerance.In these studies,freezing tolerance was generally improved by 2–3°C.While two of the lines examined(T126and T164) had an LT50that was only2–3°C better than the untrans-formed M.26plants,T166plants exhibited a signi?cantly higher level of freezing tolerance that was4–6°C better than the wild type as determined by electrolyte leakage (Fig.6).This was associated with higher levels of tran-script accumulation in T166trees compared with the other transgenic lines.(Fig.3).
Most studies have not examined the effect of overex-pression of heterologous CBF genes on native CBF gene expression when plants are initially exposed to cold acclimating conditions.In our study,PpCBF1constitutive overexpression did not affect the timing or relative abun-dance of native CBF gene expression(Fig.8a and b).The lack of an effect of constitutive overexpression of PpCBF1 on native CBF expression is surprising since it has been indicated that Arabidopsis CBF2can suppress CBF1 transcript expression,thus acting as a negative regulator (Novillo et al.2004).
Dormancy in apple trees,and other species in the Ros-aceae,tribe Pyreae,is controlled by exposure to low tem-peratures rather than SD(Heide and Prestrud2005)while dormancy in peach,and other members of the tribe Amy-gdaleae,is largely dictated by exposure to short photope-riod(Heide2008).Therefore,the onset of dormancy and leaf senescence observed in transgenic apple trees consti-tutively overexpressing PpCBF1exposed to SD(8h day/ 16h night)was quite novel and unexpected.The basis of this response is unknown and has not been previously reported as a result of constitutive CBF gene overexpres-sion.Expression of some CBF genes has been shown to be modulated by SD,delaying LT-induced CBF expression in birch(Welling and Palva2008)while enhancing expres-sion in eucalyptus(El Kayal et al.2006).However,no studies have examined the effect of SD exposure alone on CBF gene expression or have reported that CBF expression leads to SD-induced growth cessation or leaf senescence. Generally speaking,CBF gene expression has been strictly associated with low temperature(Hua2009;Thomashow et al.2001)while other DREB cis-acting transcription factors have been associated with drought and salt stress (Yamaguchi-Shinozaki and Shinozaki1994).
An evergrowing mutant of peach has been identi?ed that, under natural or controlled changes in daylength,retains its leaves,exhibits continuous terminal shoot growth(resulting in a weeping habit)and has limited ability to cold acclimate in response to LT and SD(Arora et al.1992;Rodriguez et al.1994).This mutation has been associated with dele-tions in a locus containing six MADS-box transcription factors that have been reported as candidate genes for reg-ulation of terminal bud formation(Bielenberg et al.2004, 2008).These MIKC-type Dormancy-Associated MADS-box(DAM)genes have also been associated with end-odormancy in Japanese pear(Pyrus pyrofolia)where low expression is associated with lack of dormancy(Ubi et al. 2010).Peach DAM genes have CRT/DREB response ele-ments in their promoters,except for PpDAM2and3(data not shown).Two apple Dam genes were identi?ed from the Malus NCBI EST database and have high similarity to Pyrus DAM genes described by Ubi et al.2010.One apple DAM gene promoter has a CRT-like element(CCAGAA)within the?rst500bp upstream of the coding region(data not shown).Whether the overexpression of PpCBF1in apple affects the expression of apple homologs of DAM genes remains to be determined.While the high level of PpCBF1expression present in T166may be indirectly responsible for the SD-induced dormancy observed by interaction with other transcription factors such as the DAM genes,it does not seem to be the only reason since all the transgenic lines exhibited SD-induced dormancy despite varying levels of PpCBF1overexpression(Fig.3).
The expression of CBF genes has been shown to be gated by a circadian clock(Fowler et al.2005),and more recently Iban?ez et al.(2010)have shown that genes that constitute regulatory clock components in poplar (PttLHY1,PttLHY2)play a signi?cant role in both the daylength-controlled and temperature-dependent processes of dormancy,cold hardiness,and bud burst,including the expression of poplar CBF1.These observations indicate that genes regulating dormancy and those that regulate cold hardiness may be more interactive than previously expec-ted,especially in woody plants.Sharabi-Schwanger et al. (2010a,b)found that overexpression of Arabidopsis CBF2 in Arabidopsis delayed the onset of leaf senescence and extended the life span of the plants by approximately 2weeks.While this represents the opposite effect of what we observed with the overexpression of PpCBF1in apple, it does demonstrate that CBF genes have the ability to not only affect freezing tolerance but also other developmental processes as well.Therefore,the unique phenotype that was observed with the constitutive overexpression of PpCBF1in apple may re?ect an evolved regulatory adap-tation of CBF in a woody species(Prunus persica)that has a strong interrelationship between SD and the onset of dormancy.
Acknowledgments The authors would like to thank Corrine Pierce for her assistance in developing and propagating the transgenic lines of apple and Erik Burchard for his technical assistance in all aspects of this study.
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从实践的角度探讨在日语教学中多媒体课件的应用 在今天中国的许多大学，为适应现代化，信息化的要求，建立了设备完善的适应多媒体教学的教室。许多学科的研究者及现场教员也积极致力于多媒体软件的开发和利用。在大学日语专业的教学工作中，教科书、磁带、粉笔为主流的传统教学方式差不多悄然向先进的教学手段而进展。 一、多媒体课件和精品课程的进展现状 然而，目前在专业日语教学中能够利用的教学软件并不多见。比如在中国大学日语的专业、第二外語用教科书常见的有《新编日语》（上海外语教育出版社）、《中日交流标准日本語》（初级、中级）（人民教育出版社）、《新编基础日语（初級、高級）》（上海译文出版社）、《大学日本语》（四川大学出版社）、《初级日语》《中级日语》（北京大学出版社）、《新世纪大学日语》（外语教学与研究出版社）、《综合日语》（北京大学出版社）、《新编日语教程》（华东理工大学出版社）《新编初级（中级）日本语》（吉林教育出版社）、《新大学日本语》（大连理工大学出版社）、《新大学日语》（高等教育出版社）、《现代日本语》（上海外语教育出版社）、《基础日语》（复旦大学出版社）等等。配套教材以录音磁带、教学参考、习题集为主。只有《中日交流標準日本語（初級上）》、《初級日语》、《新编日语教程》等少数教科书配备了多媒体DVD视听教材。 然而这些试听教材，有的内容为日语普及读物，并不适合专业外语课堂教学。比如《新版中日交流标准日本语（初级上）》，有的尽管DVD视听教材中有丰富的动画画面和语音练习。然而，课堂操作则花费时刻长，不利于教师重点指导，更加适合学生的课余练习。比如北京大学的《初级日语》等。在这种情形下，许多大学的日语专业致力于教材的自主开发。 其中，有些大学的还推出精品课程，取得了专门大成绩。比如天津外国语学院的《新编日语》多媒体精品课程为2007年被评为“国家级精品课”。目前已被南开大学外国语学院、成都理工大学日语系等全国40余所大学推广使用。
Unit1 Americans believe no one stands still. If you are not moving ahead, you are falling behind. This attitude results in a nation of people committed to researching, experimenting and exploring. Time is one of the two elements that Americans save carefully, the other being labor. "We are slaves to nothing but the clock,” it has been said. Time is treated as if it were something almost real. We budget it, save it, waste it, steal it, kill it, cut it, account for it; we also charge for it. It is a precious resource. Many people have a rather acute sense of the shortness of each lifetime. Once the sands have run out of a person’s hourglass, they cannot be replaced. We want every minute to count. A foreigner’s first impression of the U.S. is li kely to be that everyone is in a rush -- often under pressure. City people always appear to be hurrying to get where they are going, restlessly seeking attention in a store, or elbowing others as they try to complete their shopping. Racing through daytime meals is part of the pace
新视野三版读写B2U4Text A College sweethearts 1I smile at my two lovely daughters and they seem so much more mature than we,their parents,when we were college sweethearts.Linda,who's21,had a boyfriend in her freshman year she thought she would marry,but they're not together anymore.Melissa,who's19,hasn't had a steady boyfriend yet.My daughters wonder when they will meet"The One",their great love.They think their father and I had a classic fairy-tale romance heading for marriage from the outset.Perhaps,they're right but it didn't seem so at the time.In a way, love just happens when you least expect it.Who would have thought that Butch and I would end up getting married to each other?He became my boyfriend because of my shallow agenda:I wanted a cute boyfriend! 2We met through my college roommate at the university cafeteria.That fateful night,I was merely curious,but for him I think it was love at first sight."You have beautiful eyes",he said as he gazed at my face.He kept staring at me all night long.I really wasn't that interested for two reasons.First,he looked like he was a really wild boy,maybe even dangerous.Second,although he was very cute,he seemed a little weird. 3Riding on his bicycle,he'd ride past my dorm as if"by accident"and pretend to be surprised to see me.I liked the attention but was cautious about his wild,dynamic personality.He had a charming way with words which would charm any girl.Fear came over me when I started to fall in love.His exciting"bad boy image"was just too tempting to resist.What was it that attracted me?I always had an excellent reputation.My concentration was solely on my studies to get superior grades.But for what?College is supposed to be a time of great learning and also some fun.I had nearly achieved a great education,and graduation was just one semester away.But I hadn't had any fun;my life was stale with no component of fun!I needed a boyfriend.Not just any boyfriend.He had to be cute.My goal that semester became: Be ambitious and grab the cutest boyfriend I can find. 4I worried what he'd think of me.True,we lived in a time when a dramatic shift in sexual attitudes was taking place,but I was a traditional girl who wasn't ready for the new ways that seemed common on campus.Butch looked superb!I was not immune to his personality,but I was scared.The night when he announced to the world that I was his girlfriend,I went along
Unit 1 1学习外语是我一生中最艰苦也是最有意义的经历之一。虽然时常遭遇挫折，但却非常有价值。 2我学外语的经历始于初中的第一堂英语课。老师很慈祥耐心，时常表扬学生。由于这种积极的教学方法，我踊跃回答各种问题，从不怕答错。两年中，我的成绩一直名列前茅。 3到了高中后，我渴望继续学习英语。然而，高中时的经历与以前大不相同。以前，老师对所有的学生都很耐心，而新老师则总是惩罚答错的学生。每当有谁回答错了，她就会用长教鞭指着我们，上下挥舞大喊：“错！错！错！”没有多久，我便不再渴望回答问题了。我不仅失去了回答问题的乐趣，而且根本就不想再用英语说半个字。 4好在这种情况没持续多久。到了大学，我了解到所有学生必须上英语课。与高中老师不。大学英语老师非常耐心和蔼，而且从来不带教鞭！不过情况却远不尽如人意。由于班大，每堂课能轮到我回答的问题寥寥无几。上了几周课后，我还发现许多同学的英语说得比我要好得多。我开始产生一种畏惧感。虽然原因与高中时不同，但我却又一次不敢开口了。看来我的英语水平要永远停步不前了。 5直到几年后我有机会参加远程英语课程，情况才有所改善。这种课程的媒介是一台电脑、一条电话线和一个调制解调器。我很快配齐了必要的设备并跟一个朋友学会了电脑操作技术，于是我每周用5到7天在网上的虚拟课堂里学习英语。 6网上学习并不比普通的课堂学习容易。它需要花许多的时间，需要学习者专心自律，以跟上课程进度。我尽力达到课程的最低要求，并按时完成作业。 7我随时随地都在学习。不管去哪里，我都随身携带一本袖珍字典和笔记本，笔记本上记着我遇到的生词。我学习中出过许多错，有时是令人尴尬的错误。有时我会因挫折而哭泣，有时甚至想放弃。但我从未因别的同学英语说得比我快而感到畏惧，因为在电脑屏幕上作出回答之前，我可以根据自己的需要花时间去琢磨自己的想法。突然有一天我发现自己什么都懂了，更重要的是，我说起英语来灵活自如。尽管我还是常常出错，还有很多东西要学，但我已尝到了刻苦学习的甜头。 8学习外语对我来说是非常艰辛的经历，但它又无比珍贵。它不仅使我懂得了艰苦努力的意义，而且让我了解了不同的文化，让我以一种全新的思维去看待事物。学习一门外语最令人兴奋的收获是我能与更多的人交流。与人交谈是我最喜欢的一项活动，新的语言使我能与陌生人交往，参与他们的谈话，并建立新的难以忘怀的友谊。由于我已能说英语，别人讲英语时我不再茫然不解了。我能够参与其中，并结交朋友。我能与人交流，并能够弥合我所说的语言和所处的文化与他们的语言和文化之间的鸿沟。 III. 1. rewarding 2. communicate 3. access 4. embarrassing 5. positive 6. commitment 7. virtual 8. benefits 9. minimum 10. opportunities IV. 1. up 2. into 3. from 4. with 5. to 6. up 7. of 8. in 9. for 10.with V. 1.G 2.B 3.E 4.I 5.H 6.K 7.M 8.O 9.F 10.C Sentence Structure VI. 1. Universities in the east are better equipped, while those in the west are relatively poor. 2. Allan Clark kept talking the price up, while Wilkinson kept knocking it down. 3. The husband spent all his money drinking, while his wife saved all hers for the family. 4. Some guests spoke pleasantly and behaved politely, while others wee insulting and impolite. 5. Outwardly Sara was friendly towards all those concerned, while inwardly she was angry. VII. 1. Not only did Mr. Smith learn the Chinese language, but he also bridged the gap between his culture and ours. 2. Not only did we learn the technology through the online course, but we also learned to communicate with friends in English. 3. Not only did we lose all our money, but we also came close to losing our lives.
新大学日语简明教程课文翻译 第21课 一、我的留学生活 我从去年12月开始学习日语。已经3个月了。每天大约学30个新单词。每天学15个左右的新汉字，但总记不住。假名已经基本记住了。 简单的会话还可以，但较难的还说不了。还不能用日语发表自己的意见。既不能很好地回答老师的提问，也看不懂日语的文章。短小、简单的信写得了，但长的信写不了。 来日本不久就迎来了新年。新年时，日本的少女们穿着美丽的和服，看上去就像新娘。非常冷的时候，还是有女孩子穿着裙子和袜子走在大街上。 我在日本的第一个新年过得很愉快，因此很开心。 现在学习忙，没什么时间玩，但周末常常运动，或骑车去公园玩。有时也邀朋友一起去。虽然我有国际驾照，但没钱，买不起车。没办法，需要的时候就向朋友借车。有几个朋友愿意借车给我。 二、一个房间变成三个 从前一直认为睡在褥子上的是日本人，美国人都睡床铺，可是听说近来纽约等大都市的年轻人不睡床铺，而是睡在褥子上，是不是突然讨厌起床铺了？ 日本人自古以来就睡在褥子上，那自有它的原因。人们都说日本人的房子小，从前，很少有人在自己的房间，一家人住在一个小房间里是常有的是，今天仍然有人过着这样的生活。 在仅有的一个房间哩，如果要摆下全家人的床铺，就不能在那里吃饭了。这一点，褥子很方便。早晨，不需要褥子的时候，可以收起来。在没有了褥子的房间放上桌子，当作饭厅吃早饭。来客人的话，就在那里喝茶；孩子放学回到家里，那房间就成了书房。而后，傍晚又成为饭厅。然后收起桌子，铺上褥子，又成为了全家人睡觉的地方。 如果是床铺的话，除了睡觉的房间，还需要吃饭的房间和书房等，但如果使用褥子，一个房间就可以有各种用途。 据说从前，在纽约等大都市的大学学习的学生也租得起很大的房间。但现在房租太贵，租不起了。只能住更便宜、更小的房间。因此，似乎开始使用睡觉时作床，白天折小能成为椅子的、方便的褥子。
新视野大学英语第一册Unit 1课文翻译 学习外语是我一生中最艰苦也是最有意义的经历之一。 虽然时常遭遇挫折，但却非常有价值。 我学外语的经历始于初中的第一堂英语课。 老师很慈祥耐心，时常表扬学生。 由于这种积极的教学方法，我踊跃回答各种问题，从不怕答错。 两年中，我的成绩一直名列前茅。 到了高中后，我渴望继续学习英语。然而，高中时的经历与以前大不相同。 以前，老师对所有的学生都很耐心，而新老师则总是惩罚答错的学生。 每当有谁回答错了，她就会用长教鞭指着我们，上下挥舞大喊：“错！错！错！” 没有多久，我便不再渴望回答问题了。 我不仅失去了回答问题的乐趣，而且根本就不想再用英语说半个字。 好在这种情况没持续多久。 到了大学，我了解到所有学生必须上英语课。 与高中老师不同，大学英语老师非常耐心和蔼，而且从来不带教鞭！ 不过情况却远不尽如人意。 由于班大，每堂课能轮到我回答的问题寥寥无几。 上了几周课后，我还发现许多同学的英语说得比我要好得多。 我开始产生一种畏惧感。 虽然原因与高中时不同，但我却又一次不敢开口了。 看来我的英语水平要永远停步不前了。 直到几年后我有机会参加远程英语课程，情况才有所改善。 这种课程的媒介是一台电脑、一条电话线和一个调制解调器。 我很快配齐了必要的设备并跟一个朋友学会了电脑操作技术，于是我每周用5到7天在网上的虚拟课堂里学习英语。 网上学习并不比普通的课堂学习容易。 它需要花许多的时间，需要学习者专心自律，以跟上课程进度。 我尽力达到课程的最低要求，并按时完成作业。 我随时随地都在学习。 不管去哪里，我都随身携带一本袖珍字典和笔记本，笔记本上记着我遇到的生词。 我学习中出过许多错，有时是令人尴尬的错误。 有时我会因挫折而哭泣，有时甚至想放弃。 但我从未因别的同学英语说得比我快而感到畏惧，因为在电脑屏幕上作出回答之前，我可以根据自己的需要花时间去琢磨自己的想法。 突然有一天我发现自己什么都懂了，更重要的是，我说起英语来灵活自如。 尽管我还是常常出错，还有很多东西要学，但我已尝到了刻苦学习的甜头。 学习外语对我来说是非常艰辛的经历，但它又无比珍贵。 它不仅使我懂得了艰苦努力的意义，而且让我了解了不同的文化，让我以一种全新的思维去看待事物。 学习一门外语最令人兴奋的收获是我能与更多的人交流。 与人交谈是我最喜欢的一项活动，新的语言使我能与陌生人交往，参与他们的谈话，并建立新的难以忘怀的友谊。 由于我已能说英语，别人讲英语时我不再茫然不解了。 我能够参与其中，并结交朋友。
习惯与礼仪 我是个漫画家，对旁人细微的动作、不起眼的举止等抱有好奇。所以，我在国外只要做错一点什么，立刻会比旁人更为敏锐地感觉到那个国家的人们对此作出的反应。 譬如我多次看到过，欧美人和中国人见到我们日本人吸溜吸溜地出声喝汤而面露厌恶之色。过去，日本人坐在塌塌米上，在一张低矮的食案上用餐，餐具离嘴较远。所以，养成了把碗端至嘴边吸食的习惯。喝羹匙里的东西也象吸似的，声声作响。这并非哪一方文化高或低，只是各国的习惯、礼仪不同而已。 日本人坐在椅子上围桌用餐是1960年之后的事情。当时，还没有礼仪规矩，甚至有人盘着腿吃饭。外国人看见此景大概会一脸厌恶吧。 韩国女性就座时，单腿翘起。我认为这种姿势很美，但习惯于双膝跪坐的日本女性大概不以为然，而韩国女性恐怕也不认为跪坐为好。 日本等多数亚洲国家，常有人习惯在路上蹲着。欧美人会联想起狗排便的姿势而一脸厌恶。 日本人常常把手放在小孩的头上说“好可爱啊！”，而大部分外国人会不愿意。 如果向回教国家的人们劝食猪肉和酒，或用左手握手、递东西，会不受欢迎的。当然，饭菜也用右手抓着吃。只有从公用大盘往自己的小盘里分食用的公勺是用左手拿。一旦搞错，用黏糊糊的右手去拿，
会遭人厌恶。 在欧美，对不受欢迎的客人不说“请脱下外套”，所以电视剧中的侦探哥隆波总是穿着外套。访问日本家庭时，要在门厅外脱掉外套后进屋。穿到屋里会不受欢迎的。 这些习惯只要了解就不会出问题，如果因为不知道而遭厌恶、憎恨，实在心里难受。 过去，我曾用色彩图画和简短的文字画了一本《关键时刻的礼仪》（新潮文库）。如今越发希望用各国语言翻译这本书。以便能对在日本的外国人有所帮助。同时希望有朝一日以漫画的形式画一本“世界各国的习惯与礼仪”。 练习答案 ５、 （１）止める並んでいる見ているなる着色した （２）拾った入っていた行ったしまった始まっていた
新视野大学英语第三版第二册读写课文翻译 Unit 1 Text A 一堂难忘的英语课 1 如果我是唯一一个还在纠正小孩英语的家长，那么我儿子也许是对的。对他而言，我是一个乏味的怪物：一个他不得不听其教诲的父亲，一个还沉湎于语法规则的人，对此我儿子似乎颇为反感。 2 我觉得我是在最近偶遇我以前的一位学生时，才开始对这个问题认真起来的。这个学生刚从欧洲旅游回来。我满怀着诚挚期待问她：“欧洲之行如何？” 3 她点了三四下头，绞尽脑汁，苦苦寻找恰当的词语，然后惊呼：“真是，哇！” 4 没了。所有希腊文明和罗马建筑的辉煌居然囊括于一个浓缩的、不完整的语句之中！我的学生以“哇！”来表示她的惊叹，我只能以摇头表达比之更强烈的忧虑。 5 关于正确使用英语能力下降的问题，有许多不同的故事。学生的确本应该能够区分诸如their/there/they're之间的不同，或区别complimentary 跟complementary之间显而易见的差异。由于这些知识缺陷，他们承受着大部分不该承受的批评和指责，因为舆论认为他们应该学得更好。 6 学生并不笨，他们只是被周围所看到和听到的语言误导了。举例来说，杂货店的指示牌会把他们引向stationary（静止处），虽然便笺本、相册、和笔记本等真正的stationery（文具用品）并没有被钉在那儿。朋友和亲人常宣称They've just ate。实际上，他们应该说They've just eaten。因此，批评学生不合乎情理。 7 对这种缺乏语言功底而引起的负面指责应归咎于我们的学校。学校应对英语熟练程度制定出更高的标准。可相反，学校只教零星的语法，高级词汇更是少之又少。还有就是，学校的年轻教师显然缺乏这些重要的语言结构方面的知识，因为他们过去也没接触过。学校有责任教会年轻人进行有效的语言沟通，可他们并没把语言的基本框架——准确的语法和恰当的词汇——充分地传授给学生。
新视野大学英语1课文翻译 1下午好！作为校长，我非常自豪地欢迎你们来到这所大学。你们所取得的成就是你们自己多年努力的结果，也是你们的父母和老师们多年努力的结果。在这所大学里，我们承诺将使你们学有所成。 2在欢迎你们到来的这一刻，我想起自己高中毕业时的情景，还有妈妈为我和爸爸拍的合影。妈妈吩咐我们：“姿势自然点。”“等一等,”爸爸说，“把我递给他闹钟的情景拍下来。”在大学期间，那个闹钟每天早晨叫醒我。至今它还放在我办公室的桌子上。 3让我来告诉你们一些你们未必预料得到的事情。你们将会怀念以前的生活习惯，怀念父母曾经提醒你们要刻苦学习、取得佳绩。你们可能因为高中生活终于结束而喜极而泣，你们的父母也可能因为终于不用再给你们洗衣服而喜极而泣！但是要记住：未来是建立在过去扎实的基础上的。 4对你们而言，接下来的四年将会是无与伦比的一段时光。在这里，你们拥有丰富的资源：有来自全国各地的有趣的学生，有学识渊博又充满爱心的老师，有综合性图书馆，有完备的运动设施，还有针对不同兴趣的学生社团——从文科社团到理科社团、到社区服务等等。你们将自由地探索、学习新科目。你们要学着习惯点灯熬油，学着结交充满魅力的人，学着去追求新的爱好。我想鼓励你们充分利用这一特殊的经历，并用你们的干劲和热情去收获这一机会所带来的丰硕成果。 5有这么多课程可供选择，你可能会不知所措。你不可能选修所有的课程，但是要尽可能体验更多的课程！大学里有很多事情可做可学，每件事情都会为你提供不同视角来审视世界。如果我只能给你们一条选课建议的话，那就是：挑战自己！不要认为你早就了解自己对什么样的领域最感兴趣。选择一些你从未接触过的领域的课程。这样，你不仅会变得更加博学，而且更有可能发现一个你未曾想到的、能成就你未来的爱好。一个绝佳的例子就是时装设计师王薇薇，她最初学的是艺术史。随着时间的推移，王薇薇把艺术史研究和对时装的热爱结合起来，并将其转化为对设计的热情，从而使她成为全球闻名的设计师。 6在大学里，一下子拥有这么多新鲜体验可能不会总是令人愉快的。在你的宿舍楼里，住在你隔壁寝室的同学可能会反复播放同一首歌，令你头痛欲裂！你可能喜欢早起，而你的室友却是个夜猫子！尽管如此，你和你的室友仍然可能成
新视野大学英语2课文翻译（Unit1-Unit7） Unit 1 Section A 时间观念强的美国人 Para. 1 美国人认为没有人能停止不前。如果你不求进取，你就会落伍。这种态度造就了一个投身于研究、实验和探索的民族。时间是美国人注意节约的两个要素之一，另一个是劳力。 Para. 2 人们一直说：“只有时间才能支配我们。”人们似乎是把时间当作一个差不多是实实在在的东西来对待的。我们安排时间、节约时间、浪费时间、挤抢时间、消磨时间、缩减时间、对时间的利用作出解释；我们还要因付出时间而收取费用。时间是一种宝贵的资源，许多人都深感人生的短暂。时光一去不复返。我们应当让每一分钟都过得有意义。 Para. 3 外国人对美国的第一印象很可能是：每个人都匆匆忙忙——常常处于压力之下。城里人看上去总是在匆匆地赶往他们要去的地方，在商店里他们焦躁不安地指望店员能马上来为他们服务，或者为了赶快买完东西，用肘来推搡他人。白天吃饭时人们也都匆匆忙忙，这部分地反映出这个国家的生活节奏。工作时间被认为是宝贵的。Para. 3b 在公共用餐场所，人们都等着别人吃完后用餐，以便按时赶回去工作。你还会发现司机开车很鲁莽，人们推搡着在你身边过去。你会怀念微笑、简短的交谈以及与陌生人的随意闲聊。不要觉得这是针对你个人的，这是因为人们非常珍惜时间，而且也不喜欢他人“浪费”时间到不恰当的地步。 Para. 4 许多刚到美国的人会怀念诸如商务拜访等场合开始时的寒暄。他们也会怀念那种一边喝茶或咖啡一边进行的礼节性交流，这也许是他们自己国家的一种习俗。他们也许还会怀念在饭店或咖啡馆里谈生意时的那种轻松悠闲的交谈。一般说来，美国人是不会在如此轻松的环境里通过长时间的闲聊来评价他们的客人的，更不用说会在增进相互间信任的过程中带他们出去吃饭，或带他们去打高尔夫球。既然我们通常是通过工作而不是社交来评估和了解他人，我们就开门见山地谈正事。因此，时间老是在我们心中的耳朵里滴滴答答地响着。 Para. 5 因此，我们千方百计地节约时间。我们发明了一系列节省劳力的装置；我们通过发传真、打电话或发电子邮件与他人迅速地进行交流，而不是通过直接接触。虽然面对面接触令人愉快，但却要花更多的时间, 尤其是在马路上交通拥挤的时候。因此，我们把大多数个人拜访安排在下班以后的时间里或周末的社交聚会上。 Para. 6 就我们而言，电子交流的缺乏人情味与我们手头上事情的重要性之间很少有或完全没有关系。在有些国家, 如果没有目光接触，就做不成大生意，这需要面对面的交谈。在美国，最后协议通常也需要本人签字。然而现在人们越来越多地在电视屏幕上见面，开远程会议不仅能解决本国的问题，而且还能通过卫星解决国际问题。
新视野大学英语3第三版课文翻译 Unit 1 The Way to Success 课文A Never, ever give up! 永不言弃！ As a young boy, Britain's great Prime Minister, Sir Winston Churchill, attended a public school called Harrow. He was not a good student, and had he not been from a famous family, he probably would have been removed from the school for deviating from the rules. Thankfully, he did finish at Harrow and his errors there did not preclude him from going on to the university. He eventually had a premier army career whereby he was later elected prime minister. He achieved fame for his wit, wisdom, civic duty, and abundant courage in his refusal to surrender during the miserable dark days of World War II. His amazing determination helped motivate his entire nation and was an inspiration worldwide. Toward the end of his period as prime minister, he was invited to address the patriotic young boys at his old school, Harrow. The headmaster said, "Young gentlemen, the greatest speaker of our time, will be here in a few days to address you, and you should obey whatever sound advice he may give you." The great day arrived. Sir Winston stood up, all five feet, five inches and 107 kilos of him, and gave this short, clear-cut speech: "Young men, never give up. Never give up! Never give up! Never, never, never, never!" 英国的伟大首相温斯顿·丘吉尔爵士，小时候在哈罗公学上学。当时他可不是个好学生，要不是出身名门，他可能早就因为违反纪律被开除了。谢天谢地，他总算从哈罗毕业了，在那里犯下的错误并没影响到他上大学。后来，他凭着军旅生涯中的杰出表现当选为英国首相。他的才思、智慧、公民责任感以及在二战痛苦而黑暗的时期拒绝投降的无畏勇气，为他赢得了美名。他非凡的决心，不仅激励了整个民族，还鼓舞了全世界。 在他首相任期即将结束时，他应邀前往母校哈罗公学，为满怀报国之志的同学们作演讲。校长说：“年轻的先生们，当代最伟大的演说家过几天就会来为你们演讲，他提出的任何中肯的建议，你们都要听从。”那个激动人心的日子终于到了。温斯顿爵士站了起来——他只有5 英尺5 英寸高，体重却有107 公斤。他作了言简意赅的讲话：“年轻人，要永不放弃。永不放弃！永不放弃！永不，永不，永不，永不！” Personal history, educational opportunity, individual dilemmas - none of these can inhibit a strong spirit committed to success. No task is too hard. No amount of preparation is too long or too difficult. Take the example of two of the most scholarly scientists of our age, Albert Einstein and Thomas Edison. Both faced immense obstacles and extreme criticism. Both were called "slow to learn" and written off as idiots by their teachers. Thomas Edison ran away from school because his teacher whipped him repeatedly for asking too many questions. Einstein didn't speak fluently until he was almost nine years old and was such a poor student that some thought he was unable to learn. Yet both boys' parents believed in them. They worked intensely each day with their sons, and the boys learned to never bypass the long hours of hard work that they needed to succeed. In the end, both Einstein and Edison overcame their childhood persecution and went on to achieve magnificent discoveries that benefit the entire world today. Consider also the heroic example of Abraham Lincoln, who faced substantial hardships, failures and repeated misfortunes in his lifetime. His background was certainly not glamorous. He was raised in a very poor family with only one year of formal education. He failed in business twice, suffered a nervous breakdown when his first love died suddenly and lost eight political
Text A 课文 A The humanities: Out of date? 人文学科：过时了吗？ When the going gets tough, the tough takeaccounting. When the job market worsens, manystudents calculate they can't major in English orhistory. They have to study something that booststheir prospects of landing a job. 当形势变得困难时，强者会去选学会计。当就业市场恶化时，许多学生估算着他们不能再主修英语或历史。他们得学一些能改善他们就业前景的东西。 The data show that as students have increasingly shouldered the ever-rising c ost of tuition,they have defected from the study of the humanities and toward applied science and "hard"skills that they bet will lead to employment. In oth er words, a college education is more andmore seen as a means for economic betterment rather than a means for human betterment.This is a trend that i s likely to persist and even accelerate. 数据显示，随着学生肩负的学费不断增加，他们已从学习人文学科转向他们相信有益于将来就业的应用科学和“硬”技能。换言之，大学教育越来越被看成是改善经济而不是提升人类自身的手段。这种趋势可能会持续，甚至有加快之势。 Over the next few years, as labor markets struggle, the humanities will proba bly continue theirlong slide in succession. There already has been a nearly 50 percent decline in the portion of liberal arts majors over the past generatio n, and it is logical to think that the trend is boundto continue or even accel erate. Once the dominant pillars of university life, the humanities nowplay li ttle roles when students take their college tours. These days, labs are more vi vid and compelling than libraries. 在未来几年内，由于劳动力市场的不景气，人文学科可能会继续其长期低迷的态势。在上一代大学生中，主修文科的学生数跌幅已近50%。这种趋势会持续、甚至加速的想法是合情合理的。人文学科曾是大学生活的重要支柱，而今在学生们参观校园的时候，却只是一个小点缀。现在，实验室要比图书馆更栩栩如生、受人青睐。 Here, please allow me to stand up for and promote the true value that the h umanities add topeople's lives. 在这儿，请允许我为人文学科给人们的生活所增添的真实价值进行支持和宣传。
第10课 日本的季节 日本的一年有春、夏、秋、冬四个季节。 3月、4月和5月这三个月是春季。春季是个暖和的好季节。桃花、樱花等花儿开得很美。人们在4月去赏花。 6月到8月是夏季。夏季非常闷热。人们去北海道旅游。7月和8月是暑假，年轻人去海边或山上。也有很多人去攀登富士山。富士山是日本最高的山。 9月、10月和11月这3个月是秋季。秋季很凉爽，晴朗的日子较多。苹果、桔子等许多水果在这个季节成熟。 12月到2月是冬季。日本的南部冬天不太冷。北部非常冷，下很多雪。去年冬天东京也很冷。今年大概不会那么冷吧。如果冷的话，人们就使用暖气炉。 第12课 乡下 我爷爷住哎乡下。今天，我要去爷爷家。早上天很阴，但中午天空开始变亮，天转好了。我急急忙忙吃完午饭，坐上了电车。 现在，电车正行驶在原野上。窗外，水田、旱地连成一片。汽车在公路上奔驰。 这时，电车正行驶在大桥上。下面河水在流动。河水很清澈，可以清澈地看见河底。可以看见鱼在游动。远处，一个小孩在挥手。他身旁，牛、马在吃草。 到了爷爷居住的村子。爷爷和奶奶来到门口等着我。爷爷的房子是旧房子，但是很大。登上二楼，大海就在眼前。海岸上，很多人正在全力拉缆绳。渐渐地可以看见网了。网里有很多鱼。和城市不同，乡下的大自然真是很美。 第13课 暑假 大概没有什么比暑假更令学生感到高兴的了。大学在7月初，其他学校在二十四日左右进入暑假。暑假大约1个半月。 很多人利用这个假期去海边、山上，或者去旅行。学生中，也有人去打工。学生由于路费等只要半价，所以在学期间去各地旅行。因此，临近暑假时，去北海道的列车上就挤满了这样的人。从炎热的地方逃避到凉爽的地方去，这是很自然的事。一般在1月、最迟在2月底之前就要预定旅馆。不然的话可能会没有地方住。 暑假里，山上、海边、湖里、河里会出现死人的事，这种事故都是由于不注意引起的。大概只能每个人自己多加注意了。 在东京附近，镰仓等地的海面不起浪，因此挤满了游泳的人。也有人家只在夏季把海边的房子租下来。 暑假里，学校的老师给学生布置作业，但是有的学生叫哥哥或姐姐帮忙。 第14课 各式各样的学生 我就读的大学都有各种各样的学生入学。学生有的是中国人，有的是美国人，有的是英国人。既有年轻的，也有不年轻的。有胖的学生，也有瘦的学生。学生大多边工作边学习。因此，大家看上去都很忙。经常有人边听课边打盹。 我为了学习日本先进的科学技术和日本文化来到日本。预定在这所大学学习3年。既然特意来了日本，所以每天都很努力学习。即便如此，考试之前还是很紧张。其他学生也是这
教育界的科技革命 如果让生活在年的人来到我们这个时代，他会辨认出我们当前课堂里发生的许多事情——那盛行的讲座、对操练的强调、从基础读本到每周的拼写测试在内的教学材料和教学活动。可能除了教堂以外，很少有机构像主管下一代正规教育的学校那样缺乏变化了。 让我们把上述一贯性与校园外孩子们的经历作一番比较吧。在现代社会，孩子们有机会接触广泛的媒体，而在早些年代这些媒体简直就是奇迹。来自过去的参观者一眼就能辨认出现在的课堂，但很难适应现今一个岁孩子的校外世界。 学校——如果不是一般意义上的教育界——天生是保守的机构。我会在很大程度上为这种保守的趋势辩护。但变化在我们的世界中是如此迅速而明确，学校不可能维持现状或仅仅做一些表面的改善而生存下去。的确，如果学校不迅速、彻底地变革，就有可能被其他较灵活的机构取代。 计算机的变革力 当今时代最重要的科技事件要数计算机的崛起。计算机已渗透到我们生活的诸多方面，从交通、电讯到娱乐等等。许多学校当然不能漠视这种趋势，于是也配备了计算机和网络。在某种程度上，这些科技辅助设施已被吸纳到校园生活中，尽管他们往往只是用一种更方便、更有效的模式教授旧课程。 然而，未来将以计算机为基础组织教学。计算机将在一定程度上允许针对个人的授课，这种授课形式以往只向有钱人提供。所有的学生都会得到符合自身需要的、适合自己学习方法和进度的课程设置，以及对先前所学材料、课程的成绩记录。 毫不夸张地说，计算机科技可将世界上所有的信息置于人们的指尖。这既是幸事又是灾难。我们再也无须花费很长时间查找某个出处或某个人——现在，信息的传递是瞬时的。不久，我们甚至无须键入指令，只需大声提出问题，计算机就会打印或说出答案，这样，人们就可实现即时的"文化脱盲"。 美中不足的是，因特网没有质量控制手段；"任何人都可以拨弄"。信息和虚假信息往往混杂在一起，现在还没有将网上十分普遍的被歪曲的事实和一派胡言与真实含义区分开来的可靠手段。要识别出真的、美的、好的信息，并挑出其中那些值得知晓的, 这对人们构成巨大的挑战。 对此也许有人会说，这个世界一直充斥着错误的信息。的确如此，但以前教育当局至少能选择他们中意的课本。而今天的形势则是每个人都拥有瞬时可得的数以百万计的信息源，这种情况是史无前例的。 教育的客户化 与以往的趋势不同，从授权机构获取证书可能会变得不再重要。每个人都能在模拟的环境中自学并展示个人才能。如果一个人能像早些时候那样"读法律"，然后通过计算机模拟的实践考试展现自己的全部法律技能，为什么还要花万美元去上法学院呢？用类似的方法学开飞机或学做外科手术不同样可行吗？ 在过去，大部分教育基本是职业性的：目的是确保个人在其年富力强的整个成人阶段能可靠地从事某项工作。现在，这种设想有了缺陷。很少有人会一生只从事一种职业；许多人都会频繁地从一个职位、公司或经济部门跳到另一个。 在经济中，这些新的、迅速变换的角色的激增使教育变得大为复杂。大部分老成持重的教师和家长对帮助青年一代应对这个会经常变换工作的世界缺乏经验。由于没有先例，青少年们只有自己为快速变化的"事业之路"和生活状况作准备。