Over-expression of the apple spermidine synthase gene in pear

ORIGINAL PAPER

Over-expression of the apple spermidine synthase gene in pear confers multiple abiotic stress tolerance by altering polyamine titers

Xiao-Peng Wen ?Xiao-Ming Pang ?Narumi Matsuda ?Masayuki Kita ?Hiromichi Inoue ?Yu-Jin Hao ?

Chikako Honda ?Takaya Moriguchi

Received:11October 2006/Accepted:11April 2007/Published online:5June 2007óSpringer Science+Business Media B.V.2007

Abstract An apple spermidine synthase (SPDS)gene (MdSPDS1)was veri?ed to encode a func-tional protein by the complementation of the spe3yeast mutant,which lacks the SPDS gene.To justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well,MdSPDS1-over-expressing transgenic European pear (Pyrus

communis L.‘Ballad’)plants were created by Agrobacterium -mediated transformation.A total of 21transgenic lines showing various spermidine (Spd)titers and MdSPDS1expression levels were obtained.Selected lines were exposed to salt (150mM NaCl),osmosis (300mM mannitol),and heavy metal (500m M CuSO 4)stresses for evaluating their stress tolerances.Transgenic line no.32,which was revealed to have the highest Spd accumulation and expression level of MdSPDS1,showed the strongest tolerance to these stresses.When growth increments,electrolyte leakage (EL),and values of thiobarbituric acid reactive substances (TBARS)were monitored,line no.32showed the lowest growth inhibition and the least increase in EL or TBARS under stress conditions.Spd titers in wild-type and transgenic lines showed diverse changes upon stresses,and these changes were not consistent with the changes in MdSPDS1expressions.More-over,there were no differences in the sodium concentration in the shoots between the wild type and line no.32,whereas the copper concentration was higher in the wild type than in line no.32.Although the mechanism(s)underlying the involve-ment of polyamines in stress responses is not known,these results suggest that the over-expression of the SPDS gene substantially increased the toler-ance to multiple stresses by altering the polyamine titers in pear.Thus,MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments.

Xiao-PengWen and Xiao-Ming Pang contributed equally to this work.

X.-P.Wen

Guizhou University,Guiyang,Guizhou 550025,China X.-M.Pang áM.Kita áH.Inoue áC.Honda áT.Moriguchi (&)

National Institute of Fruit Tree Science,Tsukuba,Ibaraki 305-8605,Japan e-mail:takaya@affrc.go.jp

N.Matsuda

Department of Agro-Production Science,Yamagata General Agricultural Research Center,Sagae,Yamagata 991-0043,Japan

Y.-J.Hao

Shandong Agricultural University,Taian,Shandong 271018,China

Present Address:X.-M.Pang

Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants,Ministry of Education,Beijing Forestry University,Beijing 100083,China

Transgenic Res (2008)17:251–263DOI 10.1007/s11248-007-9098-7

Keywords Abiotic stress toleranceáEuropean pear (Pyrus communis L.‘Ballad’)áGenetic transformationáPolyamineáSpermidine synthase Introduction

The introduction of stress tolerance into plants has recently become the major focus of research.Billions of dollars is lost annually in agriculture due to adverse environmental stresses(Senaratna et al. 2003).To make it worse,plants often encounter multiple environmental stresses simultaneously.Fruit trees cannot be rotated,as can annual crops,to avoid stress.Once planted,they are generally exposed to stress injury over several years.Thus,the develop-ment of techniques for the avoidance or reduction of injury from environmental stress and/or the breeding of stress-tolerant fruit tress is important.To this end, understanding the genetic and molecular mechanisms underlying plant perception and adaptability to stresses will be of paramount relevance.Genetic resources of fruit trees showing tolerance to diverse environmental stresses are useful as tools for the elucidation of mechanisms and as materials for breeding.However,genetic resources with multiple stress tolerances are seldom available in fruit trees. Even if available,genetic improvement by conven-tional breeding in fruit trees is a very slow process due to their long life cycles and the heterogeneous genetic background.Genetic engineering using molecular techniques provides an alternative ap-proach.Several lines of evidence have revealed that transgenic plants expressing tolerance-related genes could survive or persist under stress conditions (Jaglo-Ottosen et al.1998;Liu et al.1998;Kasuga et al.1999).

Polyamines including putrescine(Put),spermi-dine(Spd),and spermine(Spm)are ubiquitous cellular polycationic compounds.The biosynthesis of Put is catalyzed either directly by ornithine decarboxylase(ODC;EC4.1.1.17)or indirectly by arginine decarboxylase(ADC;EC 4.1.1.19).With the aminopropyl moiety donated by decarboxylated S-adenosylmethionine(dcSAM),catalyzed by S-adenosylmethionine decarboxylase(SAMDC;EC 4.1.1.50),Put is converted to Spd by spermidine synthase(SPDS;EC2.5.1.16),and Spd is further changed to Spm by spermine synthase(SPMS;EC 2.5.1.22)(see review by Bagni and Tassoni2001). Recently,these polyamine biosynthetic genes have also become an issue of concern in environmental stress tolerance.Transgenic rice plants expressing the Datura stramonium adc gene produced much higher levels of Put under drought stress,resulting in higher levels of Spd and Spm synthesis,and ultimately protected the plants from drought(Capell et al.2004).The introduction of the SAMDC gene can confer stress tolerance to salt and drought in rice(Roy and Wu2002)and to salt,drought,and fungal wilts in tobacco(Waie and Rajam2003). Furthermore,the enhancement of multiple environ-mental stress tolerances by the exogenous SPDS gene has been reported in Arabidopsis(Kasukabe et al.2004)and in Ipomoea batatas(Kasukabe et al. 2006),indicating that genetic manipulation of Spd could be an effective strategy for simultaneously conferring multiple stress tolerance,which is of practical importance since plants may often suffer from several kinds of environmental stresses.There have been,however,no available achievements on the establishment of transgenic perennial plants with environmental stress tolerance,except for some forest trees.

Several polyamine biosynthetic genes have been isolated from apple(Zhang et al.2003;Hao et al. 2005a,b;Kitashiba et al.2005,2006),and we have also shown the gene expressions in the stress responses such as salt-,cold-,and dehydration stresses using an apple tissue culture system includ-ing in vitro shoots and callus cultures(Hao et al. 2005a,b;Liu et al.2006).The results suggested a pivotal role of polyamines in the tolerance of fruit trees to environmental stresses,but direct evidence for the involvement of polyamine in stress tolerance is still lacking.There is a lack of knowledge on the molecular mechanism underlying the function of polyamines in fruit trees,compared with annual plants,which is partially due to the limited successful regeneration system and/or to the very low-regener-ation ef?ciency in fruit trees.Recently,a highly ef?cient regeneration system was developed for the European pear(Pyrus communis L.)(Matsuda et al. 2005).Therefore,to address the direct involvement of polyamines in the stress tolerance of fruit trees,lines of transgenic European pears over-expressing apple SPDS(MdSPDS1)were created.In this study, MdSPDS1was introduced into an SPDS-defective

yeast mutant spe3to complement its growth for the veri?cation of its function.Subsequently,MdSPDS1 was transferred into the European pear,and its function under abiotic stress tolerances was investi-gated in transgenic lines.The results demonstrated that the over-expression of MdSPDS1could be a promising approach for conferring multiple stress tolerance in fruit trees.To our knowledge,this is the ?rst report on environmental stress tolerance,includ-ing copper stress,in transgenic fruit trees over-expressing the polyamine biosynthetic gene. Materials and methods

Complementation assay with the SPDS-defective yeast strain

The yeast mutant strain YPR069C(spe3)(Open Biosystems,Huntsville,AL,USA)is defective in SPDS production.Normal growth requires exogenous Spd,which makes it possible to characterize the functionality of apple MdSPDS1(Zhang et al.2003) by heterologous complementation.To perform the complementation assay,a full-length MdSPDS1 cDNA was recovered from pBluescript at Sma I/Kpn I sites.The inserts were then ligated into the Sma I/ Kpn I-digested pYES2plasmid(Invitrogen,Carlsbad, CA,USA)under the control of a galactose-inducible GAL1promoter.The resultant plasmid construct, namely,pYES2:pSPDS1,as well as the empty pYES2 plasmid(control),was introduced into the mutant yeast strain YPR069C using a lithium acetate trans-formation method(Gietz1992).Transformants were initially selected in an SD medium without uracil. Positively transformed single-colonies for each con-struct were con?rmed by PCR and then grown for 24h in10ml of an SD medium with or without 100m M Spd.Approximately,30m l was then subcultured in10ml of a fresh medium at intervals of24h.Cell proliferation was monitored according to Marco and Carrasco(2002).

Transformation of MdSPDS1into pear plants

MdSPDS1was excised with Bam HI/Kpn I sites from pBluescript and then ligated into a binary vector pBI121in the sense direction(pBI121:MdSPDS1).Agrobacterium tumefaciens strain LBA4404trans-formed with pBI121:MdSPDS1containing the nptII gene under the control of the CaMV35S promoter was used to transform pear(https://www.sodocs.net/doc/713137683.html,munis L.‘Ballad’). The preparation of explant materials of in vitro pear shoots,Agrobacterium inoculation,and selection procedures followed the method described by Mat-suda et al.(2005).

To con?rm transgene integration,total genomic DNA was isolated from the control and the regenerated shoots according to method described by Porebski et al.(1997)and examined by PCR.PCR was carried out using CaMV35S(50-GAT GTG ATA TCT CCA CTG ACG TAA G-30)-nosT(50-CGC AAG ACC GGC AAC AGG AT-30)primer sets and MdSPDS1 inner(50-AAT TCG GCA CGA GGC GGC ACG A-30 and50-ATC TGC TTG TTG GAT GCT ACT G-30) primer sets.The thermal condition was35cycles of 30s at948C,30s at588C,and90s at728C.

RNA isolation and RNA gel blot analysis

The RNA antisense probe was made using MdSPDS1as a template and according to the procedures supplied by the manufacture of the DIG RNA labeling kit(Roche Diagnostics,Mann-heim,Germany).Total RNA was isolated from shoots according to the procedure described by Wan and Wilkins(1994).Ten micrograms of total RNA for each sample was electrophoresed in 1.2% formaldehyde denatured agarose gel,blotted onto a Hybond N membrane(Amersham Bioscience,Pis-cataway,NJ,USA),and hybridized with a DIG-labeled RNA antisense probe.Hybridization was carried out at688C in a standard hybridization solution buffer containing0.02%SDS,5·SSC,2% blocking reagent and0.1%N-lauroylsarcosine,and 50%formamide.After hybridization,the membranes were washed twice for5min each time by 2·SSC+0.1%SDS at room temperature and then washed twice for15min each time by 0.1·SSC+0.1%SDS at688C.The detection procedures were performed according to a manual provided by the manufacturer’s instructions(Roche Diagnostics).The relative value of the expression level was corrected to the ribosomal RNA level by densitometric analysis(Scion Image,Scion Corpo-ration,Frederick,MD,USA).

Stress treatments of transgenic pear plants

For stress treatment,in vitro shoots of wild-type (control)and selected transgenic lines(no.9,no.11, and no.32)were transferred into the MS medium containing MS salts(Murashige and Skoog1962),B5 organic(Gamborg et al.1968),3%sucrose,8.0m M N6-benzylaminopurine(BA),and0.8%agar and cultured under a16-h photoperiod at258C with or without(stress-free)the supplement of150mM NaCl, 300mM mannitol or500m M CuSO4.The shoot height and fresh weight were measured at the start of the experiment and then compared with those of the wild type on the tenth day following treatment with NaCl and mannitol,and the15th day after treatment with CuSO4.The net increment(percentage)in fresh weight(FWI n)and shoot height(SHI n)during a given period was calculated with the following equation: FWI n(SHI n)=[FW(SH)at the end-FW(SH)at the start]/ FW(SH)at the start·100.The shoots were sampled with the bottom end contacting medium removed at the completion of the experiment and used for electrolyte leakage(EL)and thiobarbituric acid reactive substance(TBARS)analyses or immediately frozen and stored at-808C for the measurement of the polyamine titer and RNA gel blot analysis.

Measurement of EL

The EL measurement was performed using the method described by Liu et al.(2004).A total of 0.5g of tissues were incubated in20ml distilled water and kept at room temperature for1h,and the initial conductivity(C1)was measured by a conduc-tivity meter(DS-52,Horiba,Kyoto,Japan).The samples were then boiled for10min to induce100% leakage,followed by cooling at room temperature for 12h and measurement of the?nal electrolyte conductivity(C2).The relative conductivity(C)was expressed as a percentage of the total ion leakage and calculated as C(%)=100·C1/C2.

Measurement of TBARS

Lipid peroxidation in shoots after CuSO4stress was determined as the amount of MDA(malonyldialde-hyde)measured by the thiobarbituric acid(TBA) reaction according to methods described by Hodges et al.(1999).In vitro shoots were homogenized with sea sand in80:20(V:V)ethanol:water and centrifuged at3,000g for10min.About1ml aliquot of sample was added to a test tube with1ml of a TBA solution comprised of20%(w/v)trichloroacetic acid,0.65%TBA,and0.01%butylated hydroxytol-uene.The mixture was vortexed vigorously,heated at 958C in a block heater for30min,and then cooled on ice for10min.After centrifugation for10min at 3,000g,the absorbance of the supernatant was measured at450,532,and600nm with a UV-visible spectrophotometer(UV-1600,Shimadzu,Kyoto, Japan).The concentration of MDA was calculated as described by Liu et al.(2006).

Quanti?cation of polyamines by HPLC

Free polyamines were quanti?ed according to the method reported by He et al.(2002). Measurements of the sodium and copper concentrations

Shoots of the wild type and line no.32cultured for 15days in a medium with or without NaCl or CuSO4 were washed with distilled water.These samples were then dried at708C for2days and homogenized with a mortar and pestle.For acid digestion,a sample(ca.

0.1dry weight)was transferred into a Te?on1PFA vessel covered with a double-sealed Te?on decom-position vessel(type-P-70,SAN-AI science Co.Ltd., Nagoya,Japan)with4ml nitric acid and heated for 3min with a microwave(Kimber and Kokot1990). Digested samples were then diluted to20ml with ultra-pure water.Three replicate digestions were made for each treatment.The acid digests were analyzed using an inductively coupled plasma-atomic optical emission spectrometry(ICP-AES,model CI-ROS CCD)instrument(Spectro,Kleve,Germany).

Statistical analysis

All treatments were carried out at least in duplicate with three repeats,and data typical of the trends are presented.The data were the mean values of three repeats and are expressed as the mean±SE.The statistical analysis was performed by one-way ANO-VA analysis,taking p<0.05as signi?cant or the Tukey test for the least-squares difference(LSD).

Results

Yeast function complementation of MdSPDS1

The growth of the yeast mutant spe3was arrested in a medium without Spd but resumed by adding Spd (data not shown).Under the same conditions,the yeast transformant containing the empty pYES2 vector alone also showed arrested growth,and its growth was recovered by the supplementation of 100m M Spd,whereas the growth of the yeast transformant containing pYES2:pMdSPDS1was recovered without the addition of Spd(Fig.1).These results indicated that MdSPDS1encoded functional SPDS proteins.On the other hand,the growth pattern of yeast containing pYES2:pMdSPDS1was different from that of yeast cultured with the supplementation of Spd(pYES2+spermidine).The growth recovery of construct pYES2:pMdSPDS1took longer than that of pYES2+spermidine.The growth of the yeast transformant containing MdSPDS1was obviously improved by the continuous culture without daily subculture,whereas that of pYES2+spermidine was evident after four subcultures,i.e.,on the fourth day after culture.Production and characterization of transgenic pear plants

A transgenic approach was applied to alter the Spd level by over-expressing apple MdSPDS1in pear. Twenty-one kanamycin-resistant independent pear lines were obtained through Agrobacterium-mediated transformation,which were con?rmed by PCR using two primer pairs:CaMV35S–nosT primer sets and inner MdSPDS1sequence primer sets(data not shown).RNA gel blot analysis revealed that the expression levels of MdSPDS1varied within lines,as shown in seven randomly selected lines(Fig.2a).The wild type also had an MdSPDS1signal of a low level, indicating that pear possesses an MdSPDS1-homol-ogous sequence expressed under the given conditions. Based on the expression levels,three lines,no.

9 Fig.2(a)RNA gel blot analysis of introduced MdSPDS1in pear transformants.The lower panel shows the rRNA stained with ethidium bromide.The relative values of the expression level normalized to the ribosomal RNA level by densitometric analysis are indicated over rRNA.The hybridization signal of the wild type(WT)was set to100,and the values of the transformants were quanti?ed accordingly.(b)Morphology of wild-type(WT)and transgenic pear lines.(c)Putrescine(Put), spermidine(Spd),and spermine(Spm)titers of wild-type(WT) and transgenic pear lines at20days after transferring into a medium.Values within a group followed by the same letters for each line are not signi?cantly different at p=0.05

(low-expression level),no.11(medium),and no.32(high),were chosen for further analysis.

Morphological changes caused by the over-expres-sion of the MdSPDS1gene are shown in Fig.2b.Among the selected lines,no.32showed a very strong stem with a reduced shoot https://www.sodocs.net/doc/713137683.html,pared with the wild type,lines no.11and no.9also showed reduced shoot height although the differences were not statistically signi?cant (data not shown).

HPLC analysis demonstrated that free polyamine titers were considerably modi?ed by the expression of MdSPDS1in transgenic lines (Fig.2c).The tendency of Spd titers of these lines was consistent with their transcript levels of MdSPDS1.The Spd titers of lines no.9,no.11,and no.32were 1.25-,1.55-,and 1.89-fold higher than those of the wild type,respectively,on the 20th day after transferring into a new stress-free medium.Their Put titers were 1.25-,1.60-,and 1.07-fold higher than those of the wild type,respectively,although the increments of line no.32were not signi?cant.Similarly,the Spm titers were also increased by 0.62-,1.70-,and 0.94-fold,respectively.Thus,we produced transgenic pear lines with modi?ed morphological traits and altered polyamine levels.

Tolerance of transgenic pear plants to salt stress When 150mM NaCl was added into a medium,the growth of the transgenic lines and wild type were

greatly affected.The response of shoots to salt stress varied with the lines.On the tenth day under salt stress,there were no obvious injuries,but the shoots of the wild type and line no.9showed slightly deteri-orated features.On the 15th day,the leaves of the wild type and line no.9began to wither,while lines no.11and no.32remained vigorous (data not shown).Subsequently,necrotic damage to leaves gradually appeared in the fully expanded leaves of the wild type and lines no.9and no.11on the 25th day after treatment,whereas limited or no sign of wilting or necrosis was observed in line no.32(data not shown).Finally,on the 30th day after treatment,lines no.9and no.11and the wild type were intensively damaged,but line no.32was not (Fig.3a).

Although no obvious damage appeared within the tenth day of treatment under NaCl stress,the increments of shoot and weight of those lines tested were affected (Fig.4a).Compared with each control condition (àstress),the wild type and line no.9showed signi?cant reductions in the increments of both height and weight.Line no.11showed a sharp reduction in the height increment,whereas no signi?cant decrease in the increments of height and weight was observed in line no.32,suggesting that line no.32is possibly the candidate with the most potential for high tolerance to salt stress among all tested lines.

To assess the salt stress damage to cell mem-branes,the EL of shoots was measured.

Compared

Fig.3(a )Morphological features of in vitro wild-type (WT )and transgenic pear lines after 30days of NaCl (150mM)treatment,(b )after 10days of mannitol (300mM)

treatment,(c )after 15days of CuSO 4(500m M)treatment,and (d )after 19days of culture in a stress-free medium

following 76days of CuSO 4treatment

with the controls,the EL of lines no.9and no.11 increased signi?cantly on the third day after treat-ment,and a signi?cant difference appeared on the seventh day after treatment in the wild type(data not shown).However,no remarkable difference between the control and salt-treated shoots was detected in line no.32on the tenth day after treatment,although the EL value was slightly higher in the stressed shoots(Fig.4a).

NaCl triggered the alterations of free polyamines (Table1).The Put titers of lines no.9and no.11and the wild type signi?cantly increased on the tenth day after salt treatment,whereas that of line no.32signi?cantly decreased.The Spd titers of all tested lines increased upon salt stress,although only the increases of lines no. 11and no.32were statistically signi?cant.Salt stress also induced a high level of Spm accumulation in lines no.9and no.32.When the(Spd+Spm)/Put ratio of the transgenic lines was compared with that of the wild type,the ratios of lines no.11and no.32were signi?cantly enhanced(Table2).Tolerance of transgenic pear plants to osmotic stress

The morphological features of the tested lines were affected by300mM mannitol in the medium (Fig.3b).On the tenth day after treatment,the shoot tips of the wild type and lines no.9and no.11 withered,but those of line no.32remained intact.All wild-type and transgenic lines,compared with the controls,showed a signi?cant decrease in the incre-ment percentage of shoot height and weight,among which line no.32,however,showed the greatest increments in shoot height(6.8%)and weight (25.0%)(Fig.4b).EL also clearly showed that lines no.32and no.11were the strongest ones(Fig.4b).

Mannitol treatment also triggered alterations in free polyamines(Table1).The Put titers of lines no. 9and no.11increased on the tenth day after treatment,whereas that of line no.32decreased a little.The Spd titers of the wild type and line no.9 decreased slightly,whereas those of lines no.11

and

no.32increased by about 12and 33%,respectively,and the increase of line no.32was statistically signi?cant.Mannitol treatment also induced a signif-icant Spm accumulation in line no.32,but a slight decrease was observed in the wild type and lines no.9and no.11.The ratios of (Spd +Spm)/Put after stress treatment were higher in the transgenic lines than in the wild type;however,only that of line no.32was signi?cantly enhanced (Table 2).

Tolerance of transgenic pear plants to copper stress

The selected lines and wild type were cultured in a medium containing 500m M CuSO 4for copper stress treatment.Morphological changes on the 15th day after treatment are shown in Fig.3c.No obvious damage was observed on the 15th day,but the leaves of all plants except line no.32tended to droop.When those plants were transferred to a stress-free medium after a 76-day-CuSO 4stress treatment,lines no.11and no.32recovered shoot growth earlier than the wild type and line no.9(Fig.3d).The increments of shoot height and weight of wild-type and transgenic lines were greatly affected (Fig.4c).Compared with

Table 1Polyamine titers of wild-type (WT )and transgenic pear lines under NaCl (on the tenth day),mannitol (on the tenth day),or CuSO 4stresses (on the 15th day)Line

Put (nmol g à1FW)a Spd (nmol g à1FW)a Spm (nmol g à1FW)a àStress

+Stress

àStress

+Stress

àStress

+Stress

(NaCl)WT 303.4±35.6372.6±13.1*164.0±32.8173.4±23.943.6±12.652.4±9.3Number 9331.9±56.5449.6±90.8*194.4±43.0205.1±34.164.3±4.0110.0±9.1**Number 11186.6±65.5261.1±67.4*195.6±14.0233.2±24.0*81.3±43.172.0±7.8Number 32269.8±17.2224.6±5.1*229.7±12.9268.6±4.7**39.1±4.595.6±8.9**(Mannitol)WT 269.8±40.3294.1±56.5197.5±28.9194.5±30.659.7±5.742.2±14.4Number 9275.3±74.3348.1±20.5*218.8±38.0202.8±28.371.5±24.859.2±2.4Number 11193.5±47.6268.5±24.9*210.1±6.9234.4±28.997.6±16.875.5±15.6Number 32269.8±17.2251.1±15.7229.7±12.9305.5±24.3**39.1±4.574.8±2.7**(CuSO 4)WT 157.9±36.1115.5±28.4206.7±14.5168.4±15.5*84.4±12.663.3±12.2Number 9337.0±35.6264.2±23.2*213.5±22.2163.4±12.1*57.0±4.364.9±9.9Number 11319.5±30.4243.7±17.2*235.1±16.7244.7±13.255.8±7.843.9±11.5Number 32

249.3±17.5

139.9±16.0**

235.4±14.6

252.2±10.9

50.6±5.5

49.0±9.3

a

One or two asterisks stand for a signi?cant difference between the +Stress and àStress in the wild type and each transgenic line at p =0.05or 0.01,respectively

Table 2The (Spd +Spm)/Put Ratio of wild-type (WT)and transgenic pear lines under NaCl (on the 10th day),mannitol (on the 10th day),or CuSO4stresses (on the 15th day)Line

(Spd +Spm)/Put (%)a àStress

+Stress

(NaCl)WT

69.1±17.360.5±2.3Number 978.6±10.471.9±15.0Number 11164.6±68.7**122.9±36.0**Number 3299.7±3.3*162.7±2.4**(Mannitol)WT 85.4±30.796.4±23.3Number 983.2±14.1101.1±34.7Number 11167.0±48.4**115.8±8.3Number 3299.7±3.3151.4±1.8**(CuSO 4)WT 189.8±38.1204.5±24.8Number 980.4±3.1**86.3±1.4**Number 1191.3±5.2**118.6±9.5**Number 32

115.0±8.3**

216.7±23.4

a

One or two asterisks stand for a signi?cant difference

between the wild type and each transgenic line at p =0.05or 0.01,respectively

the controls,all transgenic lines and the wild type showed a signi?cant reduction in the percentages of height increments.The percentages of weight incre-ments were also markedly affected by CuSO4treat-ment in the wild type and lines no.11and no.9, whereas only a slight decrease was observed in line no.32.The value for TBARS,which may re?ect the damage of the cell membrane,increased after CuSO4 treatment in both transgenic lines and the wild type. The wild type and line no.11were more remarkably elevated than lines no.9and no.32(Fig.4c).These results were based on the increments of height and weight,and the TBARS values also indicate that no.32may possess a considerably high tolerance to CuSO4stress.

CuSO4stress induced the changes of free poly-amines(Table1).The Put titers of all tested lines and the wild type decreased on the15th day after treatment,although the reduction of the wild type was not statistically signi?cant.The Spd titers of the wild type and line no.9signi?cantly decreased, whereas those of lines no.11and no.32increased slightly.Except line no.9,all the tested lines showed a slight reduction in the Spm titer.The ratios of(Spd+Spm)/Put were signi?cantly reduced in lines no.9and no.11compared with their wild type(Table2).However,when compared with their control counterparts(àstress),the increment of the ratio in line no.32was remarkable after CuSO4 treatment(Table1).

Expression of MdSPDS1under stress conditions

To investigate whether in vitro transgenic pears were responsive to stress via modifying the expression of the transgene,the expression levels of MdSPDS1 were analyzed under salt,osmotic,and copper stress (Fig.5).After being cultured for10days in a medium supplemented with150mM NaCl,lines no.9and no. 32responded by increasing the transcript level of MdSPDS1,which,however,was slightly reduced in the wild type and line no.11.Upon mannitol treatment,the transcript level of MdSPDS1increased in line no.11and dropped in line no.9after stress treatment.No obvious change was observed in the wild type and line no.32.Under CuSO4stress,no obvious elevation in the transcript level was scored in the wild type,but an increase(lines no.9and no.11) and a decrease(line no.32)in the expression levels were revealed.Thus,although there were slight changes in the expression levels of the transgene after stress,these changes were not as obvious as expected;i.e.,no further inductions of MdSPDS1 were observed.Furthermore,no clear correlations with the changes in Spd titer upon stress(Table1) were obtained.

Sodium and copper concentrations of transgenic pear plants

To get an insight into the mechanism of stress tolerance in line no.32,the sodium and copper concentrations under stress treatments were com-pared with those of the wild type.Pear plants contained0.32g kgà1DW of sodium under stress-free conditions,whereas the sodium concentration was ca.90times higher under salt treatment(Table3). However,there were no signi?cant differences in the Na concentration between the wild type and line no.

32.Pear plants contained1.2mg kgà1DW of copper under stress-free conditions(Table3).After stress, the copper concentrations in the wild type and line no.32were140and100times higher than

those Fig.5RNA gel blot analysis of MdSPDS1in wild-type(WT) and transgenic pear lines after NaCl(150mM,on the tenth day),mannitol(300mM,on the10th day),or CuSO4(500m M, on the15th day)stresses.Ethidium bromide staining for equal loading of rRNA is shown below each panel.The relative values of the expression level normalized to the ribosomal RNA level by densitometric analysis are indicated under rRNA.The hybridization signal of the control in the wild type under non-stress conditions was set to100,and the values of the others were quanti?ed accordingly

under stress-free conditions,respectively.Conse-quently,the copper concentration in line no.32was considerably lower than that in the wild type. Discussion

In the present study,MdSPDS1introduced into an SPDS-defective yeast mutant spe3recovered its growth without an Spd supplement in the medium, indicating that MdSPDS1encodes functional SPDS (Fig.1).Thereby,to verify our hypothesis that MdSPDS1could be involved in the stress response and to obtain stress-tolerant fruit trees as well, MdSPDS1was used for the transformation of the European pear.Consequently,we successfully ob-tained MdSPDS1-over-expressing transgenic pear plants with various expression levels(Fig.2a)and Spd titers(Fig.2c)and evaluated their tolerance to salt,osmotic,and heavy metal stresses in terms of growth rate and membrane integrity(Fig.4).By the introduction of MdSPDS1,the selected transgenic lines(no.9,no.11,and no.32)showed higher Spd titers than the wild type(Fig.2c).However,it was noteworthy that the increase of Spd was less than expected irrespective of the use of a constitutive CaMV35S promoter.Kasukabe et al.(2004)indicated that the titers of free Spd and conjugated Spd were less than threefold in the transgenic lines than in the wild type,and similar results were also observed in tobacco(Franceschetti et al.2004).These results suggest that Spd biosynthesis should be strictly regulated(Bhatnagar et al.2002).Alternatively,a high-Spd titer might serve as a negative impact for survival;in this way,the transgenic adventitious shoots with high-Spd titer could not be regenerated or could not survive even if regenerated.Nevertheless,it is interesting that even a small increase in the Spd titer is suf?cient to confer tolerance to multiple stresses on plants,including pear.

Transgenic pear lines showed enhanced but varied tolerance to NaCl,mannitol,and CuSO4stresses than the wild type(Figs.3,4).Among the tested transgenic lines,line no.32ranked?rst in both the MdSPDS1transcript level and Spd titer and,conse-quently,showed high tolerance to various abiotic stresses,as reported in Arabidopsis lines TSP-16and TSP-15and sweet potato lines TSP-SS-1,SS-2,and SS-3(Kasukabe et al.2004,2006).The question then arises of why the transgenic line over-expressing a single transgene(SPDS)can enhance tolerance to multiple environmental stresses in plants.It has been shown that over-expression of the SPDS gene induced expressions of various stress-regulated genes,such as DREB,low-temperature-induced pro-tein78(LTI78or rd29A)in addition to many transcription factors,such as WRKY,B-box zinc ?nger proteins,NAM proteins,and MYB(Kasukabe et al.2004).A molecular process for stress responses including drought and salinity has been shown to be the speci?c regulatory network of gene expression, although some signaling pathways of these stresses share a common path(Shinozaki et al.2003;Seki et al.2003).Therefore,such transcription factors could confer a multiple environmental stresses on plants(Liu et al.1998;Kasuga et al.1999).This ?nding indicates the possibility that SPDS triggers signaling pathways for multiple environmental stres-ses.However,it is possible that,being cationic at physiological pH,the polyamines interact with anionic molecules,such as DNA,RNA,proteins, and membrane lipids(Feuerstein and Marton1989; Schuber1989),which makes it possible for polyam-ines to implicate in multiple stress tolerance.Spd could bind to these molecules and prevent them from degradation under stress conditions and affect their

Table3Sodium or copper concentrations in the wild type(WT)and transgenic line no.32incubated for15days under150mM NaCl or500m M CuSO4

Line Sodium(g kgà1DW)a Copper(g kgà1DW)a

àStress+StressàStress+Stress

WT0.33±0.0929.77±8.65 1.21±0.22171.26±17.87 Number320.32±0.0428.26±3.74 1.28±0.30124.04±11.08 NS NS NS**

a Two asterisks stand for a signi?cant difference between the wild type and line no.32at p=0.01

physiological impact.In fact,stabilization of DNA (Saminathan et al.2002)and alteration of DNA–protein interactions(Panagiotidis et al.1995)by polyamines,including Spd,have been reported. Antioxidant properties could also counteract oxida-tive damage in plants,reducing free radicals and alleviating lipid peroxidation(Singh et al.2002; Kramer and Wang1989),possibly through the induction of the activities for several antioxidant enzymes such as superoxide dismutase,catalase,and ascorbate peroxidase,as seen in the NaCl treated-Brassica juncea seedlings combined with a poly-amine application(Verma and Misha2005).

When we focused on the ratio of(Spd+Spm)/Put rather than on each change in polyamines,we found that the ratio of(Spd+Spm)/Put was always higher in the stress-treated line no.32than in that of its control counterpart(-stress)or in those of the WT and other clones(Table2).Indeed,Spd and Spm seemed to play an important role in preserving the thylakoid membrane integrity(Bestford et al.1993), whereas Put has been reported to cause depolarization of membranes(Tiburcio et al.1990).Therefore,the increase in the(Spd+Spm)/Put ratio might be one of the crucial factors for stress tolerance.

Further induction of MdSPDS1expression was not obviously observed upon stress when compared before and after stress treatments with NaCl,mannitol,or CuSO4,as shown in Fig.5.When the speci?c probe for apple MdSPDS1,which corresponds to the50-UTR region(Zhang et al.2003),was used,the same expression pro?les were obtained,indicating that the expression levels of the authentic pear SPDSs were not affected by the transgene,an apple MdSPDS1 (data not shown).In addition,the Spd titers varied with stress in wild-type and transgenic lines (Table1),but these changes were inconsistent with MdSPDS1expression.Even if Spd titers increased upon stress treatments,as seen in NaCl stress of lines no.11and no.32in Table1,MdSPDS1 expression increased little and was sometimes rather repressed(Fig.5).Theses results suggest that cellular-free Spd may not be regulated at the SPDS transcription level but at other levels,such as the catabolism of Spd/Spm by polyamine oxidase and/or acetylation by spermidine/spermine N1-acetyltrans-ferase(Del Duca et al.1995).Another possibility was that the in vitro culture itself was a kind of stress to some degree and that was the reason that no further induction of MdSPDS1expression was observed by the additional environmental stresses.It has been,however,reported that the expression of apple ADC and SAMDC was further induced and the growth of cultures was further impaired upon stress using the same culture system(Hao et al.2005a,b), indicating that the present culture system was applicable to the evaluation of the stress response of in vitro apple shoots.Thus,although the mech-anism underlying the regulation of Spd biosynthesis is yet uncertain,the transcription of MdSPDS1and metabolism of Spd might be under strict regulation.

The concentrations of sodium in pear plants subjected to stress treatment were ca.100times higher than those of plants under stress-free condi-tions;however,there were no signi?cant differences in the sodium concentration after salt stress between the wild type and line no.32,whereas the copper concentration in line no.32was much lower than that in the wild type after CuSO4treatment(Table3). These results suggest that the MdSPDS1-over-expression had no effect on the characteristics of sodium absorption/excretion;however,it might affect the allocation of sodium in the tissues/cells.Another possibility is that the cationic nature of polyamines affects membrane integrity and would,thereby, remain intact in line no.32under stresses.On the other hand,the copper concentration was somewhat lower in line no.32than in a wild plant,indicating the presence of some mechanism for promoting copper reduction in pear shoots of line no.32.Wang et al. (2006)demonstrated that endogenous Spd and Spm can promote copper tolerance in Nymphoides pelta-tum and the elevation of the(Spd+Spm)/Put ratio may be critical in improving copper tolerance of plants.In the present work,the ratios also increased signi?cantly in lines no.11and no.32after CuSO4 treatment when compared with the control(àstress). Therefore,these lines,particularly line no.32,dem-onstrated that strong tolerance to copper may be greatly attributed to the relative suitable polyamine metabolism under this stress.Lines no.11and no.32 could stand for a long period under the CuSO4stress condition,and they might,therefore,be useful for the phytoremediation of copper-contaminated soil.Thus, the polyamine biosynthetic gene should be an alter-native candidate for phytoremediation in addition to bacteria-or yeast-derived genes,such as arsenate reductase,selenocystein methyltransferase,and

mercuric ion reductase(Kra¨mer2005).Furthermore, considering the cationic nature of polyamines as discussed above,these transgenic pear plants might show tolerance to other heavy metal stresses as well.

Taken these?ndings together,we con?rmed that polyamines are closely related to the tolerance to salt, osmotic,and copper stresses in pear.MdSPDS1-over-expressing line no.32showed diverse tolerance to multiple abiotic stresses,and this is the?rst report of such a?nding in fruit trees.Therefore,it demon-strated a promising prospect in the genetic improve-ment of abiotic stress tolerance in pear.Furthermore, it extended the potential of genetic engineering using the SPDS gene to obtain plants with higher tolerance to multi-stresses from herbaceous plants to tree species.The transgenic pear,as a perennial plant, could grow under adverse environmental conditions over years,which provides an advantage over annual crops for its potential to restore environmental conditions and/or afforestation of deserts.However, it should be noted that these results came from in vitro shoot cultures;therefore,it will be necessary to carry out further experiments under potted or?eld conditions in the future.

Acknowledgments We are grateful to Drs.Ji-Hong Liu (Huazhong Agricultural University,People’s Republic of China)and Hiroyasu Kitashiba(Tohoku University,Japan) for their participation in critical discussions.This work was supported in part by a Grant-in-Aid from the Japan Society for the Promotion of Science(JSPS).This work was undertaken when X.P.Wen was a visiting scientist at NIFTS. References

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淘宝客服聊天的些技巧 淘宝客服主要工作就是和客户聊天，掌握好淘宝客服聊天技巧让你快速成交订单，下面我从五个方面假设了客服的提问和相对于的回答技巧。如果你还在对客户问你你无从回答而着急的话，快来看看吧。淘巧好，好淘巧 第一：假定准顾客已经同意购买商品，当准顾客出现购买的信号却又犹豫不决拿不定主意时，可以采用二选其一的技巧。譬如淘宝客服可以对准顾客说：“请问您要那件黑白条的还是纯黑的呢？或者说”请问是亲哪里有什么快递可以到 此种的二选其一的问话技巧，只要准顾客选中了一个，其实就是客服帮忙的主意，下决心买了！ 第二：帮助准顾客挑选：许多准顾客即使有意购买，也不喜欢快速的下单，顾客总是喜欢东挑西拣，在产品颜色规格式样交货日期不停的在打转。这个时候就需要淘宝店铺的小二们来改变策略,暂时不要谈下订单的问题，转而热情地帮助顾客挑选颜色，规格、式样等，一旦上述问题解决，您的订单也就落实了。 第三：利用怕买不到的心理，人们常对越是得不到的，买不到的东西，就越想得到它、买到它。店小二可以利用这种顾客怕买不到的心理，来促成订单的完成。譬如说，小二可以对顾客说;这个产品现在已经是最后一个了，短期内可能也不会再进新货，亲不买就没有了哦。或者说：今天是小店优惠的截止日期希望亲可以把握时间哦，明天买的话可能就买不到这种折扣价了哦！ 第四：先买一点试试看看，准顾客想要买您的单子，可是又对产品没有信心的时候，这个时候小二就可以建议顾客先买一点看看，只要您对产品有信心，虽然刚开始订单数有限，然而对方使用满意之后，就可能给您一个意想不到的单子这个一试用看看的技巧也可以帮助顾客下决心购买。 第五：在淘宝上购物有的顾客天生就喜欢优柔寡断，他虽然对店里的产品感兴趣但总是拖拖拉拉，迟迟不作决定。这时店小二就不妨等下再回顾客的信息，这个举动会让顾客早点下决心购买的。 第六:利用反问式的回答;所谓反问式的回答，就是当顾客问到淘宝店铺某种产品，不巧正好没有时，就得运用反问来促成订单。举例来说，准顾客问：掌柜家有白色的T恤吗？这时店小二不可以回答没有，而应该反问道：抱歉！我们没有生产这个型号的，不过我们有黑色红色、紫色、在这几种颜色中，亲比较喜欢哪一种呢？ 这些都是我没事的时候总结的，希望对有些新卖家会有些用，店主在此多谢了，

如何使用阿里旺旺

如何使用阿里旺旺 目录 一、教你如何玩转阿里旺旺................................................................................... - 2 - 1.1、什么是阿里旺旺....................................................................................... - 2 - 1.2、为什么要使用阿里旺旺........................................................................... - 2 - 1.3、五大秘籍教你使用阿里旺旺。............................................................... - 2 - （一）、查找添加商友。........................................................................... - 2 - （二）、分组管理客户............................................................................... - 5 - （三）、如何启用一个群........................................................................... - 9 - （四）、屏蔽垃圾信息............................................................................. - 11 - （五）、查看聊天记录............................................................................. - 13 - 二、阿里旺旺使用技巧六则................................................................................. - 14 - 2.1、如何更改阿里旺旺联系人上限............................................................. - 14 - 2.2、如何通过阿里旺旺的商友推荐添加好友............................................. - 15 - 2.3、利用阿里旺旺查找商友......................................................................... - 16 - 2.4，如何通过阿里旺旺查看资讯................................................................. - 16 - 2.5、如何通过阿里旺旺寻找热门的批发信息............................................. - 17 - 2.6、如何利用旺旺搜索供应信息................................................................. - 19 - 三、阿里旺旺使用教程之具体功能..................................................................... - 20 - 3.1、客户分组管理......................................................................................... - 20 - 3.2、使用秘密武器“阿里旺旺”来和信息发布方联系................................. - 22 - 3.3、【阿里旺旺】轻松管理商业好友........................................................... - 23 - 3.4、快捷登录阿里巴巴................................................................................. - 25 - 四、阿里旺旺遍天下让您如虎添翼..................................................................... - 25 - 五、如何利用旺旺找买家和买家洽谈................................................................. - 31 - 六、阿里旺旺快捷短语不打字也能做成生意..................................................... - 36 -

阿里旺旺卖家版 基本操作 新手必看 原创

阿里旺旺卖家版基本操作 阿里旺旺常用操作 图0.1 最下方一行，自左至右依次为： 我的店铺，卖出宝贝，店铺管理，我的淘宝，支付宝，量子恒道。 其功能分别是：我的店铺：直接进入店铺首页，并可以进行装修编辑等操作； 卖出宝贝：进入已卖出的宝贝列表，可以查看最近的成交订单； 店铺管理：进入装修界面，进行直接编辑装修； 我的淘宝：进入卖家中心，直接进行后台操作管理； 支付宝：进入支付宝管理界面，进行支付操作； 量子恒道：直接进入量子恒道数据统计界面，直接查看详细的店铺经营数据。除了以上设置的快捷进入方式之外，点击最左面的“淘”，可以出现更多的选项。 图0.2 如果想对下面的快捷栏进行自定义设置，可以按照以下步骤进行操作：

（1）点击左下角的“淘”字，弹出如图0.2所示的选项卡； （2）点击该选项卡右上角的“管理”选项命令，弹出图0.3所示对话框 图0.3 （3）将需要进行调整的快捷选项进行删除，如下图： 图0.4

（4）将需要添加的快捷方式点击添加 图0.5 （5）点击“确定”。 图0.6 3、店铺后台操作—卖家中心 进入卖家中心，有两种方法。第一种在前面已经说过，通过阿里旺旺的快捷方式进入；第二种则是通过网页上的“卖家中心”进入。进入之后，有四大方面的内容：我是卖家，账号管理，官方信息中心，卖家地图。下面是具体内容： （1）我是卖家 在页面的左侧栏，有以下内容。

图0.7 点击“我购买的服务”，出现下图所示内容： 图0.8 其中，即将到期的是特推广，限时冰点营销，以及图片空间。 【交易管理】最常用的是“已卖出的宝贝”，可以快速查看已成交的宝贝。【宝贝管理】比较常用的是下图的几项内容：

项目1任务2活动1使用阿里旺旺工具2

《客户服务》课程教学设计 授课教师： 一、阿里旺旺卖家版的主要功能及操作 提问：千牛平台有哪些功能呢？ 回答：阿里旺旺卖家版的主要功能是广交好友、买卖沟通、炫酷表情、阿 里旺旺群和文件传输。 例如：

提问：千牛平台如何添加好友呢？ 【实践】 任务一：请看书本，告诉我以下三张图片分别代表的三种添加好友的方法。 方法一：聊天时添加好友。可以点击聊天记录界面上的“加为我的好友”图标按钮进行添加。 方法二：搜索添加好友。 方法三：添加已联系人为好友。 2、阿里旺旺卖家版的主要功能及操作——群 提问：千牛平台如何新建群呢？ 教师根据图说明

【实践】 任务二：打开阿里旺旺群，请说出以下按钮的功能。 回答：阿里旺旺是一款沟通聊天工具，它的最大优势和特点就在于丰富的聊天功能，具有丰富的聊天功能按钮（如图1-23）。从左往右的功能按钮依次是：“选择表情”、“设置字体”、“发送图片”、“发送文件”、“屏幕截图”、“发送振屏”、“提醒客服评价”、“计算机”、“快捷短语”和“查看消息记录”。 3、阿里旺旺卖家版的主要功能及操作——客服插件 提问：这是什么？ 回答：客户服务插件 提问：客户服务插件有哪些？ 1、订单插件 2、商品插件 3、机器人插件 4、客户插件 教师说明： 订单插件：显示当前沟通客户在店铺的“全部”、“未完成”、“已完思考讨论完成任务 聆听 思考 讨论 记录 回答问题

成”和“已关闭”四种订单信息，方便客服了解客户订单信息，进行改单、催单、同意取消订单等操作。 商品插件：显示当前沟通客户在店铺内的商品浏览足迹，向客服展示当前店铺内的“推荐”、“热销”和“橱窗”商品，便于客服向客户快速发送商品信息和活动信息。 机器人插件：分为“半自动”和“全自动”两种模式，用于设置自动转接客户到别的客服和配置各种自动回复问题，加强了客服同时接待多位客户的能力。 客户插件：显示当前沟通客户的基本信息，如所在地、买家信誉、好评率、上次登录、注册时间和认证等信息。使客服能够更加直观的了解客户，对客户有个初步的分析和判断，便于进一步的交易沟通。 二、商品插件下按钮功能 提问：什么叫SKU？ 回答：英文全称为 stock keeping unit, 简称SKU，定义为保存库存控制的最小可用单位，可以是以件，盒，托盘等为单位。 提问：除了SKU，还有哪两个按钮？ 回答：属性：商品属性； 发送：分享商品。教师举例说明 【实践】 任务三：以两人为一个小组，分别扮演买家和卖家角色，以下图商品信息为背景，通过阿里旺旺卖家版的聊天功能进行模拟商品信息沟通。

如何使用旺旺

阿里旺旺卖家版操作指南（一） 1下载与安装 从旺旺卖家版下载页面点击下载（https://www.sodocs.net/doc/713137683.html,/seller），点击保存，将执行文件下载到本地机器上。然后双击安装文件，并点击运行。开始进入安装向导，点击"下一步"，点击“我接受”许可协议，接下来选择安装目录，您可以点击“浏览”修改安装目录。默认的路径是：C:\Program Files\淘宝网\阿里旺旺\ ，点击“安装”，再根据提示来进行操作即可。 2登陆与退出 登陆：在桌面双击打开客户端登陆界面，输入用户名和密码即可登陆。如果是子账号登陆，则用户名中间的“:”需要将输入法切换到英文状态。 退出：在旺旺客户端点击“关闭”按钮不会退出程序，退出需要在任务栏右下角的图标上点击右键退出。 3添加好友 在旺旺客户端主界面有“添加好友”的图标，点击进入好友查找界面，输入用户名，在筛选出的用户列表中，选中需要添加的联系人，点击“加为好友”按钮，如所添加好友需身份验证，请填写验证信息，然后点击“确定”，发送给对方。如好友设置不需要身份验证即可加为好友，选择将好友归类的组，点击“确定”按钮，添加的好友会收到系统消息，通知他您已添加他为好友。 如果出现提示“该用户拒绝被任何人添加”，是因为您添加的好友在阿里旺旺“系统设置”—“安全设置”—“验证设置”中设置了“拒绝任何人加我为好友”。 阿里旺旺（包括E客服账号）每天添加好友数量跟您的阿里旺旺活跃度、帐号的认证程度（进行手机号码认证或支付宝认证）、交易量等达到一定程度后会自动提升添加好友的权限了。 一、如果您想要添加更多的好友，建议您可以做如下操作提升权限： 1、进行手机号码的认证 2、进行支付宝认证 3、完成更多的交易以及提升卖家或买家信用. 最多可添加1024个好友。 注意：E客服子账号可添加的好友个数，根据主账号所拥有的权限不同而不同。 旺旺群规则介绍 1、群成员数限制和群管理员数限制： 2、群启用数量规则： 从第一次登录阿里旺旺开始即分配有1个群；活跃度达到5级，有2个群可以启用；达到9级，有3个群可以启用；达到18级，则有4个群可以启用。但一个帐号最多可以启用4个群。 3、加入群限制

阿里旺旺帐号注册与使用步骤与技巧_淘宝开店必备

阿里旺旺账号注册方法 首先，在电脑上安装阿里旺旺。 安装好后，打开登录页面。 在下方，有三个选项“找回密码”“免费注册”“网络设置”，当然选择“免费注册”。于是自动登录到淘宝网注册页。 淘宝旺旺账号注册步骤： 第一步、绑定旺旺前，请先下载安装阿里旺旺卖家版。下载成功后，双击安装程序，安装步骤提示，点击下一步即可安装成功！ 淘宝旺旺账号注册 第二步、安装程序成功后进行免费注册。

淘宝旺旺账号注册 注意：您的旺旺会员名最好与您的口碑商户用户名保持一致。 淘宝旺旺账号注册

淘宝网友解答淘宝旺旺账号注册后登陆不了遇到的几种情况： KFC3344：有一次我在淘宝旺旺账号注册后也登陆不了，我想可能是前一天晚上我在发广告，用了同一条信息的频率太多，所以第二天就不能登陆了。打电话去淘宝网也不能得到任何解释，只说我的账号是正常状态。我后来也试过换别的电脑登陆那个淘宝旺旺账号，结果可以登陆，之后又用朋友的电脑登陆，也可以登录。后来在自己的电脑上还是一直不能登录，只好把自己的淘宝旺旺卸载了，然后重新安装，淘宝旺旺账号注册也反复再操作一次。之后就正常了。 思来想去-top：可以尝试下面两点操作。 1.用修复软件修复INTER网（一般做了此步就能解决淘宝旺旺账号注册后不能登录的问题） 2.重新下载旺旺，就可以了！ 放包里的钱：一般淘宝旺旺会有提示，如果是提示密码错误，建议你修改淘宝密码，重新尝试。如果提示密码不匹配，则要清除COOKIE了，路径为浏览器-工具-Internet 选项-删除cookie/历史记录。如果还不行，则建议卸载旺旺重装或是在线升级旺旺即可。现在旺旺2009正式版已上市。

阿里旺旺快捷短语使用教程

有经验的业务员，大家会发现客户旺旺咨询的有些回答内容是重复的，于是我们就可以选择快捷短语进行编辑。制作快捷短语，用好这个功能的关键是要设计出好的快捷短语内容，并在合适的场合，发出给适当的快捷信息。这些，我会通过如下几点为大家讲解： 一、基本常用的快捷短语设置思路; 二、设置快捷短语的具体步骤，如何修改、移动位置; 三、快捷短语如何应用，导入、导出 下面给大家举几个例子介绍一下，基本常用的快捷短语设置思路，可以利用的地方当然不止这些，相信大家可以举一反三，灵活应用。可以参考一下： 1、有人来旺我们时 设计一个问候语，这点很重要，即能够显示出自己的热情，也可以显示出自己的专业。这个大家可以根据自己的语言习惯、表达方式来定。比如如下一位网友的设计： 您好!我们专门做出口绿色包装，纸浆模塑，纸托类代替吸塑包装，纸杯等纸制品，有什么可以帮您的! 2、客户询问产品质量时 可以用：这点您放心，我们的产品质量完全有保证的，原材料来自(多数客户可能都需要介绍产品情况。事先做好准备，做好快捷短语，就不会让我们每次数输入同样的话了!) 3、买家问售后保证问题 先想下对方可能会经常用到的咨询内容，然后进行设计，如：我们承诺您七天无条件退货，一年保修，具体的您可以看一下我们的产品介绍，上面有写等，当客户问到售后保证时，就可以第一时间发出这条短语，让客户放心。 4、在聊天结束时 无论对方是否购买我们的产品，给到结束语都是有必要的，让客户感觉更贴心，为下次再来提供条件。如：您要的货物我们会通过中国邮政发出，预计7-14天到，请耐心等候喔。如果有问题请随时联系XXXX(你的手机)。合作愉快，欢迎下次再次光临 在这里要提醒大家一下，快捷短语只是辅助语言，不可以用快捷短语来代替我们的主要回答，这样会给别人很生硬的感觉! 【设置快捷短语的具体步骤】

淘宝新人必看淘宝购物经验及技巧分享

淘宝购物经验及技巧分享 两岁在淘宝上购物有７年的经验了，今天总结了我的一些购物经验和技巧给大家分享下，希望对刚入淘宝的新手买家有帮助． 以前很多商家开始都是在直接做的淘宝店，在淘宝店就是一个好评一个好评的积累起来的，所心很长一段时间，信誉成为卖家特别注意的东西．这与今天说的第一点有关： 1、过分相信信誉高的卖家是好卖家． 金冠？五皇冠?四皇冠?其实信誉高不一定代表产品的质量就好．因为很多老淘友在这点上强调得厉害所以很多新人也就盲目的认为这是要买东西的第一标志．其实这也不是必然的，正确的方法是应该看淘宝买家的评价．观察其店铺的好评，中评，差评以及买家对商品的追加使用评价，这是新出来的，很能体现产品的好坏．另外还在看中差评的原理以及卖家是不是做出了相应的解释．如果一个店铺开了很久一直都没有差评或是中评这也是不正常的，毕竟买家的水平不一样，要求不一样，如果所有的人对产品都是这么好的评价，就有点虚假．要注意看他的开店时间，和评价时间．如果评价都是集中在一个时期内的说明这些评价有可能是伪造的．也就是花钱刷出来的，这样商品的真实度就可想而知了！ 不过也不是没有100%好评的店铺，因为很多小二为了自己的事业还是比较敬业的，这样每一笔交易记录都是心血，所以品质和服务都是自己追求的.擦亮眼睛，不要盲目轻信高信誉！ 2、对于描述中的图片的正确看法 两岁最开始在淘宝上买衣服的时候特别喜欢看美图，那些长得特别好看的模特，还有杂志明星美图，看着他们穿的衣服很漂亮，就跟风的买，买过两次后发现买来的衣服根本是不能看，不是像模特上面的那样漂亮．所以我们在购特的时候不要被杂志图片吸引了，这些都是做秀的．特别是很多卖瑞丽杂志的服装的大家要特别仔细看细节图，如果全是杂志图就不要买了，去别家看真实衣服的图片再确定购买吧！所以在购买的时候宝贝的描述一定要看清楚，图片也要看清楚是不是实拍的，一定要买实拍的才会有安全感的，因为实拍到手可能都有差别，更别说是杂志图片了，做了效果处理的大家都知道的，癞蛤蟆也会变天鹅的！ ３可以考虑去新店购物，但也不要贪便宜 两岁特别喜欢去关顾新店，因为要开淘宝，信誉很重要，所以刚开始买家都是保本或是赔本来赚信用的，产品的质量也是很好的，但是价格比大的卖家那里便宜很多．所以我会先去看看新店的产品，再和卖家谈，一定要谈，要看老板对产品回答专业不专业，你可以在一个大卖家那里先问过问题后再去问小卖家，这样自己心里有一个正确答案．这样知道新店回答的是不是真的！另外新店要看加消保的，不要因为一点点小便宜，１０块或是２０块的事情把自己的大头给去了，这样也是要注意的，不管怎么说，就是新店也只会做保本的生意，如果比其它店铺的同款商品的价格底很多，这多半是假的或是骗人的淘宝店，不要因小失大哦！在买东东的时候一定要多搜索几家，多比较几家，就同我们在实体店逛一样，多看看多问问，货比三家不吃亏．淘宝网里可以按商品名搜索，价格搜索，销量搜索，这些都可以帮到我们，最低价，最高价，最高销售卖家等．如果不是很在行，可以去找一些专栏的导购网或是推荐网去看，现在这类网站很多的，大家在里面可以看到各类产品网友评价和排名都很高的店铺，这样相对来说省时一点！ ４、慎用评价权利。 买卖是一个平等的交易，你付钱，商家发货，这都是建立在平等的关系上的，所以买家对卖家也要报有一颗平等的心去对待，换位思考．很多卖家觉得在一家店里买了东西就是上帝，按自己的心情来给评价，不管产品好坏，不管服务好坏，这样是不对的．如果哪天你也做了卖家，你就会体会到不易了．我们应该按真实的情况评价，

阿里巴巴诚信通的一些操作技巧分享

阿里巴巴诚信通影响客户效果揭秘之贸易通在线时长 影响客户效果是因素很多，本次给大家隆重介绍阿里旺旺在线时长对效果的影响！它可是影响效果指数的狠角色！ 为什么这么说？我们先要从效果的构成来说起—— 虽然，反馈总量是三项反馈指标的总和，但最近30天贸易通留言反馈量是占总反馈的5~6成以上。 所以，增加旺旺在线时长可以直接提升旺旺的留言反馈从而直接提升客户的反馈总量。 从相关性分析来看，旺旺在线时长是跟客户反馈量0.282,也是目前众多指标中跟总反馈量相关性最大的。 打个比方： 一个旺旺在线，另一个不在线，买家有什么问题倾向于直接问下那个在线的，所以旺旺在线时间越长，旺旺的留言反馈也会越多哦！ 我们可以这么对客户讲，两家店虽然东西都陈列得很好，但是一家店一个人都不在，连问下价格都问不了，另一家前台导购笑容可掬，客户当然选择有人接待的啦！所以最好旺旺经常在线，让你的店里服务常驻下来！ 原创文章如转载，请注明：转载自天津通用卓业https://www.sodocs.net/doc/713137683.html,/ 本文链接：https://www.sodocs.net/doc/713137683.html,/tjec/News/shangwukuaiche/

诚信通会员是否可以使用阿里巴巴诚信通的商标? 最近很多阿里巴巴诚信通客户来电话或者在贸易通上询问是否可以使用阿里巴巴诚信通的商标标识，小弟在这里给大家解释一下！ 如果您在加入阿里巴巴诚信通以后，作为诚信通会员，您是可以把阿里巴巴网站大力进行推广的诚信通商标添加到您自己企业的独立网站上作为链接或者标示的，并且还可以印在您企业的个人名片之上，以此共享阿里巴巴诚信通强大的品牌知名度，来提升您企业的形象。 原创文章如转载，请注明：转载自天津通用卓业https://www.sodocs.net/doc/713137683.html,/ 本文链接：https://www.sodocs.net/doc/713137683.html,/tjec/News/shangwukuaiche/

阿里旺旺使用规则

阿里旺旺用户（以下简称“用户”）使用阿里旺旺的各项行为均需遵守如下所列规则。阿里软件制定这些规则的目的是：维护阿里旺旺的正常运营秩序，保障用户的合法权利。在下文中，并未对这些使用规则逐一进行详细讲解，只是举例介绍了用户使用阿里旺旺的相关要求和条件。阿里软件有权根据《阿里旺旺软件使用许可协议》的约定，随时对如下所列规则进行修订。 一、禁止性行为规则： （一）有关发送非法消息、广告消息、垃圾消息方面的禁止性行为规则： 1. 以任何方式将阿里旺旺用于发表、传送、散布或传播任何法律禁止的、不当的、诈骗性的、亵渎性的、诽谤性的、淫秽的、粗俗的、暴力的标题、名称、资料或信息，如虚假的中奖消息； 2. 以任何方式将阿里旺旺用于群发任何重复的或未经请求的广告消息，如淘宝卖家店铺消息、产品或服务消息； 3. 以任何方式将阿里旺旺用于调查、竞赛、传销、兜售侵权商品、群发链式邮件、垃圾电子邮件和垃圾消息； （二）有关危害网络信息安全方面的禁止性行为规则： 1. 通过阿里旺旺进行任何危害计算机网络安全的行为，以任何方式损坏或破坏阿里旺旺或使其不能运行或超负荷或干扰第三方对阿里旺旺的使用； 2. 将阿里旺旺用于传送含有病毒、木马、不安全链接的文件和图片，或其他任何可能破坏他人电脑运行或他人财产安全的类似软件或程序； 3. 在未经授权的情况下访问任何与阿里旺旺关联的所有帐户、计算机系统或网络； 4. 以任何方式获取、收集用户并非有意披露的资料或信息，如用户ID、电子邮件地址、家庭地址等用户注册信息； 5. 在未经授权的情况下破解阿里旺旺技术，并开发与之相关的衍生产品、服务、插件、外挂软件等； 6. 制造假身份以误导、欺骗他人； （三）有关侵犯第三方合法权益方面的禁止性行为规则： 1. 以任何方式将阿里旺旺用于传送或以其他方式实现传送含有受到知识产权法律保护的图像、相片、软件或其他资料的文件，除非用户拥有或控制着相应的权利或已得到所有必要的授权； 2. 通过阿里旺旺传送文件过程中，伪造或删除任何作者署名、法律或其他正当的通告、专有设计、产地或软件来源的标记或传送的文件中包含的其他资料； 3. 通过阿里旺旺诽谤、骚扰、跟踪、威胁或以其他方式侵犯他人的合法权益； （四）违反任何适用于阿里旺旺的法律法规规定的行为。 二、处罚规则 针对用户进行上述禁止性行为，阿里软件将视情节严重程度，给予以下一到四级的处罚：一级处罚：书面警告（通过邮件、系统警告消息、阿里旺旺浮出框等方式进行警告）； 二级处罚：限制使用阿里旺旺部分功能；