ORIGINAL PAPER
The role of MrbHLH1and MrMYB1in regulating anthocyanin biosynthetic genes in tobacco and Chinese bayberry (Myrica rubra )during anthocyanin biosynthesis
Xiao-Fen Liu ?Xue-Ren Yin ?Andrew C.Allan ?Kui Lin-Wang ?Yan-Na Shi ?Yu-Ji Huang ?
Ian B.Ferguson ?Chang-Jie Xu ?Kun-Song Chen
Received:9May 2013/Accepted:20July 2013/Published online:1August 2013óSpringer Science+Business Media Dordrecht 2013
Abstract Anthocyanins,being important for both plant functions and human health,were transcriptionally regu-lated by the MYB–bHLH–WD40transcription complex.The key MYB regulator for Chinese bayberry (Myrica rubra ),MrMYB1,has been characterized in previous studies,while the speci?c bHLH partner(s)are unknown.In this study,MrbHLH1and MrbHLH2were isolated based on their homology to known plant bHLHs involved in anthocyanin biosynthesis regulation.Coordinate expression of MrbHLH1with MrMYB1and the anthocyanin biosyn-thetic genes was observed during fruit development,while MrbHLH2showed a weaker correlation.Further transient assays in tobacco leaves suggested that MrbHLH1,but not MrbHLH2,was associated with MrMYB1and triggered signi?cant anthocyanin production.The lack of function of the MrbHLH2in anthocyanin biosynthesis regulation suggested that different MrbHLH genes within the same phylogenic subfamily have different functions.Overex-pression of MrMYB1and MrbHLH1in tobacco con?rmed the crucial role of MrMYB1–MrbHLH1in anthocyanin biosynthesis and all of the structural genes from NtCHS were up-regulated by the complex.Dual luciferase assays,however,indicated that MrMYB1and MrbHLH1
selectively activated ?ve of the eight promoters of bio-synthetic genes from bayberry (MrCHI ,MrF30H ,MrDFR1,MrANS ,MrUFGT ),although expression levels of all eight biosynthetic genes including MrCHS and downstream genes were coordinately increased during fruit ripening.Moreover,the interaction between MrbHLH1and MrMYB1was con?rmed by yeast two-hybrid assay.In conclusion,MrbHLH1,but not MrbHLH2,was the essential partner of MrMYB1during anthocyanin biosynthesis regulation in tobacco and bayberry,however,the biosynthetic genes in these two species responded differently to the MrMYB1–MrbHLH1complex.
Keywords Anthocyanin áChinese bayberry (Myrica rubra )áMYB ábHLH áInteraction áTranscriptional activation
Abbreviations AbA Aureobasidin A ACE ACGT-containing element ANS Anthocyanidin synthase bHLH basic Helix-Loop-Helix BQ Biqi CHI Chalcone isomerase CHS Chalcone synthase
CTAB Cetyltrimethylammonium bromide DAFB Days after full bloom DFR Dihydro?avonol 4-reductase F3050H Flavonoid 30,50-hydroxylase F3H Flavanone 3-hydroxylase F30H Flavonoid 30-hydroxylase
LDOX Leucoanthocyanidin dioxygenase LUC Fire?y luciferase MBW MYB–bHLH–WD40MCS Multiple cloning sites
X.-F.Liu and X.-R.Yin have contributed equally to this work.X.-F.Liu áX.-R.Yin áY.-N.Shi áY.-J.Huang áC.-J.Xu (&)áK.-S.Chen
Laboratory of Fruit Quality Biology,The State Agriculture Ministry Laboratory of Horticultural Plant Growth,Development and Quality Improvement,Zhejiang University,Hangzhou 310058,People’s Republic of China e-mail:chjxu@https://www.doczj.com/doc/2c3698809.html,
A.C.Allan áK.Lin-Wang áI.
B.Ferguson
The New Zealand Institute of Plant and Food Research,Private Bag 92169,Auckland,New Zealand
Plant Cell Tiss Organ Cult (2013)115:285–298DOI 10.1007/s11240-013-0361-8
MRE MYB recognized element
OMT O-methyltransferase
ORF Open reading frame
Q-PCR Real-time quantitative PCR
RACE Rapid ampli?cation of cDNA ends
REN Renilla luciferase
SD Synthetic dropout
TF Transcription factor
UFGT UDP glucose:?avonoid3-O-glucosyltransferase UTR Untranslated region
Introduction
Anthocyanins are a class of water-soluble?avonoid pig-ments that contribute to red,blue,and purple color pig-mentation of?owers,fruits,seeds,and roots of various plant species.Consumption of anthocyanin-rich plant parts can help prevent neuronal and cardiovascular diseases, cancer,diabetes and age-related degenerative diseases in humans(Butelli et al.2008;Zhang et al.2010;Sun et al. 2012a,b,2013).Therefore,foods with high anthocyanin content are welcomed by consumers because of the rich color as well as the health bene?ts.
Anthocyanins are synthesized in the cytosol and accu-mulate in the vacuole.The anthocyanin biosynthesis pathway enzymes are encoded by a series of structural genes,including chalcone synthase(CHS),chalcone isomerase(CHI),?avanone3-hydroxylase(F3H),?avo-noid30-hydroxylase(F30H),?avonoid30,50-hydroxylase (F3050H),dihydro?avonol4-reductase(DFR),leucoantho-cyanidin dioxygenase/anthocyanidin synthase(LDOX/ ANS),and UDP glucose:?avonoid3-O-glucosyltransfer-ase(UFGT),as well as O-methyltransferase(OMT)in some plants such as grape and petunia(Holton and Cornish 1995;Grotewold2006;Hugueney et al.2009).The expression levels of these anthocyanin biosynthetic genes increases during anthocyanin accumulation and the levels of their mRNAs correspond closely to the changes in anthocyanin https://www.doczj.com/doc/2c3698809.html,ck of or low expression of these genes results in extremely total lack of,or extremely low of anthocyanin accumulation.For example,lack of CHS3 expression results in no anthocyanin accumulation in some grape cultivars(Goto-Yamamoto et al.2002),while sweet oranges and zero-red pericarp lychee fruits lack anthocy-anins due to the absence of UFGT expression(Lo Piero et al.2005;Wei et al.2011).
Research on model plants has shown that anthocyanin biosynthesis is transcriptionally regulated by R2R3-MYB and basic Helix-Loop-Helix(bHLH)transcription factors (TFs),together with WD40proteins,working as an R2R3 MYB-bHLH-WD40(MBW)heterotrimer(Broun2005; Koes et al.2005).Although there is a wealth of knowledge about the MBW complex in anthocyanin biosynthesis regulation,most attention has been focused on the MYB transcription factors(TFs),in Arabidopsis as well as in economically important fruits such as apple(Malus domestica),grape(Vitis vinifera),pear(Pyrus pyrifolia), other Rosaceae plants,lychee(Litchi chinensis),mango-steen(Garcinia mangostana)and Chinese bayberry(Myr-ica rubra)(Boss et al.1996;Takos et al.2006;Espley et al. 2007;Allan et al.2008;Palapol et al.2009;Feng et al. 2010;Lin-Wang et al.2010;Niu et al.2010;Hichri et al. 2011;Wei et al.2011;Chagne′et al.2013).Many genetic results show that the MYB component of this MBW complex appears to provide the most anthocyanin-speci?c effect,for example,a retrotransposon insertion in VvmybA1 blocks its expression,which results in white grape cultivars (Kobayashi et al.2004),a mutation of MrMYB1induces white bayberry(Niu et al.2010),and high level of MYB expression promotes anthocyanin content in red?esh apple and blood orange(Espley et al.2009;Butelli et al.2012).
Further studies on transcriptional regulation of antho-cyanin biosynthesis revealed the crucial role of a second transcription factor,a bHLH protein.It has been showed that the interaction of bHLH and R2R3MYB arose early in land plant evolution and played a key role in anthocyanin regulation.The bHLH member is essential for the activity of the R2R3MYB partner,and it also provides enhanced activity on promoters containing a cis-regulatory element conserved in several anthocyanin biosynthetic genes (Grotewold2006;Feller et al.2011).Only when both MYB and bHLH are overexpressed in tomato,do the fruit of the plants accumulate anthocyanins at substantially higher levels(Butelli et al.2008;Gonzali et al.2009).Analysis of 35S:MrMYB1transgenic Arabidopsis and tobacco plants suggested that higher expression level of an endogenous bHLH partner is required for anthocyanin production in plant tissues(Huang et al.2013a).The bHLH transcription factors,also known as MYC,regulate many cellular pro-cesses,including the biosynthetic pathway of?avonoids in plants through binding DNA either alone or as a dimer with MYB partners(Heim et al.2003;Feller et al.2011;Hichri et al.2011).The?rst bHLH TFs regulating the anthocyanin pathway were identi?ed in maize as R and B,and subse-quently in some other plants,such as AtbHLH42(AtTT8)in Arabidopsis,MdbHLH3in apple and VvMYC1and VvMYCA1from grapevine(Chandler et al.1989;Espley et al.2007;Hichri et al.2010;Matus et al.2010;Xie et al. 2012).
Here we identi?ed and characterized two bHLH mem-bers isolated from bayberry,named MrbHLH1and MrbHLH2,respectively.Phylogenic result showed that both MrbHLH1and MrbHLH2,as well as bHLH genes related to anthocyanin biosynthesis in other plants,were clustered in the IIIf subfamily of Arabidopsis bHLHs.The
results of transient assays and studies on transformed tobacco lines showed that MrbHLH1but not MrbHLH2, was the crucial partner for MrMYB1in regulating the anthocyanin biosynthesis.Dual luciferase assay indicated that MrbHLH1associating with MrMYB1activated?ve of eight anthocyanin biosynthetic genes in bayberry,while all of the anthocyanin biosynthetic genes were stimulated when over-expressing MrMYB1and MrbHLH1in trans-formed tobacco lines,indicating there are differences in regulation between these two species.
Materials and methods
Plant materials
Fruit of Chinese bayberry(M.rubra Sieb.and Zucc.), variety‘Biqi’(‘BQ’),were collected at60,67,74,78,82, and86days(commercial maturity)after full bloom (DAFB)and young leaves and stems were harvested from commercial orchards in Yuyao County,Zhejiang Province, China,during the2011season.
Three biological replicates(15fruits for fruit replicates or approximately30g for other tissue replicates)were frozen in liquid nitrogen immediately after being cut into small pieces,and stored at-80°C.
Determination of anthocyanin content
Anthocyanin content was determined by the pH differential spectro-photometry method described in Zhang et al. (2008)and Niu et al.(2010).Anthocyanins were extracted from1g samples in methanol/0.05%HCl and absorbance of the extracts at510and700nm were measured using a UV-2550spectrophotometer(SHIMADZU).
RNA extraction and cDNA synthesis
Total RNA was extracted using a modi?ed CTAB(cetyltri-methylammonium bromide)method(Shan et al.2008). Genomic DNA was removed by DNase I(Fermentas,USA), and1l g of DNA-free RNA was used to synthesize?rst-strand cDNA with Revert Aid TM First Strand cDNA Synthesis kit(Fermentas,USA)in a?nal reaction mixture of20l L following the manufacturer’s instructions.The cDNA was diluted160-fold and6.5l L of the diluted cDNA was used as template for real-time quantitative PCR(Q-PCR)analysis.
Gene cloning and sequence analysis
From a Chinese bayberry EST collection generated previ-ously by RNA-Seq(Feng et al.2012),117bHLHs were identi?ed.Based on the homology with bHLHs related to anthocyanin biosynthesis in other species,MrbHLH1and MrbHLH2were selected for further study.The MrbHLH1 sequence contains a complete open reading frame(ORF) and full30-untranslated region(UTR),while MrbHLH2 contains a partial ORF and full30-UTR.A full-length sequence of MrbHLH2was obtained by subsequent50-RACE(rapid ampli?cation of cDNA ends)with the primer 50-CGACAACTAGCTCTGAATTGAT-30using SMART RACE cDNA Ampli?cation kit(Clontech,USA)according to the manufacturer’s instructions.Sequence alignment and phylogenetic analysis were carried out with Clustal_X and MEGA v.5.0,respectively(Thompson et al.1997;Tamura et al.2011).
Real-time quantitative PCR(Q-PCR)
Real-time quantitative PCR(Q-PCR)primers(contained in Table1)used to analyze the gene expression pro?les in bayberry and transformed tobacco lines were designed with Primer3(https://www.doczj.com/doc/2c3698809.html,/cgi-bin/primer3/primer3_ www.cgi).Gene speci?cities of the primers were con?rmed by melting peaks,dissociation curves,and PCR product sequencing,according to our previous reports(Yin et al. 2008).The PCR mixture(25l L total volume)included 12.5l L SYBR Green/ROX qPCR Master Mix(Fermentas, USA),3l L of each primer(2.5l M)and6.5l L diluted cDNA.Q-PCR was performed with an iCycler iQ real-time PCR instrument(Bio-Rad,USA),initiated by steps of 2min at50°C,10min at95°C,followed by40cycles of 95°C for15s,58°C for30s,and72°C for30s.All Q-PCR reactions were normalized using the Ct value cor-responding to the actin gene(MrACT,GenBank GQ340770;NtACT,GenBank AJ421411).No-template controls and melting curve analyses were included for each gene and each PCR reaction.
Genome walking and motif analysis
The sequences of the promoter regions of the investigated genes were isolated by genome walking.Genomic DNA was extracted from young leaves of‘BQ’using the CTAB method(Porebski et al.1997),and2.5l g for each was digested with seven restriction enzymes(Dra I,Eco RV, Pvu II,Ssp I,Sca I,Sma I and Stu I)separately.All products were cleaned after digestion using DNA recovery kit(Ta-kara,Japan),and then ligated to the adaptor(Clontech, USA)to serve as templates in genomic walking PCR.The nested PCR analysis was performed with two sets of primers,one set was supplied with the kit,and another set was the gene speci?c primers contained in Table2.
Conserved cis-element motifs located in promoters were scanned by online software PLACE(http://www.dna.affrc. go.jp/PLACE/signalscan.html;Higo et al.1999)and Plant-
CARE(http://bioinformatics.psb.ugent.be/webtools/plant care/html;Lescot et al.2002).
Transient assays of gene function
Transient assays,or dual luciferase assays,were performed with tobacco(Nicotiana benthamiana or N.tabacum)as previously reported(Espley et al.2007;Palapol et al.2009;Niu et al.2010;Yin et al.2010),using pGreenII0800-LUC and pGreenII002962-SK(Hellens et al.2005).The full-length sequences of TFs,including MrMYB1,MrbHLH1, MrbHLH2and AtbHLH42,were cloned into the multiple cloning sites(MCS)of the pGreenII002962-SK vectors, while the promoters of anthocyanin biosynthetic genes were combined with the pGreenII0800-LUC vectors.The primers used for full-length TFs or promoter ampli?cation
Table1Primers for real-time
quantitative PCR
Genes GenBank ID Primers Sequences
MrCHS GQ340759Forward AGTTCAAGCGCATGTGTGAC
Reverse TGGCAGCTTCTTTGCCTAGT
MrCHI GQ340760Forward GCCATCGGGGTGTACTTAGA
Reverse GTTACCCGATAACGGCAAGA
MrF3H GQ340761Forward GTCGACATGGACCAGAAGGT
Reverse GGAGCAAGAGGGTGATGGTA
MrF30H GQ340762Forward ATGAAGCATATGGCCTGACC
Reverse CGTAGCAGTAGCACCCACAA
MrDFR1GQ340763Forward GACAATCAACGGGTTGTTAG
Reverse GACTGGCTTTTGGTGCTCTTC
MrDFR2GQ340764Forward AACGGTGAACGGGGTGTTGA
Reverse TACTTTCCTTTGGTGCTCAGA
MrANS GQ340765Forward CTAGTGGGAAGCTCGAGTGG
Reverse GCTAGCGCTCTCAGTTGCTT
MrUFGT GQ340766Forward TTTCCTCGACCAAACCAGAC
Reverse CTTACCTCCCTCCCCATCTC
MrMYB1GQ340767Forward GTGAGAAAAGGTGCCTGGACT
Reverse CATCTATTTAAGCCTGCTCTGG
MrbHLH1JX629461Forward GGAGGTGAAGAGGGCAATAAA
Reverse CACGGCTACTTCTCGATGGTA
MrbHLH2JX629462Forward ACACTCTGTCCAATCATCTGCTC
Reverse CAACAAATCAAGAAACAAAGGCT
MrACT GQ340770Forward AATGGAACTGGAATGGTCAAGGC
Reverse TGCCAGATCTTCTCCATGTCATCCCA
NtCHS AF311783Forward TTGTTCGAGCTTGTCTCTGC
Reverse AGCCCAGGAACATCTTTGAG
NtCHI AB213651Forward GTCAGGCCATTGAAAAGCTC
Reverse CTAATCGTCAATGCCCCAAC
NtF3H AB289450.1Forward CAAGGCATGTGTGGATATGG
Reverse TGTGTCGTTTCAGTCCAAGG
NtF30H AB289449.1Forward AGGCTCAACACTTCTCGT
Reverse CATCAACTTTGGGCTTCT
NtDFR EF421429.1Forward AACCAACAGTCAGGGGAATG
Reverse TTGGACATCGACAGTTCCAG
NtANS AB289447.1Forward TGGCGTTGAAGCTCATACTG
Reverse GGAATTAGGCACACACTTTGC
NtUFGT FG627024.1Forward GAGTGCATTGGATGCCTTTT
Reverse CCAGCTCCATTAGGTCCTTG
NtACT AJ421411Forward AATGGAACTGGAATGGTCAAGGC
Reverse TGCCAGATCTTCTCCATGTCATCCCA
were described in Tables3and4,respectively.All con-structs were individually electroporated into Agrobacte-rium tumefaciens GV3101(MP90).TF-promoter interactions were measured by the ratio of enzyme activi-ties of?re?y luciferase(LUC),driven by the promoter under investigation,to renilla luciferase(REN),driven by CaMV:35S.
Tobacco plants were grown in a glasshouse until six to eight leaves were available for in?ltration with Agrobac-terium(Palapol et al.2009;Yin et al.2010).In?ltrations, transient expression analysis,and enzyme activity deter-mination of LUC and REN were conducted as reported in Yin et al.(2010).3days after in?ltration,LUC and REN activities were analyzed using the Dual-Luciferase Repor-ter Assay System(Promega,USA).Measurements were carried out using a Modulus Luminometer(Promega,USA) in three independent experiments with at least four bio-logical replicates for each assay.
In a separate experiment,MrMYB1,MrbHLH1and MrbHLH2were in?ltrated into tobacco(N.tabacum) abaxial leaf surface either singly or in pairs containing MrMYB1–MrbHLH1or MrMYB1–MrbHLH2.Digital photographs of anthocyanin development in these patches were taken8days after in?ltration.
Transformation of tobacco
An overexpression binary vector(pGreenII002962-SK-MrMYB1–MrbHLH1),containing both the MrMYB1and MrbHLH1cDNAs,each under the control of the35S promoter in sense orientation,was constructed using stan-dard recombinant DNA techniques.The binary constructs were also electroporated into Agrobacterium GV3101 (MP90)and then used in tobacco(N.tabacum)transfor-mation.Plants transformed with empty vectors were set as negative controls.Transgenic plants were identi?ed by kanamycin selection as well as Q-PCR analysis(Huang et al.2013b).
Yeast two-hybrid assay
Yeast two-hybrid assay to study the interaction of MrMYB1 and MrbHLH1,was performed using Matchmaker TM Gold Yeast Two-Hybrid(Clontech,USA)according to the
Table2Primers for genome
walking
Genes GSP1(50–30)GSP2(50–30)
MrCHS CAAGTGCATGTACCGCTTCTTTA GATACAGTTGGGTGGATTGGCC
MrCHI CATTGTCACCCGCACAAACTTC GGTGGAGCCTGAATGCTTAGTC
MrF3H CTCAGCCCTCCTTCCATCTACTT GGGTCTTCTCCTCCGCTAGAGC
MrF30H ACGGGCCCCATGTGGGGCAAAT TCCCAGAAGGAGAGACAATGG
MrDFR1TGGCTCGGACAGTGTAACCGTG AACCCAGCCGCACCAGTGACG
MrDFR2ACCGTTGGCTTTATCACTTCATT GCACGGTGGCTCGGACGGTGTA
MrANS ATGGCCTCATCCTCGCAGTCAAT TGCTTGCCAGGCTCTCAACTCT
MrUFGT AAATCCTCCAATGCCAATCGTTT CAGAAGAAAGCGTCGGTCACC
Table3Primers for promoter
ampli?cation
Genes Primers Sequences
MrCHS Forward GCGGATCCATTATTGGTTCGCAGTTATG(BamH I)
Reverse ATCCATGGGTGACCGCTTGATTTAGCTC(Nco I)
MrCHI Forward ATGGATCCGCCCAATAAGCAAAGACCTAGC(BamH I)
Reverse GCCCATGGTCAAGGAGATATATATTTCCTG(Nco I)
MrF3H Forward GCGGATCCAGGATTTTAGTCAAG(BamH I)
Reverse GCCCATGGAATAGTCGTGAGCTC(Nco I)
MrF30H Forward GCGGATCCAAAAAGTCGGTAAAAC(BamH I)
Reverse ATCCATGGTTTCGGTCCTCGTATG(Nco I)
MrDFR1Forward ATGGATCCATTGAGTTGGTCGGGATG(BamH I)
Reverse ATCCATGGGCTCCTGCTCCTCAC(Nco I)
MrDFR2Forward GCGGATCCCATGGAAAGTGGTTAGG(BamH I)
Reverse ATCCATGGTCCGAACCCATGCTTTTC(Nco I)
MrANS Forward GCGGATCCTCATAGTGTTTGTTTTC(BamH I)
Reverse GCCCATGGTAATAGCAATATATG(Nco I)
MrUFGT Forward GCGGATCCATTTCGATAAACAACCACCCTC(BamH I)
Reverse AGCCATGGTTGGACAAAGTTGCTGCAGTTG(Nco I)
manufacturer’s instructions(Clontech Yeast Protocol Handbook).The ORFs of MrMYB1and MrbHLH1were inserted into the MCS of both pGBKT7and pGADT7 vectors containing the GAL4DNA-binding and activation domain respectively.The BD-MrMYB1(BD-MrbHLH1) plasmid was transformed alone or together with AD-MrbHLH1(AD-MrMYB1)into the yeast strain Y2HGold using the PEG/LiAc method.Transformed colonies of BD-MrMYB1and BD-MrbHLH1were tested on selection synthetic dropout(SD)medium lacking adenine,histidine, tryptophan,with X-a-Gal and Aureobasidin A(AbA) background,for their autoactivation.The co-transformed colonies were selected on SD medium without leucine and tryptophan(SD/-Leu/-Trp),and screened for growth on quadruple dropout SD medium lacking adenine,histidine, leucine and tryptophan(SD/-Ade/-His/-Leu/-Trp).X-a-Gal was used to assay for b-galactosidase activity to con?rm the positive interactions.
Results and discussion
Identi?cation of MrbHLH1and MrbHLH2containing
the conserved motif related to anthocyanin biosynthesis regulation
117bHLH members were identi?ed in the bayberry RNA-Seq database which was constructed with samples from stem,leaf,?ower and different developmental stages of fruit of‘BQ’bayberry(Feng et al.2012).Further sequence analysis showed that two of these members,named MrbHLH1(GenBank JX629461)and MrbHLH2(GenBank JX629462),had high similarity with the bHLHs related to anthocyanin biosynthesis in other species.The ORFs of MrbHLH1and MrbHLH2encoded710and657amino acids,respectively.Phylogenetic tree comparisons showed that most of the bHLHs recognized as regulators of anthocyanin biosynthesis belong to the group IIIf of Ara-bidopsis bHLH(Fig.1a).MrbHLH1had72and71% homology at the amino acid level with MdbHLH3 (HM122458)and AtbHLH42(NM117050),respectively, while the similarity of MrbHLH2with VvMYCA1 (ABM92332)and MdbHLH33(ABB84474)was71and 68%,respectively(Fig.1b).Although the two MrbHLHs were clustered in the same Arabidopsis bHLH gene sub-family,MrbHLH1had only58%homologous with MrbHLH2at the amino acid level(Fig.1b).
Sequence alignments of MrbHLH1,MrbHLH2and those related to anthocyanin biosynthesis in other plants showed three conserved motifs which were designed as box11,18 and13,respectively(Heim et al.2003),located in the N-terminal regions of bHLHs that interacted with MYB transcription factors during anthocyanin biosynthesis (Heim et al.2003;Fig.2a).However,although the three conserved motifs existed in both,there were differences, especially in the basic-helix1regions,between MrbHLH1 and MrbHLH2(Fig.2b).The highly conserved amino acid residues in MrbHLH1in the basic region were His5-Glu9-Arg13residues and Arg10-Arg12residues,which are required for binding to a variation of the E-box hexanu-cleotide sequence(E-box:CANNTG,variation:CACGTG) and the DNA backbone,respectively(Heim et al.2003). Different residues in these positions(His5-Asp9-Glu13and Asn10-Arg12)were present in MrbHLH2(Fig.2b).
Of the162bHLH genes in Arabidopsis and their homologies in other plants,most of those recognized as TFs regulating genes of?avonoid metabolism are in group III,especially subgroup IIIf(Heim et al.2003).For example,AtbHLH42and MdbHLH3were identi?ed as the crucial bHLH members during anthocyanin biosynthesis in Arabidopsis and apple,respectively(Nesi et al.2000; Espley et al.2007).However,in maize,the Intensi?er1, encoding a repressor of anthocyanin biosynthesis and also belonging to group III,is closely related to AtbHLH42 (Burr et al.1996).Thus,although both of them clustered in group IIIf,the functions of MrbHLH1and MrbHLH2 needed to be tested in order to establish their roles. Expression of MrbHLH1corresponds to anthocyanin accumulation during fruit development
During the development of‘BQ’fruit,the color changed from green to purple and signi?cant accumulation of anthocyanin occurred from74DAFB(Fig.3a).The anthocyanin content in fruit reached the highest level, 55.6mg/100g FW,when its color became purple at86
Table4Primers for TF full-
length ampli?cation
Genes Forward(50–30)
MrbHLH1Forward TAGAATTCATGGCTGCACCGCCGAGTAGC(EcoR I)
Reverse CGAAGCTTCTACGAGTCATTGTGGGGTAT(Hind III)
MrbHLH2Forward CGGGATCCATGGCCAATGGCACTCAAAC(BamH I)
Reverse TAGTCGACTCAACACCTGCAAGCGATTTTC(Sal I)
MrMYB1Forward TAGCGGCCGCATGGAAGGCTCTTTAGGTGTA(Not I)
Reverse GCGGTACCTTATGGATCGAGAAAATCCCA(Kpn I)
DAFB (Fig.3a).Concurrent with the accumulation of anthocyanin during bayberry fruit development,the expression levels of all eight biosynthetic genes increased
dramatically,especially during later stages from 74DAFB (Fig.3b).Expression of three transcription factors showed different patterns.The two MrbHLHs showed higher expression levels than MrMYB1during fruit development (Fig.3c).The transcript level of MrbHLH1increased during the later stages of fruit development,corresponding with the anthocyanin accumulation,while MrbHLH2expression showed a weaker correlation (Fig.3c).
To study the tissue speci?city of the expression of MrbHLH1and MrbHLH2,different tissues of ‘BQ’bay-berry including young leaves,stems and ripe fruits were collected.Anthocyanin contents in these different tissues varied signi?cantly,no anthocyanin was detectable in stems,small amounts were accumulated in young leaves,and the major accumulation was in ripe fruit (Fig.4a,b).The expression of MrMYB1showed high tissue speci?c-ity corresponding with anthocyanin content;but the expression patterns of both MrbHLH1and MrbHLH2remained constant in these tissues and were not well correlated with anthocyanin content (Fig.4c).However,the expression patterns of bHLHs might not always be helpful in identifying their functions.For example,expression pro?les of MdbHLH3and MdbHLH33from apple that regulated anthocyanin biosynthesis together with MdMYB10did not show tissue speci?city (Espley et al.2007),as shown here for MrbHLH1and MrbHLH2from bayberry.
MrbHLH1,coupled with MrMYB1,up regulates anthocyanin biosynthesis in tobacco
To verify whether MrbHLH1and/or MrbHLH2had the ability to regulate anthocyanin biosynthesis in conjunction with MrMYB1,dual luciferase transient assays in N .benthamiana and N .tabacum were carried out.When TFs were transiently transformed into N .tabacum leaves via Agrobacterium ,anthocyanin accumulation was observed 8days later with the combination of MrMYB1–MrbHLH1(Fig.5a),while MrMYB1,MrbHLH1or MrbHLH2alone or MrMYB1–MrbHLH2together induced no anthocyanin response (Fig.5a,b;Niu et al.2010).Similarly,dual luciferase assays indicated that when MrbHLH1or AtbHLH42were co-transformed with MrMYB1,the activities of the AtDFR promoter increased more than 81or 47times respectively,compared with the negative control transformed with empty vector only (Fig.5c).Furthermore,MrMYB1–MrbHLH1activated the AtDFR biosynthetic gene promoter to a greater extent than MrMYB1–AtbHLH42(Fig.5c).In contrast,MrMYB1,MrbHLH1or MrbHLH2alone as well as MrMYB1–MrbHLH2had low activity against the AtDFR promoter (Fig.5
c).
Fig.1Phylogenetic tree of bHLHs and deduced protein sequence alignment.177bHLHs,including 162from Arabidopsis and 15from other plants,were analyzed (a ).The more detailed information for group IIIf of Arabidopsis bHLHs,including bHLHs related to anthocyanin biosynthesis in other plants is showing in (b ).bHLH members isolated from Chinese bayberry were marked with circles and the genes ID of those from other species are as follows:VvMYC1(V.vinifera ,GenBank accession number EU447172),MrbHLH1(M.rubra ,JX629461),MrbHLH2(M.rubra ,JX629462),MdbHLH3(M.domestica ,HM122458),MdbHLH33(M.domestica ,DQ266451),LjTT8(Lotus japonicas ,BAH28881),AN1(Petunia hybrid ,AF260918),NtAn1a (N.tabacum ,AEE99257),NtAN1-like (N.tomentosiformis ,AEE99260),NtAn1b (N.tabacum ,AEE99258),AmDELILA (Antirrhinum majus ,AAA32663),VvMYCA1(V.vinifera ,ABM92332),L-c (Zea mays ,NM001111869)and R-S (Z.mays ,X15806)
Espley et al.(2007)found that both MdbHLH3and MdbHLH33from apple,which are closely related to MrbHLH1and MrbHLH2,respectively (Fig.1),could signi?cantly induce the transcription activity of the AtDFR promoter when MdMYB10was present,whilst MdbHLH3had a greater effect than MdbHLH33.This suggested that both two bHLH members with redundant functions were related to anthocyanin biosynthesis in apple.However,results here showed that MrbHLH1,but not MrbHLH2,could activate the AtDFR promoter together with MrMYB1(Fig.5c),suggesting the function of MrbHLH2in bayberry is not related to anthocyanin biosynthesis.The lack of function of the MrbHLH2in anthocyanin biosynthesis regulation suggested that different MrbHLH genes within the same phylogenic subfamily have different functions.Although lacking ability to stimulate promoter activity,MrbHLH2was constitutively expressed in fruit at different developmental stages and in different tissues of Chinese bayberry (Fig.3c,4c ),suggesting the importance of further research on its function.In all known plant bHLH sequences,the closest member for MrbHLH2is VvMYCA1(71%homology at the amino acid level,Fig.1b),and although the expression level of VvMYCA1gene was correlated with anthocyanin accumulation of in vitro grape plantlets (V.vinifera L.cv.Cabernet Sauvignon)grown under high salt concentrations (Matus et al.2010),VvMYCA1was
not
Fig.2Sequence alignments of two putative bHLH members and those known to be involved in anthocyanin biosynthesis from other plants.Three conserved domains in
N-terminus regions of bHLHs,which are important for interaction with R2R3-MYB protein,are present in both MrbHLH1and MrbHLH2(a ).Amino acid residues (H-E-R)at position 5,9and 13of the bHLH domains,are critical for binding DNA (b ).The amino acid sequences were obtained from The Arabidopsis
Information Resource or the National Center for
Biotechnology Information database
Fig.3Correlation of fruit color,anthocyanin content and gene expression pattern during‘BQ’bayberry fruit development.Fruit color changes signi?cantly during the six developmental stages, consistent with the anthocyanin accumulation(a).The transcript levels of anthocyanin biosynthetic genes coordinately increase in these samples(b).Expression levels of MrMYB1and MrbHLH1 showed a positive correlation with the anthocyanin accumulation, while that of MrbHLH2was weaker(c).The MrACT gene was used to normalize expression of the genes under identical conditions.Non-template reactions were set as negative control.The vertical bars represent SE of three biological
replicates Fig.4Analysis of the relationship between anthocyanin biosynthesis and gene expression pro?les in different tissues of‘BQ’bayberry. Different tissues including young leaves,stems and ripe fruit (a)accumulate different contents of anthocyanin(b).Expression pattern of MrMYB1indicates strong speci?city for different tissues, while both MrbHLH1and MrbHLH2have constant transcript levels in these samples(c).The MrACT gene was used to normalize expression of the genes under identical conditions.Non-template reactions were set as negative control.The vertical bars represent SE of three biological replicates
signi?cantly correlated at the transcriptional level with pat-terns of anthocyanin accumulation during red grape berry (V.vinifera L.cv.Alicante Bouschet)development,as it was more intensely expressed in anthocyaninless seeds than in anthocyanin-containing skin and ?esh at veraison,and it was suggested that VvMYCA1is mainly related to proanthocya-nin (PA)biosynthesis,with expression pattern being well consistent with that of PA biosynthetic genes leucoantho-cyanidin reductase 1(LAR1)and Banyuls,while plays some redundant role in anthocyanin biosynthesis regulation (Fal-ginella et al.2012).Whether MrbHLH2was involved in regulation of the PA biosynthesis in bayberry remain to be resolved.
MrMYB1–MrbHLH1activates all of the anthocyanin biosynthetic genes in transformed tobacco lines
Since the plasmids containing MrMYB1,MrbHLH1or MrMYB1–MrbHLH1were shown to be able to activate the AtDFR promoter to different extents in tobacco leaves,the mechanism for action of these genes or complexes on anthocyanin biosynthesis in tobacco were studied.Trans-formed tobacco lines overexpressing empty pGreenII 002962-SK vector,MrMYB1,MrbHLH1or MrMYB1–MrbHLH1were cultured (35S:MrMYB1construct was developed by Huang et al.2013a ;Fig.6a).Compared with the control plant transformed with empty vector,the lines over-expressing MrMYB1–MrbHLH1accumulated signi?cant amounts of anthocyanin in the whole plant (Fig.6a),while plants expressing just MrMYB1or MrbHLH1had green foliage (Fig.6a).Anthocyanin biosynthetic genes,espe-cially NtANS ,were up-regulated dramatically in MrMYB1–MrbHLH1over-expression lines compared with negative control (Fig.6b).Moreover,the endogenous bHLHs in tobacco,named An1a and An1b ,known to be related to anthocyanin regulation,were also up-regulated by the MrMYB1–MrbHLH1complex,while endogenous MYB member AN2did not respond to this complex (Fig.6c).This is quite similar with the results that the level of AtbHLH42expression could be controlled by different combinations
of
assays to probe the functions of MrbHLH1and MrbHLH2in tobacco leaves.Color
development due to transient transformation with either
MrbHLH1or MrbHLH2alone,or with MrMYB1and MrbHLH1or MrbHLH2
together in N.tabacum leaves (a ,b ).The activity of the
AtDFR promoter affected by the interaction between MrMYB1and bHLH TFs from
Arabidopsis (AtbHLH42),and Chinese bayberry (MrbHLH1and MrbHLH2),in transient tobacco transformation assays (c ).Digital images of in?ltration sites were taken 8days after in?ltration.Error bars are the SE of three independent
experiments with at least four replicate reactions
MYB (AtTT2or AtPAP1)and bHLH (AtbHLH42,AtEGL3or AtGL3)factors in planta (Baudry et al.2006).Five of eight anthocyanin biosynthetic genes from Chinese bayberry are activated by the MrMYB1–MrbHLH1complex
Since all anthocyanin biosynthetic genes from NtCHS were up-regulated by MrMYB1–MrbHLH1complex in trans-formed tobacco lines (Fig.6b),the question arises whether the concurrent expression of eight structural genes in rip-ening bayberry fruit during anthocyanin accumulation is also regulated by the complex (Fig.2b).
To study this,the promoters of these genes in bayberry were isolated and characterized.The cis -acting regulatory elements,including E-box motifs,ACEs (ACGT-contain-ing elements)and MRE (MYB recognized element),which have been reported to be targets of bHLH or MYB TFs,were found in all eight promoters (Fig.7a).However,the number of MRE motifs varied greatly in each promoter.As many as nine different kinds of MRE motifs were found in MrDFR1and MrUFGT promoters,while only two kinds were located in the promoter of MrDFR2(Fig.7a).
In vivo interactions of TF-promoters were estimated by dual luciferase assay in tobacco leaves.Activities of all eight promoters were low in the presence of only MrMYB1
or MrbHLH1(Fig.7b),while MrMYB1–MrbHLH1and MrMYB1–AtbHLH42acted as activators inducing the MrCHI,MrF30H,MrDFR1,MrANS and MrUFGT pro-moters (Fig.7b).The fold-induction elicited by MrMYB1–MrbHLH1varied greatly among these ?ve promoters ranging from 29-fold for MrANS to 146-fold for MrUFGT compared with the negative control.However,promoters of MrCHS,MrF3H and MrDFR2showed little or no response to MrMYB1–MrbHLH1or MrMYB1–AtbHLH42(Fig.7b).This result was different from that obtained in the transformed tobacco lines where all endogenous anthocyanin biosynthetic genes were up-regulated by MrMYB1–MrbHLH1(Fig.6b,c),and was also not con-sistent with the fact that the expression levels of all eight biosynthetic genes rose signi?cantly during natural color-ation as bayberry fruit ripens (Fig.1b).This may indicate that in Chinese bayberry,some other factors,not excluding other MYB or bHLH members,acting alone or together with MrMYB1–MrbHLH1,are involved in transcriptional regulation of MrCHS,MrF3H and MrDFR2.
MrbHLH1physically interacts with MrMYB1in vitro In vivo interaction of MrMYB1and MrbHLH1was iden-ti?ed in tobacco,while their in vitro interaction was
studied
lines were used to study the role of MrMYB1and MrbHLH1in anthocyanin regulation.
Changes in the color of tobacco leaves transformed with empty vector,MrMYB1,MrbHLH1or MrMYB1–MrbHLH1
overexpression construct (a ).All seven biosynthetic genes from NtCHS (b )and the
endogenous bHLH members in tobacco,named An1a and An1b (c ),were up-regulated by MrMYB1–MrbHLH1.The NtACT gene was used to normalize expression of the genes under identical conditions.Non-template reactions were set as negative control.Error bars indicate SE from three biological replicates
in yeast.The ORF of MrbHLH1was inserted into MCS of pGBKT7vector and the lack of auto-activation was veri-?ed on SD medium with X-a -Gal and 125ng/ml AbA background lacking adenine,histidine and tryptophan (data not shown).Then the bait construct carrying the BD-MrbHLH1fusion protein was co-transformed with AD-MrMYB1fusion proteins.The results showed that when MrbHLH1was used as the bait,MrMYB1and MrbHLH1could physically interact with each other in vitro (Fig.8).But when MrMYB1served as the bait,strong auto-activity was observed on SD/-Trp/-Leu medium,SD/-Ade/-His/-Trp/-Leu medium,and even with X-a -Gal adding to the medium (Fig.8).However,unidirectional interaction is widely accepted to be enough for supporting the existence of real physical interaction between two proteins (Baudry et al.2004;An et al.2012;Besseau et al.2012).Therefore,although MrMYB1had auto-activity,it does physically interact with MrbHLH1,as no self-activity was observed for MrbHLH1.In our another study,physical interaction was also observed between MrbHLH1and MrWD40-1,and between MrMYB1and MrWD40-1(Liu et al.2013).Taking together,three transcription factors,
MrbHLH1,
Fig.7In vivo interactions between transcriptional factors and promoters of anthocyanin biosynthetic genes in Chinese bayberry studied by dual luciferase assay in tobacco leaves.a Schematics of
promoters are indicated with thick lines (promoter lengths are marked),diamonds (E-box motif),squares (ACE-family),circles (MRE motif),and rectangles (LUC).Circles of different colours represent different kinds of MRE motifs.b In vivo associations of MYB,bHLHs and anthocyanin
biosynthetic gene promoters as revealed by transient assays in tobacco leaves.Error bars are the SE of three independent experiments with at least four replicate reactions
MrMYB1and MrWD40-1,interacted with each other to form the ternary complex during the anthocyanin biosyn-thesis regulation in Chinese bayberry.
Acknowledgments We thank Prof.Don Grierson from University of Nottingham (UK)for his kind discussion,suggestion,and efforts in language editing.This research was supported by the National High Technology Research and Development Program of China [2013AA102606],the National Natural Science Foundation of China [31071781],the Program of International Science and Technology Cooperation [2011DFB31580],the Special Scienti?c Research Fund of Agricultural Public Welfare Profession of China [201203089].
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五年级上册成语解释及近义词反义词和造句大全 囫囵吞枣；【解释】：囫囵：整个儿。把枣整个咽下去，不加咀嚼，不辨味道。比喻对事物不加分析考虑。【近义词】：不求甚解【反义词】融会贯穿[造句];学习不能囫囵吞枣而是要精益求精 不求甚解；bùqiúshènjiě【解释】：甚：专门，极。只求明白个大概，不求完全了解。常指学习或研究不认真、不深入【近义词】：囫囵吞枣【反义词】：精益求精 造句；1；在学习上，我们要理解透彻，不能不求甚解 2；学习科学文化知识要刻苦钻研，深入领会，不能粗枝大叶，不求甚解。 千篇一律；【解释】：一千篇文章都一个样。指文章公式化。也比喻办事按一个格式，专门机械。 【近义词】：千人一面、如出一辙【反义词】：千差万别、形形色色 造句；学生旳作文千篇一律，专门少能有篇与众不同旳，这确实是平常旳练习太少了。 倾盆大雨；qīngpéndàyǔ【解释】：雨大得象盆里旳水直往下倒。形容雨大势急。 【近义词】：大雨如柱、大雨滂沱【反义词】：细雨霏霏牛毛细雨 造句；3月旳天说变就变，瞬间下了一场倾盆大雨。今天下了一场倾盆大雨。 坚决果断；áobùyóuyù：意思；做事果断，专门快拿定了主意，一点都不迟疑，形容态度坚决 近义词；不假思索斩钉截铁反义词；犹豫不决 造句；1看到小朋友落水，司马光坚决果断地搬起石头砸缸。2我坚决果断旳承诺了她旳要求。 饥肠辘辘jīchánglùlù【近义词】：饥不择食【反义词】：丰衣足食 造句；1我放学回家已是饥肠辘辘。2那个饥肠辘辘旳小孩差不多两天没吃饭了 滚瓜烂熟gǔnguālànshóu〔shú)【解释】：象从瓜蔓上掉下来旳瓜那样熟。形容读书或背书流利纯熟。【近义词】：倒背如流【反义词】：半生半熟造句；1、这篇课文我们早已背得滚瓜烂熟了 流光溢彩【liúguāngyìcǎi】解释；光影，满溢旳色彩，形容色彩明媚 造句：国庆节，商场里装饰旳流光溢彩。 津津有味；jīnjīnyǒuwèi解释：兴趣浓厚旳模样。指吃得专门有味道或谈得专门有兴趣。 【近义词】：兴致勃勃有滋有味【反义词】：索然无味、枯燥无味 造句；1今天旳晚餐真丰富，小明吃得津津有味。 天长日久；tiānchángrìjiǔ【解释】：时刻长，生活久。【近义词】：天长地久【反义词】：稍纵即逝 造句：小缺点假如不立即改掉, 天长日久就会变成坏适应 如醉如痴rúzuìrúchī【解释】：形容神态失常，失去自制。【近义词】：如梦如醉【反义词】：恍然大悟造句；这么美妙旳音乐，我听得如醉如痴。 浮想联翩【fúxiǎngliánpiān解释】：浮想：飘浮不定旳想象；联翩：鸟飞旳模样，比喻连续不断。指许许多多旳想象不断涌现出来。【近义词】：思绪万千 造句；1他旳话让人浮想联翩。2:这幅画饱含诗情，使人浮想联翩，神游画外，得到美旳享受。 悲欢离合bēihuānlíhé解释；欢乐、离散、聚会。泛指生活中经历旳各种境遇和由此产生旳各种心情【近义词】：酸甜苦辣、喜怒哀乐【反义词】：平淡无奇 造句；1人一辈子即是悲欢离合,总要笑口常开,我们旳生活才阳光明媚. 牵肠挂肚qiānchángguàdù【解释】：牵：拉。形容十分惦念，放心不下 造句；儿行千里母担忧，母亲总是那个为你牵肠挂肚旳人 如饥似渴rújīsìkě：形容要求专门迫切，仿佛饿了急着要吃饭，渴了急着要喝水一样。 造句；我如饥似渴地一口气读完这篇文章。他对知识旳如饥似渴旳态度造就了他今天旳成功。 不言而喻bùyánéryù【解释】：喻：了解，明白。不用说话就能明白。形容道理专门明显。 【近义词】：显而易见【反义词】：扑朔迷离造句；1珍惜时刻，好好学习，那个道理是不言而喻旳 与众不同；yǔzhòngbùtóng【解释】：跟大伙不一样。 〖近义词〗别出心裁〖反义词〗平淡无奇。造句； 1从他与众不同旳解题思路中，看出他专门聪慧。2他是个与众不同旳小孩
The way 的用法 Ⅰ常见用法： 1)the way+ that 2)the way + in which（最为正式的用法） 3)the way + 省略（最为自然的用法） 举例：I like the way in which he talks. I like the way that he talks. I like the way he talks. Ⅱ习惯用法： 在当代美国英语中，the way用作为副词的对格，“the way+ 从句”实际上相当于一个状语从句来修饰整个句子。 1)The way =as I am talking to you just the way I’d talk to my own child. He did not do it the way his friends did. Most fruits are naturally sweet and we can eat them just the way they are—all we have to do is to clean and peel them. 2）The way= according to the way/ judging from the way The way you answer the question, you are an excellent student. The way most people look at you, you’d think trash man is a monster. 3）The way =how/ how much No one can imagine the way he missed her. 4）The way =because
悲惨的近义词反义词和造句 导读：悲惨的近义词 悲凉(注释:悲哀凄凉：～激越的琴声。) 悲惨的反义词 幸福(注释:个人由于理想的实现或接近而引起的一种内心满足。追求幸福是人们的普遍愿望，但剥削阶级把个人幸福看得高于一切，并把个人幸福建立在被剥削阶级的痛苦之上。无产阶级则把争取广大人民的幸福和实现全人类的解放看作最大的幸福。认为幸福不仅包括物质生活，也包括精神生活;个人幸福依赖集体幸福，集体幸福高于个人幸福;幸福不仅在于享受，而主要在于劳动和创造。) 悲惨造句 1.一个人要发现卓有成效的真理，需要千百个人在失败的探索和悲惨的错误中毁掉自己的生命。 2.贝多芬的童年尽管如是悲惨，他对这个时代和消磨这时代的地方，永远保持着一种温柔而凄凉的回忆。 3.卖火柴的小女孩在大年夜里冻死了，那情景十分悲惨。 4.他相信，他们每个人背后都有一个悲惨的故事。 5.在那次悲惨的经历之后，我深信自己绝对不是那种可以离家很远的人。 6.在人生的海洋上，最痛快的事是独断独航，但最悲惨的却是回头无岸。 7.人生是艰苦的。对不甘于平庸凡俗的人那是一场无日无夜的斗
争，往往是悲惨的、没有光华的、没有幸福的，在孤独与静寂中展开的斗争。……他们只能依靠自己，可是有时连最强的人都不免于在苦难中蹉跎。罗曼·罗兰 8.伟大的心胸，应该表现出这样的气概用笑脸来迎接悲惨的厄运，用百倍的勇气来应付开始的不幸。鲁迅人在逆境里比在在顺境里更能坚强不屈。遇厄运时比交好运时容易保全身心。 9.要抓紧时间赶快生活，因为一场莫名其妙的疾病，或者一个意外的悲惨事件，都会使生命中断。奥斯特洛夫斯基。 10.在我一生中最悲惨的一个时期，我曾经有过那类的想法：去年夏天在我回到这儿附近的地方时，这想法还缠着我;可是只有她自己的亲自说明才能使我再接受这可怕的想法。 11.他们说一个悲惨的故事是悲剧，但一千个这样的故事就只是一个统计了。 12.不要向诱惑屈服，而浪费时间去阅读别人悲惨的详细新闻。 13.那起悲惨的事件深深地铭刻在我的记忆中。 14.伟大的心胸，应该用笑脸来迎接悲惨的厄运，用百倍的勇气来应付一切的不幸。 15.一个人要发现卓有成效的真理，需要千百万个人在失败的探索和悲惨的错误中毁掉自己的生命。门捷列夫 16.生活需要爱，没有爱，那些受灾的人们生活将永远悲惨;生活需要爱，爱就像调味料，使生活这道菜充满滋味;生活需要爱，爱让生活永远充满光明。
The way的用法及其含义（二） 二、the way在句中的语法作用 the way在句中可以作主语、宾语或表语： 1.作主语 The way you are doing it is completely crazy.你这个干法简直发疯。 The way she puts on that accent really irritates me. 她故意操那种口音的样子实在令我恼火。The way she behaved towards him was utterly ruthless. 她对待他真是无情至极。 Words are important, but the way a person stands, folds his or her arms or moves his or her hands can also give us information about his or her feelings. 言语固然重要,但人的站姿,抱臂的方式和手势也回告诉我们他(她)的情感。 2.作宾语 I hate the way she stared at me.我讨厌她盯我看的样子。 We like the way that her hair hangs down.我们喜欢她的头发笔直地垂下来。 You could tell she was foreign by the way she was dressed. 从她的穿著就可以看出她是外国人。 She could not hide her amusement at the way he was dancing. 她见他跳舞的姿势，忍俊不禁。 3.作表语 This is the way the accident happened.这就是事故如何发生的。 Believe it or not, that's the way it is. 信不信由你, 反正事情就是这样。 That's the way I look at it, too. 我也是这么想。 That was the way minority nationalities were treated in old China. 那就是少数民族在旧中
定冠词the的用法： 定冠词the与指示代词this ,that同源,有“那(这)个”的意思,但较弱,可以和一个名词连用,来表示某个或某些特定的人或东西. (1)特指双方都明白的人或物 Take the medicine.把药吃了. (2)上文提到过的人或事 He bought a house.他买了幢房子. I've been to the house.我去过那幢房子. (3)指世界上独一无二的事物 the sun ,the sky ,the moon, the earth (4)单数名词连用表示一类事物 the dollar 美元 the fox 狐狸 或与形容词或分词连用,表示一类人 the rich 富人 the living 生者 (5)用在序数词和形容词最高级,及形容词等前面 Where do you live?你住在哪? I live on the second floor.我住在二楼. That's the very thing I've been looking for.那正是我要找的东西. (6)与复数名词连用,指整个群体 They are the teachers of this school.(指全体教师) They are teachers of this school.(指部分教师) (7)表示所有,相当于物主代词,用在表示身体部位的名词前 She caught me by the arm.她抓住了我的手臂. (8)用在某些有普通名词构成的国家名称,机关团体,阶级等专有名词前 the People's Republic of China 中华人民共和国 the United States 美国 (9)用在表示乐器的名词前 She plays the piano.她会弹钢琴. (10)用在姓氏的复数名词之前,表示一家人 the Greens 格林一家人(或格林夫妇) (11)用在惯用语中 in the day, in the morning... the day before yesterday, the next morning... in the sky... in the dark... in the end... on the whole, by the way...
“tｈｅwａｙ+从句”结构的意义及用法 首先让我们来看下面这个句子: Ｒead the foｌloｗｉnｇpassagｅａnd talkａbout ｉt wi ｔh your classmaｔes．Try tｏｔeｌl whaｔyｏu thiｎk ｏf Tom aｎd ｏｆｔhe ｗay the chiｌｄrenｔrｅated ｈim. 在这个句子中，ｔhe waｙ是先行词,后面是省略了关系副词that或in whｉch的定语从句。 下面我们将叙述“the ｗaｙ+从句”结构的用法。 1.tｈe wａy之后，引导定语从句的关系词是tｈat而不是hoｗ,因此，<<现代英语惯用法词典＞＞中所给出的下面两个句子是错误的：Ｔhｉs is ｔｈeｗayｈowｉtｈａppened. This is the way how he ａlwａｙs treats me. 2．在正式语体中,thaｔ可被in which所代替;在非正式语体中，ｔhat则往往省略。由此我们得到theｗay后接定语从句时的三种模式：1) tｈe ｗaｙ＋ｔhａt-从句２）the way +ｉn whiｃh－从句3) the ｗay +从句 例如：Ｔｈe way(in whiｃh ，thａt) ｔhｅｓｅｃomｒade ｓloｏｋａｔｐrobｌems is wrong．这些同志看问题的方法
不对。 Ｔｈｅｗａy(that ,ｉn ｗhiｃh)yoｕ’ｒe doinｇit is comple ｔeｌy crａzy.你这么个干法,简直发疯。 Wｅａdmiｒｅd him for thｅｗay ｉｎｗhｉch he fａｃeｓdiｆficultieｓ． Waｌlａｃe ａｎd Ｄarwiｎｇreed ｏn the way ｉｎwhi ｃh ｄｉfｆｅrｅnt forms ｏf life had ｂegｕn.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 Thｉs is ｔｈe ｗaｙ（tｈａｔ) hｅdｉd it. I lｉkeｄｔhe waｙ(ｔhat) ｓｈeｏｒganized ｔhe ｍeetｉng． 3.theｗay(tｈat)有时可以与hｏw(作“如何”解）通用。例如： That’s tｈe ｗaｙ(tｈaｔ) shｅspoke. = Tｈat’s how shｅspoke.
知己的近义词反义词及知己的造句 本文是关于知己的近义词反义词及知己的造句，感谢您的阅读！ 知己的近义词反义词及知己的造句知己 基本解释：顾名思义是了解、理解、赏识自己的人，如"知己知彼，百战不殆";更常指懂你自己的挚友或密友，它是一生难求的朋友，友情的最高境界。正所谓:"士为知己者死"。 1.谓了解、理解、赏识、懂自己。 2.彼此相知而情谊深切的人。 【知己近义词】 亲信，好友，密友，心腹，挚友，深交，相知，知交，知友，知心，知音，石友，老友，至友 【知己反义词】 仇人敌人陌路 【知己造句】 1、我们想要被人爱、想拥有知己、想历经欢乐、想要安全感。 2、朋友本应是我们的亲密知己和支持者，但对于大多数人来说，有一些朋友比起帮助我们，更多的却是阻碍。 3、那么，为什么你就认为，随着年龄的增长，比起女人来男人们的知己和丰富的人际关系更少，因此一般容易更孤独呢？ 4、他成了我的朋友、我的知己、我的顾问。 5、无论在我当州长还是总统的时候，布鲁斯都是我的密友、顾问和知己。他这样的朋友人人需要，也是所有总统必须拥有的。
6、波兰斯基有着一段声名卓著的电影生涯，也是几乎所有电影界重要人物们的挚友和同事，他们是知己，是亲密的伙伴。 7、搜索引擎变成了可以帮追我们的忏悔室，知己，信得过的朋友。 8、这样看来，奥巴马国家安全团队中最具影响力的当属盖茨了――但他却是共和党人，他不会就五角大楼以外问题发表看法或成为总统知己。 9、我们的关系在二十年前就已经和平的结束了，但在网上，我又一次成为了他精神层面上的评论家，拉拉队，以及红颜知己。 10、这位“知己”，作为拍摄者，站在距离电视屏幕几英尺的地方对比着自己年轻版的形象。 11、父亲与儿子相互被形容为对方的政治扩音筒、知己和后援。 12、这对夫妻几乎没有什么至交或知己依然在世，而他们在后纳粹时期的德国也不可能会说出实话的。 13、她把我当作知己，于是，我便将她和情人之间的争吵了解得一清二楚。 14、有一种友谊不低于爱情；关系不属于暖昧；倾诉一直推心置腹；结局总是难成眷属；这就是知己！ 15、把你的治疗师当做是可以分享一切心事的知己。 16、莉莉安对我敞开心胸，我成了她的知己。 17、据盖洛普民意调查显示，在那些自我认同的保守党人中，尽管布什仍维持72%支持率，但他在共和党领导层中似乎很少有几位知
表示“方式”、“方法”，注意以下用法： 1.表示用某种方法或按某种方式，通常用介词in(此介词有时可省略)。如： Do it (in) your own way. 按你自己的方法做吧。 Please do not talk (in) that way. 请不要那样说。 2.表示做某事的方式或方法，其后可接不定式或of doing sth。 如： It’s the best way of studying [to study] English. 这是学习英语的最好方法。 There are different ways to do [of doing] it. 做这事有不同的办法。 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句，但是其后的从句不能由how 来引导。如： 我不喜欢他说话的态度。 正：I don’t like the way he spoke. 正：I don’t like the way that he spoke. 正：I don’t like the way in which he spoke. 误：I don’t like the way how he spoke. 4.注意以下各句the way 的用法： That’s the way (=how) he spoke. 那就是他说话的方式。 Nobody else loves you the way(=as) I do. 没有人像我这样爱你。 The way (=According as) you are studying now, you won’tmake much progress. 根据你现在学习情况来看，你不会有多大的进步。 2007年陕西省高考英语中有这样一道单项填空题： ——I think he is taking an active part insocial work. ——I agree with you_____. A、in a way B、on the way C、by the way D、in the way 此题答案选A。要想弄清为什么选A，而不选其他几项，则要弄清选项中含way的四个短语的不同意义和用法，下面我们就对此作一归纳和小结。 一、in a way的用法 表示：在一定程度上，从某方面说。如： In a way he was right.在某种程度上他是对的。注：in a way也可说成in one way。 二、on the way的用法 1、表示：即将来(去)，就要来(去)。如： Spring is on the way.春天快到了。 I'd better be on my way soon.我最好还是快点儿走。 Radio forecasts said a sixth-grade wind was on the way.无线电预报说将有六级大风。 2、表示：在路上，在行进中。如： He stopped for breakfast on the way.他中途停下吃早点。 We had some good laughs on the way.我们在路上好好笑了一阵子。 3、表示：(婴儿)尚未出生。如： She has two children with another one on the way.她有两个孩子，现在还怀着一个。 She's got five children，and another one is on the way.她已经有5个孩子了，另一个又快生了。 三、by the way的用法
仿照的近义词反义词和造句 仿照的近义词 【仿制解释】：仿造：～品 【模仿解释】：个体自觉或不自觉地重复他人的行为的过程。是社会学习的重要形式之一。尤其在儿童方面，儿童的动作、语言、技能以及行为习惯、品质等的形成和发展都离不开模仿。可分为无意识模仿和有意识模仿、外部模仿和内部模仿等多种类型。 仿照的反义词 【独创解释】：独特的创造：～精神ㄧ～一格。 仿照造句 一、老师让我们仿照黑板上的图画一幅画。 二、仿照下面的句式，以“只有”开头，写一结构与之相似的复句。 三、仿照例句的句子，在下面两句的横线上补写相应的内容。 四、仿照例句，以“记忆”或“友情”开头，另写一句话。 五、仿照下面两个例句，用恰当的词语完成句子，要求前后语意关联。 六、仿照开头两句句式，通过联想，在后面两句横线上填上相应的词语。 七、仿照所给例句，用下面的词展开联想，给它一个精彩的解释。 八、仿照例句，以“你”开头，另写一个句子。 九、仿照下列句式，续写两个句子，使之与前文组成意义相关的.句子。 十、我们也仿照八股文章的笔法来一个“八股”,以毒攻毒,就叫做八大罪状吧。
十一、仿照例句，任选一种事物，写一个句子。 十二、仿照下面一句话的句式和修辞，以“时间”开头，接着写一个句子。 十三、仿照例句，以“热爱”开头，另写一句子。 十四、仿照下面的比喻形式，另写一组句子。要求选择新的本体和喻体，意思完整。 十五、根据语镜，仿照划线句子，接写两句，构成语意连贯的一段话。 十六、仿照下面句式，续写两个句式相同的比喻句。 十七、自选话题，仿照下面句子的形式和修辞，写一组排比句。 十八、仿照下面一句话的句式，仍以“人生”开头，接着写一句话。 十九、仿照例句的格式和修辞特点续写两个句子，使之与例句构成一组排比句。 二十、仿照例句，另写一个句子，要求能恰当地表达自己的愿望。 二十一、仿照下面一句话的句式，接着写一句话，使之与前面的内容、句式相对应，修辞方法相同。 二十二、仿照下面一句话的句式和修辞，以“思考”开头，接着写一个句子。 二十三、仿照下面例句，从ABCD四个英文字母中选取一个，以”青春”为话题，展开想象和联想，写一段运用了比喻修辞格、意蕴丰富的话，要求不少于30字。 二十四、仿照下面例句，另写一个句子。 二十五、仿照例句，另写一个句子。 二十六、下面是毕业前夕的班会上，数学老师为同学们写的一句赠言，请你仿照它的特点，以语文老师的身份为同学们也写一句。
The way的用法及其含义（一） 有这样一个句子：In 1770 the room was completed the way she wanted. 1770年，这间琥珀屋按照她的要求完成了。 the way在句中的语法作用是什么？其意义如何？在阅读时，学生经常会碰到一些含有the way 的句子，如：No one knows the way he invented the machine. He did not do the experiment the way his teacher told him.等等。他们对the way 的用法和含义比较模糊。在这几个句子中，the way之后的部分都是定语从句。第一句的意思是，“没人知道他是怎样发明这台机器的。”the way的意思相当于how；第二句的意思是，“他没有按照老师说的那样做实验。”the way 的意思相当于as。在In 1770 the room was completed the way she wanted.这句话中，the way也是as的含义。随着现代英语的发展，the way的用法已越来越普遍了。下面，我们从the way的语法作用和意义等方面做一考查和分析： 一、the way作先行词，后接定语从句 以下3种表达都是正确的。例如：“我喜欢她笑的样子。” 1. the way+ in which +从句 I like the way in which she smiles. 2. the way+ that +从句 I like the way that she smiles. 3. the way + 从句(省略了in which或that) I like the way she smiles. 又如：“火灾如何发生的，有好几种说法。” 1. There were several theories about the way in which the fire started. 2. There were several theories about the way that the fire started.
暗示的近义词和反义词[暗示的近义词反义词和造句] 【暗示解释】：用含蓄、间接的方式使他人的心理、行为受到影响。下面小编就给大家整理暗示的近义词，反义词和造句，供大家学习参考。 暗示近义词 默示 示意 暗指 暗意暗示反义词 明说 表明 明言暗示造句 1. 他的顶级助手已经暗示那可能就在不久之后，但是避免设定具体的日期。 2. 一些观察家甚至暗示他可能会被送到了古拉格。 3. 要有积极的心理暗示，达成目标的好处不够充分，画面不够鲜明那它对你的吸引力就不够强烈，你就不易坚持下去! 4. 不要经常去试探男人，更不要以分手做为威胁，当你经常给他这种心理暗示，他的潜意识就会做好分手的打算。 5. 向读者暗示永远不必为主角担心，他们逢凶化吉，永远会吉人天相。 6. 约她一起运动，不要一下跳跃到游泳，可以从羽毛球网球开始，展示你的体魄和运动细胞，流汗的男人毫无疑问是最性感的，有意无意的裸露与靠近，向她暗示你的运动能力。 7. 正如在上一个示例所暗示的，只有在这些对象引用内存中同一个对象时，它们才是相同的。 8. 渥太华此时打出中国牌，巧妙地对美国这种行为作出警告，暗示美国如果长期这样下去的话，美国将自食其果。 9. 团长用震撼人心的嗓音喊道，这声音对他暗示欢乐，对兵团暗示森严，对前来校阅阅兵的首长暗示迎迓之意。 10. 渥太华此时打出中国牌，巧妙地对美国这种行为作出警告，暗示美国如果长期这样下去的话，美国将自食其恶果。 11. 我们需要邀请用户，为他们描述服务产品有多少好，给他们解释为什么他们需要填那些表单并且暗示他们会因此得到利益的回报。 12. 她对暗示她在说谎的言论嗤之以鼻。 14. 在力士参孙中，整首诗都强烈暗示着弥尔顿渴望他自己也能像参孙一样，以生命为代价，与敌人同归于尽。 15. 戴维既然问将军是否看见天花板上的钉子，这就暗示着他自己已看见了，当将军做了肯定的答复后，他又说自己看不见，这显然是自相矛盾。 16. 自我暗示在我们的生活中扮演着重要角色。如果我们不加以觉察，通常它会与我们作对。相反地，如果你运用它，它的力量就可以任你使唤。 17. 纳什建议太阳招兵买马，暗示去留取决阵容实力。 18. 同时，还暗示了菊既不同流俗，就只能在此清幽高洁而又迷漾暗淡之境中任芳姿憔悴。 19. 学习哲学的最佳途径就是将它当成一个侦探故事来处理：跟踪它的每一点蛛丝马迹每一条线索与暗示，以便查出谁是真凶，谁是英雄。 20. 不要经常去试探你的伴侣，更不要以分手做为威胁，试探本身就是一种不信任，当
放弃的近义词反义词和造句 下面就给大家整理放弃的近义词，反义词和造句，供大家学习参考。 放弃的近义词【废弃解释】：抛弃不用：把～的土地变成良田ㄧ旧的规章制度要一概～。 【丢弃解释】：扔掉;抛弃：虽是旧衣服，他也舍不得～。 放弃的反义词【保存解释】：使事物、性质、意义、作风等继续存在，不受损失或不发生变化：～古迹ㄧ～实力ㄧ～自己，消灭敌人。 放弃造句(1) 运动员要一分一分地拼，不能放弃任何一次取胜的机会。 (2) 我们不要放弃每一个成功的机会。 (3) 敌军招架不住，只好放弃阵地，狼狈而逃。 (4) 为了农村的教育事业，姐姐主动放弃了调回城市的机会。 (5) 都快爬到山顶了，你却要放弃，岂不功亏一篑?(6) 纵使遇到再大的困难，我们也要勇往直前，不轻言放弃。 (7) 逆境中的他始终没有放弃努力,仍满怀信心,期待着峰回路转的那一天。 (8) 听了同学的规劝,他如梦初醒,放弃了离家出走的想法。 (9) 因寡不敌众,我军放弃了阵地。 (10) 要日本帝国主义放弃侵华野心,无异于与虎谋皮。 (11) 永不言弃固然好，但有时放弃却也很美。
(12) 他这种放弃原则、瓦鸡陶犬的行径已经被揭露出来了。 (13) 适当放弃，做出斩钉截铁的决定，才能成为人生的赢家。 (14) 他委曲求全地放弃自己的主张，采纳了对方的意见。 (17) 我们要有愚公移山一样的斗志，坚持不懈，永远不放弃，去登上梦想的彼岸!(18) 只要有希望，就不能放弃。 (19) 为了大局着想，你应该委曲求全地放弃自己的看法。 (20) 既然考试迫在眉睫，我不得不放弃做运动。 (21) 即使没有人相信你，也不要放弃希望。 (22) 无论通往成功的路途有多艰辛，我都不会放弃。 (23) 在困难面前，你是选择坚持，还是选择放弃?(24) 无论前路多么的漫长，过程多么的艰辛，我都不会放弃并坚定地走下去。 (25) 你不要因为这点小事就英雄气短,放弃出国深造的机会。 (26) 像他这样野心勃勃的政客，怎么可能放弃追求权力呢?(27) 鲁迅有感于中国人民愚昧和麻木,很需要做发聋振聩的启蒙工作,于是他放弃学医,改用笔来战斗。 (28) 我们对真理的追求应该坚持不懈，锲而不舍，绝不能随便放弃自己的理想。 (29) 感情之事不比其他，像你这样期盼东食西宿，几个男友都捨不得放弃，最后必定落得一场空。 (30) 爷爷临终前的话刻骨铭心，一直激励着我努力学习，无论是遇到多大的困难险阻，我都不曾放弃。
way 的用法 【语境展示】 1. Now I’ll show you how to do the experiment in a different way. 下面我来演示如何用一种不同的方法做这个实验。 2. The teacher had a strange way to make his classes lively and interesting. 这位老师有种奇怪的办法让他的课生动有趣。 3. Can you tell me the best way of working out this problem? 你能告诉我算出这道题的最好方法吗？ 4. I don’t know the way (that / in which) he helped her out. 我不知道他用什么方法帮助她摆脱困境的。 5. The way (that / which) he talked about to solve the problem was difficult to understand. 他所谈到的解决这个问题的方法难以理解。 6. I don’t like the way that / which is being widely used for saving water. 我不喜欢这种正在被广泛使用的节水方法。 7. They did not do it the way we do now. 他们以前的做法和我们现在不一样。 【归纳总结】 ●way作“方法，方式”讲时，如表示“以……方式”，前面常加介词in。如例1； ●way作“方法，方式”讲时，其后可接不定式to do sth.，也可接of doing sth. 作定语，表示做某事的方法。如例2，例3；
体面的近义词|反义词及造句 我们还必须迅速采取行动，为实现社会包容和人人体面工作营造有利的环境。下面是小编精选整理的体面的近义词|反义词及造句，供您参考，欢迎大家阅读。 体面的近义词： 面子、合适、美观、颜面、场合、场面、排场、得体、好看、局面 体面的反义词： 难听、寒碜、邋遢、寒酸、难看 体面造句 1、我认为，帖在墙面上的证书，并不能使你成为一个体面的人。 2、他是那里唯一的看上去很体面的人;我认为他从来没有这样好看过。 3、所有的美国人都是好的，体面的人们每天都在践行着他们的责任。 4、美国人民慷慨、强大、体面，这并非因为我们信任我们自己，而是因为我们拥有超越我们自己的信念。 5、工人就是工人，无论他们来自哪里，他们都应该受到尊重和尊敬，至少因为他们从事正当的工作而获得体面的工资。 6、然而反之有些孩子可能就是多少有些体面的父母的不良种子这一概念却令人难以接受。
7、如果奥巴马能够成就此功，并且帮助一个体面的伊拉克落稳脚跟，奥巴马和民主党不仅是结束了伊拉克战争，而是积极从战争中挽救。 8、而且，等到年纪大了退休时，他们希望能得到尊重和体面的对待。 9、爸爸，您倒对这件事处理得很体面，而我想那可能是我一生中最糟糕的一个夜晚吧。 10、有一些积极的东西，低于预期的就业损失索赔和零售销售是体面的。 11、如果你努力工作，你就能有获得一份终生工作的机会，有着体面的薪水，良好的福利，偶尔还能得到晋升。 12、体面的和生产性的工作是消除贫困和建立自给自足最有效的方法之一。 13、同时，他是一个仁慈、温和、体面的人，一个充满爱的丈夫和父亲，一个忠实的朋友。 14、几周前我们刚讨论过平板电脑是如何作为一个体面且多产的电子设备，即使它没有完整的键盘，在进行输入时会稍慢些。 15、什么才是生活体面的标准? 16、我们还必须迅速采取行动，为实现社会包容和人人体面工作营造有利的环境。 17、她告诉我人们都担心是不是必须把孩子送到国外去学习，才能保证孩子们长大后至少能过上体面正派的生活。
t h e-w a y-的用法
The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.
近义词反义词大全有关聆听的反义词近义词和造句 【聆听解释】：1.汉扬雄《法言.五百》:"聆听前世﹐清视在下﹐鉴莫近于斯矣。"后多用于书面语﹐常指仔细注意地听。下面就给大家聆听的反义词，近义词和造句，供大家学习参考。 嘱咐 [注释]1.叮嘱，吩咐 倾听 [注释]1.侧着头听。 2.细听;认真地听 凝听 [注释]1.聚精会神地听 谛听 [注释]注意地听;仔细听：侧耳谛听 细听 [注释]… 1. 学校应充分利用社会的各种，调动一切可以调动的力量，应尽一切可能请进名师专家，让教师和学生有较多的机会聆听大师的声音、与大师对话。这多少会机器他们向往大师、成为大师的冲动，多少会使他们觉得大师就在身边，大师并不遥远。 2. 今天妈妈带我来到了这个美丽的大安森林公园玩，这儿有漂亮的花儿和绿油油的树，草，看起来像一个我好想好想要的花园。树
上有小鸟儿在唱歌，有蝉声在帮忙和弦，树下有小朋友再安静仔细聆听著这最自然的交响曲。 3. 静静聆听那滴滴答答的声音，不知在为谁倾诉?她在偶尔低语，偶尔高唱，偶尔呻吟，偶尔叹息，生动灵活，充满了立体与层次之美，在心灵中轻轻一吻，便触发了思绪，这思绪时而短暂，时而绵长，时而深沉，时而悠远…… 4. 九月的手掌拂去小溪夏日的狂躁，用心聆听着秋日的私语，温顺地弹唱着九月醉人的秋歌，惹得天空湛蓝高远，碧空如洗。 5. 我光着脚丫，踩着海水，注视着波光粼粼的海面，听着哗哗的响声，那声音好像高超演奏家的激越的钢琴曲，又像歌唱家的雄浑的进行曲，那声音使胸膛激荡，热血奔涌，啊，我多么愿意聆听大海老人的谆谆教诲啊! 6. 雪花飘飘，寒风阵阵，我细细地聆听着寒风谱写成的有声有色的乐谱，为雪花的舞步增添一些光彩。寒风真像一位默默为雪花服务的人。过了一会儿，又为雪花写了一篇朗朗上口的诗歌，一会儿又为她热烈地鼓起了清脆的掌声。
way的用法总结大全 way的用法你知道多少，今天给大家带来way的用法，希望能够帮助到大家，下面就和大家分享，来欣赏一下吧。 way的用法总结大全 way的意思 n. 道路,方法,方向,某方面 adv. 远远地，大大地 way用法 way可以用作名词 way的基本意思是“路,道,街,径”,一般用来指具体的“路,道路”,也可指通向某地的“方向”“路线”或做某事所采用的手段,即“方式,方法”。way还可指“习俗,作风”“距离”“附近,周围”“某方面”等。 way作“方法,方式,手段”解时,前面常加介词in。如果way前有this, that等限定词,介词可省略,但如果放在句首,介词则不可省略。
way作“方式,方法”解时,其后可接of v -ing或to- v 作定语,也可接定语从句,引导从句的关系代词或关系副词常可省略。 way用作名词的用法例句 I am on my way to the grocery store.我正在去杂货店的路上。 We lost the way in the dark.我们在黑夜中迷路了。 He asked me the way to London.他问我去伦敦的路。 way可以用作副词 way用作副词时意思是“远远地,大大地”,通常指在程度或距离上有一定的差距。 way back表示“很久以前”。 way用作副词的用法例句 It seems like Im always way too busy with work.我工作总是太忙了。 His ideas were way ahead of his time.他的思想远远超越了他那个时代。 She finished the race way ahead of the other runners.她第一个跑到终点,远远领先于其他选手。 way用法例句
终于的近义词，反义词及造句 …终究[注释]副词。 总：总归;毕竟;最终：我们只要把事情办好，人们终究会相信我们的|腐朽的东西终究要灭…到底[注释]1.直到尽头。 2.始终;从头到尾。 3.毕竟;究竟。 …结果[注释]结果1在一定阶段，事物发展所达到的最后状态：优良的成绩，是长期刻苦学习的～ㄧ经过一番争论…终归[注释]1.传说中神兽名。 2.终究;毕竟。 …究竟终于的反义词：起初、最初、原来、依旧、起先终于的造句：(1) 盼望了一个冬天的雪花，下午终于飘飘洒洒满天飞舞般降落。 同事们也如孩子般奔向办公室的走廊，欣喜地用手去接冰凉的小雪花再用盆子收拢些飘舞的小雪花。 隔窗望去，天地之间因突然多了这白色的小精灵而愈发显得美丽。 (2) 快乐的暑假终于到了，我可以开开心心痛痛快快地玩一顿了!当然，在这之前，我也会好好安排一下我的暑假生活，使我过个既快乐又充实的暑假，我快乐的暑假生活。 (3) 坚韧，让沙石煎熬住大海的蹂躏，终于化作璀璨的珍珠;坚韧，让天空忍受住雨水倾盆的阴霾，终于看见那一道彩虹;坚韧，让泉水忘记流进山谷崎岖的历程，终于汇入蔚蓝无垠的大海。
它的叶子正面是身绿色的，反面是浅绿的，经脉上还有一些毛绒绒的小细毛。 又过了几天，含羞草的花蕾也长出来了，圆溜溜的，紫红色，还有着一些像针一样的东西，非常美丽。 (5) 漫长而炎热的夏天终于过去了，凉爽怡人的秋天来到了。 (6) 随着天空被点燃，太阳也终于露出脸来，它没有害羞，而是大大方方地把它独特的热情展示给我们，毫不吝惜地奖光芒撒向大地。 (7) 大约过了二十分钟，太阳终于挣脱了大地妈妈的怀抱，在崇山俊岭之间冉冉升起，真像个大红球，又像气得涨红了脸。 (8) 半个月的低烧，终于有了好转，高烧了……(9) 夏天的雨后，太阳冲出乌云的包围，终于露出了整张脸，此时阳光直直的，却不呆板、单调，它们穿梭在树枝之间，织成一道道金色的丝，将鱼后的水珠串成一串金黄的珍珠。 夏天少有的凉意伴着美丽的阳光，令人沁透心脾。 (10) 终于到达了果园。 我们忽然发现，果园里还种了一些菊花。 这些菊花有红的蓝的黄的白的……它们颜色不同，姿态也不同：菊花们有的含苞欲放，有的开了一半，有的已经绽开了笑脸……千姿百态，十分美丽。 (11) 发改委终于超越了统计局，说北京人均绿地是巴黎两倍。 最牛回复：“是算上了开心农场吧?。
t h e_w a y的用法大全
The way 在the way+从句中, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 如果怕弄混淆，下面的可以不看了 另外，在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮. the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的