The Purple Cauli?ower Arises from Activation of a MYB Transcription Factor1[W][OA]
Li-Wei Chiu2,Xiangjun Zhou,Sarah Burke,Xianli Wu,Ronald L.Prior,and Li Li*
Department of Plant Breeding and Genetics(L.-W.C.,X.Z.,S.B.,L.L.)and Robert W.Holley Center for Agriculture and Health,United States Department of Agriculture-Agricultural Research Service(L.L.), Cornell University,Ithaca,New York14853;and United States Department of Agriculture-Agricultural Research Service,Arkansas Children’s Nutrition Center,Little Rock,Arkansas72202(X.W.,R.L.P.)
Anthocyanins are responsible for the color of many?owers,fruits,and vegetables.An interesting and unique Purple(Pr)gene mutation in cauli?ower(Brassica oleracea var botrytis)confers an abnormal pattern of anthocyanin accumulation,giving the striking mutant phenotype of intense purple color in curds and a few other tissues.To unravel the nature of the Pr mutation in cauli?ower,we isolated the Pr gene via a combination of candidate gene analysis and?ne mapping.Pr encoded a R2R3MYB transcription factor that exhibited tissue-speci?c expression,consistent with an abnormal anthocyanin accumulation pattern in the mutant.Transgenic Arabidopsis(Arabidopsis thaliana)and cauli?ower plants expressing the Pr-D allele recapitulated the mutant phenotype,con?rming the isolation of the Pr gene.Up-regulation of Pr speci?cally activated a basic helix-loop-helix transcription factor and a subset of anthocyanin structural genes encoding?avonoid3’-hydroxylase,dihydro?avonol 4-reductase,and leucoanthocyanidin dioxygenase to confer ectopic accumulation of pigments in the purple cauli?ower.Our results indicate that the genetic variation including a Harbinger DNA transposon insertion in the upstream regulatory region of the Pr-D allele is responsible for the up-regulation of the Pr gene in inducing phenotypic change in the plant.The successful isolation of Pr provides important information on the regulatory control of anthocyanin biosynthesis in Brassica vegetables,and offers a genetic resource for development of new varieties with enhanced health-promoting properties and visual appeal.
Vegetables and fruits are fundamental components of human diets.They not only are important sources of essential vitamins and minerals,but also contain a wide variety of secondary metabolites important to human health.Colored vegetables and fruits have gained an increasing interest as functional foods, owing to their high levels of plant pigments with po-tent nutritional and health-promoting effects.Among them,purple cauli?ower(Brassica oleracea var botrytis)is a very eye-catching vegetable and available commer-cially.The purple coloration is due to the accumulation of anthocyanins.
Anthocyanins are a group of?avonoid compounds that ful?ll important biological functions in protecting plants against various biotic and abiotic stresses.All of the anthocyanin biosynthetic pathway genes and nu-merous regulatory factors have been identi?ed from studies of Arabidopsis(Arabidopsis thaliana),maize (Zea mays),petunia(Petunia hybrida),snapdragon(An-tirrhinum majus),and other plant species(Broun,2005; Dixon et al.,2005;Koes et al.,2005;Grotewold,2006). Transcriptional regulation of structural genes appears to be a major mechanism by which anthocyanin bio-synthesis is regulated in plants.R2R3MYB and basic helix-loop-helix(bHLH)transcription factors as well as WD40proteins represent the three major families of anthocyanin regulatory proteins(Paz-Ares et al.,1987; Chandler et al.,1989;Ludwig and Wessler1990;de Vetten et al.,1997;Quattrocchio et al.,1999).They form regulatory complexes to activate expression of antho-cyanin structural genes(Goff et al.,1992;Grotewold et al.,2000).In Arabidopsis,several MYB proteins, including PAP1,PAP2,MYB113,MYB114,and MYBL2 (Borevitz et al.,2000;Dubos et al.,2008;Gonzalez et al., 2008;Matsui et al.,2008),three bHLH proteins of TT8, GL3,and EGL3(Nesi et al.,2000;Payne et al.,2000; Zhang et al.,2003),and a WD40repeat protein of TTG1 (Walker et al.,1999)are involved in anthocyanin biosynthesis.While the R2R3MYB proteins of PAP1, PAP2,MYB113,and MYB114cause tissue-speci?c an-thocyanin accumulation in Arabidopsis(Borevitz et al.,2000;Gonzalez et al.,2008),the R3-MYB protein, MYBL2,acts as an inhibitor of anthocyanin biosynthe-sis(Dubos et al.,2008;Matsui et al.,2008).TT8is required for the full transcriptional activation of late anthocyanin pathway genes(Nesi et al.,2000),and is partially functionally redundant with its closest ho-
1This work was supported by the U.S.Department of Agricul-ture-Agricultural Research Service base fund.
2Present address:Pioneer Hi-Bred International Inc,4010Point Eden Way,Hayward,CA94545.
*Corresponding author;e-mail ll37@https://www.doczj.com/doc/5c14802600.html,.
The author responsible for distribution of materials integral to the ?ndings presented in this article in accordance with the policy described in the Instructions for Authors(https://www.doczj.com/doc/5c14802600.html,)is: Li Li(ll37@https://www.doczj.com/doc/5c14802600.html,).
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https://www.doczj.com/doc/5c14802600.html,/cgi/doi/10.1104/pp.110.164160
mologs,GL3and EGL3(Zhang et al.,2003).The WD40protein,TTG1,is known to physically interact with the MYB and bHLH transcription factors in controlling anthocyanin biosynthesis (Zhang et al.,2003).
R2R3MYB transcription factors have been impli-cated to play an important role for color difference in plant species.Activation of an R2R3MYB transcrip-tion factor confers anthocyanin production in a num-ber of anthocyanin-accumulating plants (Borevitz et al.,2000;Mathews et al.,2003).In contrast,loss of function of a R2R3MYB results in color loss in the normal anthocyanin-accumulating tissues.For exam-ple,a retrotransposon insertion in the promoter of Vv MYBA1and mutations in the adjacent Vv MYBA2inactivate their expression and convert red-skinned grape (Vitis vinifera )into white-skinned one (Walker et al.,2007).In Antirrhinum majus ,alteration of MYB -related gene expression controls ?oral pigmentation intensity and pattern (Schwinn et al.,2006).
Similarly,bHLH transcription factors have also been found to be responsible for color difference in plant species.Alteration of a bHLH transcription factor causes an increase in red pigment production in the aleurone of maize (Burr et al.,1996).Knockout of a bHLH function by a frame shift changes seed pericarp color from red into white in rice (Oryza sativa ;Sweeney et al.,2006).An insertion of a DNA transposon into a bHLH regulatory gene alters ?ower color of the com-mon morning glory (Ipomoea tricolor )into pale pig-mented ?owers (Park et al.,2007).
Mutant analyses have facilitated gene discovery and elucidation of the regulatory control of anthocyanin biosynthesis.Although there are large numbers of researches on the underlying mechanisms controlling anthocyanin production in ?owers,fruits,and model plants,only few have focused on vegetables.Purple cauli?ower mutant represents an interesting mutation that confers profound anthocyanin production in the otherwise low-pigmented curds and seeds—the two tissues of agricultural importance.Thus,this mutant provides an excellent opportunity to reveal regulatory control of anthocyanin biosynthesis in vegetable crops.The cauli?ower purple mutation was found to be controlled by a single,semidominant gene.We desig-
nate the symbol Pr -D for the Purple allele,and pr for its wild-type counterpart.Through a combination of can-didate gene analysis and ?ne mapping,we isolated the Pr gene and found that it encoded a R2R3MYB transcription https://www.doczj.com/doc/5c14802600.html,parison of Pr sequences from wild type and the mutant allele revealed allelic vari-ation including a Harbinger DNA transposon inser-tion in the upstream regulatory region of the Pr -D allele.Such an alteration caused an increased Pr gene transcription,which in turn up-regulated anthocyanin structural gene expression to produce the striking purple phenotype.The activation of the Pr gene,probably by introduction of new regulatory motifs in the promoter region,provides a way that anthocyanin transcriptional regulation can be switched on differ-ently in plants.
RESULTS
Phenotypic Characterization of the Purple Cauli?ower Mutant
The cauli?ower purple mutant plants grew and developed normally when compared to white cauli-?ower under normal growth conditions in ?eld and in greenhouse.The mutant exhibited a tissue-speci?c pattern of anthocyanin accumulation.Intense purple coloration was observed in curd and a few other tissues of the plant (Fig.1,A–D,F,and G),including young seedlings,very young leaves,and very young ?ower buds and siliques,as well as the endosperm of seeds,a tissue that is rarely reported to accumulate pigments in anthocyanin-accumulating mutants,e.g.in red cabbage (Brassica oleracea var capitata ;Yuan et al.,2009).The purple color was not observed in older leaves,stems,?ower petals,and older siliques (Fig.1,B,E,and F).The purple phenotype appears to be associated primarily with very young tissues,curds,and seeds,rather than in ?ower petals,the most common anthocyanin-accumulating tissue.Under the same growth conditions,the wild-type cauli?ower plants exhibited no purple hue in these tissues (Fig.1,
A–G).
Figure 1.Phenotypic comparison between wild type and the cauli?ower Pr -D mutant.A,Y oung seedlings of 5-d-old plants.B,Y oung plants of 3-week-old.C,Curds of cauli?ower plants grown in ?eld.D,Y oung ?ower buds.E,Flowers.F,Y oung (inner)and old siliques of wild type (left)and mutant (right).G,Seed endosperms.
Anthocyanin Biosynthesis in Purple Cauli?ower
Purple Mutant Predominantly Accumulates Cyanidin Glucosides
To examine the composition and content of antho-cyanins accumulated in young leaves,curds,and seeds of the purple mutant,we performed HPLC analysis and found high levels of anthocyanins in the purple tissues (Fig.2A).The curds accumulated ap-proximately 3.75mg cyanidin diglucoside equivalent g 21fresh weight,a level that was comparable with that found in blueberries (Vaccinium myrtillus ;Gao and Mazza,1994).In contrast,the samples of the wild-type control plants contained undetectable amounts of anthocyanins (data not shown),indicating that the anthocyanin pathway was biochemically quiescent under normal growth conditions.
Several different groups of anthocyanins (e.g.del-phinidin,pelargonidin,and cyanidin)exist in the plant kingdom (Tanaka et al.,2008).To identify antho-cyanin composition in the mutant,anthocyanins in curd tissue were separated and analyzed using HPLC-electrospray ionization (ESI)-tandem mass spectrome-try (MS/MS;Wu and Prior,2005b).The purple mutant contained one major peak (Fig.2B),which was iden-ti?ed as cyanidin 3-(coumaryl-caffeyl)glucoside-5-(malonyl)glucoside (Fig.2C).The other minor peaks were also identi?ed as different forms of cyanidin glycosides based on the dominant ion pairs observed in the mass spectrometry data and anthocyanin struc-tures (Mazza and Miniati,1993;Wu and Prior,2005a).
The Purple Mutation Is Controlled by a Single Semidominant Gene
To examine whether the purple mutant was con-trolled by one or multiple genes,we crossed the purple mutant Graf?ti to an inbred white cultivar Stovepipe,selfed the heterozygous F1plants,and generated a large F2population.A subpopulation of 102individ-uals was germinated in a greenhouse.Genotyping of these F2individuals was completed by visually exam-ining the absence and presence of light or dark purple color in very young leaves and curds of the progeny,followed by further con?rmation of the individual genotype in some cases by visual examination of the color with 16F3individuals.The segregation ratio of these F2plants for white:light purple:dark purple was 31:53:18.x 2test showed a good ?t with a 1:2:1ratio (P .0.05),which was consistent with the ratio ex-pected for the progeny derived from sel?ng a parent heterozygous at one locus.This result suggests that the purple phenotype is controlled by a single,semi-dominant gene,Pr .
Expression of Anthocyanin Biosynthetic and Regulatory Genes
To investigate whether Pr represented one of the biosynthetic genes that was signi?cantly up-regulated in the Pr -D mutant,we examined the expression of anthocyanin pathway genes in curds and leaves of wild type and the Pr -D mutant by northern-blot anal-ysis.The cDNAs coding chalcone synthase (CHS ),chal-cone isomerase (CHI ),?avanone 3-hydroxylase (F3H ),?avonoid 3’-hydroxylase (F3’H ),dihydro?avonol 4-reduc-tase (DFR ),leucoanthocyanidin dioxygenase (LDOX ),and UDP-glucosyltransferase (UGT )were used as probes.While the early pathway genes were expressed at similar levels between wild type and the Pr -D mutant in both leaves and curds,the late pathway genes,BoF3’H ,BoDFR ,and BoLDOX ,were dramatically up-regulated in the mutant (Fig.3A).The co-up-regulation of three structural genes suggests that Pr is unlikely a mutation of one speci?c anthocyanin pathway gene.Many regulatory genes that control anthocyanin biosynthesis have been isolated from plant species (Paz-Ares et al.,1987;Chandler et al.,1989;Ludwig and Wessler 1990;de Vetten et al.,1997;Quattrocchio et al.,1999).Since cauli?ower genes typically share high coding sequence identity with Arabidopsis genes,the available sequences of anthocyanin regula-tory genes from Arabidopsis and the
signi?cant
Figure 2.Analysis of anthocyanin content and composition in the Pr -D mutant.A,Total anthocyanin levels as cyanidin diglucoside equivalent in curds,leaves,and seeds.FW,Fresh weight.B,HPLC elution pro?le of an-thocyanins accumulated in curds.The absorbance was monitored at A 520nm.C,Major anthocyanins identi?ed by HPLC-ESI-MS/MS in curds.
Chiu et al.
amounts of Brassica sequence information in the public domains provide direct resources for designing the gene-speci?c primers for some cauli?ower homologs.In Arabidopsis,the R2R3MYB family proteins of PAP1,PAP2,MYB113,and MYB114,bHLH proteins of TT8and EGL3,and WD40protein of TTG1are known to regulate anthocyanin biosynthesis.To in-vestigate whether the Pr gene represented a mutation of one of the known regulatory genes,transcript levels of these homologous genes in wild type and the Pr -D mutant were examined by quantitative reverse tran-scription (qRT)-PCR using gene-speci?c primers (Yuan et al.,2009;Supplemental Table S1).As shown in Figure 3B,P AP -like MYB family genes,i.e.BoMYB2and BoMYB4,as well as BobHLH1,a homologous gene of Arabidopsis TT8(Nesi et al.,2000)exhibited differ-ential expression in both leaves and curds between wild type and the Pr -D mutant.
Anthocyanins in the Pr -D mutant exhibited tissue-and development-speci?c accumulation (Fig.1).To examine whether the speci?c anthocyanin accumula-tion pattern was correlated with gene expression pat-tern,we analyzed transcript levels of BoCHS and the differentially expressed genes in different tissues of wild-type and the Pr -D plants.Similar level of BoCHS
transcript was observed in tissues between wild type and the Pr -D mutant.In contrast,the transcripts of BoF3’H ,BoDFR ,and BoLDOX ,as well as BoMYB2and BobHLH1accumulated highly in very young leaves,curds,very young ?ower buds,and young siliques in the mutant.The expression patterns of these genes were consistent with the tissue-speci?c anthocyanin accumulation pattern in the Pr -D mutant.
Identi?cation of a MYB Gene That Cosegregates with Pr -D
To test whether BobHLH1or one of the PAP -like MYB family genes represented Pr ,association map-ping was carried out to investigate whether any of them cosegregated with the Pr -D locus.To develop markers for association mapping,full genomic frag-ments of BobHLH1and four BoMYB genes including promoter sequences were isolated through genomic DNA walking from both wild type and the Pr -D https://www.doczj.com/doc/5c14802600.html,parison of the DNA sequences between wild type and mutant allele revealed that there were no polymorphisms for BobHLH1and BoMYB1,sug-gesting that they were unlikely the gene representing Pr .Based on different insertion/deletion or
single
Figure 3.Expression of anthocyanin structural and regulatory genes in wild type and the Pr -D mutant.A,northern-blot analysis of transcript levels of anthocyanin pathway genes in curds and leaves.BoAct serves as equal loading control.P ,Pr -D mutant;W,wild-type control.B,qRT-PCR analysis of anthocyanin regulatory genes in curds and leaves.The identities of the ampli?ed prod-ucts were con?rmed by sequencing.Expression of genes in wild-type leaves and curds were set to 1.WL,Wild-type leaves;PL,Pr -D leaves;WC,wild-type curds;PC,Pr -D curds.BoWD40,BobHLH1,and BobHLH2are cauli?ower homologs of Arab-idopsis TTG1,TT8,and EGL3,respectively.C,qRT-PCR analysis of transcript levels of BoCHS and the differentially expressed genes during different developmental stages.Expression of genes in wild-type (WT)young leaves was set to 1.YL,Y oung leaves;OL,old leaves;C,curds;YFB,young ?ower buds;P ,petals;YS,young siliques;OS,old siliques;R,roots.qRT-PCR re-sults represent mean values +SD from three biological replicates with three technical repli-cates for each.
Anthocyanin Biosynthesis in Purple Cauli?ower
nucleotide polymorphisms in BoMYB2,BoMYB3,and BoMYB4,PCR-based markers were developed (Sup-plemental Table S1).These three MYB genes were found to cosegregate with the Pr -D locus in a mapping population of 102F2plants.
To de?ne the gene that represented Pr ,the three BoMYB genes were mapped in a large mapping pop-ulation of 1,898F2individuals.Two and seven recom-binant events were detected for BoMYB3and BoMYB4marker,respectively.In contrast,no recombinant event was observed for BoMYB2marker,indicating that BoMYB2cosegregated with the Pr -D locus and most likely the Pr gene.A high-resolution genetic map for this region is shown in Figure 4A.
Structural Analysis of Pr
The Pr gene from both wild-type and mutant plants contained an open reading frame of 744bp that en-coded a protein of 247amino acids with an estimated molecular mass of 27.99kD.Pr encoded BoMYB2that was predicted to be a nuclear protein (WoLF PSORT,https://www.doczj.com/doc/5c14802600.html,)and contained R2and R3MYB repeat domains with the signature motif of KPRPR[S/T]F,speci?c for MYB proteins that activate anthocyanin biosynthesis (Stracke et al.,2001;Fig.4B).Noticeably,the KPRPR[S/T]F motif overlaps with a TAS4-siR81target site identi?ed in Arabidopsis P AP1and P AP2(Rajagopalan et al.,2006),suggesting a possible
addi-
Figure 4.High-resolution genetic map of the Pr region,MYB sequence align-ment,and phylogenetic tree of BoMYB homologs.A,Linkage map of BoMYB2,BoMYB3,BoMYB4,and the Pr -D locus in a mapping population of 1,898F2individuals.B,Sequence alignment of cauli?ower and Arabidopsis R2R3MYB proteins.R2and R3repeat do-mains as well as the conserved KPRPR [S/T]F motif are indicated.C,Phyloge-netic tree of BoMYBs and R2R3MYBs from other plant species.Numbers along branches indicate bootstrap sup-port determined from 1,000trials.The length of the branch lines indicates the extent of divergence.The GenBank accession numbers of these proteins are provided in Supplemental Table S2.
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tion level of BoMYB2regulation.BoMYB2shared 79.8%to89.9%amino acid sequence identity to the other BoMYB proteins,and69.1%to86.3%to the Arabidopsis PAP-like proteins(Fig.4B).Alignment of the coding sequences of wild type and the mutant gene showed that there were only two single nucleo-tide differences,which resulted in two amino acid changes from Ile to Thr and Pro to Ala at position14 and159,respectively(Fig.4B).Phylogenetic analysis indicates that BoMYBs are most closely related to Arabidopsis PAP-like proteins and clustered with R2R3MYB transcription factors involved in regulating anthocyanin biosynthesis from other plant species (Fig.4C).
Functional Complementation of the Purple Mutant Phenotype in Arabidopsis and Cauli?ower
To con?rm the function of Pr,genomic fragments of the Pr gene from both wild-type and mutant plants were introduced into wild-type Arabidopsis and cau-li?ower.Over40independent transgenic Arabidopsis lines were generated for each construct.Like the vector-only control(Fig.5A,1–6),the transgenic Arab-idopsis expressing the wild-type pr allele under the control of endogenous promoter(pr pro:pr)exhibited no or very low levels of pigmentation(Fig.5C,1–6).In contrast,the Pr-D(Pr pro:Pr-D)transformants showed tissue-speci?c anthocyanin accumulation(Fig.5B, 1–6).Purple pigments accumulated mainly in young tissues of Arabidopsis,such as young leaves and young?ower buds,and in seeds,but not in old leaves,?ower petals,or mature siliques.A similar pattern of anthocyanin accumulation was also observed in trans-genic cauli?ower plants expressing the Pr-D allele (Fig.5G,1–6).Ectopic expression of the Pr-D allele under the control of endogenous promoter in both transgenic Arabidopsis and cauli?ower induced tis-sue-speci?c anthocyanin accumulation,which showed the same anthocyanin accumulation pattern as in the Pr-D mutant.Thus,these results further con?rm the isolation of the Pr gene.
Both wild-type and mutant Pr genes under the control of cauli?ower mosaic virus(CaMV)35S (35S pro:pr and35S pro:Pr-D)were also introduced into wild-type Arabidopsis and cauli?ower.Overexpres-sion of either wild-type or mutant gene resulted in the production of dark-purple transgenic plants with high levels of anthocyanin accumulation in Arabidopsis (Fig.5,D and E,1–6).Pigments accumulated in the
entire transgenic plants that included leaves,roots,?ower buds,?ower petals,siliques,and seeds.Simi-larly,when these gene constructs were introduced into wild-type cauli?ower plants,anthocyanins appeared in the entire transgenic plants(Fig.5I,1–6and J,1and 2).Interestingly,the young top curd tissues of some overexpression lines were white(Fig.5,I and J,2)with the below curd tissues purple(Fig.5I,4).These results suggest that both wild-type and mutant genes encode functional proteins.Pr Speci?cally Regulates a bHLH Transcription Factor and a Subset of Genes Involved in
Anthocyanin Production
A number of late anthocyanin pathway genes and a transcription factor BobHLH1along with Pr were expressed highly in the Pr-D mutant(Fig.3).To investigate whether expression of Pr in transgenic cauli?ower caused such speci?c activation of gene expression,transcript levels of anthocyanin
https://www.doczj.com/doc/5c14802600.html,plementation of the purple phenotype in wild-type Arabidopsis and cauli?ower.Top images:Arabidopsis transgenic lines containing empty vector(VC),the Pr-D allele with endogenous pro-moter(Pr pro:Pr-D),the wild-type pr allele with endogenous promoter (pr pro:pr),the Pr-D allele with CaMV35S promoter(35S pro:Pr-D),and the wild-type pr allele with CaMV35S promoter(35S pro:pr).The sections from top represent7-d-old seedlings,3-week-old plants, young?ower buds,?owers,older siliques,and seeds.Scale bars for seed images:0.1mm.Bottom images:Cauli?ower transgenic lines containing the same?ve constructs as top images.The different sections from top represent T0young plants,young curds,?owers,part of more mature curds,siliques,and seeds.
Anthocyanin Biosynthesis in Purple Cauli?ower
thetic genes and a number of regulatory genes in various transgenic lines were analyzed.The late path-way genes,BoF3’H ,BoDFR ,BoLDOX ,and the transcrip-tion factor BobHLH1were expressed highly in the Pr pro :Pr -D transformants as well as in 35S pro :Pr -D and 35S pro :pr transgenic lines in comparison with vector-only control and wild-type pr pro :pr lines (Fig.6).The early pathway genes showed no dramatic up-regulation.These results clearly demonstrate that Pr speci?cally activates BobHLH1and the late pathway genes in controlling anthocyanin biosynthesis in cauli?ower.
Allelic Variation at Upstream Regulatory Region of the Pr -D Allele Enhances Promoter Activity
Pr was expressed highly in the mutant and shared 99.2%nucleotide sequence identity in the coding re-gion with the wild-type gene.Thus,we hypothesized that the mutation in the promoter region controlled Pr expression in regulating anthocyanin accumulation.Analysis of the promoter sequences between the wild-type and Pr -D alleles revealed that the ?rst 2370-bp sequences were nearly identical except ?ve nucleotide differences and one extra TATA box at the wild-type promoter (Supplemental Fig.S1).A Harbinger DNA transposon insertion (giri:https://www.doczj.com/doc/5c14802600.html,)was found at 2373bp of the Pr -D mutant allele.To test whether the sequence rearrangement due to the mu-tation altered promoter activity,comparable lengths of promoter sequences from wild-type and Pr -D allele were fused to GUS gene (Fig.7A)and transformed into Arabidopsis.Interestingly,although the promoter sequences between the 2378/2373regions of Pr -D and pr were nearly identical,the mutant Pr -D 2373:GUS transformants exhibited signi?cantly higher GUS ac-tivity than the wild-type pr 2378:GUS transformants
(P ,0.01).Similarly,transformants expressing Pr -D 21079:GUS that included part of the transposon se-quence also yielded signi?cantly higher GUS activity than transgenic lines containing wild-type pr 21061:GUS construct (Fig.7B).These results indicate that the allelic alteration at the upstream regulatory region of the mutant allele enhances the promoter activity of the Pr gene in activating its expression.
DISCUSSION
The Pr gene of cauli?ower confers anthocyanin production in otherwise anthocyanin nonaccumulat-ing tissues,such as curds and seed endosperms,turning them purple.Here we report the successful isolation of the Pr gene via a combination of candidate gene analysis and ?ne mapping.The Pr gene was found to encode a R2R3MYB protein.R2R3MYB transcription factors belong to a large protein family (Stracke et al.,2001;Allan et al.,2008).In Arabidopsis,MYB transcription factor subgroup that activates an-thocyanin biosynthesis includes four genes,P AP1,MYB113,MYB114,and P AP2,with the last three local-ized in tandem on chromosome one,an arrangement also seen in grape (Walker et al.,2007).A total of
four
Figure 6.Expression of anthocyanin structural and regulatory genes in transgenic cauli?ower.qRT-PCR analysis of transcript levels of selected genes in young leaves of cauli?ower transformants containing different Pr gene constructs.The transcript levels of genes in vector control lines were set to 1.Results reported represent mean values 6SD from three biological replicate lines with three technical replicates for
each.Figure 7.Analysis of promoter activity of the Pr gene.A,GUS constructs containing different lengths of promoter sequences.The promoter regions were chosen based on transposon insertion site at 2373bp and the transposon transposase site at 21,079bp.B,GUS activity of 4-week-old stable Arabidopsis transgenic lines containing different GUS constructs.GUS activity was measured from at least four independent transgenic lines with three repeats and expressed as m mol 4-methylumbelliferone min 21mg 21protein.Error bars indicate SD (n .4plants).The letters above the bars indicate signi?cant differences (P ,0.01).
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different MYB-like genes,BoMYB1,BoMYB2,BoMYB3, and BoMYB4,were isolated from the cauli?ower genome.Three of them,i.e.BoMYB2,BoMYB3,and BoMYB4,appear to be located next to each other.High-resolution mapping and functional complementation in both cauli?ower and Arabidopsis clearly con?rm the identi?cation of BoMYB2as the Pr gene.BoMYB2 appears to be an important MYB transcription factor in regulating anthocyanin biosynthesis in Brassica ge-nomes.A previous study of red cabbage varieties also reveals that the constitutive anthocyanin production is associated with an increased expression of only BoMYB2among the four BoMYBs(Yuan et al.,2009). BoMYB2from cauli?ower shares99.6%nucleotide sequence identity with that from cabbage.
Pr and BobHLH1as well as a number of late pathway genes including BoF3’H,BoDFR,and BoLDOX,were expressed highly in the Pr-D mutant.Introduction of the Pr-D allele into wild-type cauli?ower speci?cally increased the transcript levels of BobHLH1and the same set late structural genes in the transformants.The up-regulation of BobHLH1in both the Pr-D mutant and the Pr-D transformants strongly suggests that Pr regulates the expression of BobHLH1.Although ex-pression of MdMYB10does not elevate a bHLH tran-scription factor expression in apple(Malus domestica; Espley et al.,2007),P AP1has been shown to regulate the expression of TT8in Arabidopsis(Baudry et al., 2006).Similarly,AN2and AN4,the genes encoding MYB transcription factors,activate the bHLH tran-scription factor of AN1in petunia(Spelt et al.,2000).Pr and BobHLH1likely work together to coordinately regulate several transcripts of anthocyanin late path-way genes in conferring anthocyanin accumulation in the Pr-D mutant and transformants.
Despite the fact that MYB and bHLH transcription factors share similar functions among plants,they exhibit species-speci?c differences in activating part or the entire set of anthocyanin pathway genes.For example,in maize the C1/P1family of MYB proteins and the R/B family of bHLH proteins activate the entire set of anthocyanin structural genes(Chandler et al.,1989;Ludwig and Wessler1990;Cone et al.,1993; Grotewold et al.,1994).In petunia and Arabidopsis, these two families of proteins control a subset of structural genes(Quattrocchio et al.,1993;Spelt et al.,2000;Gonzalez et al.,2008).Like the later cases, BoMYB2and BobHLH1regulated a subset of antho-cyanin structural genes in inducing tissue-speci?c expression in the Pr-D mutant.However,how Pr exhibits its unique tissue-speci?c regulation of antho-cyanin accumulation remains to be determined.
The activity of MYB-like genes has been suggested to be the primary cause of natural variation in antho-cyanin pigmentation in plants(Quattrocchio et al., 1999;Schwinn et al.,2006).Increased expression of R2R3MYB transcription factors was found to be responsible for anthocyanin production in a number of anthocyanin-accumulating mutants.For example, the constitutive up-regulation of P AP1,ANT1,and MdMYB10causes anthocyanin accumulation through-out the plant in pap1-D Arabidopsis(Borevitz et al., 2000),antl tomato(Solanum lycopersicum;Mathews et al.,2003),and red-?eshed apple(Espley et al., 2007).While overexpression of P AP1and ANT1MYB transcription factors is due to activation-tagged inser-tions in their promoter sequences(Borevitz et al.,2000; Mathews et al.,2003),the high transcript level of MdMYB10was recently found to be due to the forma-tion of a minisatellite-like structure comprising multi-ple repeats of a promoter segment that generates a novel autoregulatory motif(Espley et al.,2009).Unlike these reported anthocyanin-accumulating mutants, the Pr-D mutant appears to display a different mech-anism in activating Pr expression.
The coding regions of Pr-D and pr shared99.2% sequence identity and both of them encoded func-tional proteins.Thus,the sequence variation in the promoter region of Pr is likely responsible for the activation of Pr in controlling anthocyanin biosynthe-sis.Indeed,the promoter from wild type and the Pr-D alleles showed different capacity in activating GUS expression.For example,although the promoter of the 2378/2373regions of Pr-D and pr shared nearly identical sequences,signi?cantly higher GUS activity was observed in the transformants expressing the mutant Pr-D2373:GUS than the wild-type pr2373:GUS. Detailed examination of the Pr-D2373/pr2373sequences revealed that an insertion of two nucleotides in Pr-D2373range generated a new regulatory motif,the E-box(5#-CANNTG-3#),which is a cis-acting element with binding consensus site for bHLH proteins (Toledo-Ortiz et al.,2003;Supplemental Fig.S1).Fur-ther,both Pr-D2373and wild-type pr21061sequences contained one E-box and the transformants expressing these constructs exhibited similar promoter activity. The Pr-D21079sequence harbored two additional E-boxes and gave signi?cantly higher promoter activ-ity than wild-type pr21061sequence(Fig.7).The trans-poson insertion in the Pr-D allele introduced additional E-box cis-acting elements,which likely provide more binding sites for bHLH transcription factors to activate Pr expression,as suggested in a recent study of the DNA transposon mPing in rice (Naito et al.,2009).E-boxes are identi?ed in the pro-moter region of anthocyanin structural genes and have been shown to be required for bHLH transcription factor binding in enhancing gene expression(Shirley et al.,1992;Hartmann et al.,2005).Future experiments will help clarify the involvement of E-box ampli?cation in activating Pr expression.Nevertheless,our results reported here clearly indicate that the mutation at the upstream promoter region in the Pr-D allele resulted in activating Pr gene transcription to confer the pheno-typic change in cauli?ower.Pr exerts profound effect on anthocyanin production in plants.The successful clon-ing of Pr and the discovery of it as primary determinant of color in cauli?ower have implications for breeding vegetable crops with enhanced health-promoting prop-erties and visual appeal.
Anthocyanin Biosynthesis in Purple Cauli?ower
MATERIALS AND METHODS
Plant Materials and Growth Conditions
Purple cauli?ower(Brassica oleracea var botrytis)arose from a spontaneous mutation found in a cauli?ower?eld about20years ago.A commercial purple cauli?ower cultivar Graf?ti(Harris Seeds)and a white cultivar Stovepipe as wild-type control were used in this study.Arabidopsis(Arabidopsis thaliana) ecotype Columbia was used for genetic transformation.Cauli?ower plants were grown either in a greenhouse under14-h-light/10-h-dark photoperiod at 23°C or in a?eld.Arabidopsis plants were grown in a growth room under 14-h-light/10-h-dark photoperiod at23°C.
HPLC-ESI-MS/MS Analysis of Anthocyanins Anthocyanins in Graf?ti was extracted and analyzed following the method as described by Wu and Prior(2005b).Freeze-dried curd sample (0.5g)was ground into powder and extracted twice in total25mL methanol/water/acetic acid(85:15:0.5).The extract was diluted2-fold. Aliquot(10m L)was injected into a Zorbax stablebond analytical SB-C18 column(4.63250mm,5m m,Agilent Technologies)and separated using 5%formic acid(A)and100%methanol(B)as mobile phases on an HP1100 series HPLC.Low-resolution electrospray mass spectrometry as de-scribed(Wu and Prior,2005b)was performed to identify the major anthocyanin peaks in the purple curd sample.Quanti?cation was carried out based on peak areas and a calibration curve generated with a commercial standard of cyanidin3,5-diglucoside chloride(INDOFINE Chemical Company).
Nucleic Acid Analysis
Genomic DNA was isolated as described previously(Lu et al.,2006).Total RNA from purple and white cauli?ower tissues was extracted using Trizol reagent following the manufacturer’s instruction(Invitrogen).mRNA was isolated from total RNA using PolyATtract mRNA isolation system IV (Promega).
For northern-blot analysis,mRNA(2m g)samples were used.Prehybrid-ization and hybridization with P32-labeled probes were performed as de-scribed previously(Li et al.,2001).Probes used included CHS(U21762),CHI (U20894),F3H(U14735),and LDOX(YAY780)from Arabidopsis,as well as F3’H,DFR,UGT,and Actin ampli?ed from white cauli?ower DNA using primers as listed in Supplemental Table S1.
qRT-PCR analysis was conducted according to the requirements and guidelines described by Udvardi et al.(2008).The cDNAs were synthesized from DNase I treated total RNA(5m g)using Superscript III reverse transcriptase(Invitrogen).qRT-PCR was carried out using SYBR Green PCR master mix following the manufacturer’s instruction(Applied Biosys-tems)in an Applied Biosystems7900HT fast real-time PCR system.Gene-speci?c primers,which were con?rmed to produce speci?c gene products by sequencing(Yuan et al.,2009),are listed in Supplemental Table S1.The relative transcript levels were calculated as described previously(Lyi et al., 2007).
PCR walking on genomic DNA was performed using the Universal GenomeWalker kit following the manufacturer’s instruction as described previously(Li and Garvin,2003).Wild-type and mutant cauli?ower ge-nomic DNA(2.5m g)were digested with Hin cII,Rsa I,Sma I,Stu I,Swa I,Dra I, Eco RV,Pvu II,Sca I,and Ssp I,respectively,and ligated to the GenomeWalker adaptors to produce Genome walking libraries.PCR products of secondary nested reactions were cloned into pCR2.1vector(Invitrogen)and se-quenced.
Association Mapping of the Candidate Genes DNA of the parents and1,898F2individuals of the mapping population were extracted(Lu et al.,2006).To design PCR-based markers,the candidate genes from both mutant and wild-type plants were isolated.Based on insertions and deletions or single nucleotide polymorphisms in the two alleles of these genes,primer sets for BoMYB2,BoMYB3,and BoMYB4 markers were developed(Supplemental Table S1).BoMYB2m and BoMYB4m were codominant markers and BoMYB3m was dominant marker.Genetic linkage map was generated using MapMaker(https://www.doczj.com/doc/5c14802600.html,/ ftp/distribution/software/mapmaker3).Sequence Analysis
DNA and protein sequences were analyzed using the program of DNAStar (Lasergene).Multiple sequence alignments were produced by Vector NTI (Invitrogen)or CLC sequence viewer using default setting.The phylogenic tree was calculated by DNAStar and visualized by Treeview version1.6.6 (https://www.doczj.com/doc/5c14802600.html,/rod/treeview.html).
Plasmid Construction and Plant Transformation To create the constructs for phenotypic complementation,genomic DNA of Pr including1,755or1,061bp of promoter sequences for the mutant and wild-type allele,respectively,were ampli?ed.Despite of extensive efforts,the upstream above1,061bp promoter sequence for wild-type allele could not be obtained due to highly repeated sequence and was not found from public sequence databases.The ampli?ed products were cloned into pCAMBIA1300 vector(CAMBIA)with a nopaline synthase terminator to produce pr pro:pr and Pr pro:Pr-D constructs.To generate the overexpression constructs,comparable genomic DNA from start to stop codon of both Pr alleles were ampli?ed and inserted into pCAMBIA1300S containing CaMV35S promoter(Zhou et al., 2009)to produce35S pro:pr and35S pro:Pr-D.To create the GUS constructs,region of wild-type pr promoter(2378and21061bp),and Pr-D promoter(2373and 21,079)were ampli?ed and inserted into pSG506vector(kindly provided by Dr.Susheng Gan,Cornell University).The promoter fragments were selected based on transposable element insertion site.The fragments were then subcloned into pCAMBIA1300to produce various promoter-GUS constructs. All constructs were veri?ed by sequencing.The constructs and vector-only controls were electroporated into Agrobacterium tumefaciens strain GV3101, and transformed into wild-type Arabidopsis using a?ower-dipping method (Clough and Bent,1998),and into cauli?ower using A.tumefaciens-mediated tissue culture method(Lu et al.,2006).
GUS Activity Analysis
Quantitative analysis of GUS activity in transformants expressing different GUS constructs was carried out using?uorometric assay(Blazquez,2007) with slight modi?cation.Four-week-old Arabidopsis leaf tissues(25mg)from at least four independent transgenic lines for each construct were extracted independently in100m L GUS extraction buffer.Total proteins in the crude extract were quanti?ed using the RC DC protein assay kit(Bio-Rad).Extract (5m L)was then added to450m L GUS extraction buffer containing1m M 4-methylumbelliferyl b-D-glucuronide and incubated at37°C.Aliquot of the reaction mixture(20m L)was added into180m L stop solution(1M sodium carbonate)every10min.Fluorescence was then measured using?uorometer Fluorolite1000(DYNEX technologies)at excitation wavelength of365nm and emission wavelength of450nm.The analysis was repeated three times. The mean values for each construct were compared.
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers GU219986(BoMYB Pr-D),GU219987 (BoMYB2pr),GU219985(BoMYB1),GU219988(BoMYB3),GU219989 (BoMYB4),and GU219990(BobHLH1).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1.Nucleotide sequence comparison of the pro-moter region at2378/2373bp of the wild type and mutant Pr gene.
Supplemental Table S1.List of primers used in this study.
Supplemental Table S2.List of the GenBank accession numbers of the proteins used in the phylogenic tree of Figure4C. ACKNOWLEDGMENTS
We are especially grateful to Leon Kochian and Jim Giovannoni for valuable comments.We thank the Li lab members for helpful discussion and technical advice.We also thank Susheng Gan for providing the pSG506
Chiu et al.
vector,Don Reed for providing?eld space and assistance in growing cauli?ower,and Chrissa McFarlane for her excellent technical assistance. Received August7,2010;accepted September19,2010;published September 20,2010.
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用友T6管理软件操作手册总账日常业务处理 日常业务流程 1、进入用友企业应用平台。 T6 双击桌面上的 如设置有密码，输入密码。没有密码就直接确定。 2、填制凭证进入系统之后打开总账菜单下面的填制凭证。如下图 丄总账［演示版】国B设畫 -二疑证 i :卜0 直接双击填制凭证，然后在弹出凭证框里点增 制单日期可以根据业务情况直接修改，输入附单据数数（可以不输），凭证摘要（在后面的匝可以选择常用摘要），选择科目直接选择（不知道可以选按F2或点击后面的一）, 输入借贷方金额，凭证完后如需继续作按增加自动保存，按保存也可，再按增加 3．修改凭证 填制凭证 证 证 证 总 ￡ ■ 凭 汇 汇 流
没有审核的凭证直接在填制凭证上面直接修改，改完之后按保存。（审核、记帐了凭 证不可以修改，如需修改必须先取消记帐、取消审核）。 4．作废删除凭证只有没有审核、记帐的凭证才可以删除。在“填制凭证”第二个菜单“制单” 下面有 一个“作废恢复”，先作废，然后再到“制单”下面“整理凭证”，这样这张凭证才被彻底删除。 5．审核凭证 双击凭证里的审核凭证菜单，需用具有审核权限而且不是制单人进入审核凭证才能审核（制单单人不能审核自己做的凭证） 选择月份，确定。 再确定。 直接点击“审核”或在第二个“审核”菜单下的“成批审核” 6．取消审核 如上所述，在“成批审核”下面有一个“成批取消审核”，只有没有记帐的凭证才可 以取消审核
7．凭证记账 所有审核过的凭证才可以记帐，未审核的凭证不能记账，在“总帐——凭证——记账” 然后按照提示一步一步往下按，最后提示记帐完成。 8．取消记帐 在“总帐”—“期末”—“对帐”菜单按“ Ctrl+H ” 系统会提示“恢复记帐前状态已被激活”。然后按“总帐”——“凭证”——“恢复 记帐前状态”。最后选“月初状态”，按确定，有密码则输入密码，再确定。 10、月末结转收支 当本月所有的业务凭证全部做完，并且记账后，我们就要进行当月的期间损益结转。 点击：月末转账并选择期间损益结转。 选择要结转的月份，然后单击“全选”。点击确定后
智能窗户控制系统软件V1.0设计说明 目录 前言 (1) 第一章软件总体设计 (1) 1.1. 软件需求概括 (1) 1.2. 定义 (1) 1.3. 功能概述 (1) 1.4. 总体结构和模块接口设计 (2) 第二章控制系统的总体设计 (3) 2.1. 功能设计 (3) 第三章软件控制系统的设计与实现 (5) 3.1. RF解码过程程序设计介绍 (5) 3.2. RF对码过程设计 (6) 3.3. 通信程序设计 (8) 3.4. IIC程序设计介绍 (9) 3.5. 接近开关程序设计 (12) 3.6. 震动开关检测程序设计 (13) 3.7. 墙面按键程序设计 (15) 第四章智能窗户控制系统的设计 (17) 第五章实测与结果说明 (18) 第六章结论 (18)
前言 目的 编写详细设计说明书是软件开发过程必不可少的部分，其目的是为了使开发人员在完成概要设计说明书的基础上完成概要设计规定的各项模块的具体实现的设计工作。 第一章软件总体设计 1.1.软件需求概括 本软件采用传统的软件开发生命周期的方法，采用自顶向下，逐步细化，模块化编程的软件设计方法。 本软件主要有以下几方面的功能 (1)RF遥控解码 (2)键盘扫描 (3)通信 (4)安全检测 (5)电机驱动 1.2.定义 本项目定义为智能遥控窗户系统软件。它将实现人机互动的无缝对接，实现智能关窗，遥控开关窗户，防雨报警等功能。 1.3.功能概述 1.墙体面板按键控制窗户的开/关 2.RF遥控器控制窗户的开/关 3.具有限位，童锁等检测功能 4.实时检测大气中的温湿度，下雨关窗 5.具有防盗，防夹手等安全性能的检测
NC系统培训手册 编制单位：用友软件股份有限公司 中央大客户事业部 目录 一、NC系统登陆 .................................... 二、消息中心管理................................... 三、NC系统会计科目设置 ............................ 四、权限管理....................................... 五、打印模板设置................................... 六、打印模板分配................................... 七、财务制单....................................... 八、NC系统账簿查询 ................................ 九、辅助余额表查询................................. 十、辅助明细账查询................................. 十一、固定资产基础信息设置......................... 十二、卡片管理..................................... 十三、固定资产增加................................. 十四、固定资产变动................................. 十五、折旧计提..................................... 十六、折旧计算明细表...............................
门禁考勤管理软件 使 用 说 明 书
软件使用基本步骤
一．系统介绍―――――――――――――――――――――――――――――2二．软件的安装――――――――――――――――――――――――――――2 三．基本信息设置―――――――――――――――――――――――――――2 １）部门班组设置―――――――――――――――――――――――――3 ２）人员资料管理―――――――――――――――――――――――――3 ３）数据库维护――――――――――――――――――――――――――3 ４）用户管理―――――――――――――――――――――――――――3 四．门禁管理―――――――――――――――――――――――――――――4 １）通迅端口设置―――――――――――――――――――――――――4２）控制器管理――――――――――――――――――――――――――4３）控制器设置――――――――――――――――――――――――――6４）卡片资料管理―――――――――――――――――――――――――11 ５）卡片领用注册―――――――――――――――――――――――――12６）实时监控―――――――――――――――――――――――――――13 五．数据采集与事件查询――――――――――――――――――――――――13 六．考勤管理―――――――――――――――――――――――――――――14 1）班次信息设置――――――――――――――――――――――――――14 2）考勤参数设置――――――――――――――――――――――――――15 3）考勤排班――――――――――――――――――――――――――――15 4）节假日登记―――――――――――――――――――――――――――16 5）调休日期登记――――――――――――――――――――――――――16 6）请假/待料登记―――――――――――――――――――――――――17 7）原始数据修改――――――――――――――――――――――――――17 8）考勤数据处理分析――――――――――――――――――――――――17 9）考勤数据汇总―――――――—――――――――――――――――――18 10）考勤明细表—―――――――――――――――――――――――――18 11）考勤汇总表――――――――――――――――――――――――――18 12）日打卡查询――――――――――――――――――――――――――18 13）补卡记录查询—――――――――――――――――――――――――19
开票端操作说明 双击桌面“博思开票”图标，单击“确定”，进入开票界面： 一、开票： 日常业务——开票——选择票据类型——增加——核对票号无误后——单击“请核对票据号”——输入“缴款人或缴款单位”——选择”收费项目”、“收入标准”——单击“收费金额”。 （如需增加收费项目，可单击“增一行”） （如需加入备注栏（仅限于收款收据）），则在右侧“备注”栏内输入即可） 确认无误后，单击“打印”——“打印” 二、代收缴款书： 日常业务——代收缴款书——生成——生成缴款书——关闭——缴款——输入“专用票据号”——保存——缴款书左上角出现“已缴款”三个红字即可。 三、上报核销： 日常业务——上报核销——选择或输入核销日期的截止日期——刷新——核销。 （注意：“欠缴金额”处无论为正或为负均不可核销，解决方法见后“常见问题”）
常见问题 一、如何作废“代收缴款书” 日常业务——代收缴款书——缴款——删除“专用票据号”和“缴款日期”——保存——作废。 二、上报核销时出现欠缴金额，无法完成核销，或提示多缴。 1、首先检查有没有选择好截止日期，选择好后有没有点击“刷新”。 2、其次检查有没有做代收缴款书。注意：最后一张缴款书的日期不得晚于选择上报核 销日期。 3、若上述方法仍无效，则可能是由于以前作废过票据而未作废缴款书。解决方法： 首先作废若干张缴款书（直到不能作废为止），然后重新做一张新的缴款书。再核销。 三、打开“博思开票”时，出现“windows socket error：由于目标机器积极拒绝，无法连接。 （10061），on API’connect’” 单击“确定”，将最下面一行的连接类型“SOCKET”更换为“DCOM”，再点“连接” 即可。 四、如何设置密码 双击桌面“博思开票”，单击登录界面的右下方“改口令”输入用户编号、新密码和确认密码，单击“确认”即可。 五、更换开票人名称或增加开票人 进入开票系统——系统维护——权限管理 1、更换开票人名称：单击“用户编码”——删除“用户名”——输入新的开票人名称 ——单击“保存用户”即可。 2、增加开票人：单击“新增用户”——输入“用户编码”和“用户名”——单击“保 存用户”——单击新增的用户编码——将右边的“权限列表如下”下面的“所有”前的小方框勾上——单击右侧“保存用户权限”。 六、重装电脑系统 1、由于博思开票软件安装在D盘，所以重装电脑系统前无需做任何备份。 2、重装系统后，打开我的电脑—D盘，将“博思软件”文件夹复制到桌面上（或U盘）。 3、将安装时预留的安装光盘放入主机，打开后找到“票据核销及管理_开票端(江西欠 缴不能上报版)”（或者进入D盘----开票软件备份目录勿删文件夹里也可找到）。双 击，按提示点击“下一步”，直到“完成”。 4、双击桌面任务栏右下角“博思开票服务器”，将其关闭（或右键点击“博思开票服 务器”——“关闭服务器”）。 （这一步若找不到“博思开票服务器”，也可以用重启电脑来代替） 5、将刚才复制到桌面（或U盘）的“博思软件”再复制粘贴回D盘，若提示“此文 件夹已包含名为博思软件的文件夹”，点击下面的“全部”。 6、双击桌面“博思开票”——输入用户编码(001)——确定。 7、确认原来的票据数据没有丢失后，将桌面（或U盘）的“博思软件”文件夹删除。
控制系统使用说明 系统针对轴流风机而设计的控制系统, 系统分为上位监视及下位控制两部分 本操作为上位监控软件的使用说明: 1: 启动计算机: 按下计算机电源开关约2秒, 计算机启动指示灯点亮, 稍过大约20秒钟屏幕出现操作系统选择菜单, 通过键盘的“↑↓”键选择“windows NT 4.0”菜单，这时系统进入WINDOWS NT 4.0操作系统，进入系统的操作画面。 2：系统操作 系统共分：开机画面、停机画面、趋势画面、报警画面、主机流程画面、轴系监测画面、润滑油站画面、动力油站画面、运行工况画面、运行记录画面等十幅画面，下面就十幅画面的作用及操作进行说明 A、开机画面： 开机： 当风机开始运转前，需对各项条件进行检查，在本画面中主要对如下指标进行检查，红色为有效： 1、静叶关闭：静叶角度在14度
2、放空阀全开：放空阀指示为0% 3、润滑油压正常 4、润滑油温正常 5、动力油压正常 6、逆止阀全关 7、存储器复位：按下存储器复位按钮，即可复位，若复位不成 需查看停机画面。 8、试验开关复位：按下试验开关按钮即可，试验开关按钮在风 机启动后，将自动消失，同时试验开关也自动复位。 当以上条件达到时，按下“允许机组启动”按钮，这时机组允许启动指示变为红色，PLC机柜里的“1KA”继电器将导通。机组允许启动信号传到高压柜，等待电机启动。开始进行高压合闸操作，主电机运转，主电机运转稳定后，屏幕上主电机运行指示变红。这时静叶释放按钮变红，按下静叶释放按钮后，静叶从14度开到22度，静叶释放成功指示变红。 应继续观察风机已平稳运行后，按下自动操作按钮，启机过程结束。 B、停机画面： 停机是指极有可能对风机产生巨大危害的下列条件成立时，PLC 会让电机停止运转： 1、风机轴位移过大
用 友 T+ 软 件 系 统 操 作 手 册版本号：v1.0
目录 一、系统登录 (3) 1.1、下载T+浏览器 (3) 1.2、软件登陆 (3) 二、基础档案设置 (5) 2.1、部门、人员档案设置 (5) 2.2、往来单位设置 (6) 2.3、会计科目及结算方式设置 (6) 三、软件操作 (9) 3.1、凭证处理 (9) 3.1.1、凭证填制 (9) 3.1.2、凭证修改 (10) 3.1.3、凭证审核 (11) 3.1.4、凭证记账 (12) 3.2、月末结转 (13) 四、日常帐表查询与统计 (14) 4.1、余额表 (14) 4.2、明细账 (15) 4.3、辅助账 (16) 五、月末结账、出报表处理 (17) 5.1、总账结账 (17) 5.2、财务报表 (20)
一、系统登录 1.1、下载T+浏览器 首次登陆需要用浏览器打开软件地址，即：127.0.0.1:8000(一般服务器默认设置，具体登陆地址请参考实际配置)，第一次登陆会提示下载T+浏览器，按照提示下载安装T+浏览器，然后打开T+浏览器，输入软件登陆地址。 ，T+浏览器， 1.2、软件登陆 按键盘上的“回车键（enter）”打开软件登陆页面，如下： 选择选择“普通用户”，输入软件工程师分配的用户名和密码，选择对应的账套，以下以demo 为例，如下图：
点击登陆，进入软件，
二、基础档案设置 2.1、部门、人员档案设置 新增的部门或者人员在系统中可按照如下方法进行维护，
2.2、往来单位设置 供应商客户档案的添加方法如下： 添加往来单位分类： 2.3、会计科目及结算方式设置会计科目：
LED-ECS编辑控制系统V5.2 用 户 手 册 目录 第一章概述 (3) 1.1LED-ECS编辑控制系统介绍 (3) 1.2运行环境 (3) 第二章安装卸载 (3) 2.1安装 (3) 2.2卸载 (5) 第三章软件介绍 (5) 3.1界面介绍 (5) 3.2操作流程介绍 (13) 3.3基本概念介绍 (21) 第四章其他功能 (25) 4.1区域对齐工具栏 (25) 4.2节目对象复制、粘贴 (26) 4.3亮度调整 (26) 第五章发送 (27) 5.1发送数据 (27) 第六章常见问题解决 (28) 6.1计算机和控制卡通讯不上 (28) 6.2显示屏区域反色或亮度不够 (29)
6.3显示屏出现拖尾现象，显示屏的后面出现闪烁不稳定 (29) 6.4注意事项 (31) 6.5显示屏花屏 (31) 6.6错列现象 (32) 6.7杂点现象 (32) 第一章概述 1.1LED-ECS编辑控制系统介绍 LED-ECS编辑控制系统，是一款专门用于LED图文控制卡的配套软件。其具有功能齐全，界面直观，操作简单、方便等优点。自发布以来，受到了广大用户的一致好评。 1.2运行环境 ?操作系统 中英文Windows/2000/NT/XP ?硬件配置 CPU:奔腾600MHz以上 内存:128M 第二章安装卸载 2.1LED-ECS编辑控制系统》软件安装很简单，操作如下：双击“LED-ECS编辑控制系统”安装程序，即可弹出安装界面，如图2-1开始安装。如图所示 图2-1 单击“下一步”进入选择安装路径界面，如图2-2，如果对此不了解使用默认安装路径即可 图2-2 图2-3 单击“完成”，完成安装过程。 2.2软件卸载如图2-2 《LED-ECS编辑控制系统V5.2》提供了自动卸载功能，使您可以方便的删除《LED-ECS编辑控制系统V5.2》的所有文件、程序组件和快捷方式。用户可以在“LED-ECS编辑控制系统V5.2”组中选择“卸载LED-ECS编辑控制系统V5.2”卸载程序。也可以在“控制面板”中选择“添加/删除程序”快速卸载。卸载程序界面如图2-4，此时选择自动选项即可卸载所有文件、程序组和快捷方式。 图2-4 第三章、软件介绍
财政票据(网络版)电子化系统 开票端 操 作 说 明 福建博思软件股份有限公司
目录 1.概述 业务流程 流程说明：
1.单位到财政部门申请电子票据，由财政把单位的基本信息设置好并审核完后，财政部门给用票单位发放票据，单位进行领票确认并入库。 2.在规定时间内，单位要把开据的发票带到财政核销，然后由财政进行审核。 系统登录 登入系统界面如图： 登录日期：自动读取主服务器的日期。 所属区划：选择单位所属区划编码。【00安徽省非税收入征收管理局】 所属单位：输入单位编码。 用户编码：登录单位的用户编码【002】 用户密码：默认单位密码为【123456】 验证码：当输入错误时，会自动换一张验证码图片； 记录用户编码：勾选系统自动把用户编码保存在本地，第二次登录不需要重新输入。 填写完正确信息，点【确定】即可登入系统。 进入系统 进入系统界面如图： 当单位端票据出现变动的时候，如财政或上级直管下发票据时，才会出现此界面:
出现此界面后点击最下方的确认按钮，入库完成。 当单位端票据无变动时，直接进入界面： 2.基本编码人员管理 功能说明：对单位开票人员维护，修改开票人名称。 密码管理 修改开票人员密码，重置等操作。 收发信息 查看财政部门相关通知等。
3.日常业务 电脑开票 功能说明：是用于开票据类型为电子化的票据。 在电脑开票操作界面，点击工具栏中的【增加】按钮，系统会弹出核对票号提示框，如图： 注意：必须核对放入打印机中的票据类型、号码是否和电脑中显示的一致，如果不一致打印出来的票据为无效票据，核对完后，输入缴款人或缴款单位和收费项目等信息，全部输入完后，点【增加】按钮进行保存当前票据信息或点【打印】按钮进行保存当前票据信息并把当前的票据信息打印出来；点电脑开票操作界面工具栏中的【退出】则不保存。 在票据类型下拉单框中选择所要开票的票据类型，再点【增加】进行开票。
用友T软件系统操作手 册 Pleasure Group Office【T985AB-B866SYT-B182C-BS682T-STT18】
用 友 T+ 软 件 系 统 操 作 手 册 版本号：目录
一、系统登录 、下载T+浏览器 首次登陆需要用浏览器打开软件地址，即：(一般服务器默认设置，具体登陆地址请参考实际配置)，第一次登陆会提示下载T+浏览器，按照提示下载安装T+浏览器，然后打开T+浏览器，输入软件登陆地址。 ，T+浏览器， 、软件登陆 按键盘上的“回车键（enter）”打开软件登陆页面，如下： 选择选择“普通用户”，输入软件工程师分配的用户名和密码，选择对应的账套，以下以demo为例，如下图： 点击登陆，进入软件， 二、基础档案设置 、部门、人员档案设置 新增的部门或者人员在系统中可按照如下方法进行维护， 、往来单位设置 供应商客户档案的添加方法如下： 添加往来单位分类： 、会计科目及结算方式设置 会计科目： 系统预置170个《2013小企业会计准则》科目，如下：
结算方式，如下： 三、软件操作 、凭证处理 填制 进入总账填制凭证菜单，增加凭证，填制摘要和科目，注意有辅助核算的会计科目， 以下为点开总账的处理流程图： 如若现金流量系统指定错误，可按照以下步骤修改： 凭证在没有审核时，可以直接在当前凭证上修改，然后点击“保存”完成修改； 凭证审核 进入总审核凭证菜单下，如下图： 选择审核凭证的会计期间： 、凭证记账 进入凭证菜单下的记账菜单， 、月末结转 期间损益结转 四、日常帐表查询与统计 、余额表 用于查询统计各级科目的本期发生额、累计发生额和余额等。传统的总账，是以总账科目分页设账，而余额表则可输出某月或某几个月的所有总账科目或明细科目的期初余额、本期发生额、累计发生额、期末余额，在实行计算机记账后，我们建议用户用余额表代替总账。
创维液晶拼接控制系统 软件操作指南 【LCD-CONTROLLER12】 请在使用本产品前仔细阅读该用户指导书
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一、功能特点 二、技术参数 三、控制系统连接示意图 四、基本操作 五、故障排除 六、安全注意事项
一、功能特点创维创维--液晶液晶拼接拼接拼接控制器特点控制器特点 ★采用创维第四代V12数字阵列高速图像处理技术 视频带宽高达500MHZ，应用先进的数字高速图像处理算实时分割放大输入图像信号，在多倍分割放大处理的单屏画面上，彻底解决模/数之间转换带来的锯齿及马赛克现象，拼接画面清晰流畅，色彩鲜艳逼真。 ★具有开窗具有开窗、、漫游漫游、、叠加等功能 以屏为单元单位的前提下，真正实现图像的跨屏、开窗、画中画、缩放、叠加、漫游等个性化功能。 ★采用基于LVDS 差分传送技术差分传送技术，，增强抗干扰能力 采用并行高速总线连接技术，上位控制端发出命令后，系统能快速切换信号到命令指定的通道，实现快速响应。 采用基于LVDS 差分传送技术，提高系统抗干扰能力，外部干扰对信号的影响降到了最低，并且，抗干扰能力随频率提高而提升。★最新高速数字阵列矩阵通道切换技术 输入信号小于64路时，用户不需要再另外增加矩阵，便可以实现通道之间的任意换及显示。 ★断电前状态记忆功能 通过控制软件的提前设置，能在现场断电的情况下，重启系统后，能自动记忆设备关机前的工作模式状态。 ★全面支持全高清信号 处理器采用先进的去隔行和运动补偿算法，使得隔行信号在大屏幕拼接墙上显示更加清晰细腻，最大限度的消除了大屏幕显示的锯齿现象，图像实现了完全真正高清实时处理。纯硬件架构的视频处理模块设计，使得高清视频和高分辨率计算机信号能得到实时采样，确保了高清信号的最高视频质量，使客户看到的是高质量的完美画质。
工会经费收入专用收据(1) 福州博思软件开发有限公司 2010年6月 目录 第一部分初始安 装 ....................................................... (1) 1.1系统 安装 (1) 1.2系统登录 ............................................................ (4) 第二部分组成模块介 绍 ................................................... (4) 2.1模块组 成 (4) 2.1.1票据资料 .......................................................... (5) 2.1.2用户管 理 .......................................................... (5) 2.1.3 票据领用 (6) 2.1.4电脑开票 .......................................................... (6) 2.1.5手工开 票 .......................................................... (7) 2.1.6 手工批开票 (7) 2.1.7票据查询 .......................................................... (8) 第三部分软件操 作 ....................................................... (9) 3.2用户 管理 (10)
大屏幕控制系统软件详解说明 一软件安装 安装注意事项： 非专业人事安装：安装前请先关闭防火墙（如360安全卫士，瑞星，诺盾等），等安装完并且成功启动本软件后可重新开启防火墙； 专业人事安装：先把防火墙拦截自动处理功能改为询问后处理，第一次打开本软件时会提示一个拦截信息； 安装前请校对系统时间,安装后不能在错误的系统时间下运行/启动软件,否则会使软件注册失效，这种情况下需要重新注册； Windows 7,注意以下设置 0.1)打开控制面板 0.2) 选择系统和安全 0.3) 选择操作中心 0.4) 选择更换用户帐户控制设置 0.5)级别设置，选择成从不通知 １．软件解压后，请选择双击，进入安装界面如图１，图２ 图1
图2 ２．选择键，进入下一界面如图3 图３ 3.选中项，再按键，进入下一界面如图４
图4 4.选择键，进入下一界面如图５ 图５ ５.选中项，再选择键，进入下一界面如图６
图６ ６.选择键，进入下一界面如图７ 图８ ７.选择键，软件安装完成 二软件操作 选择WINDOWS 下开始按钮，选择程序，选择Wall Control项， 点击Wall Control软件进入大屏幕控制系统软件主界面如图9所示,整个软件分为3个区，标题区，设置区，功能区
图9 1.1标题区 大屏幕控制系统软件(只有管理员才可设置此项目) 1.2设置区 1.2.1系统 高级功能：管理员登录。 产品选型：选择拼接盒型号。 定时系统：设置定时时间。 幕墙开机：开机 幕墙关机：关机 退出：退出软件系统。 1.2.2设置 串口设置：设置使用的串口参数。 矩阵设置：设置矩阵的相关参数。 幕墙设置：幕墙设置参数。 幕墙颜色：幕墙颜色设置。 标志设置：更改幕墙名称。 系统设置：控制软件系统设置。 1.2.3工具 虚拟键盘：虚拟键盘设置。 硬件注册：可以通过时钟IC注册处理器的使用权限。 1.2.4语言 中文选择:选择软件语言类型为中文。 Ｅnglish:选择软件语言类型为英语。
用友财务管理系统操作手册 北京用友政务软件有限公司 2011年05月25日
一、账务系统： 流程：1、初始化设置及期初数装入=》2、凭证录入=》 3、凭证审核=》 4、凭证记账=》 5、月结 1、初始化设置: (1)、用自己的用户名登录【账务管理系统】=》 点击界面右边【基础资料】前的【+】号=》点击【会计科 目】前的【+】号=》双击【建立会计科目】=》设置会计科 目及挂接辅助账。(2)、点击界面右边【账务】前的【+】号 =》点击【初始建账数据】前的【+】号=》双击【期初余额 装入】=》点击【确定】=》然后对期初数据进行录入 2、凭证录入:用自己的用户名登录【账务管理系统】=》点击界 面右边【账务】前的【+】号=》点击【凭证管理】前的【+】 号=》双击【编制凭证】=》然后在【编制凭证】界面录入 收入/支出的凭证。 3、凭证审核：点击界面右边【账务】前的【+】号=》点击【凭 证管理】前的【+】号=》双击【凭证处理】=》选中需要审 核凭证的日期=》在左下角选择凭证的状态【未审核】=》 点击右键全选=》点击【审核】； 4、凭证记账：点击界面右边【账务】前的【+】号=》点击【期 末处理】前的【+】号=》双击【凭证处理】=》选中需要记 账凭证的日期=》在左下角选择凭证的状态【已审核】=》 点击右键全选=》点击【记账】； 5、月结：点击界面右边【账务】前的【+】号=》点击【期末
处理】前的【+】号=》双击【期末处理向导】=》点击【结 账向导】=》全部点击【下一步】=》下到最后点击【完成】 二、电子系统： 1、输出单位资产负债表：双击【电子报表系统】=》【管理员】 登录=》在右上角【报表数】下点击【基本户】/【专账一】 /【专账二】下前的【+】号=》双击【资产负债表】=》点击 最右上面【数据】下=》=》点击【登录数据库】=》双击【账 务系统】=》用自己的用户进行登录=》如果图片闪烁就证 明已经登录=》点击【退出】=》点击最右上角找到【插入】 功能菜单=》点击【表页】=》选择出报表的最后日期(如1 月：则时间2011年1月31日)=》选择复制指定表页 =》点击放大镜=》选择【本公司】=》选中【格式】点击【确定】=》在点【确定】=》左 下角有【第201101期】=》点击编制【眼睛图标】。=》调 试报表=》点击【保存】=》打印报表。 2、输出单位支出明细表：双击【电子报表系统】=》【管理员】 登录=》在右上角【报表数】下点击【基本户】/【专账一】 /【专账二】下前的【+】号=》双击【支出明细表】=》点击 最右上面【数据】下=》=》点击【登录数据库】=》双击【账 务系统】=》用自己的用户进行登录=》如果图片闪烁就证 明已经登录=》点击【退出】=》点击最右上角找到【插入】
目录 1，系统概述--------------------------------------------------------------------------------------------------1 1.1 系统简介---------------------------------------------------------------------------------------------2 1.2 系统主要组成---------------------------------------------------------------------------------------2 1.3 系统硬件简要连接图------------------------------------------------------------------------------3 1.4 实际连线图------------------------------------------------------------------------------------------3 2,系统软件使用软件简要说明-----------------------------------------------------------------------------5 2.1 介绍---------------------------------------------------------------------------------------------------5 2.2 操作步骤---------------------------------------------------------------------------------------------5 2.3 取景窗口---------------------------------------------------------------------------------------------7 2.4 flash/cel文件的播放--------------------------------------------------------------------------------7 注1:连接网络的相关设置修改--------------------------------------------------------------9 注2:本机IP的查询----------------------------------------------------------------------------9 注3:本机IP的修改----------------------------------------------------------------------------10 注4:控制器IP的修改-------------------------------------------------------------------------11 3,对应表制作与选择-----------------------------------------------------------------------------------------12 3.1 介绍---------------------------------------------------------------------------------------------------12 3.2 操作步骤---------------------------------------------------------------------------------------------12 4,说明-----------------------------------------------------------------------------------------------------------14 4.1 ONC1A------------------------------------------------------------------------------------------------14 4.2 ONC1B------------------------------------------------------------------------------------------------14 4.3 ONC1C------------------------------------------------------------------------------------------------15 4.4 ONC1D------------------------------------------------------------------------------------------------15 4.5 ONC1E------------------------------------------------------------------------------------------------16 4.6 ONC1F------------------------------------------------------------------------------------------------17 4.7 ONC1G------------------------------------------------------------------------------------------------17 4.8 ONC1F------------------------------------------------------------------------------------------------17 5,附件-----------------------------------------------------------------------------------------------------------19 5.1 数码按钮控制板说明--------------------------------------------------------------------------------19 5.2 象素点排列说明--------------------------------------------------------------------------------------19
政府采购网上公开信息系统供应商操作指南 福建博思软件股份有限公司 2017年06月
目录 1. CA办理 (4) 1.1 CA办理 (4) 1.2 CA盖章 (4) 2. 系统注册 (4) 2.1系统注册 (4) 2.2 注册成功，供应商登录 (5) 3.系统基础操作 (6) 3.1 CA控件下载 (6) 3.2进行政府采购活动 (9) 3.3 供应商资料维护 (10) 3.4 供应商项目报名 (11) 3.5供应商用户管理 (11) 4．投标流程 (11) 4.1项目报名 (11)
4.2 投标文件编制 (13) 4.2.1客户端安装 (13) 4.2.2投标客户端路径修改 (15) 4.2.3应答 (16) 4.2.4标书加密 (21) 4.2.5接着点击退出系统 (21) 4.3投标文件上传： (22) 4.4合同签订： (23) 4.5验收申请： (23)
1.CA办理 供应商须办理福建省CA，进行政府采购活动 1.1CA办理 可登陆https://www.doczj.com/doc/5c14802600.html,/或者联系客服0591-968975。 1.2CA盖章 CA盖章操作系统为：XP(SP2)不支持（浏览器目前测试都支持）。 2. 系统注册 2.1系统注册 登入福建省政府采购网https://www.doczj.com/doc/5c14802600.html,/，找到登陆与注册进行供应商注册
2.2 注册成功，供应商登录 注：原省网已注册的供应商已完成迁移，请各供应商使用注册时的组织机构代码登录，密码初始为1。
3.系统基础操作 3.1 CA控件下载与安装 登录之前可先进行CA控件下载 (1)打开ISignature,点击installer.exe进行安装
用友财务软件操作流程手册 系统管理 一增加操作员 1、系统管理→系统→注册→输入用户名（admin）→无密码→确定 2、单击权限→操作员→点增加→输入编号、姓名、口令→点增加 二、建新账套 1、系统管理→系统→注册→输入用户名（admin）→无密码→确定 2 单击帐套→建立→输入帐套号、帐套名称、→设置会计期间→下一步→ 输入单位名称→下一步→选择企业类型（工业类型比商业类型多产成品入库单，和材料出库单）→行业性质→选择帐套主管→在“行业性质预置科目”前面打钩则系统将预置所选行业会计科目（否则不予预置）→下一步→如需分类在项目前面方框内打钩→下一步→完成 三、分配权限 1、系统管理→系统→注册→输入用户名（admin）→无密码→确定 2、赋权限的操作顺序： A 受限，明细权限设置权限”-→“权限”菜单→首先选择所需的账套→选操作员→点增加 B 帐套主管权限设置选择所需帐套→再选操作员→在帐套主管前面直接打钩 四、修改账套 1、以“账套主管（不是admin）”身份进入“系统管理”模块（进入系统服务→系统管理→注册） 2、单击帐套→修改 五．备份 打开系统管理→系统→注册admin →帐套→备份→选择存放路径 六．恢复 系统管理→系统→注册→admin →帐套→恢复（选择本分文件的路径，lst为后缀名的文件）总帐系统 初始化 一、启用及参数设置 二、设置“系统初始化”下的各项内容（其中：最后设置会计科目和录入期初余额，其余各项从上向 下依次设置） 1 会计科目设置 1、指定科目 系统初始化→会计科目→编辑（菜单栏中的）→指定科目 现金总帐科目把现金选进以选科目 银行总帐科目把银行存款选进已选科目
动环监控软件操作 手册
深圳市通讯威科技有限公司 EP-MEVP SYSTEM 动力环境集中监控系统 安装使用说明书 版本 2.0
目录 第一章软件的安装卸载升级 ................................. 错误!未定义书签。 1.1软件安装对计算机的配置要求........................ 错误!未定义书签。 1.2软件的安装 ....................................................... 错误!未定义书签。 1.3软件的卸载 ....................................................... 错误!未定义书签。 1.4软件的升级 ....................................................... 错误!未定义书签。第二章软件的基本操作 .......................................... 错误!未定义书签。 2.1登录和进入软件操作界面................................ 错误!未定义书签。 2.2添加/设置/修改/删除硬件设备以及参数设置错误!未定义书签。 2.2.1添加/设置控制器、采集器参数................. 错误!未定义书签。 2.2.2修改/删除硬件设备 .................................... 错误!未定义书签。 2.3监控设置及记录查询 ....................................... 错误!未定义书签。 2.3.1报警方式定义 ............................................. 错误!未定义书签。 2.3.2语音电话报警 ............................................. 错误!未定义书签。 2.3.3短信报警 ..................................................... 错误!未定义书签。 2.3.4监控实时记录 ............................................. 错误!未定义书签。 2.3.5监控报警记录 ............................................. 错误!未定义书签。 2.3.6温湿度数据记录.......................................... 错误!未定义书签。 2.3.7 UPS监控数据记录 ...................................... 错误!未定义书签。 2.3.8 电话短信报警数据记录 .............................. 错误!未定义书签。 2.3.9 空调监控数据记录...................................... 错误!未定义书签。