bHLH transcription factor mediates organ,region and flower type specific signals
更新时间:2023-05-07 01:04:01 阅读量: 实用文档 文档下载
- bhlh转录因子推荐度:
- 相关推荐
The Plant Journal (1998)16(1),93–99
SHORT COMMUNICATION
A bHLH transcription factor mediates organ,region and ?ower type speci?c signals on dihydro?avonol-4-reductase (dfr )gene expression in the in?orescence of Gerbera hybrida (Asteraceae)
Paula Elomaa*,Merja Mehto,Mika Kotilainen,Yrjo ¨Helariutta,Leena Nevalainen and Teemu H.Teeri Institute of Biotechnology,PO Box 56,FIN-00014University of Helsinki,Helsinki,Finland
Summary
The angiosperm family Asteraceae is characterized by composite in?orescences,which are highly organized structures consisting of different types of ?owers.In order to approach the control of ?oral organ differentiation in Asteraceae at molecular level,we are studying regulation of ?avonoid biosynthesis in Gerbera hybrida .Dihydro?a-vonol-4-reductase (dfr )expression is regulated according to anthocyanin pigmentation patterns in all tested gerbera varieties at several anatomical levels.We have isolated a promoter for one of the dfr genes,Pgdfr2.Gerbera plants transgenic for a Pgdfr2-uidA construct reveal that the activity of the Pgdfr2promoter from one variety follows the pigmentation in other varieties which have different color patterns.It is thus evident that the observed complex regulation of dfr expression occurs in trans .In order to identify the trans -acting regulators,we isolated a cDNA (gmyc1)homologous to the previously characterized genes encoding bHLH-type regulators of the anthocyanin pathway in plants.The expression of gmyc1in different varieties suggests that it has a major role in regulating dfr activity in corolla and carpel,but not in pappus and stamen.Speci?cally in gerbera,the identical patterns of gmyc1and dfr expression in corolla tissue suggest that GMYC1also regulates dfr expression in a region and ?ower type speci?c manner.Our studies show that in gerbera GMYC1–dfr interaction is part of several develop-mental processes characteristic for Asteraceae (such as speci?cation of ?ower types across the composite in?or-escence),whereas in other processes (such as differenti-ation of sepal as pappus)other regulators control dfr expression to determine the spatial speci?city.
Received 29May 1998;revised 5August 1998;accepted 13August 1998.*For correspondence (fax ?358970859570;e-mail Paula.Elomaa@Helsinki.Fi).
?1998Blackwell Science Ltd
93
Introduction
The composite in?orescence of Asteraceae,the capitulum,is a highly organized structure of different types of ?owers which may vary in sexuality,symmetry and anthocyanin pigmentation.Also common to Asteraceae are the ?oral organs which form specialized structures like pappus hairs (sepals),and organ fusion which takes place within the petals and anthers (for details see Bremer,1994).We are interested in studying gene expression related to ?oral organ differentiation in Asteraceae,starting from the early events of organ determination and ?nally leading to differ-entiation of the complex in?orescense using an ornamental plant,Gerbera hybrida .At the molecular level,organ speci?c gene expression related to differentiation is con-trolled by distribution of common and spatially speci?c regulatory molecules,i.e.the pre-pattern (Coen et al .,1988).As an approach to understanding the molecular control of capitulum development we have identi?ed genes that sense the pre-pattern in different ways.
Different anthocyanin pigmentation patterns in gerbera varieties follow the anatomy of the capitulum and thus provide a good marker trait for our studies.In breeders’collections,varieties where pigmentation is ?ower organ speci?c,?ower type speci?c,or varies according to different regions of an organ can be found.We have previously shown that the expression of dfr ,a late gene of the anthocyanin pathway,is regulated precisely according to the anthocyanin pigmentation patterns of different gerbera varieties (Helariutta et al .,1995b).Through learning how dfr expression is controlled in gerbera at different anatomical levels,we will gain an important insight into how differen-tial gene expression is regulated in the composite in?or-escence.
In this paper we have isolated from gerbera a cDNA (gmyc1)that is homologous to the bHLH-type regulatory genes of anthocyanin pathway (Goodrich et al .,1992;Ludwig et al .,1989;Quattrocchio et al .,1998).Transient assay using particle bombardment indicates that GMYC1,together with a heterologous MYB regulator from petunia (Quattrocchio,1994),is able to activate a gerbera dfr promoter.The expression of gmyc1suggests that it has a major role in regulating anthocyanin biosynthesis in corolla and carpel,including regional as well as ?ower type levels
94Paula Elomaa et al.
during corolla development.However,in pappus and sta-men dfr ,expression is controlled by factors other than GMYC1.Results
Dfr expression is under trans regulation in gerbera We isolated a promoter from a dfr gene using the previously characterized gdfr1cDNA as a probe (Helariutta et al .,1993).The 150bp ?rst exon of the genomic clone was 98%identical to gdfr1cDNA.In line with the presence of a small gene family in gerbera genome (Helariutta et al .,1993),we concluded that this gene represents a member closely related to gdfr1,and was named Pgdfr2.Genetic analysis (see Experimental procedures)indicates that gdfr1and gdfr2are non-allelic.We transformed the 1195bp Pgdfr2promoter,fused with the uidA reporter gene,into the red gerbera variety Terra Regina.Based on ?uorometric measurements of 11individual transformants that expressed the transgene (data not shown),high GUS activity was detected in all pigmented organs (petiole,?ower scape,corolla and carpel).No or very low activity was detected in unpigmented parts (leaf,pappus,tube and stamen),indicating that the gdfr2promoter contains all essential elements that mediate the spatial and temporal speci?city needed for transcriptional regulation of dfr activ-ity in the Regina variety (Helariutta et al .,1993,1995b).In order to study the nature of dfr regulation (cis or trans )in gerbera,we crossed the Pgdfr2-uidA transgene into other genetic backgrounds.Transgenic Regina lines con-taining low copy numbers (1–2)of the transgene were crossed with the Nero and Parade varieties which have different ?oral anthocyanin pigmentation patterns (Helariutta et al .,1995b).Regina has pigmented corolla and carpel tissues.In Nero,only pappus bristles are strongly anthocyanin pigmented,while in Parade all ?oral organs except carpels are pigmented.As expected,due to the heterozygotic nature of gerbera,a range of novel pigmenta-tion patterns were displayed in the progeny both at ?ower organ and ?ower type level.From Regina ?Parade cros-sings (P-progeny)we obtained varieties with segregating stamen pigmentation (1:1;red:yellow),while carpel pig-mentation appeared to depend on multiple genes and various forms,with unpigmented to strongly pigmented carpels being found.In the P-progeny,corollas were always anthocyanin pigmented as in the parental varieties.In the N-progeny (Regina ?Nero),segregating pappus pig-mentation (1:1)was observed.Interestingly,differential pigmentation of corollas of distinct ?ower types was also observed.Often,the trans ?ower corollas were unpig-mented while the ray and disc ?ower corollas showed anthocyanin pigmentation (e.g.N5in Figure 1a).Northern analysis of selected progeny showed that uidA expression,
?Blackwell Science Ltd,The Plant Journal ,(1998),16,93–99
under the control of the gdfr 2promoter,spatially follows the new pigmentation pattern and new dfr expression instead of the Regina pattern in all cases (Figure 1).This indicates that pigmentation patterns at the ?oral organ and ?ower type levels in these varieties result from differential spatial expression of regulatory molecules that control dfr expression,rather than from different structures in the dfr promoters.
Isolation of a gerbera cDNA homologous to bHLH -type regulatory genes of anthocyanin pathway
In order to approach the nature of the trans -acting signals,we isolated a cDNA (gmyc1)from gerbera corolla cDNA pool,based on homology between the bHLH-type anthocy-anin regulatory genes,lc from maize (Ludwig et al .,1989),delila from snapdragon (Goodrich et al .,1992),and jaf13from petunia (Quattrocchio et al .,1998).gmyc1cDNA encodes a protein of 533amino acids that is highly similar to DEL,JAF13and LC at the amino acid sequence level (Figure 2).The highest similarity is shared in the N terminal regions of the proteins as well as in the C terminal ends,while the intermediate regions are less conserved.Unique to gerbera GMYC1,compared to the other regulators,is that it is approximately 100amino acids shorter and speci?cally lacks a 12amino acids long region in the conserved basic region of the bHLH motif that is suggested to be involved in DNA binding (Prendergast and Ziff,1989).Southern analysis,using the full length cDNA as a probe,under low washing stringency,showed only one hybridiz-ing fragment in Regina genomic DNA,digested with enzymes that are not cutting within the cDNA (data not shown).This indicates that in variety Regina,gmyc1is a single copy gene.
GMYC1activates a Pgdfr2-luc reporter construct in transient assay
To assess the functionality of the gmyc1cDNA as a tran-scriptional activator,we used particle bombardment for transient expression of the regulator and its putative target promoter,Pgdfr2,which was fused to the ?re?y luciferase reporter gene.Co-bombardment of gmyc1with the hetero-logous myb partner an2from petunia (Quattrocchio,1994),both under the CaMV 35S promoter,together with the Pgdfr2-luc construct into leaf tissue,activated the reporter gene (Table 1).The affector or reporter constructs alone did not give signi?cant LUC activity.The results suggest that gmyc1encodes an active regulator,and that the Pgdfr2promoter contains the sequence elements responding to the regulatory protein encoded by the gmyc1cDNA.
Spatial regulation of dfr expression in gerbera
95
Figure1.Northern analysis of the crossing progeny with novel pigmentation patterns indicates that the promoter itself is not responsible for the pattern but reacts to trans-acting signals.
uidA expression driven by the gdfr2promoter consistently follows the new anthocyanin pigmentation pattern(and dfr expression pattern)in differentially pigmented?ower types(a),stamens(b),pappus bristles(c)and carpels(d).
Expression pattern of gmyc1in gerbera suggests a regulatory role in corolla and carpel tissues
Northern analysis was performed to study the expression pattern of the putative anthocyanin regulator in gerbera. At the level of whole plant,gmyc1expression was detected in tissues where anthocyanin pigmentation is also detected: in developing in?orescence buds,in?oral stems and very faintly in bracts.No expression was detected in leaves or roots(Figure3a).Expression during ray?ower develop-ment was studied in more detail.Stages2–9correspond to stages from small buds of5–10mm in size to the completely open?owers(see Helariutta et al.,1993). Expression of gmyc1is already detectable at stage2and continues rather uniformly until stage7and fades out thereafter(data not shown).Temporally,it precedes the expression of anthocyanin biosynthetic genes,whose expression peak at stages6–7,just before anthesis (Helariutta et al.,1993,1995a).
Spatial distribution of gmyc1expression was studied in ?oral organs of different varieties.Figure3(b)shows gmyc1 expression in corolla tissue.Ray?ower corollas(stages3,5 and7)show strong gmyc1expression in the red pigmented Regina and Parade and much lower expression in Nero (yellow variety),each in accordance with the dfr expression in them(Helariutta et al.,1995b).Similarly,in carpel tissue, gmyc1expression follows the pigmentation pattern,as shown in Figure3(c).In variety Regina,carpels are clearly pigmented,in variety Nero only faint pigmentation is detected,and in variety Parade there is no pigmentation at all.Within corolla tissue,in variety Mix,pigmentation is restricted to the basal part of the corolla while the distal part
?Blackwell Science Ltd,The Plant Journal,(1998),16,93–99is unpigmented(Helariutta et al.,1995b).gmyc1expression follows the distribution of anthocyanin pigmentation,indic-ating that gmyc1is probably responsible for the regulation imposed on the dfr gene also in the different regions within the corolla ligule(Figure3b).Among the crossing progeny described above,we obtained varieties with differentially pigmented corollas in distinct?ower types.In N1with equally pigmented ray and trans?ower corollas gmyc1 expression is also detected in both?ower types,while in N5with unpigmented trans?owers gmyc1expression is detected in ray but not in trans?owers(Figure3b).
In general,gmyc1expression pattern follows the pigmentation in corolla and carpel tissues of gerbera sug-gesting a major regulatory role in these organs.Especially in corolla,gmyc1and dfr expression patterns coincidence at all anatomical levels that characterize the composite in?orescence,including the levels of?ower type and regions within a single corolla.The different gerbera variet-ies studied also vary in pigmentation of pappus and anthers.However,gmyc1expression in these organs is very low,irrespective of their pigmentation phenotype (data not shown),suggesting that pappus and stamen pigmentation is not determined by GMYC1.
Discussion
Asteraceae is characterized by complex in?orescences (capitula)where differentiation occurs not only at the level of?oral organs and their substructures,but also at the level of the capitulum,which may carry different types of ?owers(e.g.Bremer,1994).In Gerbera hybrida,spatial
96Paula Elomaa et
al.
Figure 2.Amino acid comparison of the (b)HLH-type regulator of gerbera,GMYC1with DEL of snapdragon,JAF13of petunia and LC of maize.
(*)indicates amino acids that are fully conserved in each of the proteins and (.)amino acids that GMYC1shares with at least one of the other regulators.The basic helix-loop-helix region is indicated in bold.Highest amino acid similarity (66%)is shared with snapdragon DEL.
Table 1.Co-bombardment of the gerbera gmyc1and petunia an2,both under CaMV35S promoter,activate the Pgdfr2-luc reporter construct in gerbera leaf tissue.35S-ruc was used as an internal control and was co-bombarded in each case.Ten leaves were bombarded in each case Constructs ?35S-ruc
LUC/RUC activity (SD)Pgdfr2-luc
0.023(0.011)Pgdfr2-luc ?35S-gmyc10.021(0.010)Pgdfr2-luc ?35S-an2
0.072(0.025)Pgdfr2-luc ?35S-gmyc1?35S-an2
0.382
(0.138)
patterns of anthocyanin pigmentation are regulated,in the different varieties,in diverse ways along several of these morphological units.In our earlier work we showed that dihydro?avonol-4-reductase gene expression,as a repres-entative of late biosynthetic gene expression of the antho-
?Blackwell Science Ltd,The Plant Journal ,(1998),16,
93–99
Figure 3.Expression of gmyc1in gerbera follows very precisely the anthocyanin pigmentation pattern at the level of the whole plant and in corolla and carpel tissues of individual ?owers.
The presence of anthocyanin pigmentation is indicated as (?)and absence as (–).
(a)gmyc1expression in developing in?orescences (I),bracts (B),?ower stems (S),leaves (L)and roots (R).
(b)In corolla tissue of varieties Regina,Nero,Parade,Mix,N1and N5,gmyc1expression follows the pigment distribution at ?oral organ,regional and ?ower type levels.
(c)gmyc1expression follows pigmentation in carpel tissue of varieties Regina,Nero and Parade.
cyanin pathway,precisely coincides with the pigmentation patterns,possible providing a tool for studying perception of the signals differentiating the morphological levels of the capitulum (Helariutta et al .,1995b).
We report here on a cis -and trans -acting element of dfr expression that represents part of the previously identi?ed regulatory processes.Transgenic analyses with the gdfr2promoter indicate that the promoter activity is fully consist-ent with the dfr activity previously detected by Northern analysis in different tissues of variety Regina (Helariutta et al .,1993,1995b),as well as in a crossing progeny displaying novel patterns of anthocyanin accumulation at ?oral organ and ?ower type levels.This indicates that the isolated promoter contains sequences that are responsive to various spatially speci?c signals during capitulum devel-opment.
Previous studies in maize,snapdragon and petunia on
Spatial regulation of dfr expression in gerbera97
the transcriptional regulation of(late)biosynthetic genes of anthocyanin pathway have shown that the trans-acting elements share remarkable conservation both in structure and function(reviewed by Dooner et al.,1991;Koes et al., 1994;Martin and Gerats,1993;Mol et al.,1996).Therefore, in order to approach the trans-acting signals controlling dfr expression,we isolated a gene encoding a putative bHLH-type regulator from corolla tissue of gerbera.The deduced amino acid sequence of GMYC1closely resembles those of the previously isolated regulators,suggesting that it may be functionally homologous.Indeed,although most of the conserved basic region is lacking from GMYC1,the protein is able to activate the Pgdfr2promoter in transient gene expression system,if a(heterologous)MYB partner is provided.Furthermore,previously Goff et al.(1992)have shown that only the N-terminal region of the maize bHLH type protein B,a close homologue of LC,is critical for interaction with the corresponding MYB partner,C1.Fur-ther support for the regulatory role of GMYC1is provided by its expression in corolla tissue of variety Regina,which temporally precedes and partly overlaps with the biosyn-thetic gene expression,and is thus analogous to delila expression in snapdragon(Goodrich et al.,1992)and to jaf13expression in petunia(Quattrocchio et al.,1998). These results suggest that a bHLH regulator/dfr promoter interaction analogous to one previously identi?ed in sev-eral other species plays a role during corolla development in gerbera.This will be?nally veri?ed by homologous transformation of gmyc1both in sense and antisense orientation into gerbera.However,support for the conser-vation of the regulatory interaction is provided by our other analyses.We have earlier transformed the homologous gene from snapdragon,delila,under CaMV35S promoter into gerbera and obtained transgenic lines with strongly enhanced pigmentation in?ower scapes and leaf tissue due to activation of dfr expression(Elomaa,1996).Conser-vation of the regulatory mechanism is further supported by bombardment of the maize regulators,lc(Ludwig et al., 1989)and c1(Paz-Ares et al.,1987),which together with the Pgdfr2-luc construct lead to activation of the reporter construct in leaf tissue(M.Mehto,unpublished results).In addition,transformation of the Pgdfr2-uidA construct into petunia(V26)showed that the promoter activity followed the petunia pigmentation pattern(data not shown). Spatially during corolla development,gmyc1expression follows the anthocyanin pigmentation pattern very pre-cisely in different varieties,and speci?cally in gerbera, both regionally within a single corolla(tip/base axis)and at the level of?ower type(Figure3b).This indicates that the variation in corolla pigmentation in these plants would be due to differences either in the gmyc1promoters, or higher level spatially speci?c trans-acting signals.To approach this,we are proceeding with the isolation and characterization of the promoter of gmyc1and its equiva-
?Blackwell Science Ltd,The Plant Journal,(1998),16,93–99lents in different varieties.Analogously to corolla develop-ment,gmyc1expression follows the pigmentation pattern in carpel.This suggests that gmyc1has a key role in regulating dfr expression during differentiation of both corolla and carpel.However,in pappus and stamen,where Pgdfr2is active when anthocyanins accumulate,the activa-tion is probably not mediated by GMYC1,as evidenced by the invariantly faint expression pattern.An alternate, independent regulation mechanism must exist in these tissues which still acts on the same promoter.Crossing experiments demonstrate that while corolla and carpel pigmentation are complex,polygenic traits,the inheritance of stamen and pappus pigmentation are more simple and suggests involvement of single,dominant genes.Whether the latter factors are other members of the bHLH family, or whether they are different factors that react with different parts of the Pgdfr2promoter remains to be studied.In either case,dfr regulation,which takes place along diverse developmental units in the Asteraceae capitulum,is branched and taken care of at least by two separate regulatory systems.One of these systems acts in corolla and carpel and is mediated by a(b)HLH regulatory protein.
Experimental procedures
Plant material
Gerbera hybrida varieties Regina,Nero,Parade and Mix were obtained from Terra Nigra BV,Holland.Developmental stages of the in?orescence are described by Helariutta et al.(1993) and anthocyanin accumulation patterns of different varieties in Helariutta et al.(1995b).
Isolation of plant DNA and RNA
Plant DNA was isolated using the methods of Dellaporta et al. (1983)or Doyle and Doyle(1990).Total RNA was isolated as described in Jones et al.(1985).Poly(A)?RNA was isolated using oligo(dT)cellulose af?nity chromatography(Sambrook et al., 1989).
Screening of the genomic library
Genomic library was constructed from partially Sau3A digested DNA into lambda GEM11vector.3.4?106plaques were screened according to standard methods(Sambrook et al.,1989)using gdfr1cDNA(Helariutta et al.,1993)as a probe.Washing conditions were2?SSC,0.1%SDS at68°C.Two positive clones were detected,one of which contained a gdfr promoter region(Pgdfr2). Genetic analysis of the allelic relationship between gdfr1 and gdfr2
Comparison of the150bp region common to the genomic clone and the previously isolated gdfr1cDNA indicated that the isolates differed in three nucleotides(data not shown).Using information from both the genomic clone(Pgdfr2)and the cDNA(gdfr1)we
98Paula Elomaa et al.
ampli?ed by PCR two major products from variety Regina,a longer fragment that corresponds exactly to the gdfr1cDNA and a shorter product similar to the genomic clone (M.Mehto,unpublished results).The same two PCR products were also obtained from a dihaploid Regina derivative produced via ovule culture,proving that gdfr1and gdfr2are non-allelic and correspond to two highly homologous dfr genes in gerbera.
Transformation of gerbera and crossing of the primary transformants
Transgenic gerbera plants containing the gdfr2promoter fragment (1195bp)in front of the uidA marker gene encoding for β-glucuronidase (GUS)were obtained by agrobacterium mediated transformation (Elomaa et al .,1993).The transformation construct (pHTT441)was basically similar to our previously published vec-tors (Elomaa et al .,1993;Elomaa et al .,1996)except that an intron was inserted into the Pvu II site of nptII (Elomaa,1996).Transformation was con?rmed by Southern analysis and ?uoro-metric GUS assay was performed according to Jefferson and Wilson (1991).Some of the primary transformants were reciproc-ally crossed with varieties Nero and Parade.Seeds were collected,surface sterilized and germinated on MS-medium (Murashige and Skoog,1962)in the presence of kanamycin (100mg l –1).
PCR cloning of gmyc1
First-strand cDNA was synthesized from poly(A)?RNA from ray ?oret corolla (stages 5–9,Helariutta et al .,1993)using Promega’s Riboclone cDNA synthesis kit,and used as a template for PCR ampli?cation.Partially degenerate primers GGAGGGATCCTA(T/C)AA(T/C)GGIGAI(A/G)TIAA(A/G)AC and TCTCGGATCC(A/G)TGIGC(A/G)TT(A/G)CAIA(G/A)CCA (including Bam HI sites)cor-responding to the peptides YNGDIKTRK and WLCNAH,respect-ively,were used.DNA-polymerase Dynazyme (Finnzymes)was used for ampli?cation and the conditions were:94°C/75sec,46°C/2min,72°C/3min for 30cycles.The PCR product (300bp)was cloned into pUC19(Yanisch-Perron et al .,1985)and sequenced.For 3?/5?RACE,?rst-strand cDNA was synthesized from corolla poly(A)?RNA from stages 2–4,sequence speci?c primers were designed based on the PCR fragment,and the ampli?cation of the cDNA ends was performed according to the Boehringer Mannheim Kit (no.1734792).To ensure that the fragments obtained using the RACE method originate from the same transcript,the whole cDNA (2268bp)was ampli?ed once more from corolla cDNA using Expand High Fidelity enzyme (Boehringer)and sequenced in both directions.For sequence similarity comparisons,amino acids were grouped by their chemical similarity as in Manninen and Schulman (1993)and GMYC1,DEL,JAF13and LC sequences were analyzed using PC/Gene protein analysis package (Intelligenetics Inc.)
DNA and RNA blot analyses
Ten μg of digested DNA or 10–20μg of total RNA was loaded per lane.For studies of gmyc1expression,RNA was isolated from different developmental stages of ray ?oret corollas.Pappus RNA was pooled from stages 2,3and 4,and carpel RNA from stages 5,6and 7.Stamen RNA was isolated from samples covering stages 7and 8.Electrophoresis and hybridizations were undertaken using standard protocols (Sambrook et al .,1989).Full-length cDNA of gdfr1was used as a probe for detection of dfr expression
?Blackwell Science Ltd,The Plant Journal ,(1998),16,93–99
(Helariutta et al .,1993).This probe probably detects all dfr genes active in ?oral tissue.uidA expression was detected using Sac I-Bam HI fragment (1940bp)isolated from pBI221(Jefferson,1987)as a probe.gmyc1expression was detected using a 419bp (Sal I fragment)from the 3?end as a probe.
Particle bombardment of gerbera leaf tissue and luciferase assay
For particle bombardment,BioRad PDS-1000/He equipment was used.Five mg of 0.6μm gold particles were precipitated with maximum 5μg of plasmid DNA using CaCl 2and spermidine.Plasmids containing Pgdfr2-luc ,35S-gmyc1and 35S-an2con-structs were used.35S-ruc ,encoding Renilla luciferase (Mayer-hofer et al .,1995),was used as an internal control.Leaves from in vitro plantlets were placed on water agar and bombarded on their upper epidermis using 900psi pressure plates.Leaves were kept in dark for 20h and measured for ?re?y and Renilla luciferase activities according to the protocol of the Promega Dual Luciferase Kit,except that the protein extraction buffer was the ‘modi?ed lux buffer’as described in Herrera-Estrella et al .(1994).
Acknowledgements
We thank Dr Francesca Quattrocchio for the petunia jaf13sequence and for the petunia an2plasmid,Prof.Susan Wessler for the maize lc and Prof.Udo Wienand for the c1cDNAs.Dr Anne Uimari is thanked for helpful comments on the manuscript.Eija Takala,Marja Huovila and Anu Immonen are acknowledged for excellent technical assistance.We also thank Anne Aaltonen for taking care of the plants and Eija Saarikko for the crossing experiments.The tissue culture and greenhouse facilities were provided by Kemira Agro Oy.
References
Bremer,K.(1994)Asteraceae:Cladistics and Classi?cation .Portland:Timber Press.
Coen,E.S.,Almeida,J.,Robbins,T.,Hudson,A.and Carpenter,R.(1988)Molecular analysis of genes determining spatial patterns in Antirrhinum majus .In Temporal and Spatial Regulation of Plant Genes (Verma,D.P.S.and Goldberg,R.B.,eds).Wien:Springer-Verlag,pp.63–78.
Dellaporta,S.L.,Wood,J.and Hicks,J.B.(1983)A plant DNA minipreparation:version II.Plant Mol.Biol.Rep.1,19–21.
Dooner,H.K.,Robbins,T.P.and Jorgensen,R.A.(1991)Genetic and developmental control of anthocyanin biosynthesis.Ann.Rev.Genet.25,173–199.
Doyle,J.J.and Doyle,J.L.(1990)Isolation of plant DNA from fresh tissue.Focus ,12,13–15.
Elomaa,P.(1996)Genetic modi?cation of ?avonoid pathway in ornamental plants.PhD Thesis,University of Helsinki.
Elomaa,P.,Helariutta,Y.,Kotilainen,M.and Teeri,T.H.(1996)Transformation of antisense constructs of the chalcone synthase gene superfamily into Gerbera hybrida :differential effect on the expression of family members.Mol.Breed.2,41–50.Elomaa,P.,Honkanen,J.,Puska,R.,Seppa ¨nen,P.,Helariutta,Y.,Mehto,M.,Kotilainen,M.,Nevalainen,L.and Teeri,T.H.(1993)Agrobacterium -mediated transfer of antisense chalcone synthase cDNA to Gerbera hybrida inhibits ?ower pigmentation.Bio/Technology ,11,508–511.
Goff,S.A.,Cone,K.C.and Chandler,V.L.(1992)Functional analysis
Spatial regulation of dfr expression in gerbera99
of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between two classes of regulatory proteins.Genes Dev.6,864–875.
Goodrich,J.,Carpenter,R.and Coen,E.S.(1992)A common gene regulates pigmentation pattern in diverse plants species.Cell, 68,955–964.
Helariutta,Y.,Elomaa,P.,Kotilainen,M.,Griesbach,R.J.,Schro¨der, J.and Teeri,T.H.(1995a)Chalcone synthase-like genes active during corolla development are differentially expressed and encode enzymes with different catalytic properties in Gerbera hybrida(Asteraceae).Plant Mol.Biol.28,47–60.
Helariutta,Y.,Elomaa,P.,Kotilainen,M.,Seppa¨nen,P.and Teeri, T.H.(1993)Cloning of cDNA coding for dihydro?avonol-4-reductase(DFR)and characterization of dfr expression in the corollas of Gerbera hybrida var.Regina(Compositae).Plant Mol.Biol.22,183–193.
Helariutta,Y.,Kotilainen,M.,Elomaa,P.and Teeri,T.H.(1995b) Gerbera hybrida(Asteraceae)imposes regulation at several anatomical levels during in?orescence development on the gene for dihydro?avonol-4-reductase.Plant Mol.Biol.28,935–941. Herrera-Estrella,L.,Leo′n,P.,Olsson,O.and Teeri,T.H.(1994) Reporter Genes for Plants.In Plant Mol.Biol.Manual,C2. Dordrecht:Kluwer Academic Publishers,pp.1–32.
Jefferson,R.A.(1987)Assaying chimeric genes in plants.The GUS fusion system.Plant Mol.Biol.Rep.5,387–405.
Jefferson,R.A.and Wilson,K.J.(1991)The GUS gene fusion system.In Plant Mol.Biol.Manual,B14.Dordrecht:Kluwer Academic Publishers,pp.1–33.
Jones,J.D.G.,Duinsmuir,P.and Bedbrook,J.(1985)High level expression of introduced chimeric genes in regenerated transformed plants.EMBO J.4,2411–2418.
Koes,R.E.,Quattrocchio,F.and Mol,J.N.M.(1994)The?avonoid biosynthetic pathway in plants:function and evolution. Bioessays,16,123–132.
Ludwig,S.R.,Habera,L.F.,Dellaporta,S.L.and Wessler,S.R.(1989) Lc,an member of the maize R gene family responsible for tissue-speci?c anthocyanin production,encodes a protein similar to EMBL data library accession number AJ007709.
?Blackwell Science Ltd,The Plant Journal,(1998),16,93–99
transcriptional activators and contains the myc-homology region.Proc.Natl f805dc8358fb770bf78a55f7A,86,7092–7096. Manninen,I.and Schulman, A.H.(1993)BARE-1,a copia-like retroelement in barley(Hordeum vulgare L.).Plant Mol.Biol. 22,829–846.
Martin,C.and Gerats,T.(1993)Control of pigment biosynthesis genes during petal development.Plant Cell,5,1253–1264. Mayerhofer,R.,Langridge,W.H.R.,Cormier,M.J.and Szalay,A.A. (1995)Expression of recombinant Renilla luciferase in transgenic plants result in high levels of light emission.Plant J.7,1031–1038.
Mol,J.,Jenkins,G.,Scha¨fer, E.and Weiss, D.(1996)Signal perception,transduction,and gene expression involved in anthocyanin biosynthesis.Crit Rev.Plant Sci.15,525–557. Murashige,T.and Skoog,F.(1962)A revised medium for rapid growth and bioassay with tobacco tissue cultures.Physiol. Plant.15,473–498.
Paz-Ares,J.,Ghosal,D.,Wienand,U.,Peterson,P.A.and Saedler, H.(1987)The regulatory c1locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators.EMBO J.6, 3553–3558.
Prendergast,G.C.and Ziff, E.B.(1989)Methylation-sensitive sequence-speci?c DNA binding by the c-Myc basic region. Science,251,186–189.
Quattrocchio, F.(1994)Regulatory genes controlling?ower pigmentation in Petunia hybrida.PhD Thesis.Free University of Amsterdam.
Quattrocchio, F.,Wing,J.F.,van der Woute,K.,Mol,J.N.M. and Koes,R.E.(1998)Analysis of bHLH and MYB domain proteins:species-speci?c regulatory differences are caused by divergent evolution of target anthocyanin genes.Plant J.13, 475–488.
Sambrook,J.,Fritsch, E.F.and Maniatis,T.(1989)Molecular Cloning:A Laboratory Manual.Cold Spring Harbor:Cold Spring Harbor Laboratory Press.
Yanisch-Perron,C.,Vieira,J.and Messing,J.(1985)Improved M13 phagecloning vectors and host strains:nucleotide sequences of the M13mp18and pUC19vectors.Gene,33,103–119.
正在阅读:
bHLH transcription factor mediates organ,region and flower type specific signals05-07
骨组织修复材料03-03
工程结算书(设备及安装类)11-06
中国西溪--国际湿地养生度假区概念规划09-03
幼儿园教育工作总结范本参考04-17
护理病例分析09-24
第21课第一次世界大战导学案03-08
一建学习讲义10-08
多媒体在物理教学中的作用有用09-28
三傻大闹宝莱坞影评,亲笔经典03-08
- 1自动化-Specific - Competency - DCS - Module - 图文
- 2自动化-Specific - Competency - DCS - Module - 图文
- 3the Evolution, regulation, and function of placenta-specific genes
- 4The type I interferon system in the etiopathogenesis of autoimmune diseases
- 5Getting_university_education_is_the_most_important_factor_for_success_in_daily_life
- 6Getting_university_education_is_the_most_important_factor_for_success_in_daily_life
- 7Chapter 3 Gains and Lossed from Trade in the Specific-Factors Model
- 8Synthesis of ZnO nanoparticles from microemulsions in a flow type microreactor
- 9Positron annihilation spectrum from the Galactic Center region observed by SPIINTEGRAL
- 10Single-Hole Hollow Polymer Microspheres toward Specific High
- 教学能力大赛决赛获奖-教学实施报告-(完整图文版)
- 互联网+数据中心行业分析报告
- 2017上海杨浦区高三一模数学试题及答案
- 招商部差旅接待管理制度(4-25)
- 学生游玩安全注意事项
- 学生信息管理系统(文档模板供参考)
- 叉车门架有限元分析及系统设计
- 2014帮助残疾人志愿者服务情况记录
- 叶绿体中色素的提取和分离实验
- 中国食物成分表2020年最新权威完整改进版
- 推动国土资源领域生态文明建设
- 给水管道冲洗和消毒记录
- 计算机软件专业自我评价
- 高中数学必修1-5知识点归纳
- 2018-2022年中国第五代移动通信技术(5G)产业深度分析及发展前景研究报告发展趋势(目录)
- 生产车间巡查制度
- 2018版中国光热发电行业深度研究报告目录
- (通用)2019年中考数学总复习 第一章 第四节 数的开方与二次根式课件
- 2017_2018学年高中语文第二单元第4课说数课件粤教版
- 上市新药Lumateperone(卢美哌隆)合成检索总结报告
- transcription
- mediates
- specific
- signals
- factor
- region
- flower
- organ
- bHLH
- type
- 建筑施工第四章考试题
- 音乐教案新部编本设计
- 专利独占许可合同范本
- 环境工程专业英语试题
- 行政部管理规章制度
- 新人教版七年级地理下册《八章 东半球其他的地区和国家 第四节 澳大利亚》教案_7
- 5篇介绍江苏苏州园林的导游词范文.doc
- 集控汽机运行副值班员工作标准
- 保险业系统性风险的因素及其防范措施论文.docx
- 汽车飞轮齿圈项目可行性研究报告评审方案设计(2013年发改委标准案例范文)
- 中职幼儿教育学教学计划讲课教案
- 高中语文(人教版 选修 中国小说欣赏)教师用书:第11课 《呼兰河传》——小团圆媳妇之死 Word版含答案
- “谈心式”作文评语的内在动力-文档资料
- 整数除法竖式计算练习题
- 英语教学中的互动教学模式.doc
- 最新冀教版四年级语文上册z重点知识
- 2018年中国软饮料发展现状与市场前景分析(目录)
- 浙江省台州市2017年中考试卷(数学 解析版)-真题
- 单位公司企业人员定位系统维修工操作规程
- 简述我国民航业发展中服务的重要性及改善对策