Planta (2011) 234:865–881
DOI 10.1007/s00425-011-1438-4
ORIGINAL ARTICLE
A pomegranate (Punica granatum L.) WD40-repeat gene is
a functional homologue of Arabidopsis TTG1 and is involved
in the regulation of anthocyanin biosynthesis during pomegranate fruit development
Zohar Ben-Simhon · Sylvie Judeinstein · Talia Nadler-Hassar · Taly Trainin ·
Irit Bar-Ya’akov · Hamutal Borochov-Neori · Doron Holland
Received: 24 February 2011 / Accepted: 11 May 2011 / Published online: 5 June 2011
? Springer-Verlag 2011
Abstract Anthocyanins are the major pigments responsi-ble for the pomegranate (Punica granatum L.) fruit skin color. The high variability in fruit external color in pome-granate cultivars re X ects variations in anthocyanin compo-sition. To identify genes involved in the regulation of anthocyanin biosynthesis pathway in the pomegranate fruit skin we have isolated, expressed and characterized the pomegranate homologue of the Arabidopsis thaliana TRANSPARENT TESTA GLABRA1 (TTG1), encoding a WD40-repeat protein. The TTG1 protein is a regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis, and acts by the formation of a transcriptional regulatory complex with two other regulatory proteins: bHLH and MYB. Our results reveal that the pomegranate gene, designated PgWD40, recovered the anthocyanin,PAs, trichome and seed coat mucilage phenotype in Ara-bidopsis ttg1 mutant. PgWD40 expression and anthocyanin composition in the skin were analyzed during pomegranate fruit development, in two accessions that di V er in skin color intensity and timing of appearance. The results indicate high positive correlation between the total cyanidin deriva-tives quantity (red pigments) and the expression level of PgWD40. Furthermore, strong correlation was found between the steady state levels of PgWD40 transcripts and the transcripts of pomegranate homologues of the structural genes PgDFR and PgLDOX. PgWD40, PgDFR and PgL-DOX expression also correlated with the expression of pomegranate homologues of the regulatory genes PgAn1 (bHLH) and PgAn2 (MYB). On the basis of our results we propose that PgWD40 is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit devel-opment and that expression of PgWD40, PgAn1 and PgAn2 in the pomegranate fruit skin is required to regulate the expression of downstream structural genes involved in the anthocyanin biosynthesis.
Keywords Anthocyanin biosynthesis ·
Cyanidin derivatives · Fruit development ·
MYB-bHLH-WD40 complex · Pomegranate · TTG1 Abbreviations
bHLH Basic helix–loop–helix
CHS Chalcone synthase
DFR Dihydro X avonol 4-reductase
HPLC High performance liquid chromatography LDOX Leucoanthocyanidin dioxygenase
PAs Proanthocyanidins
PG Punica granatum
RACE Rapid Ampli W cation of cDNA Ends
TTG1Transparent Testa Glabra1
Publication No. 129/2010 from the Agricultural Research Organization (ARO), the Volcani Center, Bet Dagan, Israel.
Electronic supplementary material The online version of this article (doi:10.1007/s00425-011-1438-4) contains supplementary material, which is available to authorized users.
Z. Ben-Simhon · T. Nadler-Hassar · T. Trainin ·
I. Bar-Ya’akov · D. Holland (&)
Unit of Deciduous Fruit Tree Sciences,
Newe Ya’ar Research Center, Agricultural Research Organization, P.O. Box 1021, 30095 Ramat Yishay, Israel
e-mail: vhhollan@https://www.doczj.com/doc/f79976833.html,.il
Z. Ben-Simhon
e-mail: zohar@https://www.doczj.com/doc/f79976833.html,.il
S. Judeinstein · H. Borochov-Neori
Southern Arava Research and Development,
88820 Hevel Eilot, Israel
Introduction
The color of fruits in many plant species arises from the presence of anthocyanins, water soluble X avonoid pig-ments. The latter provide a wide range of colors, from orange through red to purple/blue, and accumulate in most plant tissues (Tanaka et al. 2008). An important biological function proposed for anthocyanin pigments and other X avonoids is the recruitment of pollinators and seed dispersers. They may also have signi W cant roles in the signaling between plants and microorganisms, plant defense mechanisms, auxin transport and UV protection (Winkel-Shirley 2001; Koes et al. 2005).
High content of anthocyanins is considered valuable to human health mainly due to their high antioxidant activity (Hou et al. 2004; de Pascual-Teresa and Sanchez-Ballesta 2008). Numerous publications reported on the high antiox-idant activity in pomegranate juice (Gil et al. 2000; Noda et al. 2002; Aviram et al. 2004; Lansky and Newman 2007) and positive correlation between anthocyanins con-tent and antioxidant activity was found in juice extracted from separated arils of pomegranates (Tzulker et al. 2007). Anthocyanin accumulation in the pomegranate fruit may also be essential for protection against environmental
stress, such as high UV light and sunburns, and has a high economical value as the consumers are attracted to the fruit red color.
Pomegranate fruits are a rich source of anthocyanin, which accumulates in the skin and in the arils, the edible part of the fruit (Gil et al. 1995a; Hernandez et al. 1999; Tzulker et al. 2007). Six anthocyanin pigments were identi-W ed in pomegranate fruit, including mono- and di-gluco-sides of cyanidin (red pigments), delphinidin (purple pigment) and pelargonidin (orange pigments) (Du et al. 1975; Gil et al. 1995a). All six anthocyanin pigments were detected in Spanish, Californian, Tunisian and Italian pomegranates (Gil et al. 1995a, 1995b), with di V erences in their relative amounts, depending on variety and certain cli-matic and cultural variables (Gil et al. 1995b).
In the pomegranate fruit, the skin and arils color devel-opment are independent of one another, di V ering with respect to both timing and intensity during fruit develop-ment (Holland et al. 2009). Fruit from di V erent pomegran-ate accessions exhibit a high variability in skin and aril color. Skin color range is particularly wide, from yellow, through orange, pink and red to deep purple (Holland and Bar-Ya’akov 2008; Shwartz et al. 2009; Dafny-Yalin et al. 2010). The color variability is also re X ected in the pattern of skin color accumulation during fruit development. While some pomegranate varieties constantly accumulate antho-cyanins, others lose their color in early stages and develop it again during the W nal stages of fruit development (Holland et al. 2009).
The biochemical pathway and genetic control of antho-cyanin production is well characterized in numerous plant species and is considered as highly conserved among di V er-ent species in the plant kingdom (Winkel-Shirley 2001). Structural and regulatory genes involved in anthocyanin biosynthesis were isolated and functionally characterized from many plant species including maize, petunia, morning glory, snapdragon, Arabidopsis and more (Koes et al. 2005; Chopra et al. 2006). Common enzymatic steps shared by the anthocyanin and PA biosynthesis pathways are cata-lyzed by chalcone synthase (CHS), chalcone isomerase (CHI), X avanone 3-hydroxylase (F3H) dihydro X avonol 4-reductase (DFR), and leucoanthocyanidin dioxygenase (LDOX). These steps lead to the synthesis of common X avonoid precursors, which serve as intermediates from which the pathway diverges into several side branches, each resulting in a di V erent class of X avonoids (Fig.1; Schijlen et al. 2004).
In many plant species, anthocyanin production is basi-cally regulated at the transcriptional level, and conse-quently the anthocyanin pigment patterns are largely speci W ed by the expression patterns of relevant regulatory genes (Quattrocchio et al. 2006; Gonzalez et al. 2008). The transcriptional regulators for anthocyanin and PAs biosyn-thesis are known to include proteins containing an R2R3-MYB domain, a basic helix–loop–helix (bHLH) domain and WD40 repeats. Combinations of the R2R3-MYB, bHLH and WD40 proteins and interactions between them regulate the set of genes to be expressed (Baudry et al. 2004; Fig.
1Schematic representation of the X avonoid biosynthetic path-way leading to the production of anthocyanins and proanthocyanidins. Enzyme name abbreviations are as follows: PAL, Phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-couma-rate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, X avanone 3-hydroxylase; FLS, X avonol synthase; DFR, dihydro X avonol reductase; LAR, leucoanthocyanidin reductase; LDOX, leucoanthocyanidin oxidase/ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; GT, glucosyltransferase; AT, acyl-transferase; MT, methyltransferase
Broun 2005; Koes et al. 2005; Ramsay and Glover 2005; Morita et al. 2006; Gonzalez et al. 2008). For example, DFR and LDOX in Arabidopsis are known to be regulated at the transcriptional level by the MYB-bHLH-WD40 com-plex (Shirley et al. 1995; Pelletier et al. 1997; Nesi et al. 2001).
In the present work, we initiated a study aimed to under-stand the molecular mechanisms that control the color vari-ability of pomegranate fruit skin. A speci W c emphasis was placed on the study of TTG1-homologue gene, encoding a WD40 repeat protein (Walker et al. 1999). Homologues of this gene have been identi W ed and described from several plant species, including AN11 from Petunia hybrida (de Vetten et al. 1997), PAC1 from maize (Carey et al. 2004), InWDR1/Ca from Ipomoea nil (Morita et al. 2006), MtWD40-1 from Medicago truncatula (Pang et al. 2009) and Matthiola TTG1 from Matthiola incana (Dressel and Hemleben 2009). In all the latter reports, the isolated WD40 repeat protein was shown to be involved in anthocy-anin biosynthesis regulation. However, to date, apple (Brueggemann et al. 2010) and grapevine (Matus et al. 2010) are the only fruit trees for which the role of a TTG1 homo-logue protein in anthocyanin biosynthesis was reported.
In the present study, we have isolated and characterized a gene encoding a WD40 repeat protein closely related to the Arabidopsis TTG1, from pomegranate fruit, and further studied its involvement in anthocyanin regulation. We have compared two pomegranate accessions that di V er in their skin color and their pattern of color accumulation during fruit development and showed that the observed di V erences in color signi W cantly correlated with a di V erence in the expression of certain anthocyanin biosynthesis regulatory genes. Moreover, we show that the expression of the struc-tural genes DFR and LDOX, known to be under the control of the MYB-bHLH-WD40 complex, is highly correlated with the expression of the regulatory genes encoding MYB-like, bHLH-like and WD40 homologues proteins. Materials and methods
Plant material and growth conditions
Two pomegranate accessions, P.G.135-36 and P.G.100-1 (‘Wonderful’ landrace) were chosen from the live pomegran-ate tree collection in the Newe Ya’ar research center of the Agricultural Research Organization in Israel [regis-tered in Israel Gene Bank for agricultural crops (http:// https://www.doczj.com/doc/f79976833.html,.il)]. The trees were grown under the same envi-ronmental conditions and received similar agro-technical treatments. Flowers and fruits were collected from spring (end of April) to autumn (October), 2008. The W rst develop-mental stage was collected from unfertilized hermaphrodite X owers and the samples were taken only from the sepals that transform later into the fruit peel. Later on, developing fruits were collected at 2-week intervals. Overall, 12 di V erent stages were collected based on timing of collection starting from X ower to overripe fruit. In this study, we chose to focus on six di V erent developmental stages: X ower (stage 1), young fruit (stages 3 and 6), nearly mature fruit (stage 8), ripened fruit (stage 10), and over ripened fruit according to commer-cial practice (stage 12). For each sampling stage, 4–8 X ow-ers/fruits from three replicate trees of each pomegranate accession were collected. The fruit skin was removed and stored at ?80°C for further analysis.
Arabidopsis thaliana ttg1–9 mutant seeds (‘Columbia’background) were a gift from Prof. Richard A. Dixon (Plant Biology Division, Samuel Roberts Noble Foundation, Ard-more, OK, USA). ‘Columbia’ wild-type seeds were obtained from the Nottingham Arabidopsis Stock Centre (NASC, Nottingham, UK). Arabidopsis plants were grown in the growth room under long days (light/dark cycle of 16/ 8h), 22°C, 2–3 plants in each pot.
Nucleic acid isolation and synthesis of cDNA
Total RNA was extracted from pomegranate X ower and fruit skin as described by Meisel et al. (2005), for plants containing high amounts of polysaccharides and polyphe-nolic compounds. Total RNA was extracted also from mature leaves (green), young leaves (red), green and red stems. Each total RNA sample was incubated with 1 MBU RNase-free DNase I (EPICENTRE? Biotechnologies, Madison, WI, USA) for 30min at 37°C to remove co-extracted genomic DNA. Total RNA was quanti W ed by spectrophotometry (ND-1000 spectrophotometer, Nano-Drop Technologies, Wilmington, NC, USA), and the qual-ity was assessed by the absorbance ratios of A260/A280nm (1.8–2.2) and A260/A230nm (?1.8) and by the demonstra-tion of intact ribosomal RNA bands in agarose gel electro-phoresis. Following DNase treatment, W rst-strand cDNA was synthesized from 1.0 g of total RNA using Verso reverse transcriptase and oligo (dT) by Verso cDNA kit (Thermo scienti W c, Epson, UK).
Genomic DNA was extracted from fresh young leaves using a hexadecyl trimethyl ammonium bromide––polyvinyl pyrrolidone (CTAB-PVP) method as described in Porebski et al. (1997), a procedure suitable for plant tissues with high content of polysaccharides and polyphenolic compounds. DNA concentrations were quanti W ed by agarose gel electro-phoresis and spectrophotometrically (Nano-Drop).
Isolation of PgWD40
We used a combination of RT-PCR, 3? and 5? RACE tech-niques to isolate the full length of a TTG1 homologue gene
encoding a WD40 repeat protein, from P.G.100-1 skin tis-sue. Initially, a 340bp fragment was ampli W ed based on degenerate primers corresponding to the conserved WD40 motif of known anthocyanin regulatory proteins from other species. DNA fragments were separated on agarose gel and were cloned with the InsT/Aclone PCR cloning kit accord-ing to the manufacturer’s instructions (Fermentas, Vilnius, Lithuania). Cycle sequencing reactions were performed with the BigDye cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and were analyzed using capillary separation on an ABI3130x Genetic Analyser (Applied Biosystems). Sequence analysis enabled the design of nested primers for 3? and 5? RACE reactions (FirstChoice?RLM-RACE, Ambion, Austin, USA) for full length. All primers used for the isolation of the PgWD40 gene com-plete sequence are given in the supplemented data (Table S.1). Primers were designed using Primer3 software (Rozen and Skaletsky 2000).
Computer methods for analyzing sequence homology
The BLAST (Altschul et al. 1997) program was used to search homologues in sequence databases. Peptide sequences were searched for WD40 repeats using the protein prediction program available at the Biomolecular Engineering Research Center (Boston University; http:// https://www.doczj.com/doc/f79976833.html,/projects/wdrepeat/ ). A multiple alignment of the deduced amino acid sequences of PgWD40 and other WD40 repeat domain proteins was constructed using Clus-tal W, Version 1.83 (Thompson et al. 1994).
Cloning the PgWD40 ORF under the control of the35S promoter
Speci W c primers (Table S.1) were designed to amplify the coding region of PgWD40 from the cDNA. The primers included a Sma and Acc65 nested restriction site at the 5?region and the 3? region of PgWD40, respectively. An ampli W ed fragment spanning only the coding sequence (CDS) of PgWD40 was digested with Acc65 and Sma and cloned into the binary vector pBINaglA1, a derivative of pBI121 (GenBank entry AF485783). Direct sequencing of the inserted PgWD40 and its X anking regions was done to assure correct insertion. The W nal construct was desig-nated as pzp-WD40 and contains the PgWD40 coding region under the control of the 35S promoter and the NOS terminus.
Transformation of Arabidopsis ttg1–9 mutant Agrobacterium tumefaciens strain EHA105 containing plasmid pzp-WD40, was used to transform Arabidopsis ttg1–9 mutant (Walker et al. 1999) by the X oral dip in W ltration method (Clough and Bent 1998). Selection of transformants was conducted on 0.8% agar containing Murashige and Skoog (MS) salts (2.2g/l) and kanamycin (50 g/ml). The kanamycin-resistant seedlings were then transferred into soil to set seed. Progeny from self-fertilized primary trans-formants were grown in pots for phenotypic analyses. Young transgenic plants were inspected for restored tric-homes and anthocyanin production in vegetative tissues using a binocular. Ruthenium red staining of seed coat mucilage was performed as described by Debeaujon et al. (2000), using a Leica DM LB microscope (Leica Microsys-tems, Wetzlar, Germany), lens W1 (Nikon, Tokyo, Japan) and imaging software NIS-Element (Nikon).
Over-expression of VlMYB-A1 and PgWD40
in Arabidopsis WT and ttg1–9 mutant
Plasmid containing the grape coding region ampli W ed from full length cDNA of VlMYB-A1 (Kobayashi et al. 2002, 2004) under the control of the 35S promoter. The plasmid was transformed by Agrobacterium strain EHA105 into several types of Arabidopsis plants: (1) Intact WT plants;
(2) ttg1–9 mutant plants and (3) ttg1–9 mutant plants expressing 35S::PgWD40. Selection was done on kanamy-cin resistance. Selection of ttg1–9 transgenic mutants con-taining both VlMYB-A1 and PgWD40 was done based on the appearance of dense purple color of the cotyledons. Several dozen plants from each transformation where taken for further analyses.
HPLC analysis of anthocyanins in pomegranate X ower and fruit skin
Tissue samples were grinded in a cold mortar and homoge-nized with 80% methanol supplemented with 2mmol/l NaF (1:3 and 1:3.5 (w/v), for X ower and skin, respectively). The suspension was centrifuged and the supernatant was W ltered through a 0.45 m PTFE W lter before injection. Samples were analyzed using the LaChrom Merck Hitachi HPLC system, consisting of Pump L7100, Column oven L7350, Mixer-degasser L-7614 and Manual Injector Rheodyne, coupled with a diode array detector (DAD) with 3D feature (Multiwavelength Detector, Jasco MD-2010 Plus), inter-face (Jasco LC-Net II/ADC) and scienti W c software (EZChrom Elite? Client/Server version 3.1.6 build 3.1.6.2433). A column of Lichrospher?100 RP-18 (5 m particle size in 250￡4mm LichroCART? cartridge) with a guard column Lichrospher?100 RP-18 (5 m particle size in 4￡4mm LichroCART? cartridge) were used. The binary mobile phase consisted of phosphoric acid (0.1%, pH 2.4) (solution A), and acetonitrile (solution B). Elution was carried out with the following gradient scheme: 10% solution B at 1ml/min, 0–10min; 10–20% solution B at
1ml/min, 10–15min; 20% solution B at 1–0.6ml/min, 15–16min; and 20% Solution B at 0.6ml/min, 16–26min. Fol-lowing anthocyanin elution the column was washed with 80% solution B at 1ml/min for 10min and equilibrated with 10% solution B at 1ml/min for additional 10min. Acetonitrile was HPLC grade (LiChrosolv Merck); Col-umn-W ltered water was further distilled by Corning Mega-pure System, MP-6A, and passed through a 0.20 m Nylon membrane. Phosphoric acid and NaF were of analytical grade.
Anthocyanin standard library was constructed from del-phinidin 3,5-diglucoside, cyanidin 3,5-diglucoside, pelarg-onidin 3,5-diglucoside, malvidin 3-glucoside chloride, malvidin chloride (Apin Chemicals), delphinidin 3-gluco-side, cyanidin 3-glucoside, pelargonidin 3-glucoside (Poly-phenols Laboratories AS), pelargonidin chloride (Sigma), cyanin chloride, delphinidin chloride and cyanidin chloride (Fluka).
Anthocyanin identi W cation was performed by the soft-ware on the basis of UV/VIS absorbance spectrum and the retention time. The software also calculated peak area. A detection limit (minimal peak area) of 15,000 was consid-ered. Under the conditions employed in this study, the chromatogram peak areas linearly correlated with the con-centrations of the corresponding anthocyanins.
Semi-quantitative RT-PCR
The expression levels of several regulatory and structural genes predicted to be involved in pomegranate anthocyanin biosynthesis during fruit development was analyzed using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The pomegranate-speci W c primers used for the isolation of the anthocyanin biosynthetic genes and their corresponding PCR product lengths are listed in Table1. Total RNA was extracted from P.G.135-36 and P.G.100-1 accessions, from six di V erent developmental stages as described under “Nucleic acid isolation.” Tissues from at least three fruits from three di V erent trees of the same accession and sampling date were combined and extracted. First-strand cDNA was synthesized from 1.0 g of total RNA using Verso reverse transcriptase and oligo (dT) according to the manufacturer’s instructions (Verso cDNA kit; Thermo scienti W c). Quantity and quality of the RNA was assessed spectrophotometrically and veri W ed by appearance of intact ribosomal RNA bands on gel electro-phoresis. First-strand cDNA synthesis was carried out in triplicate for each sample (to minimize variation in RNA template levels) and then samples were pooled. Samples were normalized with 18S rRNA as a reference gene for constitutive expression.
PCR conditions were as follows: 95°C for 4min fol-lowed by 28–31 cycles of 95°C (30s), 55–60°C (30s) and 72°C (60s) with a W nal extension at 72°C for 7min and 4°C for 30min. For semi-quantitative RT-PCR assays, PCR reactions were performed with 27, 30, 33, and 40 cycles to ensure that ampli W cations were within the linear range. The number of PCR cycles appropriate for each gene speci W c primer pair was determined. Validation of the cor-rect PCR conditions to appropriately determine level of gene expression was done by making serial dilutions of the cDNA samples and consequently determining the band density on 1% agarose gels. Densitometric analyses were performed using EZQuant-Gel software (EZQuant Biology, Israel). The obtained data are expressed as the mean and standard error (SE) of W ve PCR replicates.
Statistical analyses
Correlation analyses between the expression of di V erent genes and between gene expression and anthocyanin quantity were performed using the Pearson/Spearman test, with
Table1Oligonucleotide prim-ers used for Semi-Quantitative RT-PCR experiments Gene detected Name Sequence (5? to 3?)Annealing
temperature (°C)
Size
product (bp) PgWD40TTG1-S-F GTCTCTGCTGATGGGTCGGT60350
TTG1-S-R CAGCAGAGTACATCGACATTGG
PgAN2 (MYB)AN2-S-F1CGACCTTCTTCGGAAATGTG55220
AN2-S-R2GCAATCAACGTCCATCTGT
PgAN1 (bHLH)AN1-S-F CCGTCGAGAAATGGCTGT58325
AN1-S-R CCATTCTGTCTCGGTCAGGT
PgCHS CHS-S-F2GCTTGTAGTCAGCGCAGAGA55300
CHS-S-R2CCTCCATCAAGCTCTTCTCG
PgDFR DFR-S-F GACCCTGAGAATGAAGTGATCA55250
DFR-S-R2CATCCATCCGGTCATCTTG
PgLDOX LDOX-S-F GAGGAGATGCTGCTGCAG55280
LDOX-S-R CCTCACCTTCTCCTTGTTCAC
P value less than 0.05 considered statistically signi W cant (Pearson test was calculated to parametric data and Spear-man test was calculated to non-parametric data, based on normality tests). The statistical program SPSS (IBM company) was used for the various computations. Results
The PgWD40 gene is a homolog of a regulatory gene that is involved in anthocyanin biosynthesis pathway
The full-length size of the PgWD40 gene was found to be 1,575bp. Within the full-length sequence we found an open reading frame (ORF) of 1,005-bp, predicting a protein of 335 amino acid residues. Comparison of the deduced PgWD40 protein sequence with those present in the Gen-Bank database revealed the following similarity within the WD40 protein family (Fig.2): the highest sequence simi-larity was found with TTG1 homologous proteins from fruit trees (85% identical amino acid residues), including: peach (Prunus persica) and apple (Malus x domestica). Lower sequence similarity (79–82% amino acid identity) was found to TTG1 homologues from other plants, for example: Matthiola TTG1 from M. incana, MtWD40-1 from M. truncatula, InWDR1/Ca from I. nil, TTG1 from Arabidopsis and AN11 from petunia. All these examples encode for WD40 repeat proteins that were identi W ed as regulators of the anthocyanin biosynthesis pathways in these plants.
Most of the homology was within the WD40 repeat motifs, normally a 40 amino acid tandem repeat character-ized by Gly-His (GH) and Trp-Asp (WD) doublet residues (van Nocker and Ludwig 2003). The length of the core between the GH and WD sequences in each WD40 repeat of PgWD40 protein was found to be 30–33 amino acids, which is also consistent with the consensus (Neer et al. 1994). The four hypothetical WD40 repeat motifs found in the predicted amino acid sequence of PgWD40 are indi-cated in Fig.2.
Comparison between the genomic DNA and the cDNA sequences (data not shown) indicated that PgWD40 lacked introns within the protein coding region, as is the case for TTG1 isolated from Arabidopsis (Walker et al. 1999), and other TTG1 homologues (de Vetten et al. 1997; Humphries et al. 2005; Pang et al. 2009). The pomegranate PgWD40 sequence was deposited to GenBank database under acces-sion number HQ199314.
Phenotype rescue of the Arabidopsis ttg1–9 mutant
To further study the possible function of PgWD40 as a reg-ulator of anthocyanin biosynthesis, we transformed the Arabidopsis ttg1–9 mutant with the coding sequence of PgWD40 gene under the control of the 35S promoter. The mutation in ttg1–9 (‘Columbia’ background) is a single amino acid substitution causing severe defects in trichome di V erentiation, anthocyanin pigmentation in vegetative tis-sues, seed coat pigmentation, seed coat mucilage, and for-mation of long root hair (Walker et al. 1999). Of the 70 kanamycin-resistant plants, 4 were randomly chosen, to test the presence of PgWD40 in their DNA and RNA levels. Kanamycin-resistant plants positive for the presence of PgWD40 in their DNA were veri W ed by PCR ampli W cation with primers speci W c for the CDS of PgWD40 (data not shown). RT-PCR analysis indicated that the transgenic plants expressed the CDS of PgWD40 in their leaves (data not shown). Our experimental results (Fig.3) demonstrate that PgWD40 restored the wild-type phenotype to the ttg1 mutant with respect to color accumulation and trichome biosynthesis. While the seed coat of ttg1 mutants is yellow due to the lack of proanthocyanidins, the transformed mutants had a light brown color, resembling the wild type (Fig.3a–c). In addition, the transformed mutants restored trichome growth on stems and leaves, accumulated antho-cyanin pigments in vegetative tissues (Fig.3d–f) and restored seed coat mucilage (Fig.3g–i). These W ndings were observed in all the 70 kanamycin-resistant plants.
To further study additional functional characteristics of the protein encoded by PgWD40 as an ortholog of TTG1, we investigated its dependence on functionally known MYB protein expression. The e V ect of co-expression of pomegranate PgWD40 CDS and the grape VlMYB-A1 CDS on color of Arabidopsis cotyledons was tested in three di V erent Arabidopsis lines: (1) ttg1–9 mutant; (2) trans-genic ttg1–9 mutants that expressed 35S::PgWD40 and (3) WT Arabidopsis (Fig.4). Our experimental results show that Arabidopsis ttg1–9 mutant plants over-expressing the grape VlMYB-A1 alone formed green cotyledons (Fig.4a). Plants over-expressing PgWD40 alone in ttg1–9 mutants formed green cotyledons, but were able to produce color in leaves and seed coat (Fig.3e). However, Arabidopsis ttg1–9 mutant plants over-expressing both the grape VlMYB-A1 and the pomegranate PgWD40, produced over pigmented cotyledons with purple color (Fig.4b). Similar result was obtained when WT Arabidopsis was transformed with the grape VlMYB-A1 alone (Fig.4c).
PgWD40 expression in di V erent pomegranate vegetative tissues
The steady state level of PgWD40 gene mRNA in di V erent pomegranate vegetative tissues was examined by semi-quantitative RT-PCR and the results are presented in Fig.5. Previous reports indicated that other TTG1 homologues, like AN11 from petunia (de Vetten et al. 1997) and PFWD
from Perilla frutescens (Sompornpailin et al. 2002), are expressed in various red and green tissues such as leaves. Our data indicate that the PgWD40 gene was expressed in pigmented as well as unpigmented tissues, but expression was somewhat higher in red leaves and stems compared to green leaves and stems.
Anthocyanin accumulation in pomegranate fruit skin during fruit development
In order to study the relations between anthocyanin produc-tion and gene activity, we followed the two processes in parallel during fruit development in two pomegranate accessions, P.G.135-36 and P.G.100-1. Representative X ower and fruit of the two accessions from the six develop-mental stages de W ned under “Materials and methods” are presented in Fig.6. The fruit of P.G.135-36 turned red already in stage 8 (an early anthocyanin accumulating accession), whereas P.G.100-1 remained green at this stage (a late anthocyanin accumulating accession). Once the X ower was fertilized, both accessions underwent a transi-tion from orange-red colored X owers towards the develop-ment of green fruits. Later in fruit development (stages 8 and 10), the fruit regained color accumulation and reaching maximal levels when the fruit was over ripe (stage 12).
Composition of fruit skin anthocyanins in both pome-granate accessions during fruit development was analyzed by HPLC (Figs.7, 8). Most of the anthocyanins in the
Fig.
2Alignment of deduced amino acid sequences of plant homo-
logues to the Arabidopsis TTG1 WD40-repeat protein. The WD40 re-peat domains are indicated with open boxes. Identical residues are highlighted on a black background, while similar residues and gaps are highlighted on a gray and white background, respectively. The name of the proteins, their source and GenBank accession numbers are as follows: PgWD40 isolated from Punica granatum (pomegranate)(HQ199314); PpTTG1 from Prunus persica (peach) (ACQ65867); MdTTG1 from Malus domestica (apple) (GU173813); AtTTG1 from Arabidopsis thaliana (Q9XGN1), MiTTG1 from Matthiola incana (CAE53274); MtWD40-1 from Medicago truncatula (ABW08112); GhTTG3 from Gossypium hirsutum (cotton) (AAM95645); PhAN11 from Petunia hybrida (AAC18914); PFWD from Perilla frutescens (BAB58883); ZmPAC1 from Zea mays (maize) (AAM76742)
Fig.3
Expression of pomegranate PgWD40 in Arabidopsis ttg1–9mutants. ttg1–9 mutant (a , d , g ), ttg1–9 mutant expressing 35S::PgWD40 (b , e , h )and WT Arabidopsis (c , f , i ). Seed coat pig-mentation (a–c ). Trichome phenotype and anthocyanin production in leaf (d–f ). Ruthenium red staining of seed coat mucilage (g–i ). Scale bars indicate 600 m
Fig.4
Expression of grape VlMYB -A1 in di V erent genetic back-ground of Arabidopsis plants. ttg1–9 mutant (a ), transgenic ttg1–9mutants that expressed 35S::PgWD40 (b ), WT Arabidopsis (c ).Shown plants were grown on selective media containing kanamycin.Seedlings shown in (a )were resistant to kanamycin and were green because the plants do not have normal TTG1 (ttg1–9 mutant). Seed-lings shown in (b )are the result of double transformation and were kanamycin resistant. Therefore, even the background single transfor-mants were able to grow on kanamycin and were green. All the purple plants were shown to contain both PgWD40 and VlMYB-A1. Purple seedlings shown in (c )were transgenic plants. Non transformed seed-lings were green because at the stage were the photograph was taken the non transformed plants were still viable. Later on the green back-ground seedlings died
X ower were pelargonidins (orange color) and their levels declined when the fruit began to develop (Figs.7, 8a). On the other hand, cyanidins levels (red color) were relatively low in the X ower in both accessions and accumulated dur-ing fruit development reaching their maximal levels in over ripe fruits (Figs.7, 8b). Both accessions accumulated negli-gible amounts of delphinidins (purple color) in their X owers and fruit skins (Fig.7). The anthocyanin HPLC chromato-grams reveal that in P.G.135-36 (an early anthocyanin accumulating accession) cyanidin was synthesized and accumulated already in stage 8, while at this stage P.G.100-1 (a late anthocyanin accumulating accession) did not accu-mulate signi W cant levels of anthocyanins (Fig.7). The results indicate that the red phenotype of both pomegranate accessions was due to the biosynthesis and accumulation of cyanidin and its derivatives.
Expression of regulatory genes involved in anthocyanin biosynthesis during pomegranate fruit development
The expression levels of several genes, presumably involved in pomegranate anthocyanin biosynthesis during fruit development, were analyzed. Using semi-quantitative RT-PCR, as described under “Materials and methods”, gene expression experiments were performed on the same tissue samples analyzed for anthocyanins. The expression level of three homologues of the anthocyanin biosynthesis regulatory genes, PgWD40, PgAn2 (MYB), and PgAn1 (bHLH), was measured in both accessions (Fig.9a–c). Comparison of the deduced PgAn2 (MYB) and PgAn1
(bHLH) proteins sequences with those present in the Gen-Bank database, revealed a high sequence similarity with the petunia proteins: PhAn2––a member from the MYB protein family and PhAn1––a member from the bHLH protein fam-ily (77 and 83% identical amino acid residues, respec-tively). In addition, signi W cant similarity was found also to other MYB and bHLH proteins, known as anthocyanin reg-ulatory proteins from other plants species. Alignment data with PgAn2 and PgAn1 is given in the supplemented data, including recently submitted additional sequence of puta-tive R2R3 MYB homologue from pomegranate (Fig. S1).
The expression of PgWD40 in the fruit skin gradually increased during fruit development in both accessions (Fig.9a): in young fruits (stages 3 and 6) the expression was relatively low, increasing as the fruit continued to develop and peaking in the over-ripe fruit (stage 12). Of particular interest are the expression levels in stage 8. At this stage the two accessions di V ered in their anthocyanin accumulation as well as in the mRNA steady state level of PgWD40. Expression of PgWD40 in stage 8 as well as anthocyanin accumulation were higher in accession P.G.135-36 compared to accession P.G.100-1. The patterns
Fig.
5Expression of PgWD40 in di V erent pomegranate vegetative tis-
sues. Semi quantitative RT-PCR on cDNA synthesized from Total RNA isolated from P.G.100-1 accession tissues: Mature green leaves (lane 1); young red leaves (lane 2); mature green stem (lane 3); young red stem (lane 4
). rRNA, 18S and 28S ribosomal RNA
Fig.6Flower and fruit of two pomegranate accessions, P.G.135-36
and P.G. 100-1 (a ‘wonderful’ landrace), at six di V erent developmental
stages, including: X ower (stage 1), young fruit (stages 3 and 6), nearly
mature fruit (stage 8), ripened fruit (stage 10), and over ripened fruit
according to commercial practice (stage 12). P.G.135-36 is an early
anthocyanin accumulating accession and P.G.100-1 is a late anthocya-
nin accumulating accession (see stage 8)
of PgWD40 expression and that of PgAn1 (a bHLH regula-tory gene) were similar in both accessions (Fig.9b)although higher expression levels of both genes were mea-sured in the X ower stage of P.G.100-1.
The expression of PgAn2 (a MYB regulatory gene) was relatively high in X owers (Fig.9c), declined in young developing fruits (stages 3 and 6), gradually increased again as the fruit matured and reached its peak when the fruit was over ripened. At this stage, anthocyanins content was also maximal. The di V erences in the steady state level of PgAn2 (MYB) mRNA between P.G.135-36 and P.G.100-1 in fruit developmental stage 8 were similar in trend to PgWD40 and PgAn1 (bHLH) but signi W cantly larger in magnitude (the level of PgAn2 mRNA was close to sixfold higher in P.G.135-36 compared to P.G.100-1).However, Unlike PgWD40 and PgAn1 (bHLH), the expres-sion of PgAn2 (MYB) signi W cantly decreased during fruit stages associated with low anthocyanin accumulation, i.e.stages 3 and 6.
Expression of anthocyanin structural biosynthetic genes during pomegranate fruit development
Three homologues of the key structural genes from pome-granate that were predicted to be involved in X avonoid and anthocyanin biosynthesis were studied: PgDFR , PgLDOX and PgCHS (Fig.9d–f). The genes PgDFR , PgLDOX and PgCHS are homologous to Arabidopsis and petunia (Chopra et al. 2006), grape (Boss et al. 1996; Matus et al.2010) and apple (Espley et al. 2007) genes. These genes are known as structural genes involved in anthocyanin biosyn-thesis, in the above mentioned plants. Alignment data is given in the supplemented data, including recently submit-ted additional sequences of LDOX and CHS homologues from pomegranate (Fig. S2).
Of particular interest are the expression patterns of PgDFR and PgLDOX which are very similar to one another (Fig.9d, e), and to that of the regulatory gene PgAn2(MYB) (Fig.9c). The di V erences in anthocyanin accumula-tion between stage 8 fruit of both accessions markedly cor-
7HPLC chromatograms at 520nm of pomegranate skin metha-nolic extracts in developing fruit of two di V erent accessions, P.G.135-36 and P.G.100-1. Stages 1–12 are the developmental stages from ower to over ripe fruit. Chromatograms’ peak identity: delphinidin Stage 1
Stage 3Stage 6
Stage 12Stage 8Stage 101
23
6
4
5Stage 1
Stage 3Stage 6Stage 12
Stage 8Stage 10P.G.100-1Retention time (minutes)
P.G.135-361
23
4
56
Fig.8Anthocyanin composition during fruit development in two di V erent pomegranate accessions, P.G.135-36 and P.G.100-1. a Total level of pelargonidin derivatives (orange pigments ). b Total level of cyanidin derivatives (red pigments ). Anthocyanin levels are presented in units of chromatogram peak area (proportional to pigment concen-tration under the study conditions). Stages 1–12 are developmental stages from X ower to over ripe fruit
related with the di V erences in the steady state levels of PgDFR and PgLDOX mRNA. In contrast, the expression level of PgCHS, was not correlated with the skin color and the timing of color appearance, in both accessions (Fig.9f).Statistical analysis for correlations between the expression of regulatory and structural genes and cyanidins content Most of the anthocyanins produced in developing fruit skin of both accessions were cyanidin derivatives, while the lev-els of pelargonidin derivatives were relatively low and those of delphinidin were negligible (Figs.7, 8). Therefore,statistical analyses for correlations were done only between total content of cyanidin derivatives and gene(s)expression levels, using the Pearson/Spearman test (Table 2). The highest positive correlations were obtained between total cyanidins content and the expression of the regulatory genes, PgWD40 and PgAn2 (MYB), in both accessions. In P.G.135-36 the Pearson value (calculated for parametric data) was 0.78 with PgWD40 and 0.76 with PgAn2 (MYB).In P.G.100-1 the Spearman value (calculated for non-para-metric data) was 0.77 with PgWD40 and 0.6 with PgAn2
Fig.
9Expression of regulatory genes (a–c ) and structural genes (d–f ) during fruit development in two di V erent pomegranate accessions, P.G.135-36 and P.G.100-1. Semi-quantitative RT-PCR analysis was done as described in “Materials and methods ”. Samples were nor-malized with 18S rRNA as a reference gene for constitutive expression. PCR products were separated on 1% agarose gels. Characteristic gels of the two accessions are presented for each gene product. Data
expressed as the mean of 5 PCR replicates §SE
(MYB). Low and negative correlation was obtained between total cyanidins content and the expression level of the structural gene, PgCHS , in both accessions.
In addition, correlations between the expression levels of the di V erent anthocyanin genes (regulatory and structural)were tested (Table 3). Of particular interest are the high positive correlations found between the steady state mRNA levels of the regulatory genes PgWD40 and PgAn2 (MYB)and the structural genes PgDFR and PgLDOX in both accessions (up to 0.85 in Pearson value). Negative correla-tion was found between the expression of PgCHS and PgWD40 in both accessions.
Discussion
Pomegranate cultivation develops rapidly worldwide along with the growing awareness to the health bene W ts of the pomegranate fruit (Lansky and Newman 2007; Holland and
Bar-Ya’akov 2008). One of the interesting phenomena in pomegranate is the high variability in skin color of di V erent pomegranate varieties, conferred by variations in anthocya-nin composition (Gil et al. 1995a , 1995b ). Understanding the genetic components that control natural color variation in pomegranate has both academic and practical aspects. The identi W cation of the genetic components that are responsible for observed color variability in fruit trees is specially com-plicated, since currently there are no adequate genetic sys-tems for analyzing the genetic variability in fruit trees.Pomegranate could potentially be developed into such a sys-tem because pomegranate species display high variability in fruit color, have a relatively short juvenile period and are easy to establish e Y ciently cross bred populations. From a practical aspect, the skin color determines fruit appeal to the consumers; it is one of the sources for antioxidant activity (Gil et al. 2000; Noda et al. 2002; Tzulker et al. 2007) and an important source of protective components against pests and radiation damage (Winkel-Shirley 2001; Syed et al. 2006).
Table 2Correlation between total cyanidin derivatives (red pigments) and gene expression according to the Pearson/Spearman test, in two di V er-ent pomegranate accessions, P.G.135-36 and P.G.100-1
PgWD40
PgAn2 (MYB)
PgAn1 (bHLH)
PgDFR
PgLDOX
PgCHS
P.G.135-36Pearson value 0.780.760.480.540.69?0.58Signi W cant 0.07
0.08
0.33
0.27
0.13
0.23
P.G.100-1Spearman value 0.770.600.140.600.60?0.20Signi W cant
0.07
0.21
0.79
0.21
0.21
0.70
Table 3Correlation matrix in the level of expression between di V erent anthocyanin biosynthetic genes in pomegranate accessions P.G.135-36and P.G.100-1 according to the Pearson test (r value)*P <0.05**P <0.01
P.G.135-36PgWD40PgWD401PgAn2PgAn2 (MYB)0.86*1PgAn1PgAn1 (bHLH)0.88*0.641PgDFR PgDFR 0.85*0.94**0.771PgLDOX PgLDOX 0.92**0.98**0.780.98**1PgCHS PgCHS ?0.87*?0.61
?0.72
?0.63
?0.70
1
P.G.100-1PgWD40PgWD401PgAn2PgAn2 (MYB)0.97**1PgAn1PgAn1 (bHLH)0.760.621PgDFR PgDFR 0.94**0.98**0.581PgLDOX PgLDOX 0.93**0.98**0.570.99**1PgCHS PgCHS ?0.57
?0.69
?0.13
?0.66
?0.62
1
One possible cause for the observed high variability in anthocyanin content in the fruit skin of di V erent pomegranate accessions may reside in the di V erences in expression lev-els of genes involved in the anthocyanin biosynthesis path-way. As an initial step towards understanding the regulation of anthocyanin production in pomegranate we isolated and characterized the PgWD40 gene, a homolog of TTG1 from Arabidopsis which is involved in anthocyanin biosynthesis regulation. We then studied its function in transformed Arabidopsis ttg1–9 mutant and mode of expression in the pomegranate fruit skin during develop-ment.
The PgWD40 gene is a functional homologue
of Arabidopsis TTG1 gene
Functional homologues of TTG1 from Arabidopsis were studied in many plant species including, petunia (Payne et al. 2000), maize (Carey et al. 2004), cotton (Humphries et al. 2005), M. truncatula (Pang et al. 2009) and apple (Brueggemann et al. 2010). In all of these cases the TTG1 homologous genes recovered all the Arabidopsis ttg1 mutant phenotypes. Thus, the genetic information encoding for the WD40 repeat TTG1 type of protein is retained even in plants that do not normally have properties such as tric-homes. The results of phenotype recovery with PgWD40 in transgenic ttg1 mutants indicated that PgWD40 is func-tional in Arabidopsis and is able to restore color formation in the seed coat and in vegetative tissues, trichome forma-tion in leaves and stems and seed coat mucilage (Fig.3). These results show that the isolated pomegranate PgWD40 gene is a functional homologue of Arabidopsis TTG1. The high homology of the pomegranate PgWD40 to the WD40 group (Fig.2) and its ability to rescue the phenotype of the Arabidopsis ttg1 mutant, strongly suggest that PgWD40 is a regulator of the anthocyanin biosynthesis pathway in pome-granate.
To further characterize the dependence of PgWD40 on the function of other anthocyanins biosynthesis regulatory genes we studied the e V ect of co-expressing PgWD40 and VlMYB-A1. The grape VlMYB-A1 gene (Kobayashi et al. 2002, 2004) is an ortholog of the PhAn2 gene from petunia (Quattrocchio et al. 1999), PAP1 from Arabidopsis (Bore-vitz et al. 2000) and is highly homologous to PgAn2 from pomegranate. In addition, VlMYB-A1 is one of the known fruit tree MYBs shown to be functionally active in control of anthocyanin biosynthesis in heterologous plant systems such as Arabidopsis and Tobacco (Geekiyanage 2009). The data presented in Fig.4 demonstrated that over-expression of VlMYB-A1 resulted in over production of anthocyanin in WT Arabidopsis plants that have intact TTG1 protein. On the other hand, over-expression of the same VlMYB-A1 did not cause over production of anthocyanin in plants that do not have normal TTG1 protein (Arabidopsis ttg1 mutant), indicating that Arabidopsis TTG1 protein is necessary for proper activity of VlMYB-A1 and vice versa. The ability of PgWD40 to replace the function of the original TTG1gene in Arabidopsis mutant plants that co-expressed VlMYB-A1and PgWD40 is the evidence that PgWD40 from pomegranate and the VlMYB-A1depend on each other for induction of anthocyanins biosynthesis. This W nding is additional evi-dence for the similar functional characteristics of the pome-granate PgWD40 and the Arabidopsis TTG1 protein which is known to interact also with MYB transcriptional factors such asTT2 (Baudry et al. 2004), GL1 (Larkin et al. 1999) and PAP1 (Borevitz et al. 2000).
In addition to its role in anthocyanin biosynthesis, the Arabidopsis TTG1 protein has a role in epidermal cell development, speci W cally, in trichome formation (Payne et al. 2000; Zhao et al. 2008). Pomegranates do not have trichomes in their leaves or stems but expression of the pomegranate PgWD40 CDS restored the ability to form trichomes in the Arabidopsis ttg1 mutant (Fig.3e). In view of the additional regulatory functions that both Arabidopsis TTG1 and PgWD40 exhibit in Arabidopsis, it is possible that PgWD40 has other yet unde W ned functions in pome-granates. In this respect it is interesting to note that the edi-ble X eshy part of the pomegranate seed is comprised of elongated cells that protrude from the outer epidermal cells of the testa (Fahn 1982). Such cells could be regarded as orthologous to trichomes from a botanical point of view since they are of epidermal cell origin (Fahn 1982). There-fore, PgWD40 may also play a role in regulating the devel-opment of cells that form the X eshy part of the seeds in a similar way that it controls trichome formation. Measuring the expression level of PgWD40 in arils, during fruit devel-opment in di V erent accessions, will enable us to decipher the function of PgWD40 in arils.
Anthocyanin biosynthesis in pomegranate skin is regulated during fruit development
We observed that the relative amounts of the various antho-cyanin components dramatically change during fruit devel-opment (Figs.6, 7, 8). In the pomegranate accessions tested in this study, the most prominent anthocyanin components in the X ower were pelargonidin derivatives (orange pig-ments). During the early stages of fruit development, when the color of the skin is mostly green, low levels of all antho-cyanins were detected. Whereas, cyanidin derivatives (red pigments) prevailed in the skin during the late stages of fruit development, when the color of the skin became red. Thus, the color in the fruit skin was mostly due to accumu-lation of cyanidin derivatives. The dominant presence of cyanidin derivatives and de W ciency of delphinidin deriva-tives (purple pigments) in the pomegranate fruit skin were
also reported for Spanish pomegranate cultivars (Gil et al. 1995a). Our data and the reports by Gil et al. (1995a) indi-cate that anthocyanin biosynthesis in pomegranate skin is highly regulated during fruit development with respect to both anthocyanin quantity and composition.
Regulatory and structural genes involved in anthocyanin biosynthesis pathway are transcriptionally regulated
in pomegranate skin during fruit development
In order to identify the genetic components that are involved in anthocyanin biosynthesis during pomegranate fruit development, we isolated several genes that were pre-dicted to be involved either in the regulation or biosynthesis of X avonoid and anthocyanin. We then measured their expression level (Fig.9) and did a correlation test with the content of cyanidin derivatives (Table2).
High and positive correlation was found between the expression pattern of two genes: PgWD40 and PgAn2 (MYB) with the content of cyanidin derivatives in develop-ing pomegranate fruit skin of both accessions. In addition, high and positive correlation was found between the expression level of the regulatory genes, PgWD40 and PgAn2 (MYB), and that of the structural genes, PgDFR and PgLDOX. These correlations suggest that transcriptional control of anthocyanin production in pomegranate is an important regulatory component, as demonstrated in other plants as well (Morita et al. 2006; Quattrocchio et al. 2006; Gonzalez et al. 2008; Yuan et al. 2009; Matus et al. 2010). The correlation data also suggest that in pomegranate fruit skin, PgWD40 and PgAn2 (MYB) are responsible for spe-ci W c control of cyanidin synthesis through action on DFR and LDOX and that DFR and LDOX are coordinately regu-lated. Data from several other plant systems, such as Ara-bidopsis, point at the structural genes encoding for DFR and LDOX as being the targets of a regulatory complex that consists of a WD40 protein and MYB and bHLH partners (Shirley et al. 1995; Pelletier et al. 1997; Nesi et al. 2000, 2001; Baudry et al. 2004).
The MYB and bHLH genes are known to exist in large families in the plant kingdom. In the present work we stud-ied gene representatives of these gene families. Several MYB genes were isolated from pomegranate, among them PgAn2, was found to have high sequence similarity to other anthocyanin biosynthesis promoting MYBs, including: An2 from Petunia (Quattrocchio et al. 1999), PAP1 and MYB113 from Arabidopsis (Borevitz et al. 2000; Gonzalez et al. 2008, respectively); MdMYB10 from apple (Espley et al. 2007) and VlMYB-A1 from Vitis (Kobayashi et al. 2002). Another pomegranate MYB related sequence recently submitted to GenBank was found to be homolo-gous to PgAn2 but its predicted amino acid sequence lacked the ANDV motif, which characterizes MYB proteins involved in anthocyanin biosynthesis (Lin-Wang et al. 2010). Several bHLH-like genes were also isolated from pomegranate. Among them, we focused on the PgAn1 because steady state levels of PgAn1 transcripts varied in the fruit skin during fruit development. In addition, PgAn1 was found to have high sequence similarity to other bHLH proteins involved in the regulation of anthocyanin biosyn-thesis, including: An1 from Petunia (Spelt et al. 2000), TT8 from Arabidopsis (Nesi et al. 2000), MdbHLH3 from apple (Espley et al. 2007) and VvMYC1 from grapevine (Hichri et al. 2010). Both, PgAn2 and PgAn1, positively correlated with cyanidin biosynthesis in pomegranate, but much higher correlation was found with the MYB like PgAn2. More studies with the other bHLH and MYB genes, together with PgAn2 and PgAn1 are required, in order to elucidate their role in control of anthocyanins biosynthesis.
Low correlation was found between the expression level of PgCHS and cyanidin accumulation, although negative correlation was found between the expression level of PgCHS and PgWD40; the latter, though, was signi W cant only in P.G.135-36.We do not yet have enough evidence to explain these data. It is possible that several CHS types of proteins are active in pomegranate anthocyanin biosynthe-sis. Indications that early steps of anthocyanin biosynthesis are not under the control of TTG1 homologues or that a TTG1 homologous protein controls only discrete CHS gene types were obtained in other plants, such as Japanese Morn-ing Glory. Mutation analysis in this plant revealed that the TTG1 homolog di V erentially a V ected the expression of anthocyanin structural genes and that certain CHS homo-logues were not regulated by the Japanese morning glory TTG1 homolog (Morita et al. 2006).
The genetic component responsible for the di V erences
in the timing of color appearance observed
in both accessions studied
In an attempt to understand whether PgWD40 is involved in determining the di V erences in anthocyanin accumulation timing observed in di V erent pomegranate accessions, we compared the expression of anthocyanin genes in the acces-sions P.G.135-36 (an early anthocyanin accumulating accession) and P.G.100-1 (a late anthocyanin accumulating accession). Our data indicate that in stage 8, during which the highest phenotypic di V erences were observed between the two pomegranates accessions (Fig.6), there was a sig-ni W cant di V erence in the expression level of PgWD40. A very similar pattern of expression level was also observed with PgAn1 (bHLH). Signi W cantly, larger di V erences were observed in the expression level of PgAn2 (MYB). These W ndings are not yet su Y cient to conclude whether PgWD40, PgAn2 (MYB) or PgAn1 (bHLH) are responsible for the phenotypic di V erence between P.G.135-36 and
P.G.100-1. However, the much higher expression and ear-lier rise in expression levels of PgAn2 (MYB) in accession P.G.135-36 at stage 8 insinuate that it may be involved in the earlier appearance of anthocyanins in accession P.G.135-36. The role of MYB transcriptional factor in the timing of color appearance was also described in several other studies, including fruit trees from the Rosaceae family (Espley et al. 2007; Lin-Wang et al. 2010), sweet potato (Mano et al. 2007), cabbage (Yuan et al. 2009), etc. The color change from red orange to green of the fused sepals subsequent to anthesis and in the young developing fruit (stages 3 and 6) was associated with a transient reduction in the expression of PgAn2 (MYB), PgDFR and PgLDOX in both accessions studied. This observation suggests that dur-ing the green stages of the pomegranate, transcription is controlled by the reduced expression of positive regulatory genes such as PgAn2 (MYB) and structural genes such as PgDFR and PgLDOX.
In summary, to the best of our knowledge, this research is the W rst molecular study that described the functional characterization of a genes involved in anthocyanin biosyn-thesis from pomegranate. The identi W cation of a pomegran-ate regulatory and structural genes, which a V ect anthocyanin content in pomegranate fruit skin may facili-tate our ability to study whether these genes are responsible for the high variability in color observed among di V erent pomegranate accessions. Furthermore, these results may also facilitate our ability to develop new pomegranate culti-vars with enhanced anthocyanin content by utilizing molec-ular-genetic approaches.
Acknowledgments We thank Prof. Richard A. Dixon for the kind gift of Arabidopsis ttg1–9 mutant seeds. We also thank the Israeli Min-istry of Science and Technology and Keren Kayemeth LeIsrael––Jew-ish National Fund (KKL-JNF) for W nancial support of this project. References
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123412n x A x B A B A B A n A ∈??? ????? ∈?∈?（）元素与集合的关系：属于（）和不属于（）（）集合中元素的特性：确定性、互异性、无序性集合与元素（）集合的分类：按集合中元素的个数多少分为：有限集、无限集、空集（）集合的表示方法：列举法、描述法（自然语言描述、特征性质描述）、图示法、区间法子集：若 ，则，即是的子集。、若集合中有个元素，则集合的子集有个， 注关系集合集合与集合{}00(2-1)23,,,,.4/n A A A B C A B B C A C A B A B x B x A A B A B A B A B A B x x A x B A A A A A B B A A B ??????????? ???????????≠∈?????=???=∈∈?=??=??=???真子集有个。、任何一个集合是它本身的子集，即 、对于集合如果，且那么、空集是任何集合的（真）子集。 真子集：若且（即至少存在但），则是的真子集。集合相等：且 定义：且交集性质：，，，运算{}{},/()()()-()/()()()()()()U U U U U U U U A A B B A B A B A A B x x A x B A A A A A A B B A A B A A B B A B A B B Card A B Card A Card B Card A B C A x x U x A A C A A C A A U C C A A C A B C A C B ????????=????=∈∈???=??=?=????????=???=+?=∈?=?=??==?=?，定义：或并集性质：，，，，， 定义：且补集性质：，，，， ()()()U U U C A B C A C B ????? ?? ?? ???? ?????????? ???????? ??????????????????????? ?????????????????????=??????? 集合12{,, ,}n a a a 的子集个数共有2n 个；真子集有2n –1个；非空子集有2n –1个；非空的真子集有2n –2个
《电路分析基础》作业参考解答 第一章（P26-31） 1-5 试求题1-5图中各电路中电压源、电流源及电阻的功率（须说明是吸收还是发出）。 （a ）解：标注电压如图（a ）所示。 由KVL 有 故电压源的功率为 W P 302151-=?-=（发出） 电流源的功率为 W U P 105222=?=?=（吸收） 电阻的功率为 W P 20452523=?=?=（吸收） （b ）解：标注电流如图（b ）所示。 由欧姆定律及KCL 有 A I 35 152==，A I I 123221=-=-= 故电压源的功率为 W I P 151151511-=?-=?-=（发出） 电流源的功率为 W P 302152-=?-=（发出） 电阻的功率为 W I P 459535522 23=?=?=?=（吸收） 1-8 试求题1-8图中各电路的电压U ，并分别讨论其功率平衡。 （b ）解：标注电流如图（b ）所示。 由KCL 有 故 由于电流源的功率为 电阻的功率为 外电路的功率为 且 所以电路的功率是平衡的，及电路发出的功率之和等于吸收功率之和。 1-10 电路如题1-10图所示，试求： （1）图（a ）中，1i 与ab u ； 解：如下图（a ）所示。 因为 所以 1-19 试求题1-19图所示电路中控制量1I 及电压0U 。 解：如图题1-19图所示。 由KVL 及KCL 有 整理得 解得mA A I 510531=?=-，V U 150=。
题1-19图 补充题： 1. 如图1所示电路，已知 ， ，求电阻R 。 图1 解：由题得 因为 所以 2. 如图2所示电路，求电路中的I 、R 和s U 。 图2 解：用KCL 标注各支路电流且标注回路绕行方向如图2所示。 由KVL 有 解得A I 5.0=，Ω=34R 。 故 第二章（P47-51） 2-4 求题2-4图所示各电路的等效电阻ab R ，其中Ω==121R R ，Ω==243R R ，Ω=45R ，S G G 121==， Ω=2R 。 解：如图（a ）所示。显然，4R 被短路，1R 、2R 和3R 形成并联，再与5R 串联。 如图（c ）所示。 将原电路改画成右边的电桥电路。由于Ω==23241R R R R ，所以该电路是一个平衡电桥，不管开关S 是否闭合，其所在支路均无电流流过，该支路既可开路也可短路。 故 或 如图（f ）所示。 将原电路中上边和中间的两个Y 形电路变换为?形电路，其结果如下图所示。 由此可得 2-8 求题2-8图所示各电路中对角线电压U 及总电压ab U 。 题2-8图 解：方法1。将原电路中左边的?形电路变换成Y 形电路，如下图所示： 由并联电路的分流公式可得 A I 14 12441=+?=，A I I 314412=-=-= 故 方法2。将原电路中右边的?形电路变换成Y 形电路，如下图所示： 由并联电路的分流公式可得 A I 2.16 14461=+?=，A I I 8.22.14412=-=-= 故 2-11 利用电源的等效变换，求题2-11图所示各电路的电流i 。 题2-11图 解：电源等效变换的结果如上图所示。 由此可得 V U AB 16=A I 3 2=
一、公式法化简：是利用逻辑代数的基本公式，对函数进行消项、消 因子。常用方法有： ①并项法利用公式AB+AB’=A 将两个与项合并为一个，消去其 中的一个变量。 ②吸收法利用公式A+AB=A 吸收多余的与项。 ③消因子法利用公式A+A’B=A+B 消去与项多余的因子 ④消项法利用公式AB+A’C=AB+A’C+BC 进行配项，以消去更多 的与项。 ⑤配项法利用公式A+A=A，A+A’=1配项，简化表达式。 二、卡诺图化简法 逻辑函数的卡诺图表示法 将n变量的全部最小项各用一个小方块表示，并使具有逻辑相邻性的最小项在几何位置上相邻排列，得到的图形叫做n变量最小项的卡诺图。 逻辑相邻项：仅有一个变量不同其余变量均相同的两个最小项，称为逻辑相邻项。 1.表示最小项的卡诺图 将逻辑变量分成两组，分别在两个方向用循环码形式排列出各组变量的所有取值组合，构成一个有2n个方格的图形，每一个方格对应变量的一个取值组合。具有逻辑相邻性的最小项在位置上也相邻地排列。
用卡诺图表示逻辑函数： 方法一：1、把已知逻辑函数式化为最小项之和形式。 2、将函数式中包含的最小项在卡诺图对应的方格中填 1，其余方格中填 0。 方法二：根据函数式直接填卡诺图。 用卡诺图化简逻辑函数： 化简依据：逻辑相邻性的最小项可以合并，并消去因子。 化简规则：能够合并在一起的最小项是2n个。 如何最简：圈数越少越简；圈内的最小项越多越简。 注意：卡诺图中所有的 1 都必须圈到，不能合并的 1 单独画圈。说明，一逻辑函数的化简结果可能不唯一。 合并最小项的原则： 1）任何两个相邻最小项，可以合并为一项，并消去一个变量。2）任何4个相邻的最小项，可以合并为一项，并消去2个变量。3）任何8个相邻最小项，可以合并为一项，并消去3个变量。 卡诺图化简法的步骤： 画出函数的卡诺图; 画圈（先圈孤立1格；再圈只有一个方向的最小项（1格）组合）；画圈的原则：合并个数为2n；圈尽可能大（乘积项中含因子数最少）；圈尽可能少（乘积项个数最少）；每个圈中至少有一个最小
一、选择题： 1．已知集合A ={}31<<-x x ，B ={}52≤ 电路的基本概念和基本定律 一、单项选择题 1、在图示电路中，U S ，I S 均为正值，其工作状态是 ( b )。 (a) 电压源发出功率 (b) 电流源发出功率 (c) 电压源和电流源都不发出功率 U S 图1 R 1图2 2、已知图示电路中的U S =2V ， I S = 2A 。电阻R 1和R 2 消耗的功率由( c )供 给 。 (a) 电压源 (b) 电流源 (c) 电压源和电流源 3、图示电路中，已知I SC ＝10A ，则电阻R 消耗的电功率P 为 ( c )。 (a) 600 W (b) 300 W (c) 240 W U 60 V 60 V S 2图3 U + R 图4 4、图示电阻元件R 消耗电功率10W ，则电压U 为( a )。 (a) -5 V (b) 5 V (c) 20 V 5、电路如图所示, 若R 、U S 、I S 均大于零，则电路的功率情况为 b (a) 电阻吸收功率, 电压源与电流源供出功率 (b) 电阻与电流源吸收功率, 电压源供出功率 (c) 电阻与电压源吸收功率, 电流源供出功率 (d) 电阻吸收功率，供出功率无法确定 U I S 图5 U +图6 6、图示元件供出电功率10W ，则电压U 为 ( a )。 (a) －5 V (b) 5 V (c) 20 V 7、在图示电路中，U 、I 的关系式正确的是( c )。 (a) U = U S + R 0 I (b) U = U S －R L I (c) U = U S － R 0 I R I L 8、在图示电路中，U 、I 的关系式正确的是 ( b )。 (a ) U = (I S + I )R 0 (b) U = (I S －I )R 0 (c) U = (I - I S )R 0 {}9B =,;B A =B B = )()(); U U B A B =? )()()U U B A B =? ()()card A B card A =+ ()()card B card A B - ()U A =e()U A =e13设全集，2，3，4A = {3，4，5} B = {4，7，8}, 求：（C U A ）∩ B), (C U A)(A ∪B), C U B). 有两相)(,2121x x x x <有两相等a b x x 221- ==无实根 有意义的 ①一个命题的否命题为真，它的逆 命题一定为真. （否命题?逆命 题.）②一个命题为真，则它的逆 否命题一定为真.（原命题?逆 否命题.） 4.反证法是中学数学的重要方法。 会用反证法证明一些代数命题。 充分条件与必要条件 答案见下一页 数学基础知识与典型例题(第一章集合与简易逻辑)答案 例1选A; 例2填{(2，1)} 注:方程组解的集合应是点集. 例3解:∵{}9A B =,∴9A ∈.⑴若219a -=,则5a =,此时{}{}4,9,25,9,0,4A B =-=-, {}9,4A B =-,与已知矛盾,舍去.⑵若29a =,则3a =±①当3 a =时,{}{}4,5,9,2,2,9A B =-=--.B 中有两个元素均为2-,与集合中元素的互异性矛盾,应舍去.②当3a =-时,{}{}4,7,9,9,8,4A B =--=-,符合题意.综上所述,3a =-. [点评]本题考查集合元素基本特征──确定性、互异性、无序性，切入点是分类讨论思想，由于集 合中元素用字母表示，检验必不可少。 例4C 例5C 例6①?,②ü,③ü,④ 例7填2 例8C 例9? 例10解：∵M={y|y =x 2+1，x ∈R}={y |y ≥1}，N={y|y =x +1，x ∈R}={y|y ∈R}∴ M∩N=M={y|y ≥1} 注：在集合运算之前，首先要识别集合，即认清集合中元素的特征。M 、N 均为数集，不能误认为是点集，从而解方程组。其次要化简集合。实际上，从函数角度看，本题中的M ，N 分别是二次函数和一次函数的值域。一般地，集合{y |y =f (x )，x ∈A}应看成是函数y =f (x )的值域，通过求函数值域化简集合。此集合与集合{（x ，y ）|y=x 2+1，x ∈R}是有本质差异的，后者是点集，表示抛物线y =x 2+1上的所有点，属于图形范畴。集合中元素特征与代表元素的字母无关，例如{y|y ≥1}={x |x ≥1}。 例11填?注：点集与数集的交集是φ. 例12埴?,R 例13解：∵C U A = {1，2，6，7，8} ,C U B = {1，2，3，5，6}, ∴(C U A)∩(C U B) = {1，2，6} ,(C U A)∪(C U B) = {1，2，3，5，6，7，8}, A ∪ B = {3,4,5,7,8},A∩B = {4},∴ C U (A ∪B) = {1,2,6} ,C U (A∩B) = {1,2,3,5,6,7,8} 例145,6a b ==-; 例15原不等式的解集是{}37|<<-x x 例16 53|332 2x R x x ??∈-<-+-->+?? ≥或,即3344123x x x x ? 2或x <31，∴原不等式的解集为{x | x >2或x <31}.方法2：(整体换元转化法)分析：把右边看成常数c ，就同)0(>>+c c b ax 一样∵|4x -3|>2x +1?4x -3>2x +1或4x -3<-(2x +1) ? x >2 或x < 31，∴原不等式的解集为{x | x >2或x <3 1}. 例18分析：关键是去掉绝对值. 方法1：零点分段讨论法（利用绝对值的代数定义） ①当1- 集合、简易逻辑、函数的易错点以及典型题型 1．研究集合必须注意集合元素的特征即三性(确定,互异,无序); 已知集合A={x,xy,lgxy}, 集合 B={0,｜x ｜,y},且A=B,则x+y= 2．研究集合,首先必须弄清代表元素,才能理解集合的意义。已知集合M={y ｜y=x 2 ,x ∈ R},N={y ｜y=x 2+1,x ∈R},求M ∩N ；与集合M={（x,y ）｜y=x 2 ,x ∈R},N={(x,y)｜y=x 2+1,x ∈R}，求M ∩N;以及M={x ｜y=x 2 ,x ∈R},N={y ｜y=x 2x ∈R},Q={（x,y ）｜y=x 2 ,x ∈R},求M ∩N ，M ∩Q ，Q ∩N 的区别。 3．区别?与{?}。 ?:表示空集，{?}：不是空集，是指含?的一个元素。 4．集合 A 、B ，?=?B A 时，注意“极端”情况：?=A 或?=B ；求集合子集B A ?时 否忘记?. eg. ()()012222<--+-x a x a 对一切R x ∈恒成立，求a 范围，讨论了a ＝2情况了吗？ 5．对于含有n 个元素的有限集合M, 其子集、真子集、非空子集、非空真子集的个数依次为 ，n 2， 12-n ，12-n .22-n 如满足条件}4,3,2,1{}1{??M 的集合M 共有多少个 6． 解集合问题的基本工具是韦恩图; 某文艺小组共有10名成员,每人至少会唱歌和跳舞中 的一项,其中7人会唱歌跳舞5人会,现从中选出会唱歌和会跳舞的各一人，表演一个唱歌和一个跳舞节目,问有多少种不同的选法？ 7． 两集合之间的关系。},14{},,12{Z k k x x N Z k k x x M ∈±==∈+== (C U A)∩( C U B) = C U (A ∪B) (C U A)∪( C U B) = C U (A ∩B)；B B A =I A B ??； 8.可以判断真假的语句叫做命题. 逻辑连接词有“或”、“且”和“非”. p 、q 形式的复合命题的真值表: p q P 且q P 或q 真 真 真 真 真 假 假 真 假 真 假 真 假 假 假 假 9.命题的四种形式及其相互关系 逆 互 互 互 为 互 否 逆 逆 否 否 否 原命题 若p 则q 逆命题 若q 则p 否命题 若﹃ｐ则﹃q 逆否命题 若﹃ｑ则﹃ｐ 泰安三中北校高二数学滚动测试卷3 1下列命题中的假命题是 C. V XG R,x 3 > 0 D. \/xeR,2x >0 }, B=|)j I y = x 2 f x w /?},则 AnB=() B. {xlx>0} D. 0 3?已知a>0,则xo 满足关于x 的方程ax=b 的充要条件是 (A)3x G R.—ax 1 -bx > —ax^ -bx^ (B) 3x e R.—ax 1 -bx < —axi -bx a 2 2 2 2 (C) Vx w /?, * ax 2 -bx>^ axl 一 bx () (D) Vxe ax 2 -bx < ax ： - bx 0 4. i(m<丄”是“一元二次方程兀2 + x + m = 0ff 有实数解的 4 A.充分非必要条件 C.必要非充分条件 D.罪充分必要条件 8. 函数y = J16二4^的值域是 (B) [0,4] (D) (0,4) 9. 设于（兀）为定义在/?上的奇函数，当兀》 （）时, f(x) = 2x +2x+b (b 为常数),则/(-!) = 5 ?已知函数/（X ） = log 3 x,x>0 2\x<0 A.4 1 B.— 4 C.-4 6 ?隊[数 y = . 1 = Jlog°.5(4x_3) 的定义域为 3 3 A.( -,D B(--) 4 4 7?隊|数f (%) = log 2(3X +1)的值域为 C (1, +°°) 3 D.( -,1)U (1, +8) 4 A. (0,+ 第一章半导体器件与放大电路习题 一、填空题 1. PN结的特性是单向导电性。 2.射极输出器具有：①输入电阻高，②输出电阻低，③放大倍数为1的特点。 3.互补对称功率放大电路中晶体管工作在甲乙类工作状态，主要是为了克服交越失真。 4.稳压二极管工作在反向击穿区，当外加电压撤除后，管子还是正常的，这种性能称为可逆性击穿。 二、选择题 1．电路如图1-1所示，所有二极管均为理想元件，则二极管D1、D2的工作状态为（Ｃ）。 A.D1、D2均截止 B. D1、D2均导通 C. D1导通，D2截止 D. D1截止，D2导通 图1-1 图1-2 2．电路如图1-2所示，二极管为理想元件，则电压U AB为（ A ）。 A. 6V B. 3V C. 9V D. 不确定 3.某晶体管工作在放大状态，测得三极电位分别是①～1.7V，②～1V，③～6.5V，则对三个电极的判定，( B )是正确的。 A.①～E，②～B，③～C B.①～B，②～E，③～C C.①～C，②～B，③～E D.①～B，②～C，③～E 4.若用万用表测二极管的正、反向电阻的方法来判断二极管的好坏，好的管子应为( C )。 A.正、反向电阻相等 B.正向电阻大，反向电阻小 C.反向电阻很大，正向电阻很小 D.正、反向电阻都等于无穷大 5.在N型半导体中参与导电的多数载流子是( A )。 A.自由电子 B.空穴 C.正离子 D.负离子 6.当温度升高时，晶体管的穿透电流I CEO ( A)。 A.增大 B.减小 C.不变 D.无法确定 7. 对于三极管放大作用的实质，下列说法正确的是（D）。 A.三极管可以把小能量放大成大能量 B.三极管可以把小电流放大成大电流 C.三极管可以把小电压放大成大电压 第一章 集合与常用逻辑用语知识结构 【知识概要】 一、集合的概念、关系与运算 1. 集合中元素的特性：确定性、互异性、无序性. 在应用集合的概念求解集合问题时，要特别注意这三个性质在解题中的应用，元素的互异性往往就是检验的重要依椐。 2. 集合的表示方法：列举法、描述法. 有的集合还可用Venn 图表示，用专用符号表示，如,,,,,,N N N Z R Q φ*+等。 3. 元素与集合的关系：我们把研究对象统称为元素，把一些元素组成的总体叫做集合，若元素x 是集合A 的元素，则x A ∈，否则x A ?。 4. 集合与集合之间的关系： ①子集：若x A ∈，则x B ∈，此时称集合A 是集合B 的子集，记作A B ?。 ②真子集：若A B ?，且存在元素x B ∈，且x A ?，则称A 是B 的真子集，记作：A B . ③相等：若A B ?，且A B ?，则称集合A 与B 相等，记作A ＝B .。 5. 集合的基本运算： ①交集：{}A B x x A x B =∈∈I 且 ②并集：{}A B x x A x B =∈∈U 或 ③补集：{|,}U C A x x U x A =∈?且，其中U 为全集，A U ?。 6. 集合运算中常用结论： ①,,A A A A A B B A φφ===I I I I ，A B A A B =??I 。 ②,,A A A A A A B B A φ===U U U U ，A B A B A =??U 。 ③()U A C A U =U ，()U C A A ?=I ， ()()()U U U C A B C A C B =I U ，()()()U U U C A B C A C B =U I 。 ④由n 个元素所组成的集合，其子集个数为2n 个。 集合、逻辑及函数 1、已知集合{}m A ,3,1=，{ }m B ,1=，A B A =?，则m= 2、设集合{}41<<=x x A ，集合{} 0322≤--=x x x B ，则()=?B C A R 3、已知集合{}32<+∈=x R x A ，集合()(){}02<--∈=x m x R x B ，且),1(n B A -=?，则m= ，n= 4、已知集合(){}(){},0,,2P x y x y Q x y x y =+==-=，则P Q ?= 5、设集合(){} {}25,log 3,,,(,)A a B a b a b R =+=∈，若{}1A B ?=，则A B ?= 6、已知集合{}1,0,2A =-， {} 2a B =，若B A ?，则实数a 的值为 7、已知集合{}220,A x x x x R =-≤∈，{} B x x a =≥，若A B B ?=，则实数a 的取值范围是 8、已知集合{}230A x x x =-≤，[]{} 22,2,1B y y x x ==-+∈-，则A B ?= 9、命题“若a b >，则221a b >-”的否命题为 10、 “1x >”是“2x x >”的 条件 11、设函数()f x =A ，则集合A Z I 中元素的个数是 12、函数y =的定义域是 13、已知全集U R =，函数 y =A ，函数()2log 2y x =+的定义域为集合B ，则集合()U C A B =I 14、函数()f x =的定义域为 15、已知集合{}1A x x =≤，{}222B y y x x ==++，则A B =I 16、方程()2log 121x -=-的解x = 17、已知集合{}2log 2,(,)A x x B a =≤=-∞，若A B ?，则实数a 的取值范围是(),c +∞，其中c = 18、已知函数()()1,23,2 x f x x f x x -?+≤?=? >??，则()3log 2f 的值为 19、（12苏州期中调研，6）设函数()()???>-≤=-0 ,10,31x x f x x f x ，则()()=-1f f 20、设0a >且1a ≠，则“函数()x f x a =在R 上是减函数”是“函数()()32g x a x =-在R 上是增函数”的 条件 21、设R ?∈，则“0?=”是“()()()cos f x x x R ?=+∈为偶函数”的 条件 22、（常州市2013届高三期末）函数22()log (4)f x x =-的值域为 ． 第1章 直流电路习题 一、单项选择题 1．图示电阻元件R 消耗电功率10W ，则电压U 为( )。 A ）-5V B ）5V C ）20V U I 2 A － + R 题1图 2．图示电路中，A 点的电位V A 为( )。 A ）2 V B ）-4 V C ） -2 V . A －6V 12V . 2 k 7 k ΩΩ 题2图 3．图示电路中，U 、I 的关系式正确的是( )。 A ）U = (I S + I )R 0 B ）U = (I S －I )R 0 C ）U = (I - I S )R 0 I I R U R S 0L . . . . +－ 题3图 10A 342Ω Ω ΩI I 12 10 A 6 A . . . 题4图 4．图示电路中电流I 2为( )。 A ）7A B ）3A C ）-3A 5．理想电流源的外接电阻越大，则它的端电压( )。 A ）越高 B ）越低 C ）不能确定 6．把图1所示的电路改为图2的电路，其负载电流I 1和I 2将( )。 A ）增大 B ）不变 C ）减小 2A I I I I 1 2 122V 1Ω 1Ω 1Ω 1Ω 2V 2A 图 1 图 2 + 题6图 7．图示电路中，供出功率的电源是( )。 A ）理想电压源 B ）理想电流源 C ）理想电压源与理想电流源 8．在图示电路中，各电阻值和U S 值均已知。欲用支路电流法求解流过电阻R G 的电流I G ，需列出独立的电流方程数和电压方程数分别为( )。 A ）4和3 B ）3和3 C ）3和4 I R U 2A 1 4V S S 1Ω 1Ω. . +题7图 R R R R R I U 1 2 3 4 G G S . . .. +题8图 9．在计算线性电阻电路的电压和电流时，用叠加原理。在计算线性电阻电路的功率时，加原理( )。 A ）可以用 B ）不可以用 C ）有条件地使用 10．在图示电路中，已知U S =12V ，I S =2A 。A 、B 两点间的电压U AB 为( )。 A ）-18V B ）18V C ）-6V U I A B S S Ω 3+ 题10图 . －6V S A . 1k 1k Ω Ω 题11图 11．在图示电路中，当开关S 闭合时A 点的电位V A ( )。 A ） -3V B ）3V C ）0V 12．图示电路中，理想电流源I S1发出的电功率P 为( )。 A ）-3W B ）21W C ）3W I I U S 1 S 2 3 A 1 A 4 V 1 ΩS 2. . +题12图 I I 12A 2363Ω ΩΩ Ω S . . .. 题13图 13．图示电路中，电流I 是( )。 A ）3A B ）0A C ）6A 二、分析计算题 1.1 （1）试求如图所示电路中的432,,U I I 。 （2）求如图所示电路中A 点电位。 （集合与简易逻辑） 一、选择题：本大题共12小题，每小题5分，共60分，在每小题给出的四个选项中，只有一项是符合题目要求的。 1、设P，Q为两个非空实数集合，定义集合P+Q={a+b|, a∈P，b∈Q}，若P={0，2，5}，Q={1，2，6}，则P+Q中元素的个数是（） A.9 B.8 C.7 D.6 2、若集合M=｛y| y=｝，P=｛y| y=｝，则M∩P= （） A｛y| y>1｝B｛y| y≥1｝C｛y| y>0｝D｛y| y≥0｝ 3、下列四个集合中，是空集的是( ) A . B . C. { D .. 4、若关于x的不等式<1的解集为{x|x <1或x > 2},则实数a的值为( ) A.1 B.0 C.2 D. 5、已知集合M=｛a2, a+1,-3｝, N=｛a-3, 2a-1, a2+1｝, 若M∩N=｛-3｝, 则a的值是( ) A -1 B 0 C 1 D 2 6、设集合A={x| < 0},B={x||x-1| A.35 B.25 C.28 D.15 8、一元二次方程有一个正根和一个负根的充分不必要条件是：（） A．B．C．D． 9、若二次不等式ax2+bx+c > 0的解集是{x| < x <},那么不等式2cx2-2bx-a < 0的解集是( ) A.{x|x< -10或x > 1} B.{x|－< x <} C.{x|4< x <5} D.{x|-5< x < -4} 10、已知函数f(x)在(-∞,+∞)上为增函数,a,b∈R,对于命题“若a+b≥0，则f(a)+f(b)≥f(-a)+f(-b)”有下列结论: ①此命题的逆命题为真命题②此命题的否命题为真命题 ③此命题的逆否命题为真命题④此命题的逆命题和否命题有且只有一个真命题 其中正确结论的个数为( ) A.1个 B.2个 C.3个 D.4个 11、对任意实数, 若不等式恒成立, 则实数的取值范围是( ) A k≥1 B k <1 C k≤1 D k >1 12、若集合A B, A C, B=｛0,1,2,3,4,7,8｝, C=｛0,3,4,7,8｝, 则满足条件的集合A 的个数为( ) A. 16 B 15 C 32 D 31 二、填空题：本大题共4小题；每小题4分，共16分，把答案填在题中的横线上。 练习一、1．分别计算图示电路中各电源的端电压和它们的功率。 (5分) 解：(a ) …………2分 (b ) 电流源： ……….1.5分 电压源： …………1.5分 2．利用电源等效变换化简各二端网络。 (6分) 解: (a) 3 ．计算图示电路在开关S 打开和闭合时a 点的电位?=a U (5分) 解：开关S 打开：mA I 25.11 121 6=++-= ……..1分 V U a 25.225.111=?+= …………………1.5分 开关S 闭合：V U 8.22 141212 131=+++ = ….1分 V U U a 9.11 11 111=+- ? += ….1.5分 4．求图示电路中的电流1I 和电压ab U 。(5分) 解：A I 25 10 9.01== A A I 222.29 20 1== ……….2.5分 (a ) (b ) W U P V U 5051052=?==?=发W U P V U 1052=?==发W P A I V U 6323252=?==-==吸 ，方向向下 a b a b b b b a U b A A I I U ab 889.09 8 4)9.0(11== ?-= ……..2.5分 5.电路如图所示，求X I 。(5分) 解： 电压源单独作用：4)42(-='+X I A I X 3 2 - ='? ……..1.5分 电流源单独作用：A I X 3 2 2422=?+= '' ……..1.5分 故原图中的03 2 32=+- =''+'=X X X I I I ………..2分 6、计算图四（4）所示二端网络吸收的有功功率、无功功率，并计算其视在功率和功率因数。其中,())(2sin 25V t t u =, R 1=1Ω, R 2=2Ω。 解：端口电压为?∠=05U . 对于2=ω, X C = - 2j , X L =1j . 端口阻抗为 ()()()()() Ω-=-++-+= j j j j j Z 34221221 (2分) 端口电流： ?-∠=-=4.18953.3345 j I A （2分） 有功功率：()W P 75.18]4.180cos[953.35=?--??= （2分） 无功功率：VA Q 24.64.18sin 953.35=??= （2分） 视在功率：VA Q P S 764.1922=+=（1分） 功率因数：95.0cos == S P ?（1分） Ω 2Ω 2Ω 2 第2章逻辑函数及其化简 内容提要 本章是数字逻辑电路的基础，主要内容包含： （1）基本逻辑概念，逻辑代数中的三种基本运算（与、或、非）及其复合运算（与非、或非、与或非、同或、异或等）。 （2）逻辑代数运算的基本规律（变量和常量的关系、交换律、结合律、分配律、重叠律、反演律、调换律等）。 （3）逻辑代数基本运算公式及三个规则（代入规则、反演规则和对偶规则）。 （4）逻辑函数的五种表示方法（真值表法、表达式法、卡诺图法、逻辑图法及硬件描述语言）及其之间关系。本章主要讲述了前三种。（5）逻辑函数的三种化简方法（公式化简法、卡诺图法和Q–M法）。教学基本要求 要求掌握： （1）逻辑代数的基本定律和定理。 （2）逻辑问题的描述方法。 （3）逻辑函数的化简方法。 重点与难点 本章重点： （1）逻辑代数中的基本公式、基本定理和基本定律。 （2）常用公式。 （3）逻辑函数的真值表、表达式、卡诺图表示方法及其相互转换。 （4）最小项和最大项概念。 （5）逻辑函数公式化简法和卡诺图化简法。主要教学内容 2.1 逻辑代数中的三种基本运算和复合运算2.1.1 三种基本运算 2.1.2 复合运算 2.2 逻辑代数运算的基本规律 2.3 逻辑代数的常用运算公式和三个规则2. 3.1 逻辑代数的常用运算公式 2.3.2 逻辑代数的三个规则 2.4 逻辑函数及其描述方法 2.4.1 逻辑函数 2.4.2 逻辑函数及其描述方法 2.4.3 逻辑函数的标准形式 2.4.4 逻辑函数的同或、异或表达式 2.5 逻辑函数化简 2.5.1 公式法化简 2.5.2 卡诺图化简 2.1 逻辑代数中的三种基本运算和复合运算 2.1.1 三种基本运算 1. 与运算（逻辑乘） 2. 或运算（逻辑加） 3. 非运算（逻辑非） 2.1.2 复合运算 1. 与非运算 与非运算是与运算和非运算的组合，先进行与运算，再进行非运算。 2. 或非运算 高二文科数学月考检测 一 选择题 1. 集合}log ,2{3a M =，},{b a N =，若}1{=?N M ，则N M U =（ ） A 、{0，1，2} B 、{0，1，3} C 、{0，2，3} D 、{1，2，3} 2. 已知命题p 、q ，“p ?为 真”是“p q ∧为假”的 ( ) A ．充分不必要条件 B ．必要不充分条件 C ．充要条件 D. 既不充分也不必要条件 3.下列函数中与函数y x =是同一函数的是 ( ) A ．()2y x = B.33y x = C.2 y x = D.2 x y x = 4.下列命题中，真命题是 A ．存在,0x x e ∈≤R B ．1,1a b >>是1ab >的充分条件 C ．任意2,2x x x ∈>R D ．0a b +=的充要条件是1a b =- 5.已知)(x f 是定义在R 上的奇函数，对任意R x ∈，都有)()4(x f x f =+，若2)1(=-f ，则)2013(f 等于（ ） A 、-2 B 、2 C 、2013 D 、2012 6.当(0,)x ∈+∞时，幂函数21(1)m y m m x --=--为减函数，则实数m =（ ） A ．m=2 B ．m=-1 C ．m=2或m=1 D ． 152 m +≠ 7. 函数y=x ln(1-x)的定义域为（ ） A ．（0,1） B.[ 0,1) C.( 0,1] D.[ 0,1] 8.函数sin ((,0)(0,))x y x x =∈-π?π的图象大致是 9.设()lg(101)x f x ax =++是偶函数，4()2x x b g x -=是奇函数，那么a ＋b 的值为 A ．1 B ．－1 C ．21 D ．－2 1 10.定义方程f (x )＝f ′(x )的实数根x 0叫做函数f (x )的“新驻点”，若函数g (x )＝2x ，h (x )＝ln x ，φ(x )＝x 3(x ≠0)的“新驻点”分别为a ，b ，c ，则a ，b ，c 的大小关系为( ) A ．a >b >c B ．c >b >a C ．a >c >b D ．b >a >c 二 填空题 11. 命题“?x ∈R ，x 2>4”的否定是____ _____． 12.设函数32)(+=x x f ，)()2(x f x g =+，则=)(x g 。 13.曲线 22y x x =+-在点()1,0处的切线方程为 14.已知函数???≥-<=, 1),1(,1,2)(x x f x x f x 则=)8(log 2f 15. 定义在R 上的偶函数)(x f 满足：)()1(x f x f -=+，且在[－1，0]上是增函数，下列关于)(x f 的判断：①)(x f 是周期函数；②)(x f 的图象关于直线2=x 对称；③)(x f 在[0，1]上是增函数；④)(x f 在[1，2]上是减函数；⑤)0()4(f f = 其中判断正确的序号是 。 三 解答题 16.命题p ：关于x 的不等式a 2240x ax ++>对一切R x ∈恒成立；命题q ：函数()(32)x f x a =-是增函数,若p 或q 为真，p 且q 为假，求实数a 的取值范围. 高中数学集合、逻辑、函数、向量、数列、不等式、立体 几何 综合测试题 一、选择题：本大题共12小题，每小题5分，共60分．在每小题给出的四个选项中，只有一项是符合题目要求的．每小题选出答案后，请填涂在答题卡上． 1. 若非空集合}5,4,3,2,1{?S ，且若S a ∈，则必有S a ∈-6，则所有满足上述条件的集合S 共有 A ．6个 B ．7个 C ．8个 D ．9个 2. 命题P ：若函数()f x 有反函数，则()f x 为单调函数；命题Q ： 111 222 a b c a b c == 是不等式21110a x b x c ++>与2 2220a x b x c ++>(121212a a b b c c ，，，，，均不为零)同解的充要条件，则以下是真命 题的为 A ．P ?且Q B ．P 且Q C ．P ?或Q D ．P 或Q 3. 若函数)10(log )(<<=a x x f a 在区间]2,[a a 上的最大值是最小值的3倍，则a = A ． 42 B ．22 C ．41 D ．2 1 4. 如图，一个空间几何体的三视图如图所示，其中，主视图中ABC ?是边长为2的正三角形，俯视图为正六边形，那么该几何体的体积为 C. 32 D. 3 左视图 主视图俯视图 5. 已知函数bx x x f +=2 )(的图象在点))1(,1(f A 处的切线l 与直线0223=+-y x 平行，若数列}) (1 { n f 的前n 项和为n S , 则2012S 的值为 A ．20102009 B ．20112010 C ．20122011 D ．2013 2012 6. 若m b a m a f 2)13()(-+-=，当]1,0[∈m 时，1)(≤a f 恒成立，则b a +的最大值为 A ． 31 B ．32 C ．35 D ．3 7 7. 已知a 、b 是不共线的向量，()AB AC R λμλμ=+=+∈， ，a b a b ，那么A B C 、、三点共线的充要条件为 A ．1λμ= B ．1λμ=- C ．1=-μλ D ．2λμ+= 8. 设平面上有四个互异的点A 、B 、C 、D ，已知（,0)()2=-?-+则ABC ?的形状是 A ．直角三角形 B ．等腰三角形 C ．等腰直角三角形 D ．等边三角形 9. 设函数()(sin cos )(02011),x f x e x x x π=-≤≤则函数()f x 的各极大值之和为1.《电工技术基础》复习题-电路定律 (2)
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