cloning and characterization of a novel r1-myb

Cloning and characterization of a novel R1-MYB transcriptionfactor in maize

Guanqing Jia, Bo Li, Dengfeng Zhang, Tifu Zhang, Zhiyong Li, Jingrui Dai, Shoucai Wang *

National Maize Improvement Center of China, Key Laboratory of Crop Genomics and Genetic Improvement of Agriculture Ministry,

Department of Plant Genetics & Breeding, China Agricultural University, Beijing 100193, China

Received 23 December 2008; received in revised form 16 January 2009; accepted 6 February 2009

Abstract

R1-MYB transcription regulatory factors play vital roles in transcriptional modification during higher plant metabolism and devel-opment. In the present study, an R1-MYB gene was isolated from the maize (Zea mays L.) inbred line C8605-2 based on expressedsequence tags (ESTs), which expressed differentially between the hybrid C8605-2 �W1445 and its two parents in an oligonucleotidemicroarray. The full-length cDNA, designated as ZmMYBE1 (GeneBank Accession No. FJ024049), consists of 1992 nucleotides andcontains a 1296-bp open reading frame. One conserved MYB domain was detected near the N-terminus of the deduced amino acidsequence, where an acidic Ser/Thr-rich area was also observed in the downstream region. The sequence of ZmMYBE1, mapped to chro-mosome 5 (bin 5.03), was revealed belonging to a multi-gene family in the maize genome. Subcellular localization analysis determinedthat ZmMYBE1 was located in the nucleus. Expression analysis showed that ZmMYBE1 transcripts accumulated in various tissuesexamined, with high expression levels in the immature ear and low levels in the tassel, root, and stem. The expression analysis forZmMYBE1 in immature ears showed the highest expression level at the earliest vegetative developmental stage of the plant. Expressionof ZmMYBE1 higher than that of the parents was observed in the hybrid C8605-2 �W1445. And also the expression level similar to thelowest of the parents in the hybrid C8605-2 �W245 was observed, revealing different expression patterns for the same gene in varioushybrids combined from diversified inbred lines. Transgenic Arabidopsis overexpressing ZmMYBE1 implies that ZmMYBE1 has animportant role in developmental regulation in maize.� 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science inChina Press. All rights reserved.

Keywords: MYB transcription factor; Differential expression; Maize

1. Introduction

Transcription factors, also known as trans-acting pro-teins, are defined as regulatory proteins that control theearly transcription course of various functional genes,whose physiological roles have been widely verified in plantdevelopment, tissue organization, compound metabolism,and stimulus response. In Arabidopsis, over 5% of the geno-mic regions code for more than 1500 trans-acting proteins,

and over 1300 proteins have been classified as transcrip-tional regulators in rice. MYB proteins constitute a hugefamily with the largest number of members in plants: 190have been deduced in Arabidopsis, 156 in rice, and morethan 200 estimated in maize [1–3].

MYB transcriptional regulators contain conservedDNA-binding domains that are usually composed of one,two, or three imperfect 51 or 52 residue repeats (R1, R2,and R3). Each repeat encodes three a-helices, with the sec-ond and third helices forming a helix–turn–helix (HTH)structure when bound to DNA [4,5]. In plants, the over-whelming majority of MYB proteins are classified into a

1002-0071/$ - see front matter � 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited

and Science in China Press. All rights reserved.

doi:10.1016/j.pnsc.2009.02.004

* Corresponding author. Tel./fax: +86 010 62732409.E-mail address: [email protected] (S. Wang).

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Progress in Natural Science 19 (2009) 1089–1096

subfamily characterized by the presence of R2R3 motifs. Incontrast to animals, proteins containing one (R1) or three(R1R2R3) motifs, which constitute MYB-family proteinsalong with R2R3-MYB trans-actors, are also widelyobserved in plant cells [6–9]. Functional analysis of MYBproteins has determined that these genes may play tran-scription regulatory roles in cellular morphogenesis, sec-ondary metabolism, the accommodation of responses todiseases and hormones, modulation of cell cycles [10,11],and especially maintenance of chromosome structure,responses to light rhythms, and formation of root hairsfor R1-MYB proteins in plants [12–14].

A handful of MYB proteins have been functionally ver-ified in terms of the largest number of associations forMYB transcription factors in plants. The trials introducedhere aimed to isolate the full-length sequence of a new R1-MYB gene ZmMYBE1 in maize, whose expression rulesand positions on the chromosome linkage map andthrough the cell organelles were systematically analyzed.Arabidopsis lines overexpressing ZmMYBE1 were identi-fied and used to examine ZmMYBE1 in plants, and the roleof ZmMYBE1 in the grain yield of maize is discussed withregard to the formation of premature ears.

2. Materials and methods

2.1. Plant materials

Previous studies on maize hybrids have been carried outby the authors, consisting of an incomplete diallel crossdesign containing 33 hybrids propagated from 12 inbredmaize lines and examination of yields arranged in random-ized blocks for 3 years. Two of these hybrids (C8605-2 �W1445, C8605-2 �W245), sharing one commonfemale parent, and having higher and lower yields, respec-tively, among all the hybrids, were selected for in-depthanalysis [15].

Immature ears at the spikelet branch primordial stagefrom the three inbred lines C8605-2, W1445, and W245and the two hybrids C8605-2 �W1445 and C8605-2 �W245 were used. The hybrid C8605-2 �W1445 hadsuperior grain yield heterosis compared to C8605-2 �W245. The root (aerial root), stalk (below the highestknob), leaf (at knob where the ear grew), tassel, and imma-ture ear of C8605-2 at the joining stage (when immature earwas at the stage of spikelet branch primordial) and imma-ture ears at four different developmental stages (i.e., spike-let branch primordial, spikelet differentiation, early flowerdifferentiation, and sexual organ maturation) fromC8605-2 were utilized for gene cloning and transcriptomevariation analysis. C8605-2 has been widely used in Chinaas an elite inbred line, and W1445 and W245 are new elitelines that were developed in the authors’ laboratory.

All the materials were collected and pooled from groupsof at least 10 individuals at the same time of day (8:00–10:00 AM), and were immediately frozen and stored at�80 �C until RNA extraction.

2.2. Cloning of full-length cDNA and genomic DNA

Total RNA isolated from immature ears of C8605-2using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA)was reverse transcribed using M-MLV Reverse Transcrip-tase (Promega, Madison, WI, USA), following the manu-facturer’s instructions. The three nested gene-specificprimers 5’-CCATTTCCTTGCCCTTGCC-3’, 5’-CTCCCTCCGTTCCTCACG-3’, and 5’CCGGAGAAGGGACCGAATT-3’, designed from the differentially expressedEST (ID in TIGR: TM00022889), and Oligo dT-Adapterprimer were used to obtain the 3’ end fragment of thegene. The genomic DNA was obtained by primer 5’-TAAATCCGTCTACTGCTTCTATGC-3’ (forward) and5’-AGGTCACCCGCATCTTACATTC-3’ (reverse) usingLA tag (TaKaRa). The polymerase chain reaction (PCR)products were purified and cloned into the pGEM-Teasyvector (Promega) for sequencing. Sequence analysiswas performed using SMART [16], clustalx1.83 [17], DNA-MAN (Version 5.2.2, Lynnon Biosoft, Canada), andPromoter Finder [18] programs.

2.3. Semi-quantitative reverse transcriptase (RT)-PCR andreal-time PCR analysis

Total RNA from all the materials digested with RQ1RNase-free DNase (Promega) was used for first strandcDNA synthesis with a negative control obtained withoutthe addition of reverse transcriptase. The specific primersfor amplification of the full-length open reading frame ofZmMYBE1 were 5’-AAGACAGTTATCATCCCAGACCCA-3’ (forward) and 5’-AACCGTAGTAAGGCCACCACAT-3’ (reverse). The gene GAPDH was used as aninternal control in maize, and was amplified with primers5’-CCCTTCATCACCACGGACTAC-3’ (forward) and5’-AACCTTCTTGGCACCACCCT-3’ (reverse). In Ara-

bidopsis, the absence of contaminating DNA in theRNA samples was confirmed using the actin primer sets5’-CATCAGGAAGGACTTGTACGG-3’ and 5’-GATGGACCTGACTCGTCATAC-3’ spanning the introns.Various PCR cycles were tested to ensure that the reactiondid not reach the plateau for RT-PCR. Finally, three RT-PCR replications were conducted using the followingamplification profile: a 5-min denaturation step at 94 �Cwas followed by different cycles (30 and 28 cycles for maize,and 28 cycles for Arabidopsis) of 45 s at 94 �C, 45 s at56 �C, 45 s at 72 �C, and a final extension for 10 min at72 �C. Gel images were analyzed by software ImageToolversion 3.0. The nine (three biological and three technical)replications for real-time PCR were conducted using thefollowing amplification profile: a 5-min incubation step at94 �C was followed by 39 cycles of 30 s at 94 �C, 30 s at56 �C, 30 s at 72 �C, reading of the plate before and after1 s incubation at 81.5 �C, a final extension for 5 min at72 �C, and then reading of the melting curve from 65 to98 �C every 0.2 �C with each temperature held for 1 s.Analysis of the results of real-time PCR was carried out

1090 G. Jia et al. / Progress in Natural Science 19 (2009) 1089–1096

according to the operations manual for the Chromo4TM sys-tem by Opticon 3 software (MJ Research, Watertown,MA).

2.4. Electronic in silico mapping and subcellular localization

of ZmMYBE1

The genomic sequence of ZmMYBE1 was analyzedusing the BLASTN (Basic Local Alignment Search Tool)algorithm [19] in NCBI (http://www.ncbi.nlm.nih.gov/),the result of which was submitted to the maize FPC Map(http://www.genome.arizona.edu/fpc/WebAGCoL/maize/WebFPC) [20] to search for the nearest molecular marker,and finally the chromosomal genetic linkage map forZmMYBE1 was obtained from MaizeGDB (http://www.maizegdb.org/).

Full-length ZmMYBE1 was amplified using PCR incor-porating a unique XbaI restriction site immediatelyupstream from the translational start codon (5’-TCTAGACAAGAAGAGCCTACCAAGGTG-3’) and a uniqueBamH I restriction site immediately downstream from thetermination codon (5’-GGATCCGGCGATTCGACCTTACA-3’). The amplified fragment was cloned intopGEM-Teasy, sequenced, and subcloned into pMON30049[21] to generate a ZmMYBE1:GFP translational fusioncassette under the control of the 35S promoter ofcauliflower mosaic virus. The resulting construct,pMON30049-ZmMYBE1, was bombarded into onionepidermal cells, and green fluorescent protein (GFP)fluorescence was visualized using a confocal microscope(Bio-Rad, MRC1024, USA) under 488 nm exciting light.

2.5. Overexpression of ZmMYBE1 in Arabidopsis

The ZmMYBE1 coding sequence was inserted into thepCAMBIA3301 vector (CAMBIA, Canberra, Australia)with Bgl II and BstE II restriction sites amplified by theprimers 5’-TGAGATCTAAGAGCCTACCAAGGTG-3’(forward) and 5’-AGGTCACCCGCATCTTACATTC-3’(reverse). Vector pCAMBIA3301 carries a selective markergene (bar), which is used to screen transgenic plants withphosphinothricin (PPT). These constructs were mobilizedinto Agrobacterium tumefaciens GV3101 and then intro-duced into wild-type Arabidopsis using floral dip methods[22]. The plants were grown at 22 �C under long-day condi-tions (16 h light/8 h dark cycle) throughout the study.

3. Results

3.1. Cloning and characterization of the maize ZmMYBE1

gene

A maize oligonucleotide array (version 1.3) from Ari-zona containing 55,000 oligonucleotides was used to detectdifferentially expressed genes among maize inbred linesC8605-2 and W1445, and their hybrid C8605-2 �W1445[15]. An expressed sequence tag (EST, ID: TM00022889)

coding for a putative MYB trans-acting protein showed ahigher expression level in the hybrid compared with thetwo parents. The 3’ region fragment was obtained throughRT-PCR with three specific primers designed from basesdownloaded from the Internet (http://www.tigr.org/).mRNA from C8605-2 was used as a template.

Sequence analysis revealed that ZmMYBE1 consists of1992 nucleotides and contains a 1296-bp open readingframe, a 228-bp 5’ untranslated region, and a 465-bp 3’untranslated region. The putative ZmMYBE1 protein con-tains 432 amino acids and has a calculated molecularweight of 46.9 kDa with a pI of 5.34. Compared with itscorresponding genomic DNA, six exons and five intronswere identified and a TAAAATAT box was found nearthe transcription start site (Fig. 1(a) and (b)) [23]. Onestructurally conserved MYB domain was characterizednear the N-terminal of ZmMYBE1, which was conservedamong different species with regularly spaced Trp andAla residues (Fig. 1(c)). Full-length amino acid sequencesfrom different species were compared and a phylogenetictree showed similarity to the actual relationships amongspecies, which implies that ZmMYBE1 may code for a typ-ical R1-MYB protein (Fig. 1(d)). Several worthy featureswere identified: a nuclear localization signal (NLS)sequence was found immediately downstream of the R1domain, and an acidic Ser/Thr-rich area was observed inthe downstream region of the NLS fragment. Based onthese results, ZmMYBE1 may be localized to the nucleusand act as an activated domain for DNA transcriptionand four casein kinase II phosphorylation motifs (T/S-X-X-D/E) (Fig. 1(b)).

3.2. Analysis of expression rules for ZmMYBE1 in maize

Expression analysis of ZmMYBE1 in the inbred lineC8605-2 revealed that there was tissue-specific expressionamong the root, leaf, stalk, immature ear, and tassel, indi-cating that ZmMYBE1 transcripts accumulated differentlyin various tissues, with the highest expression level in theimmature ear, lower levels in the leaf, root, stalk and tassel,which suggests that ZmMYBE1 may play a more impor-tant role in the immature ear. Expression analysis of imma-ture ears at four different developmental stages (i.e.,spikelet branch primordial, spikelet differentiation, earlyflower differentiation, and sexual organ maturation) indi-cates that ZmMYBE1 is more significant at the earliestdevelopmental stages of the ears (Fig. 2(a)). To investigatethe role of ZmMYBE1 in early formation of heterosis,immature ears at the spikelet branch primordial stage fromthe three inbred lines C8605-2, W1445, and W245, and thetwo hybrids C8605-2 �W1445 and C8605-2 �W245 wereused to detect transcript accumulations of ZmMYBE1. Therelative expression levels of ZmMYBE1 were 0.24 ± 0.04for W1445, 0.38 ± 0.05 for C8605-2, 0.10 ± 0.02 forW245, 0.44 ± 0.09 for C8605-2 �W1445, and 0.14 ± 0.02for C8605-2 �W245, indicating expression above theparental level in hybrid C8605-2 �W1445 and expression

G. Jia et al. / Progress in Natural Science 19 (2009) 1089–1096 1091

similar to the lowest of the parents in hybrid C8605-2 �W245 (Fig. 2(b)).

3.3. In silico mapping and subcellular localization of

ZmMYBE1

The similarity of the amino acid sequence to other MYBtranscription factors indicated that ZmMYBE1 might

localize to the nucleus. To test this possibility, a CaMV35S-ZmMYBE1-green fluorescent protein (GFP) fusion cas-sette was constructed, followed by transient transfectionassays in onion epidermal cells. Epifluorescence analysisshowed that the ZmMYBE1-GFP fusion protein was local-ized exclusively to the nucleus (Fig. 3).

The genomic sequence of the isolated ZmMYBE1 wasanalyzed by searching for its corresponding genomicDNA against the GenBank databases using the BLASTN(Basic Local Alignment Search Tool) algorithm. The out-put result (GenBank ID: AC206634.2) displayedZmMYBE1 in maize, compared with the maize FPCMap (Clone ID: b0285F15), the SSR marker umc1870was identified near ZmMYBE1 in bin 5.03 of the geneticlinkage map IBM2 (Fig. 4) downloaded from the Internet(http://www.maizegdb.org/). Several yield-related quanti-tative trait loci (QTLs) have been mapped to the samechromosome region in previous studies.

3.4. Functional analysis through overexpression of

ZmMYBE1 in Arabidopsis

To investigate the potential physiological role ofZmMYBE1, transgenic Arabidopsis plants overexpressingZmMYBE1 were studied. A plasmid vector containing aCaMV35S promoter plus ZmMYBE1 was constructedand integrated using Agrobacterium into the genome ofthe Columbia wild type of Arabidopsis by floral dipmethods.

Five transgenic lines, screened using phosphinothricin(PPT), were obtained and the expression of ZmMYBE1

was confirmed by RT-PCR analysis. Positive plants wereplanted at 22 �C under long-day conditions (16 h light/8 hdark cycle) throughout the study. Three individual linesshowed obvious and identical changed phenotypes: aslower growth rate at the vegetative development stage, a

Fig. 1. Isolation and sequence analysis of ZmMYBE1. (a) Gene structureof ZmMYBE1. Filled boxes indicate exons and lines between boxesindicate introns. The two filled triangles mark the TATA box detected andthe transcriptional start site. Numbers show the relative positions on thegenomic DNA: +455 indicates the initiation codon ATG and +2617indicates the stop codon TAA. 2163 bp represents DNA sequence lengthrelative to the cDNA coding regions of ZmMYBE1. (b) Nuclear anddeduced amino acid sequences of ZmMYBE1. The initiation and stopcodon are underlined and the stop codon is marked with an asterisk. Boldblack bars mark the R1 domain. The nuclear localization signals (NLS)are underlined with broken lines and a Ser/Thr area is underlined. (c)Alignment of the amino acid sequences of the R1 domain of ZmMYBE1

and R1-MYB proteins from grap (GeneBank ID: CAN68733), medick(GeneBank ID: ABO83505), tomato (GeneBank ID: AAX44342), peri-winkle (GeneBank ID: ABL63123), soybean (GeneBank ID: ABH02924),rice (GeneBank ID: NP_001058645), and Arabidopsis (GeneBank ID:NP_198542) using the CLUSTALX1.83 program. Identical amino acidresidues are colored; asterisks represent the positions of conserved Trp andAla residues in the MYB domain. (d) The phylogenetic relationship ofZmMYBE1 and seven other R1-MYB proteins based on full-lengthsequences using the DNAMAN program.

3


shorter stem, later florescence (Fig. 5(a)), and a longergrowing period (Fig. 5(b)), which are inferred to relate toessential roles of ZmMYBE1 in plants. To validate obser-vations of the T1 generation lines, positive T2 generationlines for each of the three positive T1 plants were also con-firmed using RT-PCR and observed under the same growthconditions, the records for which showed identical pheno-types to their counterpart T1 lines compared with the CKlines (Table 1). After two generations of observations, itcan be confirmed that the flowering time for transgenicplants was extended for nearly 2 months beyond when

the growing time for the CK lines had ended. The expres-sion of ZmMYBE1 in two independent transgenic linesshowed that obvious phenotype changes were confirmedby RT-PCR analysis with wild-type lines as the control(Fig. 5(c)).

4. Discussion

An R1-MYB protein gene ZmMYBE1 with an entireopen reading frame was isolated using 3’ RACE in thisstudy. The structurally conserved R1 MYB domain sug-gests its important role in maize compared with other spe-cies [10,24], and the existence of the acidic Ser/Thr-richarea reveals its function in transcriptional regulation.

As the carrier for grain yield, immature ears of hybridsmay represent an early morphological manifestation ofgrowth vigor in maize. Several investigations have been car-ried out on the immature ears from two parents and thehybrid at different developmental stages using variousresearch methods at the expression level [25–28]. In thisstudy, ZmMYBE1 in the immature ear showed the highestexpression level among various tissues, and there werehigher transcriptional levels in immature ears at the earliestdevelopmental stages compared with later ones, whichimplies more important roles for ZmMYBE1 in immatureears at the earliest developmental phases. Two hybrids shar-ing one common female parent were used to evaluate theexpression level of ZmMYBE1 in different combinations,

Fig. 2. Transcriptom variation analysis of ZmMYBE1. (a) Expression analysis of different tissues (left) and in immature ears from various developmentalstages (right) by RT-PCR. Histograms below showed relative expression levels of ZmMYBE1 compared with GAPDH for three repeats analyzed bysoftware Imagetool. (b) Varying expression levels in immature ears among hybrids (C8605-2 �W1445, C8605-2 �W245) and parents (W1445, C8605-2,W245) by real-time PCR. GAPDH from maize was amplified as a control.

Fig. 3. Localization of ZmMYBE1 in onion epidermal cells. Epifluores-cence analysis showing GFP fluorescence in the nucleus. (Left) view under488 nm exciting light; (right) view under white light. The red arrowsindicate the nucleus.


and had obviously different grain yields and displayed verydifferent levels of midparent heterosis. The superior hybridhad a higher expression level than the parents and the infe-rior one had expression similar to that of the lowest parent.These results suggest that ZmMYBE1 may be involved inthe degree of grain yield heterosis at the transcription level.In tomato, the heterochromic differential expression offw2.2 clearly influences fruit size [29] and transcriptionaldiversity of specific sets of genes may influence heterosisfor different traits in maize [30]; however, studies on specificfunctional genes may be useful to provide molecular expla-nations for heterosis.

The Maize Genome Sequencing Project revealed the firstdraft genomic sequence of maize at the 50th Maize Genet-ics Conference, and the integration of genetic linkage andgenomic physical maps in maize has been carried outthrough the maize FPC Map. ZmMYBE1 was mapped tobin 5.03, and several yield-related QTLs have also beenlocated to the same chromosomal region in previous stud-ies, which supports the potentially important role ofZmMYBE1 in maize. The present work to localizeZmMYBE1 in organelles suggests its trans-acting role inthe nucleus.

Proteins containing only one MYB motif play vital rolesin chromosomal structural maintenance and light cycleresponse in plants. In maize, only one gene (IBP1) with asingle MYB domain has been released [6], which encodesa telomeric DNA binding protein [10] and affects develop-ing cells from the root or shoot apex and the gibberellinhormone balance [31]. RTBP1, an R3-MYB protein clonedfrom rice, has been identified as playing important roles inplant telomere function in vivo [8]. In Arabidopsis, severalR1-MYB proteins, such as EPR1 [14], LHY [32], andCCA1 [33], have been isolated and confirmed as circadianoscillators involved in developmental modulations inplants. The phenotype of Arabidopsis lines overexpressing

Fig. 4. In silico mapping of ZmMYBE1 to the IBM2 linkage map of bin5.03. The red bar shows the map region between molecular markerumc1355 and umc1389 where ZmMYBE1 was mapped.3

Fig. 5. Overexpression of ZmMYBE1 in Arabidopsis Columbia wide type. (a) Transgenic plants showed a slower growth rate at the vegetativedevelopment stage, shorter stem, and later florescence compared with CK. (b) A longer growth period was observed in comparing the transgenic lines withCK. (c) RT-PCR analysis of wide-type Columbia (Col) and transgenic lines overexpressing ZmMYBE1. CK represents wide-type Arabidopsis, T-1 and T-2represent two positive transgenic Arabidopsis lines. Actin was amplified as a control.


ZmMYBE1 compared with CK illustrates that ZmMYBE1

may be involved in the regulation of growth rate and per-iod in plants. Maize with a slower growth rate at the vege-tative developmental stage supplies an adequate vegetativebasis for the reproductive growth phase and a longergrowth time, which provides enough time for seed-fillingto achieve a higher grain yield. Several interesting, simulta-neously changed phenotypes were observed in transgenicArabidopsis lines, which also elucidate the relationshipsbetween flowering time and growth period and the compli-cated role of ZmMYBE1 in the development of plantheight.

Large-scale detections of transcriptome variationsbetween hybrids and parents identified many functionalcandidate genes, exploration of which in the future couldunveil the mysteries of grain yield in maize and benefitplant molecular breeding programs and ultimately, agricul-tural production.

Acknowledgement

This work was supported by the National Basic Re-search Program of China (Grant No. 2007CB109006).The authors thank Prof. Guoying Wang for providingthe pCAMBIA3301.

References

[1] Riechmann JL, Heard J, Martin G, et al. Arabidopsis transcriptionfactors: genome-wide comparative analysis among eukaryotes. Sci-ence 2000;290:2105–10.

[2] Goff SA, Ricke D, Lan TH, et al. A draft sequence of the rice genome(oryza sativa L. ssp. japonica). Science 2002;296:92–100.

[3] Dias AP, Braun EL, McMullen MD, et al. Recent duplicated maizeR2R3 Myb genes provide evidence for distinct mechanisms ofevolutionary divergence after duplication. Plant Physiol2003;131:610–20.

[4] Gabrielsen OS, Sentenac A, Fromageot P. Specific DNA binding byc-Myb: evidence for a double helix-turn-helix-related motif. Science1991;253:1140–3.

[5] Ogata K, Morikawa S, Nakamura H, et al. Solution structure of aspecific DNA complex of the Myb DNA-binding domain withcooperative recognition helices. Cell 1994;79:639–48.

[6] Lugert T, Werr W. A novel DNA-binding domain in the Shrunken

initiator-binding protein (IBP1). Plant Mol Biol 1994;25:493–506.[7] Wang ZY, Kenigsbuch D, Sun L, et al. A Myb-related transcription

factor is involved in the phytochrome regulation of an Arabidopsis

Lhcb gene. Plant Cell 1997;9:491–507.[8] Yu EY, Kim JH, Ko JH, et al. Sequence-specific DNA recognition by

the Myb-like domain of plant telomeric protein RTBP1. J Biol Chem2000;275:24208–14.

[9] Kranz H, Scholz K, Weisshaar B. C-MYB oncogene-like genesencoding three MYB repeats occur in all major plant lineages. Plant J2000;21:231–5.

[10] Jin H, Martin C. Multifunctionality and diversity within the plantMYB-gene family. Plant Mol Biol 1999;41:577–85.

[11] Abe H, Urao T, Ito T, et al. Arabidopsis AtMYC2 (bHLH) andAtMYB2 (MYB) function as transcriptional activators in abscisicacid signaling. Plant Cell 2003;15:63–78.

[12] Bilaud T, Koering CE, Binet-Brasselet E, et al. The telobox, a myb-related telomeric DNA binding motif found in proteins from yeast,plants and human. Nucleic Acids Res 1996;24:1294–303.

[13] Lee MM, Schiefelbein J. WEREWOLF, a MYB-related protein inArabidopsis, is a position-dependent regulator of epidermal cellpatterning. Cell 1999;99:473–83.

[14] Kuno N, Moller SG, Shinomura T, et al. The novel MYB proteinEARLY-PHYTOCHROME-RESPONSIVE1 is a component of aslave circadian oscillator in Arabidopsis. Plant Cell 2003;15:2476–88.

[15] Li B, Zhang D, Jia G, et al. Genome-wide comparisons of geneexpression for yield heterosis in maize. Plant Mol Biol Rep DOI10.1007/s11105-008-0068-x.

[16] Schultz J, Milpetz F, Bork P, et al. SMART, a simple moleculararchitecture research tool: Identification of signaling domains. ProcNatl Acad Sci USA 1998;95:5857–64.

[17] Thompson JD, Gibson TJ, Plewniak F, et al. The ClustalX windowsinterface: flexible strategies for multiple sequence alignment aided byquality analysis tools. Nucleic Acids Res 1997;25:4876–82.

[18] Reese MG. Application of a time-delay neural network to promoterannotation in the Drosophila melanogaster genome. Comput Chem2001;26:51–6.

[19] Altschul SF, Gish W, Miller W, et al. Basic local alignment searchtool. J Mol Biol 1990;215:403–10.

[20] Cone KC, McMullen MD, Bi IV, et al. Genetic, physical, andinformatics resources for maize: On the road to an integrated map.Plant Physiol 2002;130:1598–605.

[21] Pang SZ, Deboer DL, Wan Y, et al. An improved green fluorescentprotein gene as a vital marker in plants. Plant Physiol1996;112:893–900.

[22] Clough SJ, Bent AF. Floral dip: a simplified method for Agrobac-

terium-mediated transformation of Arabidopsis thaliana. Plant J1998;16:735–43.

[23] Joshi CP. An inspection of the domain between putative TATA boxand translation start site in 79 plant genes. Nucleic Acids Res1987;15:6643–53.

[24] Lipsick JS. One billion years of MYB. Oncogene 1996;13:223–35.[25] Stupar RM, Springer NM. Cis-transcriptional variation in maize

inbred lines B73 and Mo17 leads to additive expression patterns inthe F1 hybrid. Genetics 2006;173:2199–210.

[26] Guo M, Rupe MA, Yang X, et al. Genome-wide transcript analysisof maize hybrids: allelic additive gene expression and yield heterosis.Theor Appl Genet 2006;113:831–45.

[27] Pea G, Ferron S, Gianfranceschi L, et al. Gene expression non-additivity in immature ears of a heterotic F1 maize hybrid. Plant Sci2008;174:17–24.

[28] Guo M, Yang S, Rupe M, et al. Genome-wide allele-specificexpression analysis using Massively Parallel Signature Sequencing(MPSSTM) reveals cis- and trans-effect on gene expression in maizehybrid meristem tissue. Plant Mol Biol 2008;66:551–63.

[29] Cong B, Liu J, Tanksley SD. Natural alleles at a tomato fruit sizequantitative trait locus differ by heterochromic regulatory mutations.Proc Natl Acad Sci USA 2002;99:13606–11.

Table 1Trait observations for 3 transgenic lines overexpressing ZmMYBE1 fortwo generations.

Arabidopsis

linesLinesnumbers

Traits for observation

Flowering time(days)

Plant height(CM)

Growth time(days)

T-1a 1 83 4.5 110T1b 24 84 4.5 110

T-2a 1 85 4.1 112T2b 22 85 4 111

T-3a 1 80 5.5 105T3b 21 81 5 103

CK 35 13.5 75

CK represents wide-type Arabidopsis lines.a Observed records for the single line in the T1 generation.b Average observed records for positive T2 generation lines.


[30] Stupar RM, Gardiner JM, Oldre AG, et al. Gene expression analysesin maize inbreds and hybrids with varying levels of heterosis. BMCPlant Biol 2008;8:33.

[31] Klinge B, Lange T, Werr W. The IBP gene of maize are expressed innon-meristematic, elongating cells of the seedling and in abortivefloral organs. Mol Gen Genet 1997;255:248–57.

[32] Schaffer R, Ramsay N, Samach A, et al. The late elongated hypocotylmutation of Arabidopsis disrupts circadian rhythms and the photo-periodic control of flowering. Cell 1998;93:1219–29.

[33] Wang ZY, Tobin EM. Constitutive expression of the CIRCADIANCLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythmsand suppresses its own expression. Cell 1998;93:1207–17.


cloning and characterization of a novel r1-myb

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