全 文 ：DEVELOPMENTAL & REPRODUCTIVE BIOLOGY ISSN1004-6453/CN11-3569/Q
Vol.10 No.1 pp:69~75 Jun. 2001 Ó2001 Chin. Dev. & Reprod. Biol. Socs.
Vol.10 No.1 (2001) 69
Isolation and Expression of HAP2, A Homolog of AP2 in
Hyacinthus orientalis L.1
LI Quan-zi, LI Xing-guo, BAI Shu-nong*,
LU Wen-liang** and ZHANG Xian-sheng2
College of Life Sciences, Shandong Agricultural University, Taian 271018, China
* College of Life Sciences, Peking University, Beijing 100871, China
** Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
ABSTRACT By precisely controlling exogenous hormones applied in the cultural process,
particular type of floral organs, such as tapels, stamens, or ovules, can be regenerated from
perianth explant of Hyacinthus orientalis L. To further study the molecular mechanism of
the specific organgenesis in the system, an AP2 homolog, HAP2 was isolated from regenerated
tepals by using RT-PCR. Results from RT-PCR combined with Southern hybridization
showed that HAP2 was expressed in leaves, perianth, regenerated tepals and regenerated
stamens. The possible functions of HAP2 on hormone-regulated floral organ regeneration
are discussed.
Key words: Hyacinthus orientalis, regenerated floral organ, HAP2
1. INTRODUCTION
In recent years, detailed genetic analysis on floral organ development of Arabidopsis and
Antirrhinum led to the proposal of the “ABC” model[1,2], which describes how three classes of
homeotic genes act combinatorially in discrete domains to specify the identity of floral organ types.
Homeotic genes that affect the floral organ identities have been isolated from many plant
species[3,4]. In Arabidopsis, floral organ identities were determined by the activity of genes
APETALA1 (AP1), APETALA2 (AP2), APETALA3(AP3), PISTILLA (PI) and AGAMOUS (AG)[4].
Among them, AP2 plays an important role in the regulation of flower development. In
Hyacinthus orientalis, the floral organ differentiation of regenerated floral buds could be regulated
by the age of explants and exogenous hormones in vitro[5]. At a high concentration of hormones
in the medium, tapel differentiation occurs from the regenerated floral buds. At a lower
concentration of hormones, stamens and ovules could be induced respectively[6]. It is obvious
that the hormones are involved in the differentiation of floral organs[5]. This phenomenon
addresses an interesting question that whether the hormonal regulation of the particular organ
differentiation is mediated, directly or indirectly of the expression of “ABC” genes. To test this
possibility, we isolated several homologs of “ABC” genes from Hyacinthus orientalis and showed
that HAG1, a homolog of AGAMOUS, expressed differently in regenerated stamen and regenerated
tepals[7]. In this study, we isolated a homeotic gene HAP2 from the regenerated tepals and
determined its expression.

1 Foundation item: Pandeng project and National Natural Science Foundation of China (No. 39970076).
2 Author for correspondence.
LI Quan-zi …
Developmental & Reproductive Biology70
2. MATERIALS AND METHODS
2.1. Plant materials
Bulbs (three-year-old) of Hyacinthus orientalis (cv. Delft blue) were used as experimental
materials. In early November, bulbs were planted in the soil, and floral buds were collected at
right stages.
2.2. Induction and culture of regenerated tapels and stamens
Suitable perianth were selected as the explants. Isolation and sterilization of the explants were
performed as described previously[8]. The induction and culture of regenerated tapels and
stamens were conducted as described previously[5,8].
2.3. Reverse transcription and PCR
Total RNA isolation was conducted according to the method of Cathala et al.[9] 2 mg total RNA
was reversely transcripted into cDNA. Based on the conserved domains of AP2 homologs, two
degenerated primers were designed for PCR amplification. 5’ and 3’ oligonucleotides were 5’ -
TGGGA(A/G)TC(G/C/T)CA(C/T)AT(C/T)TG GGA-3’ and 5’ -TCCCA(A/G/C)(C/T)(G/T)(A/G)
CC(A/G)CA(C/T)TT (A/G)TG-3’ , respectively. PCR amplification was conducted in a total
volume of 50 mL with 5 mL 10´ reaction buffer, 4 mL of 2.5 mM dNTP, 4 mL of 5 mM of the two
primers, respectively, and 0.5 mL Taq polymerase. Thermocycling was performed at 94 for 60
sec, 56 for 60 sec and 72 for 120 sec for 35 cycles. 5 and 3’ RACE were conducted by
using GIBCO BRL Kit.
2.4. Gene cloning and sequencing
Target gene cloning was performed according to the protocol of pGEM-T Easy Vector Systems
(Promega). Sequencing was carried out by using ABI PRISMTM 377 DNA Sequencer with
BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elemer).
2.5. RT-PCR and Southern hybridization
Total RNA isolated from leave, perianth, regenerated tepals and regenerated stamens were
digested with DNase respectively, then 2mg total RNA was reversely transcripted into cDNA.
According to the sequence of HAP2, two specific primers were designed. RT-PCR was
conducted with control lacking reverse transcriptase, to determine whether there is contamination
of genomic DNA. After amplification, 8 ml PCR product was electrophoresed in 1.0 % agarose
gel for 6 h, and DNA was blotted onto a nylon membrane. The probe for hybridization was
labeled by using random priming method (Promega). Blots were hybridized at 65 overnight in
5 SSPE, 10% dextran sulfate, 1% SDS, and 0.2 mg/mL denatured salmon sperm DNA. Blots
were washed once at room temperature for 30 min in 0.2 SSC, 0.5% SDS and twice at 55 for
Isolation and Expression of HAP2, A Homolog of AP2 in …
Vol.10 No.1 (2001) 71
60 min in 0.1 SSC, 0.5% SDS. Autoradiography was performed at -70 for 1 day.
3. RESULTS
3.1. Induction and regeneration of tapels and stamens
Floral buds could be regenerated from the perianth explants in the medium containing high
concentration of hormones. Maintaining this level of exogenous hormones in the medium, the
tepals can be continuously induced from the regenerated floral buds. However, when the level of
the exogeneous hormones was decreased, tepal differentiation is inhibited, and stamen
differentiation occurs. Detailed descriptions can be found in our previous studies[5~7 ].
3.2. HAP2 gene cloning and sequence analysis
According to previous work in Arabidopsis, AP2 is involved in determining sepal and petal
identities[10], so we tried to isolate the homologs of AP2 from the floral organs of Hyacinthus
orientalis. RT-PCR amplification was conducted by using total RNA of regenerated tepal
primodia as templates. Using degenerated primers, a fragment of 280 bp was amplified from the
total RNA of regenerated tepals. The sequencing and blast search results showed that the
fragment has 89% to AP2 at amino acid sequence level, which demonstrated that a fragment
homologous to AP2 had been isolated from Hyacinthus orientalis. Further, the 5’ - and 3- end
sequences were isolated by using 5’ and 3’ RACE and a full length cDNA was obtained. This
gene was designated HAP2 (for Hyacinthus AP2), and its accession number in GenBank is
AF134116. Fig.1 shows both nucleotide and amino acid sequences of HAP2. The HAP2 gene
is 1597 bp in full length, and encodes a putative polypeptide of 368 amino acids.
AP2 homologs contain two copies of 68-amino acid repeat sequences that are referred to as the
AP2 domain, both of which contain about 18 amino acid residues capable of forming amphipathic
a-helic structures that may participate in protein-protein interactions[11]. In Arabidopsis, these
two domains are designated as AP2-R1 and AP2-R2, respectively[11]. As Fig.2 shows, HAP2 also
contains the two repeats, HAP2-R1 and HAP2-R2. The residues capable of forming amphipathic
a-helice structure between HAP2-R1 and AP2-R1 is 100% identical, but the residues capable of
forming amphipathic a-helice structure in HAP2-R2 lack 9 amino acids compared with those in
AP2-R2. Besides, AP2 homologs have a highly basic 10-amino acid domain that includes a
putative nuclear located sequence KKSR[12,13], suggesting that AP2 may function in the nucleus[11],
and this structure was also found in HAP2. The 5’ end of coding region of AP2 contains a
serine-rich acidic domain analogous to regions that function as activation domains in a number of
RNA polymerase II transcription factors[14], but this structure is not evident in HAP2. Taken
together, these findings suggest that, samilar to AP2, HAP2 also encodes a putative nuclear
protein.
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Developmental & Reproductive Biology72
TGAAGAGAAGCAAGTGACTGTGATGCCCAAACTCAGAGCAGAGAGCACAGAGAAAGAC 58
AGTGTGAAGACTGAATAGAGAGCGAGAGAGCAAAAATAACAGCAAGTTTTCTTCTTTTCT 118
TCTTTCTCTCTCCGTCTTCAAGAAGCAAATCTTCTGCTGATGTTTTTCTCTGGAGATGGA 178
AAATGACAAAAAGGTGGGATCTTGGTGATCAGAAGCTCCTCAGTTGGAGAAAAGCAGCGG 238
CAGGGTAAATAGAGAGATTGGGAGCGAGAGAGAAGAAAAGGGCATCGGTTTCTCTATCTG 298
GGGAGTTGGAAATTGATAAAAAGGAGGGATCTTGGTGAGAGGTTCCGGTGGTTGCGGGAT 358
TTCAACGGCTTGCCCTAGAGAATGGGGG TCCCGTCACCTTCCAGTTCTTTCCTGTCGGT 418
M**G**D**P**V**T**F**Q**F**F**P**V**G
GAGCCGGAGGAGGAGGAGAGCCCTGGTGGTGAGTCTACCGTCGCCGCCGGAGGAGCGCCG 478
E**P**E**E**E**E**S**P**G**G**E**S**T**V**A**A**G**G**A**P
CGGGGCCACTGGGCGGAGGTGAAGTTTGTTGAGAAGGAGGCGGACGTGTCGCCGGCGATC 538
R**G**H**W**A**E**V**K**F**V**E**K**E**A**D**V**S**P**A**I
AAGAAGAGCCGGCGGGGGCCGAGGTCTAGGAGCTCGCAGTACAGGGGAGTGACTTTCTAC 598
K**K**S**R**R**G**P**R**S**K**S**S**Q**Y**R**G**V**T**F**Y
AGGAGGACTGGGAGATGGGAGTCGCATATTTGGGATTGCGGCAAACAGGTCTATTTGGGA 658
R**R**T**G**R**W**E**S**H**I**W**D**C**G**K**Q**V**Y**L**G
GGATTTGACACTGCTCATGCTGCTGCAAGAGCTTACGATCGAGCTGCGATTAAGTTTCGA 718
G**F**D**T**A**H**A**A A**R**A**Y**D**R**A**A**I**K**F**R
GGGGTTGATGCTGATATCAATTTCAATCTCAGTGATTATAATGAGGATCTGAAGCAGATG 778
*G**V**D**A**D I**N**F**N**L**S**D**Y**N**E**D**L**K**Q**M
ATGAACTTAGCCAAAGAGGAGTTTGTTCACATCCTTCGAAGGCAGAGCACTGGGTTTTCG 838
*M**N**L**A**K**E**E**F**V**H**I**L**R**R**Q**S**T**G**F**S
AGGGGCAGCTCGAAATATCGAGGGGTGACTCTCCACAAGTGCGGCCGCTGGGAAGCTAGG 898
*R**G**S**S**K**Y**R**G**V**T**L**H**K**C**G**H**W**E**A**R
ATGGGCCAATTCCTTGGCAAAAAGGCTTATGACAAAGCGGCTATCAAGAGCAGTGGAAGG 958
*M**G**Q**F**L**G**K**K**A**Y**D**K**A**A**I**K**S**S**G**R
GAAGCTGTAACCAATTTTGAGCCAAGTTCTTATGAACGGGAAGTGCTCACTGAGGCAGAT1018
*E**A**V**T**N**F**E**P**S**S**Y**E**R**E**V**L**T**E**A**D
AGTGATGCCATTGGCCATGACATCGATCTGAACTTGAGGATATCTCAACCGAATGTTAGC1078
*S**D**A**I**G**H**D**I**D**L**N**L**R**I**S**Q**P**N**V**S
AGTCCAAAGAGGCGTGATAATCCAGTTGGCGTCCAGTTCCATTTGGGCTCCTTCGAATCG1138
*S**P**K**R**R**D**N**P**V**G**V**Q**F**H**L**G**S**F**E**S
TCTGACGCCAAGAAAGCTATGATTGACACCCATTCATCAATTTTGGTTGGTCAGCCCCAT1198
*S**D**A**K**K**A**M**I**D**T**H**S**S**I**L**V**G**Q**P**H
ACGGCGGCAATGACATCCGAGGCTTCTCGAGTATGGTCTGCCCTTTATCCTGGATTCTAT1258
*T**A**A**M**T**S**E**A**S**R**V**W**S**A**L**Y**P**G**F**Y
CCCCCTATTGAGCTACGAGCCAAAGACAAGATGTCTATGGTCGGTTCAGCAGCCCTACCA1318
*P**P**I**E**L**R**A**K**D**K**M**S**M**V**G**S**A**A**L**P
AATTGGACATGGCAATTGCACGGGCCAATGCCATCGCCGATGTTCACTTCTGCAGCATCA1378
*N**W**T**W**Q**L**H**G**P**M**P**S**P**M**F**T**S**A**A**S
TCAGGATTCGCCACCACCCATGCCACATCCTCCATTACCCCCCCTGCCAGCCATCATCTT1438
S**G**F**A**T**T**H**A**T**S**S**I**T**P**P**A**S**H**H**L
CAGTTCCCTCCACCACCCAACACCAACTACTATTCCAGGAGCTGAAGCCGATGGAAATGC1498
*Q**F**P**P**P**P**N**T**N**Y**Y**S**R**S**
ATTCTAATGGTACATACATGTGTCATTGTATGTAAAGCACAAATTCGGGAGTAAAGCAGG1558
ATGGTGCATGTTTTTACTGACAAAAAAAAAAAAAAAAAA 1597
Fig.1 Nucleotides and predicted amino acid sequences of HAP2.
Isolation and Expression of HAP2, A Homolog of AP2 in …
Vol.10 No.1 (2001) 73
HAP2 M--------------------------------------------------------GDP
AP2 *WDLNDAPHQTQREEESEEFCYSSPSKRVGSFSNSSSSAVVIEDGSDDDELNRVRPNNPL
PHAP2B M---FDLNLCFEDEEELQFDNHNNSTETSNNSSSIINNIETTTTSSTCDDHEYISYSNNE
HAP2 VTFQFFPVGEPEEEESPGGESTVAAGGAPRGHWAEVKFVEKE---------------ADV
AP2 **H****EMDSNGGG--------V*S*F**A**FG***CQSDLATGSSAGKATNVAA*V*
PHAP2B YNNNSFVDFLKTDNNDQFLDSKELFPLSNGGEMAAPVNVYGNYGGTMEQRIIIPVQQQQQ
HAP2 SPA--IKKSRRGPRSKSSQYRGVTFYRRTGRWESHIWDCGKQVYLGGFDTAHAAARAYDR
AP2 E**QPL*********R********************************************
PHAP2B QQQQQV******************************************************
HAP2 AAIKFRGVDADINFNLSDYNEDLKQMMNLAKEEFVHILRRQSTGFSRGSSKYRGVTLHKC
AP2 ********E******ID**DD*****T**T******V********P**************
PHAP2B ***************V***QD*****T*FT********************Q*********
HAP2 GHWEARMGQFLGKK---------- AYDKAAIKSSGRE VTNFEPSSYEREVLTEA
AP2 *R************YVYLGLFDTEVEAAR********CN KD *D**I*DE*LNA*S
PHAP2B *R**S*********YIYLGLFDSEIEAAR**Y*****CN * **L*T**G*LS***
HAP2 DSDAIGHDIDLNLRISQPNVSSPKRRDNPVGV---QFHLGSFESSDAKKAMIDTHSSILV
AP2 SGNPTTPQ-*H**DL*LG*SANS*HKSQDMRL---RMNQQQQD*LHSNEVLGLGQTGM*N
PHAP2B *NGGAS*NL****G*ASSSIAD-DQH**TCLIGNSE*QCA*IGLPEYRG**NSPCTTMGS
HAP2 GQPHTAAMTSEASRVWSALYPGFYPPIELRAKDKMSMVGSAALPNWTWQ---LHGPMPS-
AP2 HT*NSNHQFPGS*NIG*GGGFSLF*AA*NHRF*GRASTNQV-------------------
PHAP2B KM**GRHL------L*NGVNTSVF*TFKGT*IG*GME*D**--******DQN*Y*GSS*V
HAP2 PMFTSAASSGFATT-----HAT---SSITPPASHHLQ---FPPPPNTN---YYSRS#
AP2 -LTNA******SPH-----*HNQIFN*TST*HQNW**TNG*Q**-------LMRP*#
PHAP2B *L*ST*******NSTTADV*QH---YFS*G*LPY*-H---S*SLA*M*FAQ**C**#
Fig.2 Alignment of the deduced amino acid sequences for HAP2 cDNA with AP2 homologs
from Arabidopsis (AP2) and Petunia (PHAP2B).
The asterisks designate the same amino acid. The underlined amino acids indicated HAP2
domain repeated motif HAP2-R1 and HAP2-R2, in which the residues capable of forming
amphipathic a– helices are boxed. A 10-amino acid basic region that contains a putative nuclear
located signal sequence (KKSR) is also underlined.
3.3. HAP2 expression analysis
Since the expression of HAP2 was hard to be detected by Northern hybridization, we combined
RT-PCR and Southern hybridization to determine its expression. Based on the sequence of
HAP2, two specific primers were designed, and RT-PCR results is shown in Fig.3. A specific
900 bp size DNA fragment can be amplified from leaves, perianth, regenerated tepals and
LI Quan-zi …
Developmental & Reproductive Biology74
regenerated stamens. Further, when the 3’end cDNA was used as a probe, the PCR Southern
hybridization result is consistent with that of RT-PCR. HAP2 is expressed in both non-floral and
floral organs in vivo, which is the same as the pattern of AP2[10,11].
Fig.3 HAP2 expression analysis by using RT-PCR combined with Southern hybridization.
(a) RT-PCR result. (b) Southern hybridization. Lane 1 and 2, mature regenerated stamens; 3
and 4, young regenerated stamens; 5 and 6, mature regenerated tepals; 7 and 8, young regenerated
tepals; 9 and 10, perianth; 11 and 12, leaves; 2, 4, 6, 8, 10 and 12, controls without reverse
transcriptase.
4. DISCUSSION
In Arabidopsis, AP2 plays a critical role in the regulation of flower development. According to
the “ABC” model, AP2 and AG are mutually antagonistic genes[1,2]. AP2 negatively regulates AG
expression in sepal and petal, and conversely AG negatively regulated AP2 gene expression in
stamens and carpels. Our result revealed that HAP2 is also expressed in stamens, suggesting
HAP2 expression in stamen is not repressed at RNA level by AG-like genes. Similar data were
also reported of organogenesis on a single organ type without interference of other organs or
tissues. Recently we have isolated several homeotic genes, such as HAG1, HPI1 and HPI2[7,16].
Further using molecular methods such as in situ hybridization to analyze their expression in
regenerated flower primordia and floral organs may provide important information on
understanding their function and relationship and especially the mechanism of the regulation of
floral development by hormones.
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提要
风信子花器官中 HAP2 基因的分离与表达研究
李全梓 李兴国 白书农* 陆文梁** 张宪省
山东农业大学生命科学学院, 泰安 271018
* 北京大学生命科学学院, 北京 100871
** 中国科学院植物研究所, 北京 100093
在离体条件下 以风信子花被片为外植体 通过控制激素的浓度可诱导花被片 雄蕊
或胚珠的再生 近年来 在拟南芥和金鱼草等模式植物中已经分离出了许多控制花器官发
育的同源异形基因 如 AG AP1 AP2 AP3 等 其中 AP2 在控制花萼和花瓣形成过程中
起重要作用 因此本文从风信子中分离 AP2 的同源基因 并对它在风信子再生系统中的表
达进行了分析
根据 AP2同源基因功能域的保守序列设计一对简并引物 5’ -TGGGA A/G TC G/T/C
CA C/T AT C/T TGGA-3’和 5’ -TCCCA(AGC)(CT)(GT)(AG)CC(AG) CA(CT)TT(AG)TG-3’ ,
以再生的花被片为材料进行 RT-PCR 扩增出大小约 300bp 的片段 序列分析表明该片段
的氨基酸序列与 AP2同源性高达 89% 进而 利用 5 和 3 Race PCR 得到全长的 cDNA
该基因命名为 HAP2 GenBank登记号为 AF134116 该基因全长 1597bp 编码 368个氨基
酸(Fig.1 与 AP2相比 HAP2也含有 10个氨基酸长的碱性功能域 其中 KKSR为核定位
信号 此外 HAP2也含有两个序列重复的 68个氨基酸长的功能域 HAP2-R1 HAP2-R2
HAP2-R1也含有能形成a-螺旋结构的核心区域 且与 AP2-R1中的核心序列 100%同源 而
HAP2-R2中的核心区域与 AP2-R2相比, 缺少 9个氨基酸(Fig.2 RT-PCR结合 Southern 杂
交结果表明(Fig.3 HAP2 在叶 花被片中表达 其表达方式与 AP2 一样 此外 在再生
花被片和再生雄蕊中也检测到 HAP2 的表达 进一步 利用原位杂交技术分析 HAP2 在风
信子花器官再生过程中的表达将对于激素调节的花发育的机理有更深入的理解