全 文 ：植 物 分 类 学 报 44 (4): 353–361(2006) doi:10.1360/aps050071
Acta Phytotaxonomica Sinica http://www.plantsystematics.com
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Received: 21 April 2005 Accepted: 2 December 2005
Supported by the National Natural Science Foundation of China, Grant Nos. 30121003, 30270093.
* Author for correspondence. E-mail: . .
Isolation and sequence analysis of two CYC-like genes,
SiCYC1A and SiCYC1B, from zygomorphic and
actinomorphic cultivars of Saintpaulia ionantha
(Gesneriaceae)
WANG Lei GAO Qiu WANG Yin-Zheng* LIN Qi-Bing
(State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, the Chinese Academy of Sciences,
Beijing 100093, China)
Abstract Using the mTAIL-PCR method, we have isolated the two CYC-like genes,
SiCYC1A and SiCYC1B, from zygomorphic and actinomorphic cultivars of Saintpaulia
ionantha respectively in Gesneriaceae. The two genes, SiCYC1A and SiCYC1B, from the
zygomorphic cultivar both contain the whole regulation domain, i.e. TCP and R domains.
Therefore, they should be functional in the floral symmetry establishment, homologous with
CYC in Antirrhinum majus. Unexpectedly, the two genes from the actinomorphic cultivar are
identical to those from the zygomorphic in DNA sequence, respectively. Based on
comparative analysis of the molecular alteration at CYC-like genes, which are responsible for
the morphological transformation from zygomorphy to actinomorphy, we suggest that the two
closely related genes SiCYC1A and SiCYC1B might be regulated by a common upstream
regulator, whose change would result in silence of both SiCYC1A and SiCYC1B in controlling
the development of the adaxial and lateral organs in a flower. In addition, an mTAIL-PCR
method was shown to have the technological advantages of the unknown sequence for
isolation.
Key words CYC-like gene, whole gene, Saintpaulia ionantha, mTAIL-PCR, upstream
regulator.
During the evolution of angiosperms, zygomorphy has very likely arisen from
actinomorphy many times independently, but there also appear to have been reversals from
zygomorphy to actinomorphy (Donoghue et al., 1998). In Lamiales s.l., the zygomorphic
flower is believed to be ancestral while the actinomorphic one secondary (Endress, 1998).
However, research in how modifications of development lead to the transformation between
zygomorphy and actinomorphy during evolution is not a well unexplored field at molecular
developmental level in Lamiales, except for Antirrhinum majus L. and a few allied species in
Antirrhineae (Veronicaceae).
In the model species Antirrhinum majus, the asymmetrical expressions of CYCLOIDEA/
DICHOTOMA (CYC/DICH) genes control the zygomorphy of the flower (Luo et al., 1996,
1999). The CYC gene activity in the adaxial floral organs in a flower promotes the growth of
adaxial petal in whorl two, but arrests the growth of the adaxial stamen in whorl three (Luo et
al., 1996). The local expression of DICH accompanied with CYC activity determines the
internal asymmetry of adaxial petals (Luo et al., 1999). Both CYC and DICH belong to the
TCP gene family, including TB1 (TEOSINTE BRANCHED1) from maize, PCF1 and PCF2
from rice and TCP1-9 isolated from Arabidopsis (Doebley et al., 1995, 1997; Kosugi &
Acta Phytotaxonomica Sinica Vol. 44 354
Ohashi, 1997, 2002; Cubas et al., 1999a; Cubas, 2004). All members of this gene family have
the TCP and R domains.
Beside the model species, a lot of CYC-like genes have been isolated with partial
sequences of Open Reading Frame (ORF) in the clades with zygomorphic flower in Lamiales
s.l., and studies have been focusing on the molecular evolution of CYC-like genes, such as
Veronicaceae, Scrophulariaceae s.l., Gesneriaceae and Fabaceae (Möller et al., 1999; Citerne
et al., 2000, 2003; Picó et al., 2002; Fukuda et al., 2003; Gubitz et al., 2003; Hileman &
Baum, 2003a; Smith et al., 2004). However, the isolation of the whole ORF of these genes
and studies on their expression pattern have been only limited in a few species within
Antirrhineae (Veronicaceae) in Lamiales up to date (Luo et al., 1996, 1999; Cubas et al.,
1999b; Hileman et al., 2003).
Gesneriaceae is the most basal family of Lamiales s.l., an interesting group in the respect
of evolution of floral symmetry (Endress, 1998). In this family, the corolla tends to be weakly
bilateral and the adaxial stamen is less reduced than in other Lamiales (Endress, 1998, 1999).
It is therefore possible that this family represents an ancient and not too elaborated form of
floral bilateral symmetry within Lamiales s.l. In Gesneriaceae, several CYC-like genes with
60%–70% of their ORF have been isolated in up to 54 species, and analyses for the phylogeny
and molecular evolution of these genes were reported (Möller et al., 1999; Citerne et al.,
2000; Smith et al., 2004). These studies reveal that GCYC in Gesneriaceae has undergone
several duplication and putative gene loss events during the evolution of Gesneriaceae. Two
closely related genes, GCYC1A and GCYC1B have been isolated only in the two closely
related genera, i.e. Streptocarpus Lindl. and Saintpaulia Wendl. GCYC1A and GCYC1B are
probably derived from the gene GCYC1 that is widely distributed in Gesneriaceae (Möller et
al., 1999; Citerne et al., 2000). However, all these isolated genes lack the whole TCP domain
and the 5′ region of ORF that are necessary for further studies in the function and expression
pattern of these CYC-like genes in Gesneriaceae.
Saintpaulia is one of the smaller genera in the Old World Gesneriaceae (Burtt, 1958).
Most of species in Saintpaulia have been in cultivation for many years. Saintpaulia ionantha
Wendl. is the most popular one of species known as“The African violet”in the world, and its
cultivated descendants are the basis of the African violet commercial sales. Its wild-type has a
zygomorphic corolla with the adaxial and lateral stamens aborted. Both zygomorphic and
actinomorphic cultivars are found in S. ionantha. To analyse if the sequence variation of
CYC-like genes between the zygomorphic and actinomorphic cultivars is the cause for the
morphological transformation, we have isolated the ORF of two CYC-like genes from S.
ionantha, SiCYC1A and SiCYC1B, with modified TAIL-PCR. The sequences of SiCYC1A and
SiCYC1B, containing complete TCP domain, R domain and 5′ region, enable us to analyse the
molecular mechanism in the morphological transformation between zygomorphic and
actinomorphic flowers, which would shed light on further research on their genetic control
and molecular mechanism related to this transformation in floral symmetry.
1 Material and methods
1.1 DNA extraction and PCR amplification
Fresh leaves of zygomorphic cultivar (ZC) and actinomorphic cultivar (AC) of
Saintpaulia ionantha were collected from greenhouse of IBCAS (Institute of Botany, the
Chinese Academy of Sciences). Genomic DNA of ZC and AC were isolated by the CTAB
method (Doyle & Doyle, 1987). The primers (forward FS 5-ATGCTAGGTTTCGA-
CAAGCC-3, reverse R 5-ATGAATTTGTGCTGATCCAAAATG-3) were used to amplify
70% SiCYC sequences from zygomorphic cultivar (Möller et al., 1999; Citerne et al., 2000;
No. 4 WANG Lei et al.: Isolation and sequence analysis of CYC-like genes in Saintpaulia ionantha 355
Smith et al., 2004) (Fig. 1). PCR was performed with 32–36 cycles each with denaturation at
94 ℃ for 30 s, annealing at 52 ℃ for 30 s and extension at 72 ℃ for 1 min. PCR products
were run on 1% agarose gels, and bands near the expected length (ca. 620 bp for GCYC loci)
were excised. Excised PCR products of SiCYC from zygomorphic cultivar were cloned and
sequenced.

Fig. 1. Schematic outline of the procedures used to isolate SiCYC 5′ flanking regions from Saintpaulia ionantha. First,
70% coding region of SiCYC was amplified by primer pairs FS and R. On the basis of these sequence amplified, three
gene-specific primers iFSa, iFSb and iFSc were designed and used in combination with a 10-mers primer (AP) for
TAIL-PCR to obtain the 5′ flanking region.


1.2 Modified TAIL-PCR procedure
In order to clone the unknown 5′ flanking sequence beyond the partial sequence of
SiCYC from zygomorphic cultivar, a modified TAIL-PCR (mTAIL-PCR) was conducted, by
using of 10 mers random primers instead of degenerate 16 mers as the short primer (Liu &
Whittier, 1995; Liu et al., 1995; Terauchi & Kahl, 2000). Three gene-specific primers in
nested positions close to the 5 end of the coding regions were designed and synthesized (Fig.
1). iFSA (5-AGAGCTAGAGCTCTTCTTTG-3), iFSB (5-CGTTAACAGCCACTCAAG-
3), and iFSC (5-CGGCTTGTCGAAACCTAGCAT-3). Through the primary and secondary
rounds of mTAIL-PCR, one of the arbitrary 10 mers primers (S82: 5-GGCACTGAGG-3) for
SiCYC was chosen from the 10 mers random primer sets (Sangon, Shanghai). mTAIL-PCR
amplification was performed on Biometra TGRADIENT thermocycler. Cycling parameters for
the primary, secondary, and tertiary rounds are shown in Table 1.
1.3 Isolation of the 5 region of the SiCYC genes in S. ionantha
The primer SiF (5-TTGAGCCCTCCATCCCACA-3) was designed from 5 region
sequence of SiCYC amplified from zygomorphic cultivar by mTAIL-PCR and was used to
isolate the complete ORF of the SiCYC genes with reverse primer R
(5-ATGAATTTGTGGTGATCCAAAATG-3) from both zygomorphic cultivar and
actinomorphic cultivar. PCR was performed with 32 cycles each with denaturation at 94 ℃
for 30 s, annealing at 52 ℃ for 30 s and extension at 72 ℃ for 1.5 min. PCR products were
run on 1% agarose gels, excised bands near the expected length (900 bp). Excised PCR
products were extracted and cloned into the pGEM-T Easy vector (Promega, Madison, Wis).
Sixteen to 24 clones per ligation reaction were sequenced with the vector specific primers T7
and SP6. Sequencing was performed on an ABI PRISM 377 DNA Sequencer according to
Acta Phytotaxonomica Sinica Vol. 44 356
manufacturer’s instructions (Applied Biosystems, Foster City, Calif). Sequences alignments
were performed with CLUSTAL W (Thompson et al., 1994) and adjusted manually. The
secondary structure of TCP domain and R domain prediction for SiCYC1A/SiCYC1B, CYC
and LCYC were plotted with PROTEAN 5.0.

Table 1 mTAIL–PCR cycle settings for this study
Reaction Number of cycles Thermal settings
1 95 ℃, 5 min
5 94 ℃, 30 s; 54 ℃, 1 min; 72 ℃, 2.5 min
1 94 ℃, 30 s; 25 ℃, ramping to 72 ℃ in 3 min; 72 ℃, 2.5 min
94 ℃, 30 s; 54 ℃, 3.5 min
94 ℃, 30 s; 54 ℃, 3.5 min
15
94 ℃, 30 s; 42 ℃, 1 min; 72 ℃, 2.5 min
Primary
(iFSa-S82)
1 72 ℃, 5 min; 4 ℃ hold
94 ℃, 30 s; 54 ℃, 3.5 min
94 ℃, 30 s; 54 ℃, 3.5 min
12
94 ℃, 30 s; 42 ℃, 1 min; 72 ℃, 2.5 min
Secondary
(iFSb-S82)
1 72 ℃, 5 min; 4 ℃ hold
30 94 ℃, 30 s; 42 ℃, 1 min; 72 ℃, 2.5 min Tertiary
(iFSc-S82) 1 72 ℃, 5 min; 4 ℃ hold
Primary, secondary, and tertiary nested PCR reactions are performed sequentially. The primary PCR reaction consists of 15
TAIL cycles, while the secondary reaction contains 12 TAIL cycles. Primers iFSa, iFSb and iFSc are nested gene-specific
primers while S82 is a 10-mers RAPD primer.

2 Results and discussion
2.1 Floral morphology
Flowers in the zygomorphic
cultivar are identical to those of the wild
species of Saintpaulia ionantha. The
bilabiate corolla has a lower (abaxial)
trilobed lip that is longer than the upper
(adaxial) bilobed lip (Fig. 2: A). The
androecium consists of two abaxial
stamens and three staminodes in lateral
and adaxial positions (Fig. 2: A, C). The
actinomorphic cultivar has five almost
equal petals with a short corolla tube,
and five fertile stamens, i.e. one adaxial,
two lateral and two abaxial stamens.
Among the five stamens, the two abaxial
stamens are larger than the adaxial and
lateral ones (Fig. 2: B, C).
2.2 Analysis of DNA and deduced
amino acid sequences
We conducted mTAIL-PCR to
amplify the 5 region sequence of
CYC-like genes from zygomorphic
cultivar in S. ionantha base on their
partial sequences in the coding region
(Citerne et al., 2000). The sequence of a
Fig. 2. Two types of floral morphology (A, B) and diagram (C)
in zygomorphic cultivar (ZC) and actinomorphic cultivar (AC) of
Saintpaulia ionantha. In a ZC flower, the upper lip with two
adaxial petals is shorter than the lower lip with one abaxial and
two lateral petals, with two abaxial stamens plus three staminodes
(A, C, ZC). In an AC flower, there are five almost equal petals
with five stamens (B, C, AC). C, carpel; B, abaxial petal; D,
adaxial petal; S, sepal; L, lateral petal; Sb, abaxial stamen; Sd,
adaxial stamen; Sl, lateral stamen; Std, adaxial staminode; Stl,
lateral staminodes.
No. 4 WANG Lei et al.: Isolation and sequence analysis of CYC-like genes in Saintpaulia ionantha 357
420 bp fragment obtained from 5 upstream by mTAIL-PCR was 20 bp overlapping with the
5-end of the partial sequence of SiCYC from zygomorphic cultivar of S. ionantha. The longer
SiCYC sequences with a complete 5 ends were further amplified and cloned, using the
primers which were designed based on the two partial sequences. Sequencing results show
that there are two copies of CYC-like genes in the zygomorphic cultivar of S. ionantha, i.e.
SiCYC1A and SiCYC1B. The two genes SiCYC1A and SiCYC1B are further cloned from the
actinomorphic cultivar. It is unexpected that the DNA and deduced protein sequences of
SiCYC1A and SiCYC1B from the actinomorphic cultivar are identical to those of zygomorphic
cultivar, respectively (Fig. 3). The total length of sequence is 951 bp in SiCYC1A and 963 bp
in SiCYC1b. The protein sequences of SiCYC1A and SiCYC1B are 316 aa and 320 aa,
respectively. Like other CYC homologues, SiCYC sequences lack intron in the coding regions
(Luo et al., 1996, 1999; Cubas et al., 1999b).
Alignment of DNA and amino acid sequences shows that the similarity is 88% between
SiCYC1A and SiCYC1B at DNA level, while 84% at protein level. The substitutions between
the two genes usually occur outside the conserved regions, such as TCP domain, R domain
and End box. The length of TCP domain is 59 aa and R domain is 18 aa both in SiCYC1A and
SiCYC1B respectively, which have the same length as the ones from CYC and LCYC in
Antirrhineae (Luo et al., 1996; Cubas et al., 1999a, b). Alignment of the amino acid sequence
from Antirrhinum majus CYC, Linaria vulgaris LCYC and SiCYC1A/SiCYC1B shows that
there are five amino acid substitutions in TCP domain of SiCYC (Fig. 4). The secondary
structure prediction for SiCYC1A/SiCYC1B, CYC and LCYC plotted with PROTEAN 5.0
shows that the amino acid substitutions in TCP domain SiCYC do not change the secondary
structure (data not shown).
Homology assessment from phylogenetic analysis suggests that CYC-like genes in
Gesneriaceae have undergone several duplication and putative gene loss events during the
evolution of Gesneriaceae (Citerne et al., 2000). According to the phylogenetic analysis, the
SiCYC homologues GCYC1A and GCYC1B limited within the two closely related clades of
the African Streptocarpus and Saintpaulia have been suggested as a secondary and more
recent duplication, which might have evolved from the gene GCYC1, a gene widely
distributed in Gesneriaceae (Möller et al., 1999; Citerne et al., 2000; Smith et al., 2004).
Therefore, we consider that SiCYC1A/SiCYC1B might have functionally differentiated from
GCYC1 as two redundant genes in controlling the floral symmetry during evolution.
2.3 The morphological transformation from zygomorphy to actinomorphy in
Saintpaulia ionantha
In the zygomorphic cultivar, flowers are identical to those of the wild plants of S.
ionantha in floral symmetry. However, there are five almost equal petals and five fertile
stamens in the actinomorphic cultivar of S. ionantha. This morphological change from
zygomorphic to actinomorphic flower should be related to partial or complete loss of function
of SiCYC1A/SiCYC1B during the floral development. Nevertheless, the identical sequence of
both SiCYC1A and SiCYC1B between zygomorphic and actinomorphic cultivars raises an
interesting question about what lead to this morphological transformation without sequence
alteration at the two CYC-like genes. In the model species Antirrhinum majus, the transposon
insertion at cis or trans-regulation region makes CYC complete loss of function, disrupting the
normal expression of CYC transcription in the adaxial and lateral regions, which leads to the
peloric mutants (Luo et al., 1996, 1999). However, the loss of function of LCYC, the only
homologue of Antirrhinum CYC in Linaria Miller, in actinomorphic variation is not caused by
a cis-regulator disrupting or DNA change but is correlated with extensive methylation (Cubas
et al., 1999b). In another case, the single GCYC1 gene from the actinomorphic cultivar of
Sinningia speciosa Benth. & Hook. (Gesneriaceae) carries a frame-shift mutation that gives
Acta Phytotaxonomica Sinica Vol. 44 358

Fig. 3. Alignment of the protein sequence of SiCYC1A, SiCYC1B in zygomorphic cultivar (ZC) and actinomorphic
cultivar (AC) of Saintpaulia ionantha. The length of TCP domain is 59 aa and R domain is 18 aa both in SiCYC1A and
SiCYC1B (underlined).

rise to a truncated protein, which might be responsible for the loss of function of GCYC1 in
this actinomorphic cultivar (Citerne & Cronk, 1999; Möller et al., 1999; Citerne et al., 2000;
Cubas, 2004).
In all three cases mentioned above, alteration of CYC-like gene expression or function is
responsible for the morphological transformation from zygomorphy to actinomorphy. The
first is the disruption or change in their cis or trans-regulation elements, as observed in peloric
mutants in the model species snapdragon. Another is the modified coding region of CYC-like
genes without DNA change, such as the methylation of the LCYC gene sequence in the
actinomorphic variation of Linaria. And the third is due to the substitution, insertion or
No. 4 WANG Lei et al.: Isolation and sequence analysis of CYC-like genes in Saintpaulia ionantha 359

Fig. 4. Alignment of the amino acid sequence of TCP domain of Antirrhinum majus CYC, Linaria vulgaris LCYC and
Saintpaulia ionantha SiCYC1A/SiCYC1B.


deletion taking place in CYC-like genes at DNA level, such as the deletion resulting in the
frame-shift in GCYC1 sequence from the actinomorphic cultivar of Sinningia speciosa.
Because the DNA and protein sequences of both SiCYC1A and SiCYC1B are identical
between zygomorphic and actinomorphic cultivars of S. ionantha, it is possible that the
methylation of the SiCYC gene sequence leads to the morphological transformation from
zygomorphic to actinomorphic flowers. However, since the two redundant genes SiCYC1A
and SiCYC1B are recently duplicated from GCYC1 (Citerne et al., 2000), they should have a
very similar function in controlling zygomorphy like CYC/DICH in snapdragon. Therefore,
the morphological transformation from zygomorphy to actinomorphy might be resulted from
double silence of the two genes, SiCYC1A and SiCYC1B. If this is true, the above explanation
would require an additional hypothesis that the methylation of SiCYC genes as two separate
events would co-occur at the same time. Alternatively and most likely, these would be a
single factor which is responsible for this morphological transformation. Therefore, we
consider that the two closely related genes SiCYC1A and SiCYC1B might be regulated by a
common upstream regulator, whose change would result in silence of both SiCYC1A and
SiCYC1B in controlling the adaxial and lateral organs in a flower. It is necessary to carry out
an intensive study on expression pattern and upstream regulator of the two genes SiCYC1A
and SiCYC1B, which would be helpful to understand the developmental pathways and
molecular mechanism for the morphological transformation from zygomorphy to
actinomorphy in S. ionantha.
2.4 Technological advantages of the modified TAIL-PCR
Modified PCR procedures to isolate the DNA fragments adjacent to known sequences
are powerful strategies for chromosome walking. Several modified PCR methods are
available for this purpose, for example: inverse PCR, ligation-mediated PCR (LM-PCR) and
randomly primed PCR (RP-PCR) (Frohman et al., 1988; Ohara et al., 1989; Huang, 1994;
Sterky et al., 1998; Akiyama et al., 2000; Dai et al., 2000). The TAIL-PCR (thermal
asymmetric interlaced PCR) method developed by Liu and Whittier (1995) is a simple, but
sometimes not efficient, technique for genomic walking which does not require any restriction
or ligation steps. In this study, using the random 10 mers originally developed for RAPD
analysis (Williams et al., 1990) as the short arbitrary primers instead of three degenerate
16-mer primers as described in the original TAIL procedure, we have successfully isolated the
5′-flanking regions of SiCYC genes. The mTAIL-PCR is more efficient and controllable.
Using the random 10 mers primers, we are able to obtain the right size of the prospective PCR
products from 200 to 1500 bp. Therefore, the random 10 mers primers may be much suitable
to isolate adjacent fragments from some new genes, where the sequence information around
Acta Phytotaxonomica Sinica Vol. 44 360
the conserved region is often limited.
Acknowledgements We thank Prof. ZHANG Zuo-Shuang, Beijing Botanical Garden, for
providing the zygomorphic and actinomorphic cultivars for this research.
References
Akiyama K, Watanabe H, Tsukada S, Sasai H. 2000. A novel method for constructing gene-targeting vectors.
Nucleic Acids Research 28: e77.
Burtt B L. 1958. Studies on the Gesneriaceae of the Old World XV: the genus Saintpaulia. Notes from the
Royal Botanic Garden Edinburgh 22: 547–568.
Citerne H, Cronk Q. 1999. The origin of the peloric Sinningia. The New Plantsman 6: 219–222.
Citerne H, Luo D, Pennington R, Coen E, Cronk Q. 2003. A phylogenomic investigation of CYCLOIDEA-like
TCP genes in the Leguminosae. Plant Physiology 131: 1042–1053.
Citerne H, Möller M, Cronk Q. 2000. Diversity of CYCLOIDEA-like genes in Gesneriaceae in relation to
floral symmetry. Annals of Botany 86: 167–176.
Cubas P. 2004. Floral zygomorphy, the recurring evolution of a successful trait. BioEssays 26: 1175–1184.
Cubas P, Lauter N, Doebley J, Coen E. 1999a. The TCP domain: a motif found in proteins regulating plant
growth and development. The Plant Journal 18: 215–222.
Cubas P, Vincent C, Coen E. 1999b. An epigenetic mutation responsible for natural variation in floral
symmetry. Nature 401: 157–161.
Dai S M, Chen H-H, Chang C, Riggs A D, Flanagan S D. 2000. Ligation-mediated PCR for quantitative in
vivo footprinting. Nature Biotechnology 18: 1108–1111.
Doebley J, Stec A, Gustus C. 1995. TEOSINTE BRANCHED 1 and the origin of maize: evidence for epistasis
and the evolution of dominance. Genetics 141: 333–346.
Doebley J, Stec A, Hubbard L. 1997. The evolution of apical dominance in maize. Nature 386: 485–488.
Donoghue M, Ree R, Baum D. 1998. Phylogeny and the evolution of flower symmetry in the Asteridae.
Trends in Plant Science 3: 311–317.
Doyle J J, Doyle J L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue.
Phytochemistry Bulletin 19: 11–15.
Endress P K. 1998. Antirrhinum and Asteridae—evolutionary changes of floral symmetry. Symposia of the
Society for Experimental Biology 51: 133–140.
Endress P K. 1999. Symmetry in flowers: diversity and evolution. International Journal of Plant Sciences 160:
S3–S23.
Frohman M A, Dush M K, Martin G R. 1988. Rapid production of full-length cDNAs from rare transcripts:
amplification using a single gene-specific oligonucleotide primer. Proceedings of the National Academy
of Sciences, USA 85: 8998–9002.
Fukuda T, Yokoyama J, Maki M. 2003. Molecular evolution of CYCLOIDEA like genes in Fabaceae. Journal
of Molecular Evolution 57: 588–597.
Gubitz T, Caldwell A, Hudson A. 2003. Rapid molecular evolution of CYCLOIDEA-like genes in Antirrhinum
and its relatives. Molecular Biology and Evolution 20: 1537–1544.
Hileman L C, Baum D A. 2003. Why do paralogs persist? Molecular evolution of CYCLOIDEA and related
floral symmetry genes in Antirrhineae (Veronicaceae). Molecular Biology and Evolution 20: 591–600.
Hileman L C, Kramer E M, Baum D A. 2003. Differential regulation of symmetry genes and the evolution of
floral morphologies. Proceedings of the National Academy of Sciences, USA 100: 12814–12819.
Huang S H. 1994. Inverse polymerase chain reaction: an efficient approach to cloning cDNA ends. Molecular
Biotechnology 2: 15–22.
Kosugi S, Ohashi Y. 1997. PCF1 and PCF2 specifically bind to cis elements in the rice proliferating cell
nuclear antigen gene. The Plant Cell 9: 1607–1619.
Kosugi S, Ohashi Y. 2002. DNA binding and dimerization specificity and potential targets for the TCP protein
family. The Plant Journal 30: 337–348.
Liu Y G, Whittier R F. 1995. Thermal asymmetric interlaced PCR: automatable amplification and sequencing
of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25: 674–681.
Liu Y G, Mitsukawa N, Oosumi T, Whittier R F. 1995. Efficient isolation and mapping of Arabidopsis
thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. The Plant Journal 8: 457–463.
Luo D, Carpenter R, Vincent C, Copsey L, Coen E. 1996. Origin of floral asymmetry in Antirrhinum. Nature
383: 794–799.
No. 4 WANG Lei et al.: Isolation and sequence analysis of CYC-like genes in Saintpaulia ionantha 361
Luo D, Carpenter R, Copsey L, Vincent C, Clark J, Coen E. 1999. Control of organ asymmetry in flowers of
Antirrhinum. Cell 99: 367–376.
Möller M, Clokie M, Cubas P, Cronk Q. 1999. Integrating molecular phylogenies and developmental genetics:
a Gesneriaceae case study. In: Hollingsworth P M, Bateman R J, Gornall R J eds. Molecular Systematics
and Plant Evolution. London: Taylor & Francis. 375–402.
Ohara O, Dorit R E, Gilbert W. 1989. One-sided polymerase chain reaction: the amplification of cDNA.
Proceedings of the National Academy of Sciences, USA 86: 5673–5677.
Picó F, Möller M, Ouborg N, Cronk Q. 2002. Single nucleotide polymorphisms in the coding region of the
developmental gene GCYC in natural populations of the relict Ramonda myconi (Gesneriaceae). Plant
Biology 4: 625–629.
Smith J F, Hileman L, Powell M P, Baum D. 2004. Evolution of GCYC, a Gesneriaceae homolog of
CYCLOIDEA, within Gesnerioideae (Gesneriaceae). Molecular Phylogenetics and Evolution 31:
765–779.
Sterky F, Holmberg A, Alexandersson G, Lundeberg J, Uhlen M. 1998. Direct sequencing of bacterial artificial
chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR. Journal of Biotechnology 60:
119–129.
Terauchi R, Kahl G. 2000. Rapid isolation of promoter sequences by TAIL-PCR: The 5-flanking regions of
Pal and Pgi genes from yams (Dioscorea). Molecular and General Genetics 263: 554–560.
Thompson J D, Higgins D G, Gibson T J. 1994. CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight
matrix choice. Nucleic Acids Research 22: 4673–4680.
Williams K, Kubelik A R, Livak J, Rafalski J A, Tingey S V. 1990. DNA polymorphisms amplified by
arbitrary primers are useful as genetic markers. Nucleic Acids Research 18: 6531–6535.
非洲紫罗兰两侧与辐射对称花中两个 CYC类完整
基因的分离和序列分析
王 磊 高 秋 王印政* 林启冰
(系统与进化植物学国家重点实验室, 中国科学院植物研究所 北京 100093)

摘要 在已知GCYC基因部分序列基础上, 通过改进的mTAIL-PCR方法克隆非洲紫罗兰Saintpaulia
ionantha两侧对称栽培种中CYC类基因的5′未知序列, 并进而从两侧与辐射对称栽培种中分离得到苦苣
苔科Gesneriaceae中第一组完整基因: SiCYC1A与SiCYC1B。对以上基因的核酸和氨基酸序列比较发现,
SiCYC1A与SiCYC1B序列同源性很高, 均含有完整的功能调控区域(即TCP domain和R domain)并与模式
植物金鱼草Antirrhinum majus中CYC基因同源。因此, 这两个基因应具有正常功能, 是功能上互补的冗
余基因。令人意外的是在辐射对称花栽培品种中的这两个基因和两侧对称花栽培品种中对应基因的序
列完全相同。经过对金鱼草以及相关类群辐射对称花突变体中CYC类基因序列的比较分析, 推论在非
洲紫罗兰中, SiCYC1A与SiCYC1B基因可能受上游未知的共同调控因子调控, 该调控因子的改变是导致
栽培品种中花对称性发生变化的主要原因。另外, 对改进后的TAIL-PCR(mTAIL-PCR)的方法和过程进
行了详细叙述, 并对其技术特征和优势开展了简单的论述。
关键词 CYC类基因; 完整基因; 非洲紫罗兰; mTAIL-PCR; 调控因子