全 文 ：　 　第 37卷　第 2期
1998年　 3月
中山大学学报 (自然科学版 )
ACTA　 SCIEN TIARUM　 N ATURALIUM
UNIVE RSITATIS　 SUNYATSENI
　
Vo l. 37　 No . 2
Ma r.　 1998
　
Sequence Analyses of ITS Regions of nrDNA from
Siphocranion nudipes Kudo (Lamiaceae)

Shi Suhua
1)　Wen Jun2)　 Zhu Lingfeng3)　 Zhu Zhongbao3)　 Huang Lingying3)
( 1) Schooo l of Life Sciences, Zhong shan Univ ersity, Guang zhou 510275;
2) Department of Bio logy , Colo rado Sta te Universi ty , For t Co llins, CO 80523, U SA;
3) Department of Fo rest ry , South-Central Fo rest ry Univ ersi ty, Zhuzhou 412000)
Abstract　 The sequence o f internal tr anscribed spacer ( IT S) r eg ions o f nuclear ribo somal
DN A ( nrDN A) w as determined for the endemic species Siphocranion nudipes ( Hemsl. ) Kudo
( Lamiaceae) fr om China. This is the fir st sequence repor t o f the ITS regions fo r Lamiaceae.
The entire ITS and 5. 8S regions of Siphocranion nudipes a re 609 bases long with an ITS-1 o f
207 bp, an ITS-2 o f 236 bp and a 5. 8S o f 166 bp. The ove rall G+ C content w as 59. 11% with
64. 25% in ITS-1, 57. 63% in IT S-2, and 53. 61% in 5. 8S. The numerous copies of the ITS se-
quences w ithin an indiv idual in Siphocranion nudipes showed a high lev el o f sequence homogene-
ity and only th ree nucleo tide po sitio ns w ere ambiguous. This sequence w ill serv e as a ba sis for
determining th e phy lo g enetic status o f Siphocranion nudipes within Lamiaceae and fo r indepth
studies on the conse rva tion o f this endemic species from subt ropical ar ea s o f China.
Keywords　 sequence , Siphocranion nudipes, interna l transcribed spacer regions
Classification number　 Q 949. 777. 6
Siphocranion Kudo is a genus f rom subtropical a reas of China, no rth er ns of India,
Myanmar and Viet-Nam
[1 ]
. It consists of tw o species Siphocranion nudipes ( Hemsl. ) Ku-
do dist ributed in subtropical regions of China, Yunnan, Sichuan, Hubei, Guizhou,
Guangdong , Jiangxi and Fujian provinces, and S.macranthum ( Hook. f. ) Wu
[2 ]
dist ribut-
ed in southw estern China, India, Myanma r and Viet　 Nam. The type-species o f
Siphocranion , S.nudipes Kudo w as previously described as Plectranthus nudipes by Hemsl
in 1890 and then as Hancea nudipes by Dunn in 1913. Kudo
[3 ]
put i t in the genus
Siphocranion and Wu
[ 2]
too by a perennial herb w ith a ligneous subterranean stem , almost
w ithout hai r, v erticellaeters 2-terminal, f low ered, racemes, solida ry o r sometimes 3 to-
gether. Caly x broadly campanula te in blo om, 5-clef t subregular, and distinct ly 2-lipped in
f rui t ( 3 /2 type) . Co ro lla-tube st raight , na rrow and elongate; stameus included, filaments
g labrous.
Siphocranion nudipes is an endemic species and was previously recorded from Wushan
in Sichuan Province
[1 ] . Recently several populations w ere found in the Doupengshan forest
region wi th an eleva tion above 1800 meters in the Dagong shan Natural Preserv e, Sang zhi
county , Hunan Prov ince. The discovery of the addi tional populations a tt racted recent
studies on the eco logy and conserva tion of this species. How ever the phylo genetic and evo-
lutiona ry sta tus of Siphocranion nudipes wi thin Lamiaceae has not been w ell inv estigated.
国家八五 ( 85-910-11-01) 课题基金和国家自然科学基金 ( 39570052) 资助项目
收稿日期: 1997-08-09　　施苏华 , 女 , 41岁 , 教授
The internal t ranscribed spacer ( ITS) sequences of nrDN A have been commonly used
fo r phy logenetic studies at the interspecific and int rafami lial level in plants
[4～ 7 ]
. At present
there has been no repo rt fo r the I TS sequences f rom Lamiaceae. In this study we examined
the characteristics o f the sequence o f the ITS regions f rom Siphocranion nudipes and com-
pared this sequence wi th that of o ther plant g enera.
1　Materials and methods
Leaves of Siphocranion nudipes were collected f rom Doupengshan of the Dagongshan
Na tural Preserv e, Sang zhi county, Hunan Province. Voucher specimens ( Zhu 079) w ere
deposi ted in South-Central Fo rest ry Universi ty Herbarium , Hunan.
1. 1　 DNA isolation and purif ication
Total DN A was isola ted f rom 0. 2～ 0. 3 g dried leaves using the acetone-po tassium ac-
eta te method
[ 8]
, and purified w ith glass powder.
1. 2　 Amplif ication of the ITS regions
The enti re ITS regions of rDN A was amplified fo r Siphocranion nudipes via the po ly-
merase chain reaction ( PCR) . Double st randed DN A and sing le-st randed DN A ampli fica-
tions were performed following the procedure o f Wen
[7 ]
. C26A and N-nc18S10 ( Tab. 1)
w ere used as amplification primers. The sing le-st randed PCR products w ere puri fied wi th
ult raf ree-MC fil ters ( Mi llipo re ) to eliminate the non-incorporated primers and nu-
cleo tides.
Tab. 1　 Primers used for DNA amplif icat ion and sequencing
Primer Sequence Use Designer Refer ence
C26A 5* -GT TTCT TT TCC TCCGCT-3* Ampification Suh Y modified from [7 ]
N-nc18S10 5* -AGGAGAAGTCGTAACAAG-3* Ampification Bult C [7 ]
C5. 8S 5* -TGCGT TC AAAGACTCGAT-3* Sequencing Suh Y [6 ]
IT S4 5* -TCC TCCGCTT ATTGATATGC-3* Sequencing White et al [5 ]
1. 3　 Sequencing the single-stranded DNA
The ITS regions w ere sequenced fo llowing the dideoxy chain temina tion method
[9 ]
us-
ing the Sequenase V ersion 2. 0 DN A Sequencing Ki t ( US70770, Amersham ) and alpha
35S-dAT P as a radioactiv e tracer. C5. 8S and ITS4 primers ( Tab. 1) w ere used as sequenc-
ing primers to obtain the ITS-1 and ITS-2 regions, respectively. Products of the sequenc-
ing reactions w ere separated on a 6% polyacrylamide gel a t 50 W. Gels w ere t ransferred to
3 mm Whatman paper, dried in an oven at 45℃ for 1. 5 h, and exposed to an X-ray fi lm
( Kodak XAR) fo r 36～ 48 h at room temperature. About 325 nucleo tides were obtained
per sequencing run.
1. 4　 Sequence analysis
The sequences obtained w ere assembled and the boundaries betw een the coding and
spacer regions w ere determined by compa ring with those o f Acanthopanax o f Araliaceae
[ 7] .
The G+ C content o f ITS-1, 5. 8S and I TS-2 regions was calcula ted for Siphocranion
nudipes and 10 species of o ther plant fami lies. The GC percentag e of Siphocranion nudipes
was based upon unambiguous sequence. Nucleatides of ambiguous posi tions were added to
the respectiv e ca tego ry af terwa rds.
2　 Results and discussion
Fig. 1 shows the sequence and the boundary of IT S-1, 5. 8S, and ITS-2 regions o f
Siphocranion nudipes. The general o rg aniza tion o f a nrDN A repeat is presented in Fig. 2.
53第 2期　　　　　　　施苏华等: 光柄筒冠花核糖体 DNA ITS区序列
Ribo somal DN A is ar ranged in numerous tandem repeats in one o r a few chromosomal
lo ci. Each repea t of the nrDN A of plants consists o f th ree major genes ( 18S, 26S and
5. 8S) and a few spacer regions.
18S|IT S-1　　 TCGAAACCTG CAAAGCAGAC CGCGAACACG TGT T TAACAC CGT CGGCACG GCCTCGTCGT
　　 GATCGT CCGC CGCT GCG TAG CCCCCGCTT C GCATCGT GCG GGCT AACGAA CCCCGGCGGA
　　 ATGCGCCAAG GAAAACT TAA TGGAGCGTCG GCCCCCT GCC ACCCCGT TCG CGGT GCGT GC　　 IT S-1|5. 8S　　 GGGGGGAGCG GATGT CT ATC GAATGT CAAA ACGACTCTCG GCAACGGAT A TCT CGCGTCT
　　 CGCATCGATG AAGAACGT AG CCAAA TGCGA T ACTT GG TGT GAAT TGCAGA TCCCGT GAAC
　　 TCGAGT CT TT CGAAGCAAGT TGCGCCGAAG CCAT TA AGGC CGAGGCACGT CTGCCT TGGG　　 5. 8S| ITS-2　　 CGTCAGCATT GCGTCGT CCC CCTAT TGCCC CGTGCCCAAA CAACGCTT GG GAT GGGGGAA
　　 CGGATAT TGG CCTCCGGTGC ACCCCGGT TG CGM GGCTGGC CAAAATGCGA TCCCTCGGCG
　　 ACTCT TGTCA CGACAT GTGG TGGT TGAAAA T TTCAATCTC GCT T TGTGCC GT GCT TTCGA
　　 GTCGT TCG TA AGGGM TT CAA AAAAYGACCC AATGGCGTCG ACGCGACCGA CCTGGCCGCG
IT S-2|26S　　 CGCT TT CGAC AGCGACCCC
Fig. 1　 Sequence of internal transcribed spacer regions f rom Siphocran ion nudipes
N-nc18S10 C26A→ ←
　 IGS　
18S rDN A
ITS-1
207 bp
5. 8S
ITS-2
236 bp
28S rDNA
IGS
←　
C5. 8S
←　
ITS4
Fig. 2　Organization of nuclear ribosomal genes and spacer regions (modif ied from Hamby and Zimmer,
1991) with the length of ITS regions of Siphocranion nud ipes and primer position. IGS represents
intergenic spacer and ITS is the abbreviation for the internal transcribed spacer regions.
2. 1　 The length of ITS regions in Siphocranion nudipes
The leng th of the enti re ITS and 5. 8S regions in Siphocranion nudipes are 609 bases
w ith an ITS-1 region o f 207 bp, an I TS-2 region of 236 bp and a 5. 8S region o f 166 bp
( Fig . 2, Tab. 2) , wi th I TS1 region sho rter than ITS2 region. Among the ITS sequences
reported so fa r, ITS-1 region w as found to be sho rter than ITS-2 region in Lamiaceae and
in Betulaceae[ 10] , Scrophula riaceae[11 ] . ITS-1 reg ion is longer in Asteraceae[4 ] , Ona-
g raceae
[12 ] , and Winteraceae
[6 ] . The tw o spacers are nearly equal in leng th in Araliaceae
[7 ]
and So lanaceae
[ 13] . In Fabaceae and Poaceae, the rela tiv e leng th of ITS-1 and I TS-2 re-
gions va ries among taxa
[14, 15 ] . ITS-1 and I TS-2 regions are each less than 300 bp in all the
f low ering plant ( ITS-1 from 187 to 298 bp, ITS-2 from 187 to 252 bp, rev iew ed in Bald-
w in et al. , 1995) , in contrast to much longer spacers in some gymnosperms ( e. g. ,
pines
[ 16] ) and in some vertebrates
[17 ] . The leng th o f the 5. 8S region is usually 163 o r 164
bp and show s li t tle va ria tion among angio sperms. The leng th o f ITS regions in Siphocran-
ion nudipes fa lls wi thin the narrow range of that of f low ering plants.
2. 2　 The G+ C content
Percentg e G+ C content in Siphocranion nudipes were 64. 25% in ITS-1 region,
57. 63% in ITS-2, and 53. 61% in 5. 8S region, and the G+ C content fo r the enti re ITS
and 5. 8S regions was 59. 11% . Overall the G+ C content of ITS regions va ries widely
across angio sperm species, f rom 47% in Madinae of Asteraceae to 75% inOryza sat iva o f
Poaceae ( Tab. 2) . Siphocranion nudipes falls in this range, but has a slight ly higher ITS G
+ C content ( only nex t to Oryza sat iva ) . The G+ C content of ITS regions are usually
higher than tha t o f 5. 8S, 26S and 18S.
54 中山大学学报 (自然科学版 ) 　　　　　　　　　　　　　第 37卷
Tab. 2　 Sizes and G+ C content of ITS-1, 5. 8S and ITS-2 of
S iphocranion nudipes and other angiosperms
Taxon Family ITS-1 5. 8S ITS-2 To tal Refe rence
Siphocran-
ion
Lamiaceae a
1)
b2)
207
64. 25
166
53. 61
236
57. 63
609
59. 11 This study
Aralia Araliaceae
a
b
220～ 221
60. 2～ 62 16354～ 54. 6 221～ 22462. 1～ 63. 8 614～ 60859. 2～ 61 [ 7]
Betula Betulaceae a
b
214～ 219
62～ 63 226～ 23163～ 65 [10]
Daucus Apiaceae
a
b
215
49
224
52
[15]
Epilobium Onag raceae a
b
240～ 244
57. 4～ 58. 7 16452. 0～ 53. 4 211～ 21656. 3～ 56. 9 616～ 62255. 9～ 56. 4 [12]
Madinae Aste raceae
a
b
255～ 261
47. 7～ 51. 4 16451. 2～ 53. 7 216～ 22349. 5～ 53. 6 635～ 64746. 9～ 52. 2 [ 4]
Mimulus Sc rophu-
lariaceae
a
b
189～ 214
44～ 49 203～ 22545～ 48 [11]
Nicotiana Solanaceae
a
b
216
69
217
65
[13]
Oryza Poaceae a
b
94
72. 2
233
77. 3
[14]
Panax Araliaceae
a
b
220～ 221
61. 1～ 61. 2 16351. 5～ 54. 1 222～ 22457. 8～ 63. 2 606～ 60859. 2～ 61. 1 [ 7]
Vicia Fabaceae a
b
235
52
208
50
[15]
　　 1) size /bp; 2)w ( G+ C) /%
2. 3　 Sequence homogeneity
Overall sequence homogenei ty o f the IT S regions w as detected in Siphocranion
nudipes, wi th only th ree ambiguous nucleo tide po sitions ( I TS po si tio ns 453, 555, and
565, Fig. 1) . This homogenei ty is also found in ITS sequences of most f low ering plants,
al though a high lev el of po lymo rphism w as reported f rom Winteraceae
[ 6] . This most re-
ma rkable fea ture o f rDN A, the overall int raspeci fic sequence homogenei ty among members
o f the mul tig ene fami ly, has been referred to as concer ted evo lution.
This is the first repo rt o f I TS sequence of Lamiaceae. Bo th the leng th and G+ C con-
tent percentag e o f IT S regions in Siphocranion nudipes fall w ithin the range of tho se o f
f low ering plants. The ITS sequence o f Siphocranion nudipes is relativ ely homogeneous and
only th ree nucleo tide po si tio ns w ere ambiguous. This sequence may serv e as a basis for
further phylog enetic and compa ra tiv e analysis o f Lamiaceae. The relativ e ef feciency of se-
quencing ITS regions and the lack o f int raspeci fic sequence polymorphism makes this an
a tt ractiv e phylog enetic ma rker fo r this family.
Acknoledgement　 This study w as suppo r ted by the Colo rado State Univ er sity Car eer Advancement
Awa rd and th e labo rato ry set- up funds to JW , and by the Nationa l Natural Science Foundation o f China,
the Foundation o f th e Na tional Education Committee the Natura l Science Foundation o f Guangdong
Prov ince and the Na tural Science Foundation o f Zhongshan Univ e rsity to SS. We thank Chen Yueqin, Qiu
Xiao zhong and Tang Shaoqing fo r DN A ex trac tion and Liao Wenbo for litera ture search.
Ref erences
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55第 2期　　　　　　　施苏华等: 光柄筒冠花核糖体 DNA ITS区序列
3　 Kudo . Siphocranion. Mem Faci Agr , T aihoku Univ , 1929 ( 2): 53
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7　 Wen J, Zimmer E A. Phylog eny and biogeog raphy of Panax L . ( the g inseng genus, A raliaceae):
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12　 Baum D A, Sy tsma K J, Hoch P C. A phy log enetic analy sis o f Epilobium ( Onag raceae) based on
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13　 Venka tesw ar lu K, Naza r R. A conserv ed co re str uc ture in the 18～ 25S rRN A inter genic r eg ion
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光柄筒冠花核糖体 DNA ITS区序列
施苏华 　文　军　朱林峰　朱忠保　黄玲英
摘　要　测定了中国特有植物光柄筒冠花 ( Siphocranion nudipes)的 IT S区序列 , 分析了其结构特征、 G
+ C百分含质量 (w ( G+ C) /% )及序列变异性 , 并比较了光柄筒冠花与其他被子植物的 ITS区序列特征 .
光柄筒冠花的 ITS区和 5. 8S DN A的总长度为 609个核苷酸 , 其中 ITS-1为 207 bp, ITS-2为 236 bp,
5. 8SrDN A为 166 bp; 总的 w ( G+ C)为 59. 11% , 其中 ITS-1为 64. 25% 、 ITS-2为 57. 63%和 5. 8S rDNA为
53. 61% . 这一序列的测定为唇形科植物的 ITS区序列分析及多态性研究打下了基础 , 同时对进一步研究
这一我国特有植物的生态学及物种保护学有着重要意义 . 这是唇形科 ITS区序列的首次报道 .
关键词　序列 , 光柄筒冠花 , ITS区
56 中山大学学报 (自然科学版 ) 　　　　　　　　　　　　　第 37卷
中山大学生命科学学院 , 广州 510275