Cloning and functional analysis of a flavonoid-3-O-glucosyltransferas gene from <i>Indosasa hispida</i>-文献传递-植物通论文库

作者：王毅^1,2, 王晨晨^1,3, 周旭^1,3, 毕玮^1,2, 杨宇明^1,2, 王娟^1,2*

摘要：类黄酮-3-O-葡萄糖基转移酶(Flavonoid-3-O-glucosyltransferase,3GT)是花青苷(Anthocyanins)生物合成途径中的关键酶,它主要负责将不稳定的花色素转变为稳定的花色素苷,虽然目前已经从其他植物中克隆获得类黄酮-3-O-葡萄糖基转移酶,但是对竹子中的类黄酮-3-O-葡萄糖基转移酶并不清楚。该文以产生花青素的七彩红竹(Indosasa hispida McClure cv. Rainbow)为材料,首先通过3GT的同源比对后设计3GT基因特异引物,获得3GT基因片段; 然后运用RT-PCR及RACE技术从七彩红竹茎中克隆得到完整的3GT基因(Ih3GT)。结果表明:Ih3GT基因的cDNA全长序列为1 730 bp,含有1个1 425 bp的开放阅读框(ORF),编码474个氨基酸; 系统进化分析显示,七彩红竹3GT与其他禾本科植物的3GT聚类到同一个分支; 该基因推断的蛋白与水稻(Oryza sativa)3GT蛋白的相似性为69%,与二穗短柄草(Brachypodium distachyon)3GT的相似性为67%; 经氨基酸序列比对,推断七彩红竹3GT含有糖基转移酶基因家族特有的结构域PSPG-box; 半定量PCR的结果显示,七彩红竹3GT基因在微红的幼茎中大量表达,而在其他组织中并不表达,说明Ih3GT具有组织表达特异性。该结果为今后深入研究七彩红竹花色苷的形成机理、鉴定Ih3GT酶活性以及利用Ih3GT基因培育竹子新品种奠定了基础。

Abstract:Flavonoid -3-O- glucosyltransferase(3GT)is the key enzyme in anthocyanins biosynthesis,which transforms unstable anthocyanidins into stable anthocyanins. The function of Flavonoid -3-O- glucosyltransferase in bamboo is unclear,although many flavonoid -3-O- glucosyltransferases from other plant had been reported,so far. Indosasa hispida McClure cv. Rain bow is of few bamboo species which can produce anthocyanins in clum. Therefore,I. hispida McClure cv. Rain bow is very important material to reveal the function of Flavonoid -3-O- glucosyltransferase in bamboo and the mechanism of anthocyanins biosynthesis in bamboo. Therefore,it is first step to clone 3GT gene from I. hispida McClure cv. Rain bow. First,gene special primers of 3GT were obtained based on the homology analysis of reported flavonoid -3-O- glucosyltransferase,and the RNA was extracted from yound red clum which produce anthocyanins part in I. hispida McClure cv. Rain bow by Trizol method. And then gene fragment of I. hispida McClure cv. Rainbow was cloned with special primers of 3GT. According to the obtained fragment sequence,the primers were designed,which were used in rapid amplification of cDNA ends(RACE). Next,the 3‘ end and 5‘ end sequence of 3GT were obtained by RACE,the full length gene of Ih3GT was assembled by ATG software. Finally,the full length gene of Ih3GT was cloned from I. hispida McClure cv. Rainbow by reverse transcription-polymerase chain reaction(RT-PCR). ORF analysis program was used to confirm open reading frame,MEGA software was used to construct phylogenetic tree. the DNAman software was used in Homology analysis,and semi RT-PCR was applied in gene expression profile. The results showed that the cDNA sequence of Ih3GT consisted of 1 425 bp open reading frame(ORF)which encodes 474 amino acid, Ih3GT and 3GT from Poaceae were grouped in same clade,the deduced protein of 3GT from I. hispida McClure cv. Rain bow shared 69% identities with 3GT of Oryza sativa and shared 67% identities with 3GT of Brachypodium distachyon. Homology analysis showed that deduced Ih3GT protein had a glycosyltransferase signature domain PSPG-box. Expression profiling with semi RT-PCR analysis revealed that Ih3GT was expressed in young red culm and was not expressed in old culm,old leaf,young leaf and shoot. This implied that the expression of Ih3GT from I. hispida McClure cv. Rain bow showed obvious tissue specificity. This study will provide useful information to reveal mechanism of anthocyanins biosynthesis in I. hispida McClure cv. Rainbow in future. Ih3GT gene can be transformed into Eschera coli,and heterologous expression obtain Ih3GT protein and detect the enzyme activity of Ih3GT,and it can also be transformed into Arabidopsis thaliana or Oryza sativa,the function of Ih3GT will be confirmed by heterologous expression. The obtained Ih3GT also can been implied in bamboo breeding or other horticultural plants by genetic engineering.

全文：广西植物 Guihaia Apr． 2015，35(2):282 － 284 http:/ / journal． gxzw． gxib． cn
王发松，温铁龙，牛苗，等．中国隔距兰属(兰科)一新记录［J］．广西植物，2015，35(2):282 －284
Wang FS，Wen TL，Niu M，et al． A new record of Cleisostoma(Orchidaceae)from China［J］． Guihaia，2015，35(2):282 －284
A new record of Cleisostoma (Orchidaceae)from China
WANG Fa-Song1，2，WEN Tie-Long3，NIU Miao3，4，LI Lin3*
(1． Key Laboratory of Biologic Ｒesources Protection and Utilization of Hubei Province，Enshi 445000，China;2． College
of Chemistry and Environmental Engineering，Hubei University for Nationalities，Enshi 445000，China;
3． South China Botanical Garden，Chinese Academy of Sciences，Guangzhou 510650，China;
4． University of Chinese Academy of Sciences，Beijing 100049，China)
Abstract:Cleisostoma Bl． is an orchid genus with approximately 100 accepted species widely distributed in tropical and
subtropical areas． There are about 16 species in China (4 endemic)before． Cleisostoma aspersum (Ｒchb． f．)Garay，
so far known only from Yunnan，is reported from China． A detailed description and photos are provided． This species is
easily distinguished from its relatives by bilobulate leaves，fat and obtuse midlobe of the lip，conical rostellum with an
oblique apex，and beaked operculum． It is similar to Cleisostoma discolor Lindl． in floral morphology，but the latter dif-
fers by having a lip with thin spathulate truncate midlobe，with erose front edge．
Key words:Cleisostoma;Cleisostoma aspersum;Orchidaceae;new record;China
CLC number:Q949． 71 Document code:A Article ID:1000-3142(2015)02-0282-03
中国隔距兰属(兰科)一新记录
王发松1，2，温铁龙3，牛苗3，4，李琳3*
(1．生物资源保护与利用湖北省重点实验室，湖北恩施 445000;2．湖北民族学院化学与环境工程学院，
湖北恩施 445000;3．中国科学院华南植物园，广州 510650;4．中国科学院大学，北京 100049 )
摘要:隔距兰属(Cleisostoma Bl．)是兰科植物中较大的一个属，约有 100种，广泛分布于热带、亚热带地区。中
国约有 16种，其中 4种为特有。该文报道了该属一中国新记录———二齿叶隔距兰［Cleisostoma aspersum (Ｒchb．
f．)Garay］，并提供描述和照片，目前该种仅见于云南。与近缘种类相比，本种具有以下鉴别特征:叶齿状二裂;
唇瓣中裂片肥厚，前缘钝;蕊喙圆锥状，先端歪斜;药帽喙状等。在花部形态上，该种与 Cleisostoma discolor Lindl．
相似，然而后者的唇瓣中裂片明显较薄，匙状方形，并且具有啮齿状边缘，易于区别。
关键词:隔距兰属;二齿叶隔距兰;兰科;新记录;中国
Cleisostoma Bl． was established by Blume
(1825)． This is a large genus with about 100 species
and also a highly variable and heterogeneous group af-
ter clarification of much synonymy and after moving
many species to some new and old genera，where fur-
ther separations may be expected (Seidenfaden，1992;
Seidenfaden et al．，1992)． It is widespread from Sri
Lanka，India，SE Asia，Malaysia，Indonesia，Philip-
pines，New Guinea，and Pacific Islands to Australia
(Pearce et al．，2002)． There were about 16 species in
收稿日期:2014-04-25 修回日期:2014-07-18
基金项目:国家自然科学基金 (31100149)
作者简介:王发松(1969-)，男，湖北巴东人，博士，主要研究方向为药用植物资源与化学，(E-mail)zsuwangfasong@ aliyun． com。
* 通讯作者:李琳，博士，副研究员，主要从事植物分类学、园林植物与观赏园艺方向的研究，(E-mail)lilin@ scib． ac． cn。
DOI:10.11931/guihaia.gxzw201403056
Fig． 1 Cleisostoma aspersum (Ｒchb． f．)Garay A． Habitat;B． Inflorescence;C． Front view of a single flower;D． Operculum;
E． Pollinarium;F． Lateral view of column and lip;G． Lateral view of lip;H． Front view of column with anther in situ;
I． Lateral view of column with anther ex situ． (D-E． Bar =1 mm;F-I． Bar =2 mm)．
China (4 endemic)and 12 in Yunnan，China (Su，
2000;Chen et al．，2009)before．
During a field tour in Yunnan Province in 2012，
an orchid species was collected and introduced in the
nursery of South China Botanical Garden，Chinese A-
cademy of Sciences． In August，2013，the species
bloomed． After extensive morphological comparison，it
was identified as Cleisostoma aspersum (Ｒchb． f．)Ga-
ray． Subsequently，the identity was confirmed in con-
sultation with relevant literatures (Ｒeichenbach，1865;
Hooker，1890;King et al．，1898;Garay，1972;Seiden-
faden，1975;Seidenfaden，1992;Pearce et al．，2002;
Lucksom，2007)and type materials． A thorough scruti-
ny of literature revealed that this species was a new re-
cord to the Chinese orchid flora (Chen et al．，1998;
Su，2000;Chen et al．，2009)and here reported．
Cleisostoma aspersum (Ｒchb． f．)Garay in Bot．
Mus． Leafl． 23(4):164． 1972．———Sarcanthus aspersus
Ｒchb． f．，Hamburger Garten-Blumenzeitung 21:297．
1865． Type:Myanmar，Moulmein，Veitch 39 (W，pho-
to!)．
———Cleisostoma bicuspidatum Hook． f．，Fl． Brit．
India 6(1):75． 1890．———Stereochillus bicuspidatum
(Hook． f．)King ＆ Pantl． in Ann．Ｒoy． Bot． Gard．
3822期王发松等:中国隔距兰属(兰科)一新记录
Calcutta 8:236． t． 314． 1898． Type:India，Khasia
Hills，Mann 56 (K，photo!)．
Cleisostoma aspersum (Ｒchb． f) Garay
(Fig． 1)
A miniature to small sized epiphytic plant，10 － 20
cm tall． Stem short，1 － 5 cm long，branched with nu-
merous roots at base，covered by leaf sheaths;sheaths
overlapping． Leaves 7 － 9，coriaceous，linear-oblong，
sharply and obliquely bi-lobed at apex，6 － 15 cm long，
0． 8 － 1． 5 cm wide，the lobules often erose，pale-green
to dark green，sheathing at base． Inflorescence 2 － 3，
axillary，from base of leaf sheaths，10 － 20 cm long，
shortly branched，peduncle slender，glabrous，dark-pur-
ple，5 － 12 cm long;floral bracts ovate-lanceolate，suba-
cute，1 － 1． 5 mm long，distantly placed;rachis 3 － 10
cm long;pedicel and ovary glabrous，ribbed，3． 5 － 5
mm long． Flowers 6． 5 － 8 mm across，spreading or re-
flexed;Sepals and petals brownish，with rose markings
along midribs;dorsal sepal ovate，3． 5 － 4 mm long，2 －
2． 5 mm wide，obtuse;lateral sepals broadly elliptic，3．
8 － 4 mm long，2 － 2． 5 mm wide，recurved． Petals ob-
long-obovate，oblique，3． 8 － 3． 9 mm long，1． 6 － 1． 8
mm wide，obtuse． Lip adnate to the column，3-lobed，
ca． 3 mm long，produced to a prominent spur at base，
infundibulate，fleshy，white，speckled or flushed with
rose and yellow;spur ca． 4 mm long，cylindric，parallel
to the ovary;lateral lobes truncate，erose at edges;mid-
lobe broadly ovate，concave，curving upwards，acute，
base with 2 cushion-like calli occluding the entrance to
the spur． Column short，ca． 1． 5 mm long，white;rostel-
lum large，decurved and slightly hooked at apex． Oper-
culum cordate，depressed and beaked at apex，1． 5 － 1．
8 mm long． Pollinia 4，orbicular，compressed attached
by pairs;stipes ca． 1． 5 mm long，thickened at the mid-
dle and tapered at apex and also at base;gland small，
oval． Flowering in July － August．
Distribution:Sikkim Himalayas，NE India，Bhu-
tan，Nepal，Myanmar，Thailand and Vietnam． New re-
cord to China．
China:Yunnan)，Wenshan，Zhuang and Miao Au-
tonomous Prefecture，Epiphytic on the trunk in humid
sparse woods，tropical rain forest，alt． 1 020 m． 12 Feb．
2012，L． Li 158 (IBSC)．
It is similar to Cleisostoma discolor Lindl．，but dif-
fers by having a lip with fat midlobe directed upwards，
without erose front edge．
Acknowledgments We thank Dr． Cao Ming
(Guangxi Institute of Botany，CAS)for his help in the
review of the manuscript．
Ｒeferences:
Blume CL． 1825． Bijdragen tot de Flora van Nederlandsch Indie
［M］． Batavia:Ter Lands Drukkerij:6
Chen SC，Tsi ZH． 1998． The Orchids of China［M］． Beijing:China
Forestry Publishing House:61 －62
Chen SC，Wood JJ． 2009． Orchidaceae［M］/ /Wu ZY，Peter HＲ，
Hong DY (eds)． Flora of China． Beijing:Science Press;St．
Louis:Missouri Botanical Garden Press，25:458 －463
Garay LA． 1972． On the systematics of the monopodial orchids I
［J］． Bot Mus Leafl Harv Univ，23(4):149 －212
Hooker JD． 1890． The Flora of British India (Orchideae)［M］．
London:L．Ｒeeve ＆ Co．，The Oast House，Brook，Ashford，
Kent，6:75
King G，Pantling Ｒ． 1898． The orchids of the Sikkim-Himalaya
［J］． Ann Ｒoy Bot Gard，(Calcutta) ，8:236，314
Lucksom S Z． 2007． The Orchids of Sikkim and North East Hima-
laya［M］． Siliguri:Lucksom:812 －816
Pearce N，Cribb PJ． 2002． The orchids of Bhutan，in Flora of Bhu-
tan Vol． 3 part 3［M］． Edinburgh:Ｒoyal Botanic Garden，Edin-
burgh and Ｒoyal Government of Bhutan:507 －514
Ｒeichenbach HG． 1865． Ueber einigen Garten-Orchideen［J］．
Hamburger Garten-und Blumenzeitung，21:293 －301
Seidenfaden G． 1975． Orchid genera in Thailand II:Cleisostoma
Bl．［J］． Dansk Bot Ark，29(3):1 －80
Seidenfaden G． 1992． The orchids of Indochina［J］． Opera Botani-
ca，114:385 －395
Seidenfaden G，Wood JJ． 1992． The Orchids of Peninsular Malayi-
sia and Singapore［M］． Denmark:Olsen ＆ Olsen:619 －631
Su HJ，Huang TC． 2000． Flora of Taiwan(2nd edition)［M］． Taipei:
Editorial Committee of the Flora of Taiwan，5:806 －808
Tsi ZH． 1999． Zeuxine Lind1［M］/ /Lang KY，Chen SC，Luo YB
(eds)． Flora Ｒepulica Popularis Sinicae． Bejing:Science Press，
19:311 －330
482 广西植物 35卷

Cloning and functional analysis of a flavonoid-3-O-glucosyltransferas gene from Indosasa hispida

中国隔距兰属(兰科)一新记录(英文)

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