全 文 ：第27卷 第 3期 西 南 师 范 大 学 学 报(自然科学版) 2002年 6月
Vol.2 7 No.3 Journal of Southwest China Normal University (Natural Science ) Jun. 2002
文章编号:1000 5471(2002)03 0433 04
Molecular Cloning of Lectin Gene
from Zephyranthes Candida
①
PANG Yong-zhen1 , 2 , YAO Jian-hong1 , SHEN Guo-an1 ,
TANG Ke-xuan1＊ , TAN Feng2
1.State Key Laboratory of Genetic Engineering , Institute of Genetics , School of Life Sciences , Fudan University , Shanghai 200433 , China ;
2.School of Life Science , Southwest China Normal University , Chongqing 400715 , China
Abstract:The full-length cDNA of Zephyranthes candida lectin gene was cloned from leaves by using RACE-
PCR.Through comparative analysis of Zephyranthes candida lectin gene and its deduced amino acid sequence
with those of other Amaryllidaceae species , Zephyranthes candida lectin gene was found to encode a precursor
lectin with signal peptide.Zephyranthes candida lectin was a mannose-binding lectin with three mannose-binding
boxes like lectins from other species of Amaryllidaceae.
Key words:cDNA;lectin gene;RACE-PCR;Zephyranthes candida
CLC number:Q789 Document code:A
Using pest-resistant varieties to control pests in various crop species is very economical , effective and safe compared
with using pesticides.However , conventional breeding of such plants is a difficult and long process , which has not been
successful for insect resistance.The very important groups of insects that suck the phloem of plants(including aphids and
planthoppers)have proven difficult to control by conventional plant breeding , a matter made worse by their importance as
vectors of plant viruses[ 1] .Recent studies show that some plant lectins are toxic to sap-sucking insects in artificial diet
assay[ 2 ,3] , among which the lectin(GNA)from snowdrop(Galanthus nivalis), encoded by the gna gene , is the most
toxic.Transgenic tobacco and rice expressing GNA showed significant insecticidal activity towards the peach potato aphids
(Myzus persicae Sulzer)[ 4] and rice brown planthopper(BPH , Nilaparvata lugens)[ 5] respectively in bioassay and feeding
tests.
Interestingly , up to now , most of the lectin genes that show more or less inhibition to sap-sucking insects including
aphids and planthopper by artificial diet assays were cloned from species of Amaryllidaceae such as Galanthus nivalis ,
Narcissus pseudonarcissus and Hippeastrum
[ 3] .As Zephyranthes candida belongs to Amaryllidaceae , it is speculated that
Zephyranthes candida lectin also has similar inhibitory effect on sap-sucking insects like other lectins from Amarylli-
daceae and may play a role in controlling sap-sucking insects by genetic engineering.Up to now , there is no report on the
molecular cloning of lectin gene from Zephyranthes candida .In this paper , the cloning of lectin gene from Zephyranthes
candida was presented , which enables us to test its effect on aphids and planthoppers by transferring the gene into tobac-
① 收稿日期:2001 11 29
作者简介:庞永珍(1976-), 女 , 河南洛阳人 , 硕士研究生 , 主要从事植物分子遗传学研究.
DOI :10.13718/j.cnki.xsxb.2002.03.035
co and rice in the future.
1 Material and Method
1.1 Plant material
The young leaves of Zephyranthes candida , collected from Shanghai Chinese Medical University.
1.2 Template RNA
The leaves were used as the starting material for RNA isolation according to the manufacturers instructions(TRIzol
Reagents , Gibco , USA).
Approximately 120 ng of RNA was used as the template in the RACE-PCR reaction for the cloning of the lectin gene
of Zephyranthes candida using the protocol described by the manufacturer (RACE PCR Kit , Gibco , USA).
1.3 PCR Amplification
Several primers , designed according to the conservative amino acid sequences of Amaryllidaceae , were used in 3
and 5RACE-PCR amplifications of the lectin gene.PCR was carried out by denaturing at 94℃for 5min followed by 35
cycles of amplification(94℃ for 1 min , 60℃ for 1 min , 72℃ for 2 min)and by 10 min at 72℃.
Firstly , 3RACE-PCR reaction was conducted using the pair of primers:AP and CLF3(5-CTCATTTTGGCCC-
CGCCATCTTCC-3).The CL3product(Fig.1 , 492bp)was collected and ligated , after purification , to T-easy vector
prior to sequencing using ABI 377 Sequencer(Perkin-Elmer , USA).Then , 5RACE-PCR amplification was carried out
to amplify the 5end of the lectin gene by firstly using primers AAP and CLR1 (5-GCTTTATTATCCCAGCGGTGC-
3)followed by using the following pair of primers:AUAP and CLR2(5-CAGTCCTCCTGCATGATGAAAG-3).The
CL5product(Fig.2 , 256bp)was ligated to T-easy vector followed by sequencing.By comparing and aligning the se-
quences of CL3and CL5, the full-length cDNA sequence(Fig.3 , 647bp)of Zephyranthes candida lectin genewas ob-
tained.Finally , RT-PCR reaction was carried out by the use of the following pair of primers CLF1 (5-TCGAAA-
CAAGCAGATAATATA-3)and CLR3 (5-AATCTAGCAGAAGTGAAGTCT -3)and the full-length cDNA of the
lectin gene was amplified.The full-length cDNA of Zephyranthes candida lectin was then analyzed for the presentation of
lectin blocks , signal peptide and mannose-binding sites as described before[ 6] .
Fig.1 CL3product Fig.2 CL5product Fig.3 Full-length cDNA Sequence
2 Results and Discussion
The cloned full-length cDNA of Zephyranthes candida lectin was 647bp , a size very similar to those of other Amarylli-
daceae species[6 ,7] (Table 1).The deduced amino acid sequence (159aa)of the lectin gene is given below(Table 2).
434 西南师范大学学报(自然科学版) 第 27卷
Table 1 The Full-Length cDNA Sequence of Zephyranthes Candida Lectin Gene
1 TCGAAACAAG CAGATAATAT AAGCACAAAC TGCAAAATGA CTAGGATAAC CTTCCTAGTT
61 GTGGCCGCCA TCTTCCTTGG TGTGATCACA CCATCTTGCC TCAGTGTCTG TGACAGCATC
121 CTGTACTCCG GCGAGACTCT CTCCGCCGGC CAATCCCTCA ACAACGGGCC GTACACTTTC
181 ATCATGCAGG AGGACTGCAA TCTAGTCCTG TACAATGTAG ACAAGCCGAT CTGGGCGTCA
241 AACACGGGCG GCCTCGCACG GGGCTGCCAC CTCAGCATGC AGAGGGATGG AAACCTGGTC
301 GTGTACAGCC AGAGGAACCG TCCCATTTGG GCAAGCAACA CCGGGGGCCA CAATGCTGCA
361 AACTACGTGC TGATTCTTCA GAAAGATCGC AACGTCGTGA TCTATGGACC TGCGAAGTGG
421 GCTACCGGGA CTTATACCGG GGCTGTTGGA ATTTCCGGGT TCGAAACTGG CACCGCTGGG
481 ATAATAAAGC CAGTGACCGT CAAGTCAGCC AAGTAATGGC AAGTGTGAAG CTTGCATGCA
541 GGTGTGAAGC GTACGTAATA AGTGCACTTG TGGTTATTGC ACATGTACCC TCGTACTACT
601 GTAGTGGCTC CACTCTTTAA TAGTAGAGAC TTCACTTCTG CTAGATT
Table 2 The Deduced Amino Acid Sequence of Zephyranthes candida Lectin Gene
1 MTRITFLVVA AIFLGVITPS CLSVC ↑DSILY SGETLSAGQS LNNGPYTFIM
51 QEDCNLVLYN VDKPIWASNT GGLARGCHLS MQRDGNLVVY SQRNRPIWAS
101 NTGGHNAANY VLILQKDRNV VIYGPAKWAT GTYTGAVGIS GFETGTAGII
151 KPVTVKSAK
The arrow indicated the cleavage site of signal peptide(between C and D).
Blocksanalysis revealed that the deduced amino acid sequence of Zephyranthes candida lectin had the functional do-
mains specific for lectin(boxed sequences of 26th-51st , 66th-100th and 105th-124th), indicating the cloned gene was
a lectin gene (Table 3).
Table 3 The Result of BlocksAnalysis of the Deduced Amino Acid Sequence of
Zephyranthes candida Lectin Gene
1 MTRITFLVVA AIFLGVITPS CLSVC DSILY SGETLSAGQS LNNGPYTFIM
51 QEDCNLVLYN VDKPIWASNT GGLARGCHLS MQRDGNLVVY SQRNRPIWAS
101 NTGG HNAANY VLILQKDRNV VIYGPAKWAT GTYTGAVGIS GFETGTAGII
151 KPVTVKSASN GKCEACMQV
Comparative analysis of cDNA and amino acid sequences of Zephyranthes candida lectin with those of other Amaryl-
lidaceae lectins indicated that Zephyranthes candida lectin gene encoded a precursor lectin with a signal peptide.Accord-
ing to the rule of predicting lectin signal peptide[ 8] , the cleavage site of signal peptide of Zephyranthes candida precursor
lectin was between Cys25 (C) and Asp26 (D)(beforeDSIL), which was in good agreement with those of lectins from
other Amaryllidaceae species such as Galanthus nivalis , Clivia miniata , Narcissus pseudonarcissus and Hippeastrum[ 6]
(Table 2).
Sugar-binding analysis revealed that like other lectins of Amaryllidaceae such as Galanthus nivalis , Clivia miniata
and Narcissus pseudonarcissus , Zephyranthes candida lectin also had three specific conservative mannose-binding boxes
(QDNY), indicating Zephyranthes candida lectin was a mannose-specific lectin(Table 4).
Table 4 The Sugar-binding Boxes(QDNY)of Zephyranthes candida lectin
1 MTRITFLVVA AIFLGVITPS CLSVCDSILY SGETLSAGQS LNNGPYTFIM
51 QE DC NLVL YN VDKPIWASNT GGLARGCHLS M QR DG NLVV Y SQRNRPIWAS
101 NTGGHNAANY VLIL QK DR NV VI YGPAKWAT GTYTGAVGIS GFETGTAGII
151 KPVTVKSASN GKCEACMQV
The cloning of Zephyranthes candida lectin gene enables us to test its potential role in controlling sap-sucking in-
sects by transferring the gene into tobacco and rice.The genetic transformation of tobacco and rice using the lectin gene
is undergoing.
435第 3期 Molecular Cloning of Lectin Gene from Zephyranthes Candida
Reference:
[ 1] Mochida O , Wahyu A , Surjani TK.Some Considerations on Screening Resistant Cultivars or Lines of Rice Plant to the Brown Planthop-
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[ 2] Powell KS , Gatehouse AMR , Hilder VA , et al.Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal
stages of two important rice pest , Nilaparvata lugens and Nephotettix cinciteps [ J] .Entomol Exp Appl , 1993 , 66(2):119-126.
[ 3] Powell KS , Gatehouse AMR , Hilder VA , et al.Antifeedant effects of plant lectins and an enzymes on the adult stage of the rice brown
planthopper , Nilaparvata lugens [ J] .Entomol Exp Appl , 1995 , 75(1):51-59.
[ 4] Hilder VA , Powell KS , Gatehouse AMR , et al.Expression of snowdrop lectin in transgenic tobacco plants results in added protection a-
gainst aphids [ J] .Transgenic Res , 1995 , 4(1):18-25.
[ 5] Rao KV , Rathore KS , Hodges TK , et al.Expression of snowdrop lectin (GNA)in transgenic rice plants confers resistance to rice
brown planthopper [ J] .Plant J , 1998 , 15(4):469-477.
[ 6] Van Damme ELM , Goldstein IJ , Vercammen G , et al.Lectins of members of the Amaryllidaceae are encoded by multigene families
which show extensive homologies [ J] .Physiol Plant , 1992 , 86:245-252.
[ 7] Van Damme ELM , Smeets K , Van Leuven F , et al.Molecular cloning of mannose-binding lectins from Clivia miniata [ J] .Plant Mol
Biol , 1994 , 24(5):825-830.
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葱莲凝集素基因的克隆
庞永珍1 , 2 , 姚剑虹1 , 申国安1 , 唐克轩1 , 谈 锋2
1.复旦大学生命科学学院遗传学研究所遗传工程国家重点实验室 , 上海 200433;
2.西南师范大学生命科学学院 , 重庆 400715
摘要:利用 RACE-PCR技术从葱莲叶片中克隆出葱莲凝集素的全长 cDNA.通过比较葱莲同其它石蒜科植物的凝集
素基因序列和推测的氨基酸序列 ,发现葱莲凝集素基因编码一具有信号肽的前体蛋白.葱莲凝集素同其它石蒜科植
物凝集素一样为具有 3个甘露糖专一结合盒的凝集素.
关 键 词:cDNA;凝集素基因;RACE-PCR;葱莲
责任编辑 汤兴华
436 西南师范大学学报(自然科学版) 第 27卷