用SSH结合cDNA芯片分离、鉴定淡色库蚊溴氰菊酯抗性相关基因*
|
摘要
杀虫剂一直是人类杀灭害虫的重要武器，尤其是40年代
有机合成杀虫剂问世以后，在病媒防制等方面发挥了巨大作
用。然而时至今日，虫媒病无论在分布范围还是感染人数上，
都呈明显的增长势头。究其主要原因，是连续、大量地使用
杀虫剂导致媒介昆虫产生了抗药性（以下简称抗性）。
目前已知，昆虫抗性是可遗传的生物进化现象，是由基
因决定的，在绝大多数情况下，抗性为多基因遗传。
对媒介抗性基因的研究，早期通常首先寻找杀虫剂的作
用靶标（受体）或杀虫剂代谢相关酶类，然后再根据发现的
特异蛋白质（酶）来克隆相应基因。如与有机磷类和氨基甲
酸酯类杀虫剂抗性相关的酯酶基因，与拟除虫菊酯类杀虫剂
抗性相关的钠通道基因与细胞色素P450基因等。以上经典研
究途径仅能分离单一基因，难以从总体上认识抗性、阐明抗
性机理。这也是抗性研究长期进展缓慢的主要原因。当前分
子生物学技术的快速发展使得大通量筛选抗性相关基因成为
可能。
淡色库蚊（Culex pipiens pallens）是我国重要的家栖
性病媒蚊种，近年来各地主要采用拟除虫菊酯类的溴氰菊酯
进行防治，已陆续有敏感性显著下降的报告。
本研究采用正向和反向抑制性差减杂交法（suppression
D 南京医科大学博士学位论文D
subtractive hybridization，SSH）分离淡色库 氰菊酯 l
l 抗除和敏感品系高表这或 固，并呆用 cDNA芯片、l
叹 Northern和 Northern blot g 同时，构建了淡 D
D 色库诌d亢性品系cDNA支J享，从中筛 出目 困的cDNA D
Dde＆｝＋列。D
11．用SSH分离淡色库蚊涣氰菊酯抗，队R兢因l
D 糊化3天内来吸血的淡色库狮隆成蚊，以对跟菊D
D 酯坑姓品系为鹏d组k ter）、对鳅菊＠郭劲感品系为g孵 D
D 组Uriver），进行正向 SSH，以 性品系高表达或特异 D
D 表腿因；以咖品系为tester、抗怯品系为driver，断
l 反向SSH，以获取敏感品系高表达或特异表达基固。分别忖影粑l
D 2品系蚊虫的总RNA、InRNA，碾一cDNA第嘴，置换 D
19：vsa．第二链，然后经Rsa酶切、接头连接、24kL减杂交 l
D 和2轮叽R，获得正、反向差减杂交严物。朵交严物纯化后将D
立9。降入质陇一PinPoint“Xa－IT－－Vector，转化入大
D 肠杆菌Wil 09。以趴片段两侧徽体序列PiflPoint和SP6为D
D51物PCR扩增，琼脂糖凝胶电帷…X段大小。D
D 结果：N、反向昭H分一瓣523和286个差献隆，D
l 构建了 2个el］含 5乃和 286个重组子的正、反向差减M。l
D 枫片段大小约3m6。初步提示淡色犀刎没氰菊酯的D
D 抗性可能涉及的并非个别基因，而是多基因或基因群；抗性D
D 不贩抗贻因控制，而且可5腑…基因。D
D．二．D
南京医科大学搏士学位论文D
2．用cDNA芯片鉴定淡色库蚊抗性和敏感品系差减支隆l
用PCR扩增棚一一的8 09个差献隆，PCR产l
物经贱、变性处理，用自动点 左制于尼龙膜戴体上，7
制成cDNA芯片。性品系和敏感品系IllRNA，反转轭经D
同赈‘？ 己为抗注探针和敏感探针。将两 与2 D
剃目同的芯片杂交，通过跟、压片、栅自显影扫和唁号扫D
描鞭弧的杂妮号。D
结果：CDNA芯片共获得差异表达克隆 2 21个，其中抗性 D
品系高表铂隆197个，卜3顾3倍以上高雄克降分另fi有D
155个和42个；髓性品系高表贩隆24个，2刁顾3倍1
以上高表达克o有15个和9个：郴卜脯2个差减氛D
怯品系中特异表达。拥究结果进一命支持本文第一I
D 部分的撕，即淡色库附涣氰菊酯的抉注可能涉及的并非D
Dc知个别基因，6是多基因或基因群；抗性不仅受抗赈因D
D 控制，而且可s腑眺基因，抗性可能航踉困8裘达
D 和特异表达、敏感基因低表达综合作用的结果。
3．用叹 Northern和 Northern blot j｝L－步跟淡色犀颜
D 性牙一铣感品系差减。寺。隆
l 逆 Northern：＄先 PCR扩增 cDNA芯片中抗性品系 3倍
］以上高一克隆16、43、30、6、38和敏感品系3倍以上
l 高表达的兑隆 33、27、5L 45、4的插入基因片段，琼脂糖
I 凝胶《膝梆至尼溅。然后分别 恰品B一品
D 系
Cloning and identification of deltamethrin resistance associated genes of Cukx p4iens pallens by combining suppression subtractive hybridization and eDNA chip*
ABSTRACT
Insecticides have played an important role in the control of pests since the early 20th century. Although important advances continue to be made in the development of alternative control measures, insecticides will remain a vital part in the integrated control programs of vectors for the foreseeable future. Unfortunately, the remarkable ability of vector populations to evolve resistance to every class of insecticide has been developed. Now it has been recognized that a change in the genetic composition of a population is a direct result of the selective effects of a toxicant An understanding of the insecticide-resistant mechanisms can lead to better management of pest resistance to pesticides. By strategy of functional cloning, several genes have been reported to be related with insecticide resistance, such as cytochrome P450 gene, sodium channel gene, esterase gene and so on. How many genes contribute in the mechanisms of insecticide resistance? Are there other genes acting on the resistance? In terms of the strategy of “phenotype cloning ” differentially expressed genes between the deltamethrin-resistant
-8-
and suscePtible ed of Culexgh~ were cfoned by the
method named SUPpessha bo hybM (SSffi, and
wer Med by N chiP, reverse nOrthem bfot and nOH
bwt A ffe mp libop was construtriby be clonin
teCboc using the deltametbrinesiStan strain of Cx tw
N as samPle and a full lengIh cDNA gen named twsin
sen was pe by SCreenin the COnStrUctin ffe mp
bo
l Isolation of drially mpssed pe betwee
deWtw and SUSeePtile hans of Cx ptw
peuens by SSH
The deWsusCePtible bo of Cx plPbo the was
origined from the Shangh Insect Ihatitu of Chinse Academ
of Science and the LC50 value was 0.0008mgh. W the
MresiM H was M by seleCted wtth
uefor ana the LC50 ds was 0.32mrt. In orta to set
the gens tha wer high and rpecilically exPreSsed in the
resiStan strain, the - poPulation ofresistan sha was used
as M and that ofsuseqle nd as the The total RNA and
M wer isolated rerpectiVely from the both for adults that
had no been fed wh blood within three-da edosion. The singl
wt and Hle ed cDNAs wer Synthsed. Then theSe
dOUble Stran cDNAs wer digested by the An I.The teste
- 9 -
dscDNAs were -- added to adaPo l and adaPtO 2K
bo, the de dscDNAs were mp with the excess
be oc An bo the op pe were ~ by
-- PCh The PCR Produds were cfoned into im
~ vectors dri were ds bo E coli mloo. Ai
wt 523 subwt clones were pe.
hi de to get the pe ta were highly ana peifically
exPwt in the Hle tw the m poPulation of
Hle ni was used as teSter and that ofresStan ni as
driVer The SSH was wi as above and 286 subwt clones
were pe in the ena.
The length of SSH be inserted into Pinpofor vector was
N 300600 base pairs by PCR detoCon.
The results aboVe SUggested tha man gens Inay play roles on
the dehosistance orcx twpouas anu that no o*
the retw W may H on insecticide-resStanCe, bot also the
Wle pe.
2 hidendfication ofsSH clones by cDNA chiP
A M chiP containin 809 SSH clones was mad. Briefly the
bo in the hafor pedd vecto were amnlified by Pcn
using seqUenCes flankin the cfonin site as PriIners. The PCR
wt were viW on l% Wse ge to ensure adW
PCR ammpcation Prior to being robecally Printed onto nyon
- l0 -
meInbran. The un frOm the both ch were repe
rnad as resistan Probe and SUSW Probe by reverse
W in the M of a ?3P dCTP
scanning, W and subSeqUen -- were
-- It was that l55 and 42 pe were re~ 2-3 fod
and >3 fold OVeWed in the resed tw l5 and 9 pe
wer rerpeCtively 2-3 fold and >3 fod OVeWssed in the
He the. In additio there wer 2 gens gncifically
Wssed in the resiStan sha.
ACcoding tD the results, we Med that vecto reSStane to
insectiCde may be rethed ed OVeWssed. lowWsed and
sPecifically exPrssed gens in insecticideresiSha sha.
3 M Mdri of SSH clones rewt by reverse
nota
有机合成杀虫剂问世以后，在病媒防制等方面发挥了巨大作
用。然而时至今日，虫媒病无论在分布范围还是感染人数上，
都呈明显的增长势头。究其主要原因，是连续、大量地使用
杀虫剂导致媒介昆虫产生了抗药性（以下简称抗性）。
目前已知，昆虫抗性是可遗传的生物进化现象，是由基
因决定的，在绝大多数情况下，抗性为多基因遗传。
对媒介抗性基因的研究，早期通常首先寻找杀虫剂的作
用靶标（受体）或杀虫剂代谢相关酶类，然后再根据发现的
特异蛋白质（酶）来克隆相应基因。如与有机磷类和氨基甲
酸酯类杀虫剂抗性相关的酯酶基因，与拟除虫菊酯类杀虫剂
抗性相关的钠通道基因与细胞色素P450基因等。以上经典研
究途径仅能分离单一基因，难以从总体上认识抗性、阐明抗
性机理。这也是抗性研究长期进展缓慢的主要原因。当前分
子生物学技术的快速发展使得大通量筛选抗性相关基因成为
可能。
淡色库蚊（Culex pipiens pallens）是我国重要的家栖
性病媒蚊种，近年来各地主要采用拟除虫菊酯类的溴氰菊酯
进行防治，已陆续有敏感性显著下降的报告。
本研究采用正向和反向抑制性差减杂交法（suppression
D 南京医科大学博士学位论文D
subtractive hybridization，SSH）分离淡色库 氰菊酯 l
l 抗除和敏感品系高表这或 固，并呆用 cDNA芯片、l
叹 Northern和 Northern blot g 同时，构建了淡 D
D 色库诌d亢性品系cDNA支J享，从中筛 出目 困的cDNA D
Dde＆｝＋列。D
11．用SSH分离淡色库蚊涣氰菊酯抗，队R兢因l
D 糊化3天内来吸血的淡色库狮隆成蚊，以对跟菊D
D 酯坑姓品系为鹏d组k ter）、对鳅菊＠郭劲感品系为g孵 D
D 组Uriver），进行正向 SSH，以 性品系高表达或特异 D
D 表腿因；以咖品系为tester、抗怯品系为driver，断
l 反向SSH，以获取敏感品系高表达或特异表达基固。分别忖影粑l
D 2品系蚊虫的总RNA、InRNA，碾一cDNA第嘴，置换 D
19：vsa．第二链，然后经Rsa酶切、接头连接、24kL减杂交 l
D 和2轮叽R，获得正、反向差减杂交严物。朵交严物纯化后将D
立9。降入质陇一PinPoint“Xa－IT－－Vector，转化入大
D 肠杆菌Wil 09。以趴片段两侧徽体序列PiflPoint和SP6为D
D51物PCR扩增，琼脂糖凝胶电帷…X段大小。D
D 结果：N、反向昭H分一瓣523和286个差献隆，D
l 构建了 2个el］含 5乃和 286个重组子的正、反向差减M。l
D 枫片段大小约3m6。初步提示淡色犀刎没氰菊酯的D
D 抗性可能涉及的并非个别基因，而是多基因或基因群；抗性D
D 不贩抗贻因控制，而且可5腑…基因。D
D．二．D
南京医科大学搏士学位论文D
2．用cDNA芯片鉴定淡色库蚊抗性和敏感品系差减支隆l
用PCR扩增棚一一的8 09个差献隆，PCR产l
物经贱、变性处理，用自动点 左制于尼龙膜戴体上，7
制成cDNA芯片。性品系和敏感品系IllRNA，反转轭经D
同赈‘？ 己为抗注探针和敏感探针。将两 与2 D
剃目同的芯片杂交，通过跟、压片、栅自显影扫和唁号扫D
描鞭弧的杂妮号。D
结果：CDNA芯片共获得差异表达克隆 2 21个，其中抗性 D
品系高表铂隆197个，卜3顾3倍以上高雄克降分另fi有D
155个和42个；髓性品系高表贩隆24个，2刁顾3倍1
以上高表达克o有15个和9个：郴卜脯2个差减氛D
怯品系中特异表达。拥究结果进一命支持本文第一I
D 部分的撕，即淡色库附涣氰菊酯的抉注可能涉及的并非D
Dc知个别基因，6是多基因或基因群；抗性不仅受抗赈因D
D 控制，而且可s腑眺基因，抗性可能航踉困8裘达
D 和特异表达、敏感基因低表达综合作用的结果。
3．用叹 Northern和 Northern blot j｝L－步跟淡色犀颜
D 性牙一铣感品系差减。寺。隆
l 逆 Northern：＄先 PCR扩增 cDNA芯片中抗性品系 3倍
］以上高一克隆16、43、30、6、38和敏感品系3倍以上
l 高表达的兑隆 33、27、5L 45、4的插入基因片段，琼脂糖
I 凝胶《膝梆至尼溅。然后分别 恰品B一品
D 系
Cloning and identification of deltamethrin resistance associated genes of Cukx p4iens pallens by combining suppression subtractive hybridization and eDNA chip*
ABSTRACT
Insecticides have played an important role in the control of pests since the early 20th century. Although important advances continue to be made in the development of alternative control measures, insecticides will remain a vital part in the integrated control programs of vectors for the foreseeable future. Unfortunately, the remarkable ability of vector populations to evolve resistance to every class of insecticide has been developed. Now it has been recognized that a change in the genetic composition of a population is a direct result of the selective effects of a toxicant An understanding of the insecticide-resistant mechanisms can lead to better management of pest resistance to pesticides. By strategy of functional cloning, several genes have been reported to be related with insecticide resistance, such as cytochrome P450 gene, sodium channel gene, esterase gene and so on. How many genes contribute in the mechanisms of insecticide resistance? Are there other genes acting on the resistance? In terms of the strategy of “phenotype cloning ” differentially expressed genes between the deltamethrin-resistant
-8-
and suscePtible ed of Culexgh~ were cfoned by the
method named SUPpessha bo hybM (SSffi, and
wer Med by N chiP, reverse nOrthem bfot and nOH
bwt A ffe mp libop was construtriby be clonin
teCboc using the deltametbrinesiStan strain of Cx tw
N as samPle and a full lengIh cDNA gen named twsin
sen was pe by SCreenin the COnStrUctin ffe mp
bo
l Isolation of drially mpssed pe betwee
deWtw and SUSeePtile hans of Cx ptw
peuens by SSH
The deWsusCePtible bo of Cx plPbo the was
origined from the Shangh Insect Ihatitu of Chinse Academ
of Science and the LC50 value was 0.0008mgh. W the
MresiM H was M by seleCted wtth
uefor ana the LC50 ds was 0.32mrt. In orta to set
the gens tha wer high and rpecilically exPreSsed in the
resiStan strain, the - poPulation ofresistan sha was used
as M and that ofsuseqle nd as the The total RNA and
M wer isolated rerpectiVely from the both for adults that
had no been fed wh blood within three-da edosion. The singl
wt and Hle ed cDNAs wer Synthsed. Then theSe
dOUble Stran cDNAs wer digested by the An I.The teste
- 9 -
dscDNAs were -- added to adaPo l and adaPtO 2K
bo, the de dscDNAs were mp with the excess
be oc An bo the op pe were ~ by
-- PCh The PCR Produds were cfoned into im
~ vectors dri were ds bo E coli mloo. Ai
wt 523 subwt clones were pe.
hi de to get the pe ta were highly ana peifically
exPwt in the Hle tw the m poPulation of
Hle ni was used as teSter and that ofresStan ni as
driVer The SSH was wi as above and 286 subwt clones
were pe in the ena.
The length of SSH be inserted into Pinpofor vector was
N 300600 base pairs by PCR detoCon.
The results aboVe SUggested tha man gens Inay play roles on
the dehosistance orcx twpouas anu that no o*
the retw W may H on insecticide-resStanCe, bot also the
Wle pe.
2 hidendfication ofsSH clones by cDNA chiP
A M chiP containin 809 SSH clones was mad. Briefly the
bo in the hafor pedd vecto were amnlified by Pcn
using seqUenCes flankin the cfonin site as PriIners. The PCR
wt were viW on l% Wse ge to ensure adW
PCR ammpcation Prior to being robecally Printed onto nyon
- l0 -
meInbran. The un frOm the both ch were repe
rnad as resistan Probe and SUSW Probe by reverse
W in the M of a ?3P dCTP
scanning, W and subSeqUen -- were
-- It was that l55 and 42 pe were re~ 2-3 fod
and >3 fold OVeWed in the resed tw l5 and 9 pe
wer rerpeCtively 2-3 fold and >3 fod OVeWssed in the
He the. In additio there wer 2 gens gncifically
Wssed in the resiStan sha.
ACcoding tD the results, we Med that vecto reSStane to
insectiCde may be rethed ed OVeWssed. lowWsed and
sPecifically exPrssed gens in insecticideresiSha sha.
3 M Mdri of SSH clones rewt by reverse
nota
引文
1 WHO. Vector resistance to pesticides. Fifteenth report of the expert committee on vector biology and control. In WHO Tech Rep Ser. 1992; 818:1-55．
2 Barry JB and William CM. The biology of disease vectors. University Press of Colorado, Colorado, 1996; P:512-529．
3 唐振华．昆虫抗药性及其治理．农业出版社，北京 , 1993; P: 9-40．
4 Brawn AWA. Insecticide resistance in mosquitoes: a pragmatic review. J Am Mosq Control Assoc. 1986; 2:123-140．
5 Devonshire AL and Sawicki RM. Insecticide-resistant Myzus persicae as an example of evolution by gene duplication. Nature. 1979;280:140-141．
6 Oppenoorth FJ. Biochemistry and physiology of resitance. In: GA Kerkut and LI Gilbert, editors, Comprehensive insert physiology, biochemistry and pharmacology, Vol.12． Oxford: Pergamon 1985;P:731-773．
7 Takken FL. A functional cloning strategy, based on a binary PVX-expression vector, to isolate HR-inducing cDNAs of plant pathogens. Plant J. 2000; 24 :275-283．
8 Soderlund DM and Bloomquist JR. Molecular mechanisms
of insecticide resistance In:B.E. Tabashnik and RT Roush, editors, Pesticide resistance in arthropods. New York: Chapman and Hall 1990． P:58-96．
9 Mutero A, PralavorioM, Bride JM, FournierD. Resistance-associated point mutations in insecticide-insensitive acetylcholinesterase. Proc Natl Acad Sci USA. 1994;91:5922-5926．
10 Fournier D, Bride JM, Hoffmann F, et al. Acetylcholinesterase: two types of modifications confer resistant to insecticides. J Biol Chem 1992a; 267:14270-14274．
11 Williamson MS, Denholm I, Bell CA, et al. Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly {Musca domestica). Mol Gen Genet 1993;240:17-22．
12 Pittendrigh B,Reenan R, French-Constant RH, et al. Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides. Mol Gen Genet. 1997 ;256:602-610．
13 Ranson H, Jensen B, Vulule JM, et al. Identification of a point mutation in the voltage-gated sodium channel gene of Kenyan Anopheles gambiae associated with resistance to DDT and pyrethroids. Insect Mol Biol. 2000;9:491-497．
14 Casida JE and Quistad GB. Golden age of insecticide resistance: past, present, or future? Annu Rev Entomol. 1998;43:1-16.
15 Mouches C, Pasteur N, Berge JB, et al. Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito. Science. 1986;233:778-780.
16 Devonshire AL and Field LM. Gene amplification and insecticide resistance. Ann Rev Entolmol. 1991;36:1-23
17 Tomita T, Liu N, Smith FF, et al. Molecular mechanisms involved in increased expression of a cytochrome P450 responsible for pyrethroid resistance in the housefly, Musca domestica. Insect Molec Biol. 1995:4:135-140.
18 Barry JB and William CM. The biology of disease vectors. University Press of Colorado, Colorado, 1996; P:523-526.
19 苏寿氏，叶炳辉主编。现代医学昆虫学．高等教育出版社，北京．1996；P179-182．
20 Zlotkin E. The insect voltage-gated sodium channel as target of insecticides. Annu Rev Entomol. 1999;44:429-455.
21 Feyereisen IL Insect P450 enzymes. Annu Rev Entomol.1999;44:507-533.
22 朱昌亮，李玉兰，孙立新等．淡色库蚊对溴氰菊酯抗性的遗传分析．中国寄生虫学与寄生虫病杂志．1998；16：32-35．
23 Jonsson JJ and Weissman SM. From mutation mapping to phenotype cloning. Proc Natl Acad Sci USA. 1995 ;92:83-85．
24 Diatchenko L, Lau YF, Campbell AP, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA. 1996; 93:6025-6030．
25 Villalva C, Trempat P, Zenou RC, et al. Gene expression profiling in lymphomas by suppression subtractive hybridization. Bull Cancer. 2001 ;88:315-319．
26 Wang H, Zhan Y, Xu L, et al. Use of suppression subtractive hybridization for differential gene expression in stroke: discovery of CD44 gene expression and localization in permanent focal stroke in rats. Stroke. 2001;32:1020-1027．
27 Chen I, Hsieh T, Thomas T, et al. Identification of estrogen-induced genes downregulated by AhR agonists in MCF-7 breast cancer cells using suppression subtractive hybridization. Gene. 2001;262:207-214．
28 Dessens JT, Margos G, Rodriguez MC, et al. Identification of differentially regulated genes of Plasmodium by suppression subtractive hybridization. Parasitol Today. 2000; 16:354-356．
29 田海生，李秀兰，马磊等．淡色库蚊对溴氰菊酯抗性和敏感品系差异表达蛋白的研究．南京医科大学学报．
2001; 21: 93-95．
30 Zhu CL, Li YL,Tian HS, et al. Study on the dynamics and mechanism of deltamethrin resistance of Culex pipiens pollens Coquillett, 1898 (diptera:culicidae) in China The annals of medical entomology. 1995; 4:1-4．
31 Duguid JR, Rohwer RG and Seed B. Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library. Proc Natl Acad sci USA. 1988; 85 : 5738-5742．
32 Sive HL and St John T. A simple subtractive hybridization technique employing photoactivatable biotin and phenol extraction. Nucleic Acid Res. 1988; 16 :10937-10938．
33 Hubank M and Schatz DG. Identifying differences in mRNA egression by representative difference analysis of cDNA. Nucleic Acid Res. 1994;22:5640-5648．
34 Zeng J, Gorski RA and Hamer D. Differential cDNA cloning by enzymatic degrading subtraction (EDS). Nucleic Acid Res. 1994; 22:4381-4385．
35 Siebert PD, Chenchik A, Kellogg DE, et al. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acid Res.l995;23:1087-1088．
36 Schena M. Genome analysis with gene expression microarrays. Bioessays. 1996; 18:427-431．
37 Anderson KM, Alrefai WA, Bonomi P, et al. Altered oncogene, tumor suppressor and cell-cycle gene expression in PANC-1 cells cultured with the pleiotrophic 5-lipoxygenase inhibitor, MK886, assessed with a gene chip. Anticancer Res. 1999; 19:3873-3887．
38 Kumar A and Liang Z. Chemical nanoprinting: a novel method for fabricating DNA microchips. Nucleic Acids Res. 2001 ;29:2-5．
39 Liu H, He Z and Rosenwaks Z. Application of complementary DNA microarray (DNA chip) technology in the study of gene expression profiles during folliculogenesis. Fertil Steril. 2001;75:947-955．
40 Schena M, Shalon D, Dais RW, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. 1995; 270:467-470．
41 Schena M, Shalon D, Heller R, et al. Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.Proc Natl Acad Sci U S A. 1996;93 :10614-10619．
42 Geschwind DH, Ou J, Easterday MC, et al. A genetic analysis of neural progenitor differentiation. Neuron. 2001;29:325-339．
43 Alizadeh A, Eisen M, Davis R, et al. Distinct types of
diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403:503-511．
44 Wang K, Gan I, Jeffery E, et al. Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray. Gene. 1999;229:101-109．
45 Yang GP, Ross DT, Kuang WW, et al. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nucleic Acids Res. 1999 ;27:1517-1523．
46 Sandhu H, Dehnen W, Roller M, et al. mRNA expression patterns in different stages of asbestos-induced carcinogenesis in rats. Carcinogenesis. 2000; 21:1023-1029．
47 Li JY, Boado RJ and Pardridge WM. Blood-brain barrier genomics. J Cereb Blood Flow Metab. 2001 ;21:61-68．
48 Voiblet C, Duplessis S, Encelot N, et al. Identification of symbiosis-regulated genes in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza by differential hybridization of arrayed cDNAs. Plant J.2001;25:181-191．
49 Porkka KP and Visakorpi T. Detection of differentially expressed genes in prostate cancer by combining suppression subtractive hybridization and cDNA library array. J Pathol. 2001 ;193:73-79．
50 Carlisle AJ, Prabhu W, Elkahloun A, et al. Development of
a prostate cDNA microarray and statistical gene expression analysis package. Mol Carcinog. 2000;28:12-22．
51 Zhao YL, Piao CQ, Wu LJ, et al. Differentially expressed genes in asbestos-induced tumorigenic human bronchial epithelial cells:implication for mechanism. Carcinogenesis. 2000;21: 2005-2010．
52 Devonshire AL and Sawicki RM. Insecticide-resistant Myzus Persicae as an example of evolution by gene duplication. Nature. 1979; 280:140-141．
53 Amichot M, Castella C, Cuany A, et al. Target modification as a molecular mechenism of pyrethroid resistance in Drosophila melanogaster. Pestic Biochem Physiol. 1992; 44:183-190．
54 Carino FA, Koener JF, Plapp FW, et al. Constitutive overexpression of the cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to insecticides. Insect Biochem Mol Biol. 1994;24:411-418．
55 Lukyanov KA, Matz MV, Bogdanova EA, et al. Molecule by molecule PCR amplification of complex DNA mixtures for direct sequencing : an approach to in vitro cloning. Nucleic Acids Res.1996;24:2194-2195．
56 Chaib H, Cockrell EK, Rubin MA, et al. Profiling and Verification of Gene Expression Patterns in Normal and
Malignant Human Prostate Tissues by cDNA Microarray Analysis. Neoplasia, 2001;3:43-52．
57 Madder SA and Eberwine JH. Cellular adaptation to opiates alters ion-channel mRNA levels. Proc Natl Acad Sci U S A. 1994． 91:385-389．
58 Jung MH, Kim SC, Jeon GA, et al. Identification of differentially expressed genes in normal and tumor human gastric tissue. Genomics. 2000;69:281-286．
59 Mouches C, Pasteur N, Berge JB, et al. Amplification of an esterase geme is responsible for insecticide resistance in a California Culex mosquito. Science. 1986;233:779-780．
60 Mouches C, Magnin M, Berge JB, et al. Overproduction of detoxifying esterases in organophosphate-resistant Culex mosquitoes and their presence in other insects. Proc Natl Acad Sci USA. 1987;84:2113-2116．
61 Fournier D and Berg JB. Resistance to insecticide: detection of a mutation in the acetylcholinesterase gene from Drosophila melanogaster. Molecular Insect Science. New York : Plenum Press. 1990;P302-318．
62 Wang JY, McCommas S, Syvanen M. Molecular cloning of a glutathions S-transferase overproduced in an insecticide-resistant strain of the housefly. Mol Gen Gent. 1991; 227:260-266．
63 Ffench-Constant RH, Mortlock DP, Shaffer CD, et al. Molecular cloning and transformation of cyclodience resistance in Drosophila: an invertebrate GABA receptor locus. Proc Natl Acad Sci U S A. 1991;88:7209-7215.
64 Anderson JF, Utermohlen JG and Feyereisen R. Expression of house fly CYP6A1 and DNAPH-cytochrome P450 reductase in Escherichia coli and reconstritution of an insecticide-metabolizing P450 system. Biochemistry. 1994;33:2171-2177.
65 Harris AJ, Shaddock JG, Manjanatha MG, et al. Identification of differentially expressed genes in aflatoxin B1-treated cultured primary rat hepatocytes and Fischer 344 rats. Carcinogenesis. 1998;19:1451-1458.
66 Tonsgard JH, Tung B, Komafel KS, et al. Environmentally induced differential amplification of mitochondrial populations. Biochem J. 1990;270:511-518.
67 Thon VJ, Vigneron-Lesens C, Marianne-Pepin T, et al. Coordinate regulation of glycogen metabolism in the yeast Saccharomyces cerevisiae. Induction of glycogen branching enzyme. J Biol Chem. 1992;267:15224-15228.
68 李瑶，裘敏燕，吴超群等．用基因表达谱芯片研究人正常肝和肝细胞癌中差异表达的基因．遗传学报．2000；27：138-145．
69 Wang K, Gan I, Jeffery E, et al. Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray. Gene. 1999; 229:101-108.
70 Wang Q, Yang C, Zhou J, et al. Cloning and characterization of full-length human ribosomal protein L15 cDNA which was overexpressed in esophageal cancer. Gene. 2001; 263:205-209.
71 Grunstein M and Hogness DS.Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Biotechnology. 1975; 24:117-21.
72 Haas MJ and fleming DJ. Use of biotinylated DNA probes in colony hybridization. Nucleic Acids Res. 1986; 14:3976-3978.
73 唐振华，吴士雄著．昆虫抗药性的遗传与进化．上海科学技术文献出版社，上海．2000，P92．
74 沈斌，高荣，朱锦等．猪囊尾蚴cDNA表达文库构建及抗原基因筛选．中国人兽共患病杂志，1999，15：57-60．
75 Williams, JG. The preparation and screening of a cDNA clone bank. In Genetic engineering. Academic Press, London, 1981, P1-18.
76 王琳芳，杨克恭主编．蛋白质与核酸．北京医科大学中国医科大学联合出版社．北京，1998，P70-86．
77 Lemos FJ, Comel AJ and Jacobs-Lorena M Trypsin and aminopeptidase gene expression is affected by age and food composition in Anopheles gambiae. Insect Biochem Molec Biol.
1996;26:651-658．
78 Shahabuddin M, Toyoshima T, Aikawa M, et al. Transmission-blocking activity of a chitinase inhibitor and activation of malarial parasite chitinase by mosquito protease. Proc Nad Acad Sci U S A. 1993;90:4266-4270．
79 Shahabuddin M. Plasmodium ookinete development in the mosquito midgut: a case of reciprocal manipulation. Parasitology. 1998;116:83-93．
80 Thompson JD, Gibson TJ, Plewniak F, et al. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research. 1997;24:4876-4882．
81 Muller HM, Catteruccia F, Vizioli J, et al. Constitutive and blood meal-induced trypsin genes in Anopheles gambiae. Experiment Parasitol. 1995; 81:371-385．
82 Takumi T and Lodish HF. Rapid cDNA cloning by PCR screening. BioTechniques. 1994; 17:443-444．
83 Frohman MA, Dush Mk, Martin GR, et al. Rapid production of full-lengths cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA. 1988;85:8998-9002．
84 Borson ND, Salo WL, Drews LR, et al. A Lock-docking
oligo(dT) primer for 5' and 3' RACE PCR. PCR Methods Appl. 1992;2:144-148．
85 Mayer H, Breuss J, Ziegler S, et al. Molecular characterization and tissue-specific expression of a murine putative G-protein-coupled receptor. Biochim Biophys Acta. 1998;1399:51-56．
2 Barry JB and William CM. The biology of disease vectors. University Press of Colorado, Colorado, 1996; P:512-529．
3 唐振华．昆虫抗药性及其治理．农业出版社，北京 , 1993; P: 9-40．
4 Brawn AWA. Insecticide resistance in mosquitoes: a pragmatic review. J Am Mosq Control Assoc. 1986; 2:123-140．
5 Devonshire AL and Sawicki RM. Insecticide-resistant Myzus persicae as an example of evolution by gene duplication. Nature. 1979;280:140-141．
6 Oppenoorth FJ. Biochemistry and physiology of resitance. In: GA Kerkut and LI Gilbert, editors, Comprehensive insert physiology, biochemistry and pharmacology, Vol.12． Oxford: Pergamon 1985;P:731-773．
7 Takken FL. A functional cloning strategy, based on a binary PVX-expression vector, to isolate HR-inducing cDNAs of plant pathogens. Plant J. 2000; 24 :275-283．
8 Soderlund DM and Bloomquist JR. Molecular mechanisms
of insecticide resistance In:B.E. Tabashnik and RT Roush, editors, Pesticide resistance in arthropods. New York: Chapman and Hall 1990． P:58-96．
9 Mutero A, PralavorioM, Bride JM, FournierD. Resistance-associated point mutations in insecticide-insensitive acetylcholinesterase. Proc Natl Acad Sci USA. 1994;91:5922-5926．
10 Fournier D, Bride JM, Hoffmann F, et al. Acetylcholinesterase: two types of modifications confer resistant to insecticides. J Biol Chem 1992a; 267:14270-14274．
11 Williamson MS, Denholm I, Bell CA, et al. Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly {Musca domestica). Mol Gen Genet 1993;240:17-22．
12 Pittendrigh B,Reenan R, French-Constant RH, et al. Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides. Mol Gen Genet. 1997 ;256:602-610．
13 Ranson H, Jensen B, Vulule JM, et al. Identification of a point mutation in the voltage-gated sodium channel gene of Kenyan Anopheles gambiae associated with resistance to DDT and pyrethroids. Insect Mol Biol. 2000;9:491-497．
14 Casida JE and Quistad GB. Golden age of insecticide resistance: past, present, or future? Annu Rev Entomol. 1998;43:1-16.
15 Mouches C, Pasteur N, Berge JB, et al. Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito. Science. 1986;233:778-780.
16 Devonshire AL and Field LM. Gene amplification and insecticide resistance. Ann Rev Entolmol. 1991;36:1-23
17 Tomita T, Liu N, Smith FF, et al. Molecular mechanisms involved in increased expression of a cytochrome P450 responsible for pyrethroid resistance in the housefly, Musca domestica. Insect Molec Biol. 1995:4:135-140.
18 Barry JB and William CM. The biology of disease vectors. University Press of Colorado, Colorado, 1996; P:523-526.
19 苏寿氏，叶炳辉主编。现代医学昆虫学．高等教育出版社，北京．1996；P179-182．
20 Zlotkin E. The insect voltage-gated sodium channel as target of insecticides. Annu Rev Entomol. 1999;44:429-455.
21 Feyereisen IL Insect P450 enzymes. Annu Rev Entomol.1999;44:507-533.
22 朱昌亮，李玉兰，孙立新等．淡色库蚊对溴氰菊酯抗性的遗传分析．中国寄生虫学与寄生虫病杂志．1998；16：32-35．
23 Jonsson JJ and Weissman SM. From mutation mapping to phenotype cloning. Proc Natl Acad Sci USA. 1995 ;92:83-85．
24 Diatchenko L, Lau YF, Campbell AP, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA. 1996; 93:6025-6030．
25 Villalva C, Trempat P, Zenou RC, et al. Gene expression profiling in lymphomas by suppression subtractive hybridization. Bull Cancer. 2001 ;88:315-319．
26 Wang H, Zhan Y, Xu L, et al. Use of suppression subtractive hybridization for differential gene expression in stroke: discovery of CD44 gene expression and localization in permanent focal stroke in rats. Stroke. 2001;32:1020-1027．
27 Chen I, Hsieh T, Thomas T, et al. Identification of estrogen-induced genes downregulated by AhR agonists in MCF-7 breast cancer cells using suppression subtractive hybridization. Gene. 2001;262:207-214．
28 Dessens JT, Margos G, Rodriguez MC, et al. Identification of differentially regulated genes of Plasmodium by suppression subtractive hybridization. Parasitol Today. 2000; 16:354-356．
29 田海生，李秀兰，马磊等．淡色库蚊对溴氰菊酯抗性和敏感品系差异表达蛋白的研究．南京医科大学学报．
2001; 21: 93-95．
30 Zhu CL, Li YL,Tian HS, et al. Study on the dynamics and mechanism of deltamethrin resistance of Culex pipiens pollens Coquillett, 1898 (diptera:culicidae) in China The annals of medical entomology. 1995; 4:1-4．
31 Duguid JR, Rohwer RG and Seed B. Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library. Proc Natl Acad sci USA. 1988; 85 : 5738-5742．
32 Sive HL and St John T. A simple subtractive hybridization technique employing photoactivatable biotin and phenol extraction. Nucleic Acid Res. 1988; 16 :10937-10938．
33 Hubank M and Schatz DG. Identifying differences in mRNA egression by representative difference analysis of cDNA. Nucleic Acid Res. 1994;22:5640-5648．
34 Zeng J, Gorski RA and Hamer D. Differential cDNA cloning by enzymatic degrading subtraction (EDS). Nucleic Acid Res. 1994; 22:4381-4385．
35 Siebert PD, Chenchik A, Kellogg DE, et al. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acid Res.l995;23:1087-1088．
36 Schena M. Genome analysis with gene expression microarrays. Bioessays. 1996; 18:427-431．
37 Anderson KM, Alrefai WA, Bonomi P, et al. Altered oncogene, tumor suppressor and cell-cycle gene expression in PANC-1 cells cultured with the pleiotrophic 5-lipoxygenase inhibitor, MK886, assessed with a gene chip. Anticancer Res. 1999; 19:3873-3887．
38 Kumar A and Liang Z. Chemical nanoprinting: a novel method for fabricating DNA microchips. Nucleic Acids Res. 2001 ;29:2-5．
39 Liu H, He Z and Rosenwaks Z. Application of complementary DNA microarray (DNA chip) technology in the study of gene expression profiles during folliculogenesis. Fertil Steril. 2001;75:947-955．
40 Schena M, Shalon D, Dais RW, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. 1995; 270:467-470．
41 Schena M, Shalon D, Heller R, et al. Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.Proc Natl Acad Sci U S A. 1996;93 :10614-10619．
42 Geschwind DH, Ou J, Easterday MC, et al. A genetic analysis of neural progenitor differentiation. Neuron. 2001;29:325-339．
43 Alizadeh A, Eisen M, Davis R, et al. Distinct types of
diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403:503-511．
44 Wang K, Gan I, Jeffery E, et al. Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray. Gene. 1999;229:101-109．
45 Yang GP, Ross DT, Kuang WW, et al. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nucleic Acids Res. 1999 ;27:1517-1523．
46 Sandhu H, Dehnen W, Roller M, et al. mRNA expression patterns in different stages of asbestos-induced carcinogenesis in rats. Carcinogenesis. 2000; 21:1023-1029．
47 Li JY, Boado RJ and Pardridge WM. Blood-brain barrier genomics. J Cereb Blood Flow Metab. 2001 ;21:61-68．
48 Voiblet C, Duplessis S, Encelot N, et al. Identification of symbiosis-regulated genes in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza by differential hybridization of arrayed cDNAs. Plant J.2001;25:181-191．
49 Porkka KP and Visakorpi T. Detection of differentially expressed genes in prostate cancer by combining suppression subtractive hybridization and cDNA library array. J Pathol. 2001 ;193:73-79．
50 Carlisle AJ, Prabhu W, Elkahloun A, et al. Development of
a prostate cDNA microarray and statistical gene expression analysis package. Mol Carcinog. 2000;28:12-22．
51 Zhao YL, Piao CQ, Wu LJ, et al. Differentially expressed genes in asbestos-induced tumorigenic human bronchial epithelial cells:implication for mechanism. Carcinogenesis. 2000;21: 2005-2010．
52 Devonshire AL and Sawicki RM. Insecticide-resistant Myzus Persicae as an example of evolution by gene duplication. Nature. 1979; 280:140-141．
53 Amichot M, Castella C, Cuany A, et al. Target modification as a molecular mechenism of pyrethroid resistance in Drosophila melanogaster. Pestic Biochem Physiol. 1992; 44:183-190．
54 Carino FA, Koener JF, Plapp FW, et al. Constitutive overexpression of the cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to insecticides. Insect Biochem Mol Biol. 1994;24:411-418．
55 Lukyanov KA, Matz MV, Bogdanova EA, et al. Molecule by molecule PCR amplification of complex DNA mixtures for direct sequencing : an approach to in vitro cloning. Nucleic Acids Res.1996;24:2194-2195．
56 Chaib H, Cockrell EK, Rubin MA, et al. Profiling and Verification of Gene Expression Patterns in Normal and
Malignant Human Prostate Tissues by cDNA Microarray Analysis. Neoplasia, 2001;3:43-52．
57 Madder SA and Eberwine JH. Cellular adaptation to opiates alters ion-channel mRNA levels. Proc Natl Acad Sci U S A. 1994． 91:385-389．
58 Jung MH, Kim SC, Jeon GA, et al. Identification of differentially expressed genes in normal and tumor human gastric tissue. Genomics. 2000;69:281-286．
59 Mouches C, Pasteur N, Berge JB, et al. Amplification of an esterase geme is responsible for insecticide resistance in a California Culex mosquito. Science. 1986;233:779-780．
60 Mouches C, Magnin M, Berge JB, et al. Overproduction of detoxifying esterases in organophosphate-resistant Culex mosquitoes and their presence in other insects. Proc Natl Acad Sci USA. 1987;84:2113-2116．
61 Fournier D and Berg JB. Resistance to insecticide: detection of a mutation in the acetylcholinesterase gene from Drosophila melanogaster. Molecular Insect Science. New York : Plenum Press. 1990;P302-318．
62 Wang JY, McCommas S, Syvanen M. Molecular cloning of a glutathions S-transferase overproduced in an insecticide-resistant strain of the housefly. Mol Gen Gent. 1991; 227:260-266．
63 Ffench-Constant RH, Mortlock DP, Shaffer CD, et al. Molecular cloning and transformation of cyclodience resistance in Drosophila: an invertebrate GABA receptor locus. Proc Natl Acad Sci U S A. 1991;88:7209-7215.
64 Anderson JF, Utermohlen JG and Feyereisen R. Expression of house fly CYP6A1 and DNAPH-cytochrome P450 reductase in Escherichia coli and reconstritution of an insecticide-metabolizing P450 system. Biochemistry. 1994;33:2171-2177.
65 Harris AJ, Shaddock JG, Manjanatha MG, et al. Identification of differentially expressed genes in aflatoxin B1-treated cultured primary rat hepatocytes and Fischer 344 rats. Carcinogenesis. 1998;19:1451-1458.
66 Tonsgard JH, Tung B, Komafel KS, et al. Environmentally induced differential amplification of mitochondrial populations. Biochem J. 1990;270:511-518.
67 Thon VJ, Vigneron-Lesens C, Marianne-Pepin T, et al. Coordinate regulation of glycogen metabolism in the yeast Saccharomyces cerevisiae. Induction of glycogen branching enzyme. J Biol Chem. 1992;267:15224-15228.
68 李瑶，裘敏燕，吴超群等．用基因表达谱芯片研究人正常肝和肝细胞癌中差异表达的基因．遗传学报．2000；27：138-145．
69 Wang K, Gan I, Jeffery E, et al. Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray. Gene. 1999; 229:101-108.
70 Wang Q, Yang C, Zhou J, et al. Cloning and characterization of full-length human ribosomal protein L15 cDNA which was overexpressed in esophageal cancer. Gene. 2001; 263:205-209.
71 Grunstein M and Hogness DS.Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Biotechnology. 1975; 24:117-21.
72 Haas MJ and fleming DJ. Use of biotinylated DNA probes in colony hybridization. Nucleic Acids Res. 1986; 14:3976-3978.
73 唐振华，吴士雄著．昆虫抗药性的遗传与进化．上海科学技术文献出版社，上海．2000，P92．
74 沈斌，高荣，朱锦等．猪囊尾蚴cDNA表达文库构建及抗原基因筛选．中国人兽共患病杂志，1999，15：57-60．
75 Williams, JG. The preparation and screening of a cDNA clone bank. In Genetic engineering. Academic Press, London, 1981, P1-18.
76 王琳芳，杨克恭主编．蛋白质与核酸．北京医科大学中国医科大学联合出版社．北京，1998，P70-86．
77 Lemos FJ, Comel AJ and Jacobs-Lorena M Trypsin and aminopeptidase gene expression is affected by age and food composition in Anopheles gambiae. Insect Biochem Molec Biol.
1996;26:651-658．
78 Shahabuddin M, Toyoshima T, Aikawa M, et al. Transmission-blocking activity of a chitinase inhibitor and activation of malarial parasite chitinase by mosquito protease. Proc Nad Acad Sci U S A. 1993;90:4266-4270．
79 Shahabuddin M. Plasmodium ookinete development in the mosquito midgut: a case of reciprocal manipulation. Parasitology. 1998;116:83-93．
80 Thompson JD, Gibson TJ, Plewniak F, et al. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research. 1997;24:4876-4882．
81 Muller HM, Catteruccia F, Vizioli J, et al. Constitutive and blood meal-induced trypsin genes in Anopheles gambiae. Experiment Parasitol. 1995; 81:371-385．
82 Takumi T and Lodish HF. Rapid cDNA cloning by PCR screening. BioTechniques. 1994; 17:443-444．
83 Frohman MA, Dush Mk, Martin GR, et al. Rapid production of full-lengths cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA. 1988;85:8998-9002．
84 Borson ND, Salo WL, Drews LR, et al. A Lock-docking
oligo(dT) primer for 5' and 3' RACE PCR. PCR Methods Appl. 1992;2:144-148．
85 Mayer H, Breuss J, Ziegler S, et al. Molecular characterization and tissue-specific expression of a murine putative G-protein-coupled receptor. Biochim Biophys Acta. 1998;1399:51-56．