Research and application of plant immunity

Research and Application of Plant Immunity

Institute of Plant ProtectionChinese Academy of Agricultural Sciences

Dewen Qiu

主要内容

Application of plant immunity Application of plant immunity

Mechanism of plant immunityMechanism of plant immunity

Concept of plant immunityConcept of plant immunity

Mammals immunity Plant disease resistance and immunity: L. Yarwood (), “Mechanism of acquired immunity to a plant rust” Proc Natl

Acad Sci U S A. 1954, 40(6): pp. 374–377 KUC J, ULLSTRUP AJ, QUACKENBUSH FW, Production of fungistatic

substances by plant tissue after inoculation, Science. 1955,Dec16;122(3181):1186-7

Ross, AF . Systemic acquired resistance induced by localized virus infections in plants. Virology 1961, 14, 340-358.

Plant immunity conceptPlant immunity concept

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Cell

Plant immunology- Hot Areas

New discovery

Jen Sheen, Researchers discover mechanism of plant resistance to pathogens, Plants have effective mechanisms aimed at protecting themselves against bacteria and fungi. Science Daily Feb. 28, 2002.

Jonathan D. G. Jones1 & Jeffery L. Dangl,The plant immune system,Nature 444, 323-329 (16 November 2006)

Shen QH, et al.Nuclear Activity of MLA Immune Receptors Links Isolate-Specific and Basal Disease-Resistance Responses, Science, 23 February 2007: Vol. 315 no. 5815 pp. 1098-1103

Sang-Wook Park, Evans Kaimoyo, Dhirendra Kumar*, Stephen Mosher† and Daniel F. Klessig. Methyl Salicylate is a Critical Mobile Signal for Plant Systemic Acquired Resistance. Science, 5 October 2007: Vol. 318 no. 5847 pp. 113-116

New research results

Liqun Du1, Gul S. Ali3, Kayla A. Simons1, Jingguo Hou2,4, Tianbao Yang1, A. S. N. Reddy3 & B. W. Poovaiah1, Ca2+/calmodulin regulates salicylic-acid-mediated plant immunity. Nature 457, 1154-1158 (26 February 2009)

Thomas Boller1,* and Sheng Yang He, Innate Immunity in Plants: An Arms Race Between Pattern Recognition Receptors in Plants and Effectors in Microbial Pathogens. Science 8 May 2009: Vol. 324 no. 5928 pp. 742-744

Boudsocq M, Willmann MR, McCormack M, Lee H, Shan L, He P, Bush J, Cheng SH, Sheen J, Differential innate immune signalling via Ca(2+) sensor protein kinases. Nature. 2010 Mar 18;464(7287):418-22

Presidential Awards by EPA in 2001

Messenger --Cornell University and EDEN Bioscience,

主要内容

Application of plant immunity Application of plant immunity

Mechanism of plant immunityMechanism of plant immunity

Concept of plant immunityConcept of plant immunity

Panstruga R, Parker JE, Schulze-Lefert P. SnapShot: Plant immune response pathways.Cell. 2009 Mar 6;136(5):978.e1-3.

Plant Growth Promoting Rhizobacteria

Trichoderma and Rhizobacteria -induced plant immunity

SA→PR →induced plant immune (SAR)? JA/ET→LOX →PAL → induced plant immune（ISR）?JA →MAPK?

Plant immunity mechanismPlant immunity mechanism

Multiple and complex effects (Plant stress and disease resistance

Plant growth promoting)

Multiple and complex effects (Plant stress and disease resistance

Plant growth promoting)

Plant immunity signaling ( Protein elicitor and

Chitosan Oligosaccharide )

Plant immunity signaling ( Protein elicitor and

Chitosan Oligosaccharide )

Salicylic acid Jasmonic acid Ethylene NO

多年来我们一直试图寻找专化性高效免疫诱抗剂。

经研究表明蛋白质、寡糖、枯草芽孢杆菌及木霉菌等免疫诱抗剂所诱导的免疫反应基本比较一致，主要集中在水杨酸、茉莉酸和乙烯的途径。

本人分析认为植物的免疫作用可能与哺乳动物或昆虫等动物的免疫反应不同， 不形成IGG或YGG等免疫结构，植物的免疫是一个原始初级的免疫反应，植物的免疫反应通过大致相同的路径来抵御病虫害的侵入，也就是植物诱抗剂所诱导的免疫反应具有广谱性或多功能。

植物免疫的诱导或激活是一个初级的免疫反应

主要内容

Application of plant immunity Application of plant immunity

Mechanism of plant immunityMechanism of plant immunity

Concept of plant immunityConcept of plant immunity

Products of Elicitor

Eden Bioscienes, “Messenger® ” Redox Chemicals, “Oxycom” Morse, “ KeyPlex” Syngenta , “Actigard” Nippon Kayaku Co Ltd, “NCI” “Chitosan”

Application of Elicitor

Messenger, Promotes root growth, vigor, disease and stress resistence, increased flowering and fruit set.

“Oxycom”, “KeyPlex”, for control of leaf spot disease in Orange and Banana.

“Chitosan”, used in seed treatment and plant growth enhancer, against fungal infections, control of diseases vegetables.

“Actigard”, for downy mildew in leafy vegetables, bacterial leaf spots in tomatoes and blue mold in tobacco.

HR –inducing protein elicitorPevD1;Hrip1--- Extracellular

Non HR protein elicitorPeaT1;PebC1;PemG1- Intracellular

Protein elicitor from fungi

Disease resistance and growth were enhanced by protein elicitor

75.5%

11.09%

Treatment Control

11.9%

PeaT1

CK

SAR against TMV induced by protein elicitor PeaT1

Purification of protein

Gene clone

1

2

3

protein application

Protein expression and function

Application

Mechanism4

5

Alternaria tenuissima

Botrytis cinerea

Magnaporthe grisea

Penicillium spp

Aspergillus Flarres

Rhizoctonia solani

Trichoderma spp

Fusarium spp

Fungi species

PeaT1

一、Protein purification

PebC1

PemG1PeaT1

PebC1

Purification of PevD1

HR

HP Q HiTrap™ Ion exchange chromatography

hydrophobic chromatography

SDS-PAGE

2

1, 2, 3, 4, 5 and 6 represents pH 2, 4, 6, 8, 11 and 13

HR –inducing protein elicitorPevD1;Hrip1--- Extracellular

Non HR protein elicitorPeaT1;PebC1;PemG1- Intracellular

Fungi elicitor protein

PeaT1

Purification of Protein elicitor

PebC1

PemG1PeaT1

PebC1

Purification of Protein elicitor

HR2

HP Q HiTrap™ Ion exchange chromatography

hydrophobic chromatography

SDS-PAGE

1, 2, 3, 4, 5 and 6 represents pH 2, 4, 6, 8, 11 and 13

离子交换的色谱 分子筛的色谱和电泳图

目的蛋白

PeaT1蛋白纯化与检测

peptides fingerprint

Trypsin T Chromtrypsin CTGlu-C V8

Gene cloning of protein elicitor

The mass-spectrometry/proteomic experiment

1. NILFVINKPDVYKSPSSNTWIIFGEAK2. DIELVMQQASVSR3. ALKENDNDIVNSIMALSI4. RPKNILFVINQPDVYK

PeaT1 protein sequence

1. NILFVINKPDVYKSPSSNTWIIFGEAK2. DIELVMQQASVSR3. ALKENDNDIVNSIMALSI4. RPKNILFVINQPDVYK

3‘RACE获得该基因的3’全长

交链孢菌激活蛋白的全基因序列207 amono acids MANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVAVHSRNEKKARKAIAKLGLKHIDGITRVTLRRPKNILFVINQPDVYKSPSSNTWIIFGEAKIEDLNSQAQASAAQQLAQAEAASHDHAGHDHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQQASVSRKKAVKALKENDNDIVNSIMALSI

NAC Domain:49-108aa

Alternaria tenuissima NAC protein sequence

1 MSNPRIEELP DNEEPTKQQV TAEDEGSDSS DSEAEGEEVA GIPAGSQVAF SRNEKKARKS

61 IAKLGLTRVP GITRVTLRRP KNILFVINQP EVYKSPTSNT YIVFGEAKIE DLNSQAQASA

121 AAQLAAQESH DHAGHDHSGH DHSHDHGKGK AVDTEEKKEE EEDDTEEVDA TGLEDKDIEL

181 VMTQASVSRN KAVKALKEND NDIVNSIMAL SI

Botrytis cinerea protein sequence

Fusarium spp αNAC和βNACαNAC

βNAC

MSNPRVEELPDEEPKKTTVQEHEDDSSDDSEVEEVGEGQLPAGSTVIHNRNEKKARKALEKLHLTRIPGITRVTLRRPKNILFVINTPEVYKSPNSNTYIVFGEAKIEDVNAAAQQAAAAQLASQNAEDHSGHNHGEPSKAVEADEKKEDKEDDEDEEEEEEEEVDASGLEDKDIELVMTQANVSRNKAVKALKENDNDIVNSIMALSI(210aa)

MSDVQERLKKLGLGARTGESNKLIYNTGGKGTPRRKVKRAPARSGADDKKLQLALKKLNTQPIQAIEEVNMFKQDGNVIHFAAPKVHAAVPSNTFAIYGNGEDKELTELVPGILNQLGPDSLASLRKLAESYQNLQKEKGEDDDEIPDLVEGENFEGEPKVE (162aa)

Gene:624bp

ATGGCCAACCCCCGCATTGAAGAGCTCCCCGACGAGCCCGAGAAGAAGAACGTCCAGATCGAGGAGGATGAGTCCAGCGACGAGTCTGAGGGCGAGGAGGGCGAGGTCAGCGTACCCGCGGGCTCCTCCGTCGCTGTCCACTCCCGCAACGAGAAGAAGGCTCGCAAGGCCATCGCCAAGCTCGGCCTGAAGCACATCGACGGCATCACACGCGTCACCCTCCGCCGACCCAAGAACATCCTCTTTGTCATCAACCAGCCCGACGTCTACAAGTCCCCTTCAAGCAACACCTGGATCATCTTCGGTGAGGCCAAGATCGAGGACCTCAACTCCCAGGCTCAGGCTTCCGCCGCCCAGCAGCTTGCTCAGGCCGAGGCCGCATCCCACGACCACGCCGGCCACGACCACGGCGACGAGGCCAGCAAGGGCAAGGGCAAGGCTGTCGAGGACAAGAAGGACGAGGAGGAGGAGGATGACGATGAGGAGATTGACGACTCTGGCCTTGAGGCCAAGGACATCGAGCTCGTCATGCAGCAGGCCAGCGTTTCGCGGAAGAAGGCCGTCAAGGCCCTCAAGGAGAACGATAACGATATAGTCAACTCCATCATGGCGCTGAGCATATAG

Protein:207aa

MANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVAVHSRNEKKARKAIAKLGLKHIDGITRVTLRRPKNILFVINQPDVYKSPSSNTWIIFGEAKIEDLNSQAQASAAQQLAQAEAASHDHAGHDHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQQASVSRKKAVKALKENDNDIVNSIMALSI

PeaT1 (Alternaria tenuissima )

Doman analysis of PeaT1

Structure of protein PeaT1

207Amino acidMANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVAVHSRNEKKARKAIAKLGLKHIDGITRVTLRRPKNILFVINQPDVYKSPSSNTWIIFGEAKIEDLNSQAQASAAQQLAQAEAASHDHAGHDHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQQASVSRKKAVKALKENDNDIVNSIMALSI

NAC Domain:49-108aa

Genes Fungi GenBank

Accession No Patens

pemG1 Magnaporthe spp EF062504 ZL200510011580.5 peaT1 Alternaria tenuissima EF030819 ZL200610152700.8 pebC1 Botrytis cinerea FJ748868 -- PevD1 Verticillium dahliae HQ540585 201010593488.5 Hrip1 Alternaria tenuissima HQ713431 201110144389.3

Genes and patents

1. PET-28a/PeaT1

2. PET-28a

3. Marker

SDS-PAGE

Expression of protein elicitor

Pichia pastoris

E.coli

PeaT1

PeaT1

PeaT1的X-射线晶体学

Crystallization X-ray diffraction

Conditions：20% PEG 3350，0.2M MgCl2，0.1M Mes pH5.5 20℃

分辨率：2.4Å

Crystal and X-ray diffraction

a-Helix b-Sheet 0.161 0.307

Structure of protein elicitor （Peat1）

NAC domain was constructed. NAC domain formed a homodimerfrom reverse direction. This structure outline was similar with a barrel, with a hole at the center and 6 intercrossed β strands surrounded

Structure of NAC domain in PeaT1

TCTP1 TCTP2

Intermediate file

据目前研究表明：TCTP家族蛋白功能：在人体内为胞外作用，引起过敏反应，与免疫有关

TCTP1：暴露在蛋白的表面，亲水性强，结构无固定规则----为可能的活性位点

TCTP2：无规则卷曲，固定在在两个折叠之间

1. PET-28a/PeaT1

2. PET-28a

3. Marker

SDS-PAGE

Expression of protein elicitor

Pichia pastoris

E.coli

PeaT1

PeaT1

PeaT1

CK

SAR against TMV induced by protein elicitor PeaT1

Protein elicitor products

Certificate for Bio-fertilizer

Certificate for pesticide

Treatment

Before

treated

Treated 3 times Treated 4 times

7d 15d 7d 15d

Disease

index

Disease

index

Effect

（%）

Disease

index

Effect

（%）

Disease

index

Effect

（%）

Disease

index

Effect

（%）

Bingdu A 9.48 34.80 37.01 35.64 40.75 36.51 42.09 36.89 44.87 99zhibao 11.73 25.09 54.58 25.17 58.16 22.78 63.87 21.95 67.15 Protein 11.02 21.55 60.99 20.10 66.59 18.75 70.26 17.02 74.53 Protein

+Bingdu A 9.82 20.95 62.08 19.21 68.07 17.71 71.91 15.84 76.29

Protein+

99 zhibao 10.63 55.24 65.11 17.75 70.49 13.24 79.00 11.60 82.64

ck 10.79 ＼ 60.16 ＼ 63.04 ＼ 66.81 ＼

Control of pepper virus disease

Increase pepper yield

Treatment

Treated 3 times Treated 4 times

7d 15d 7d 15d

Disease

index

Effect

（%）

Disease

index

Effeact

（%）

Disease

index

Effect

（%）

Disease

index

Effect

（%）

Bingdu A 47.68 40.15 47.31 46.29 44.84 50.32 39.35 58.16

99 Zhibao 40.24 49.79 38.69 56.08 27.59 69.43 25.99 72.37

Protein 29.08 63.72 28.07 68.14 18.73 79.25 14.44 84.65

Protein+

Bingdu A 31.39 60.83 23.61 73.20 16.82 81.36 11.36 87.92

Protein+

99 Zhibao 17.20 78.54 12.41 85.91 6.16 93.18 3.56 96.21

CK 80.15 ＼ 88.09 ＼ 90.26 ＼ 94.05 ＼

Hengyang, Hunan

Control of virus disease in tomato

Treatment

Before

treated

Treated 3 times Treated 4 times

7d 15d 7d 15d

Disease

index

Disease

index

Effect

（%）

Disease

index

Effect

（%）

Disease

index

Effect

（%）

Disease

index

Effect

（%）

Bingdu A 9.48 34.80 37.01 35.64 40.75 36.51 42.09 36.89 44.87 99zhibao 11.73 25.09 54.58 25.17 58.16 22.78 63.87 21.95 67.15 Protein 11.02 21.55 60.99 20.10 66.59 18.75 70.26 17.02 74.53 Protein

+Bingdu A 9.82 20.95 62.08 19.21 68.07 17.71 71.91 15.84 76.29

Protein+

99 zhibao 10.63 55.24 65.11 17.75 70.49 13.24 79.00 11.60 82.64

ck 10.79 ＼ 60.16 ＼ 63.04 ＼ 66.81 ＼

Control of pepper virus disease

Treatment

Treated 3 times Treated 4 times

7d 15d 7d 15d

Disease

index

Effect

（%）

Disease

index

Effeact

（%）

Disease

index

Effect

（%）

Disease

index

Effect

（%）

Bingdu A 47.68 40.15 47.31 46.29 44.84 50.32 39.35 58.16

99 Zhibao 40.24 49.79 38.69 56.08 27.59 69.43 25.99 72.37

Protein 29.08 63.72 28.07 68.14 18.73 79.25 14.44 84.65

Protein+

Bingdu A 31.39 60.83 23.61 73.20 16.82 81.36 11.36 87.92

Protein+

99 Zhibao 17.20 78.54 12.41 85.91 6.16 93.18 3.56 96.21

CK 80.15 ＼ 88.09 ＼ 90.26 ＼ 94.05 ＼

Hengyang, Hunan

Control of virus disease in tomato

Treatment 20 days after treatment 45 days after treatment

Morbidity(%)

Disease index

Effect（%）

Morbidity(%)

Disease index

Effect （%）

Activator WP

8.07 3.95 72.87 9.1 4.32 73.93

bingdubike 8.35 401 71.84 9.56 4.95 70.13

shengwujunsu

11.65 5.72 60.71 14.75 7.23 56.37

Water 27.11 14.56 — 32.25 16.57 —

Control of tobacco virus disease

CK 处理

60%

40%65%

Treatment Control

Treatment Control Treatment Control

Increase disease resistance and improve yields of strawberry and grape

Chinese cabbage, Cucumber, Pepper, Celery, Rice, Peach

TreatmentControl

Protein gene expressed in trangenical rice and cotton

pemG1转基因水稻：对稻瘟病防治效果为 42.53%

利用农杆菌介导的方法，成功地将激发子基因pemG1、peaT1和pebC1转入不同水稻品种（粳稻、恢复系）

水稻

A:转化 B:棉铃 C:卡那筛选 D:抗性棉苗

2号

对照 0

100

200

300

400

500

对照 2号

取食

面积

/平方

毫米

取食量

棉铃虫取食量差异 卷叶螟取食量差异

棉花

花粉管通道法：获得转PeaT1抗性棉株

茎粗增加，棉铃数增加，表现出一定抗虫性

农杆菌介导法转化棉花下胚轴： Coker312和CCRI24获得再生棉苗

无菌苗

继代

侵染

共培养

胚状体

成苗

胚性愈伤

转昆虫蜕皮基因烟草饲喂棉铃虫1天后的生测结果

转昆虫蜕皮基因烟草饲喂棉铃虫1天后的生测结果

转昆虫蜕皮基因烟草饲喂棉铃虫5天后的生测结果

昆虫取食后5天脱皮体式显微镜图

Steinernema glaserisurface coat protein suppresses the

immune response of Popilliajaponica larvae

Cuticle protein on gel

昆虫病原线虫：

多种害虫寄生天敌

表皮蛋白

抑制昆虫免疫

一种什么蛋白，编码基因，到目前为止还没有报道

昆虫免疫抑制蛋白

Small-scale laboratory entomopathogenic nematode culture

The insect great wax moth is used as a culture medium

(a) Healthy (cream);(b) Steinernema feltiae - infected (brown);

(c) Steinernema carpocapsae - infected (tan) and (d) Heterorhabditis bacteriophora -

infected (red) larvae of Galleria mellonella.

3-5days

10 days

抗昆虫免疫蛋白抗昆虫免疫蛋白

The nematode culture room

Vegetal medium: soybean powders,flour, egg powder, lard, termoamyl

Semi-vegetal medium:soybean powder, flou r, egg powder, lard, insect pupa homogenate,

Animal source medium:Pig liver extract, eggs, lard

Wash the nematodes out from sponge

Wash residue culture medium using the “The Big funnel”

Infective stage

0.5% sodium hypochlorite for 15minStore in distilled water with bubbled air for 2 days

Extraction in ice-cold ethanol

for 30 mins and lyophilized

Dissolve the freeze-dry powder in millQ water and incubate on ice for 30 mins with gently vortex

Centrifuge at 13000 rpm, 4 .c

Lyophilize the extraction

Lipid layer

The “sandwich structure”

Supernatant:

Protein solution

Pellet:

Surface coat debris

12% SDS-PAGE

Dissolve in 1% oNG

8% native PAGE

Marker

97kd,66kd,44kd,22kd,14kd

How do they correspond?

pH4 pH7

2-D results of 35% EtOH extraction proteins

IEF with ampholine

SDS-PAGEpH4 pH6

新昆虫免疫抑制蛋白：分离纯化、基因克隆及表达

72kDa

15% sds page 2-DE

72kDa55kDa

XeGroEL (from Xenorhabdus ehlersii) 共生菌 1647bp Xbecotin (from Xenorhabdus bovienii) 共生菌 483bp Haemocoel Insecticidal toxin

（from Xenorhabdus budapestensis D43）共生菌

ATGAAAAAGTATCTACTTCCTTTAGCCGCTATGATGGTTTCGGTTTCTACTTTTGCTCAGGCTGATAAGAAACTTGAAGATGTCGCTCCTTATCCTAAAGCTTCAGAGGGAATGGTACGCAATGTCATTGACCTTGCACCAGAAGAGGATGAAGGCGATTACATGGTTGAACTGATGATTGGCAAGGATATAAAAGTAGACTGTAATCATCACTGGTTTGGTGGGCAACTTGAAACAAAAACCTTGGAGGGGTGGGGATACGATTATTATGTATTGAATAATGTCACCGGCCCAGCGTCAACCCACATGGGTTGTTCAGGACAAAAAGAAACCGTGCGCTTTGTTCAAGTGCAGTTAGGCAAAGATGCGCTTATACGCTATAACAGCAAATTACCTATCGTGGTATATACGCCAAAATCAATGACAGTCAAATATCGTATTTGGCAGGCATTGGATGATGTCGATAATGCAGTAATCAAATAA

Xbecotin：483bp

1.0 *107 coupled Microspherses(3ul)

In vivo phagocytosis test

Microinjection

Cherry red microspheres in larva body

12 hours

Bleeding

Grace insect medium

DAPI treatment

(cell nucleolus stain)

Fluorescent microscope imaging

Engulfment of microspheresKeratin and Enolase protein coupled microspheres

BSA coupled microspheres

Transparent blue: Cell nucleolus

Bright red: Microspheres

Lipid layerSupernatant:

Protein solutionPellet:

Surface coat debris

Bioassay抑制昆虫血细胞免

疫BSA SCP2

Phagocytosis

2D electrophoresis

蛋白斑点

MAIDI-TOF-MS/MSLC-MS/MS

YGLDATAVGDEGGFAPNIQDNKAAVPSGASTGIHEALELRACNCLLLK

秀丽隐杆线虫、单一异尖线虫、旋毛线虫、刺尾蝎、拟南芥、番茄、油菜

GenBank:enolase

eno1: 1311bpEno1: 436aa

GenBank: EU333864

1 MPITRIHARQIYDSRGNPTVEVDLHTEKGVFRAAVPSGAS

41 TGIHEALELRDQDKAVHHGKGVEKAVANVIEKIAPALIAK

81 NFDVTDQVAIDKFMIELDGTENKSSLGANAILGVSLAVAK

121 AGAVHKGVPLYKHLADLAGVSKVVLPVPAFNVINGGSHAG

161 NKLAMQEFMILPVGAKSFKEAMRMGSEIYHHLKAEIKKRY

201 GLDATAVGDEGGFAPNIQDNKEGLDLLNTAIKLAGYTGLV

241 SIGMDVAASEFYKENEKKYDLDFKNPNSDPSKWINGDELA

281 ALYQSFIKDYPVVSIEDAFDQDDWANWSKLMGNTSIQLVG

321 DDLTVTNPKRIQMAVDQKACNCLLLKVNQIGSITESIEAA

361 KLSRANGWGVMVSHRSGETEDCFIADLVVGLATGQIKTGA

401 PCRSERLAKYNQLLRIEEELGADAVYAGENFRNPQI

专利申请200910119907.9

表达蛋白：具有抑制昆虫免疫的活性

格式线虫enolase基因序列： 5’UTR GACTCACGCGGCAACCCAACTGTTGAAGTCGACCTCCACACCGAGAAGGGTGAGTCTTTGC

TTGTCTTTGCAGTAGAATAACTGAATCGATTTAGGTGTGTTCCGCGCTGCTGTCCCCAGCGGAGCCTCCACTGGCATCCACGAGGCTCTGGAACTCCGCGACCAGGACAAGGCTGTCCACCACGGAAAGGGAGTCGAGAAGGCCGTCGCCAACGTCATCGAGAAGATCGCGCCGGCCCTCATCGCCAAGAACTTCGATGTGACCGACCAGGTCGCCATCGACAAGTTC

ORF:1032bp: ATGATCGAGCTTGACGGAACCGAGAACAAGTCGAGCCTCGGAGCCAACGCCATCCTCGGC

GTGTCGCTCGCCGTCGCCAAGGCCGGTGCCGTGCACAAGGGAGTGCCCCTCTACAAGCACTTGGCCGATCTCGCCGGAGTGAGCAAGGTTGTCCTCCCCGTTCCTGCCTTCAACGTCATCAACGGAGGCTCGCACGCCGGAAACAAGCTCGCCATGCAGGAGTTCATGATCCTCCCCGTCGGAGCCAAGAGCTTCAAGGAGGCCATGCGCATGGGATCCGAGATCTACCACCACCTCAAGGCCGAGATCAAGAAGCGCTACGGACTCGACGCCACCGCCGTCGGAGATGAGGGAGGATTCGCCCCCAACATCCAGGACAACAAGGAGGGTCTTGACCTCCTCAACACCGCTATCAAGCTTGCCGGATATACCGGACTGGTGTCCATCGGCATGGACGTCGCCGCCTCCGAGTTCTACAAGGAGAACGAGAAGAAGTACGACTTGGACTTCAAGAACCCCAACTCCGACCCCAGCAAGTGGATCAACGGAGACGAGCTCGCCGCGCTCTACCAGTCGTTCATCAAGGACTATCCCGTCGTCTCCATCGAGGACGCCTTCGACCAGGACGACTGGGCGAACTGGAGCAAGCTGATGGGCAACACCTCCATCCAGCTCGTCGGAGATGATCTCACCGTCACCAACCCCAAGCGCATCCAGATGGCCGTCGACCAGAAGGCGTGCAACTGCCTTCTCCTCAAGGTCAACCAGATCGGATCCATCACCGAGTCCATCGAGGCCGCCAAGCTGTCCCGCGCCAACGGATGGGGCGTCATGGTTTCCCACCGTTCTGGAGAAACCGAAGACTGCTTCATCGCTGATCTTGTTGTTGGCCTCGCTACTGGACAGATCAAGACCGGAGCCCCTTGCCGATCTGAGCGTCTCGCCAAGTACAACCAGCTTCTTCGCATCGAGGAGGAGCTCGGAGCCGATGCCGTGTACGCCGGAGAGAACTTCCGCAACCCCCAGATCTAA

3’UTR-98bp: ATGACCACCGTACTTTAATCTATCGTGTAGTTTGTAGTTTGACATTGTGATGAGTGGTTAAT

TGTATAATACGTAATTGTTGGTCAAAAAAAAAAAAA

蛋白序列：MIELDGTENKSSLGANAILGVSLAVAKAGAVHK

GVPLYKHLADLAGVSKVVLPVPAFNVINGGSHAGNKLAMQEFMILPVGAKSFKEAMRMGSEIYHHLKAEIKKRYGLDATAVGDEGGFAPNIQDNKEGLDLLNTAIKLAGYTGLVSIGMDVAASEFYKENEKKYDLDFKNPNSDPSKWINGDELAALYQSFIKDYPVVSIEDAFDQDDWANWSKLMGNTSIQLVGDDLTVTNPKRIQMAVDQKACNCLLLKVNQIGSITESIEAAKLSRANGWGVMVSHRSGETEDCFIADLVVGLATGQIKTGAPCRSERLAKYNQLLRIEEELGADAVYAGENFRNPQI

昆虫拒食蛋白 CB6蛋白对大蜡螟的生物活性测定： 1）大蜡螟的注射活性

取总蛋白5微升进行注射大蜡螟（5龄老虫），总蛋白浓度：1.890mg/mL,大约不到5分钟，大蜡螟体色很快变成黑灰色，表现为中毒现象。

取GeAB蛋白回收条带III,也同样进行大蜡螟注射活性验证得到和总

蛋白相同的现象。怀疑致使大蜡螟血腔毒素蛋白即可能就是这一单个蛋白（约为62kD左右）。

毒素III的胃毒活性还在进行中，待定。

处理 CK

双向电泳与结果分析： 1）采用GE公司的IPGphor-3等电聚焦仪进行第一相，具体的操作

由刘华指导完成。

60kD

60kD

Acknowledgement