FOODCROPS.VN.Research and application of plant immunity CAAS

1. Research and Application of
Plant Immunity
Dewen Qiu
Institute of Plant Protection
Chinese Academy of Agricultural Sciences

2. 主要内容
Concept of plant immunity
Mechanism of plant immunity
Application of plant immunity

3. Plant immunity concept
 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.

4. Plant immunology- Hot Areas
Science
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1991-1995 1996-2000 2001-2005 2006-2010 1991-1995 1996-2000 2001-2005 2006-2010

5. 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

6. 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

7. Messenger --Cornell University and EDEN Bioscience,
Presidential Awards by EPA in 2001

8. 主要内容
Concept of plant immunity
Mechanism of plant immunity
Application of plant immunity

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

10. Plant Growth Promoting Rhizobacteria

11. Trichoderma and Rhizobacteria -induced plant immunity
SA→PR →induced plant immune (SAR)?
JA/ET→LOX →PAL → induced plant immune（ISR）?
JA →MAPK?

13. Plant immunity mechanism
Plant immunity signaling
( Protein elicitor and
Chitosan Oligosaccharide )
Salicylic acid Jasmonic acid Ethylene NO
Multiple and complex effects
(Plant stress and disease resistance
Plant growth promoting)

14. 植物免疫的诱导或激活是一个初级的免疫反应
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。

15. 主要内容
Concept of plant immunity
Mechanism of plant immunity
Application of plant immunity

16. Products of Elicitor
 Eden Bioscienes, “Messenger® ”
 Redox Chemicals, “Oxycom”
 Morse, “ KeyPlex”
 Syngenta , “Actigard”
 Nippon Kayaku Co Ltd, “NCI”
 “Chitosan”

17. 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.

18. Protein elicitor from fungi
 HR –inducing protein elicitor
PevD1;Hrip1--- Extracellular
 Non HR protein elicitor
PeaT1;PebC1;PemG1- Intracellular

19. Disease resistance and growth were enhanced
by protein elicitor
75.5% Treatment Control
11.09
%
11.9%

20. SAR against TMV induced by protein elicitor PeaT1
C
P K
e
a
T
1

21. protein application
1 Purification of protein
2 Gene clone
3 Protein expression and function
4 Mechanism
5 Application

22. Fungi species
 Alternaria tenuissima
 Botrytis cinerea
 Magnaporthe grisea
 Penicillium spp
 Aspergillus Flarres
 Rhizoctonia solani
 Trichoderma spp
 Fusarium spp

23. 一、Protein purification
PeaT1
PeaT1 PemG1
PebC1
PebC1

24. Purification of PevD1
HP Q HiTrap™ Ion exchange chromatography
SDS-PAGE
HR
2
1, 2, 3, 4, 5
and 6
represents
pH 2, 4, 6, 8,
11 and 13 hydrophobic chromatography

25. Fungi elicitor protein
 HR –inducing protein elicitor
PevD1;Hrip1--- Extracellular
 Non HR protein elicitor
PeaT1;PebC1;PemG1- Intracellular

26. Purification of Protein elicitor
PeaT1
PeaT1 PemG1
PebC1
PebC1

27. Purification of Protein elicitor
HP Q HiTrap™ Ion exchange chromatography
SDS-PAGE
HR
2
1, 2, 3, 4, 5
and 6
represents
pH 2, 4, 6, 8,
11 and 13
hydrophobic chromatography

28. PeaT1蛋白纯化与检测
目的蛋白
离子交换的色谱 分子筛的色谱和电泳图

29. peptides fingerprint
Trypsin T
Chromtrypsin CT
Glu-C V8

30. Gene cloning of protein elicitor
The mass-spectrometry/proteomic
experiment
1. NILFVINKPDVYKSPSSNTWIIFGEAK
2. DIELVMQQASVSR
3. ALKENDNDIVNSIMALSI
4. RPKNILFVINQPDVYK

31. PeaT1 protein sequence
1. NILFVINKPDVYKSPSSNTWIIFGEAK
2. DIELVMQQASVSR
3. ALKENDNDIVNSIMALSI
4. RPKNILFVINQPDVYK

32. 3‘RACE获得该基因的3’全长

33. Alternaria tenuissima NAC protein sequence
交链孢菌激活蛋白的全基因序列
207 amono acids
MANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVA
VHSRNEKKARKAIAKLGLKHIDGITRVTLRRPKNILFVINQPDVY
KSPSSNTWIIFGEAKIEDLNSQAQASAAQQLAQAEAASHDHAGH
DHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQ
QASVSRKKAVKALKENDNDIVNSIMALSI
NAC Domain:49-108aa

34. Botrytis cinerea 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

35. Fusarium spp αNAC和βNAC
αNAC
MSNPRVEELPDEEPKKTTVQEHEDDSSDDSEVEEVGEGQLPAGSTVIHNRNE
KKARKALEKLHLTRIPGITRVTLRRPKNILFVINTPEVYKSPNSNTYIVFGEAKIE
DVNAAAQQAAAAQLASQNAEDHSGHNHGEPSKAVEADEKKEDKEDDEDEEE
EEEEEVDASGLEDKDIELVMTQANVSRNKAVKALKENDNDIVNSIMALSI
(210aa)
βNAC
MSDVQERLKKLGLGARTGESNKLIYNTGGKGTPRRKVKRAPARSGADDKK
LQLALKKLNTQPIQAIEEVNMFKQDGNVIHFAAPKVHAAVPSNTFAIYGNGED
KELTELVPGILNQLGPDSLASLRKLAESYQNLQKEKGEDDDEIPDLVEGENF
EGEPKVE (162aa)

36. PeaT1 (Alternaria tenuissima )
Gene:624bp
ATGGCCAACCCCCGCATTGAAGAGCTCCCCGACGAGCCCGAGAAGAAGAACGTCCAGATCG
AGGAGGATGAGTCCAGCGACGAGTCTGAGGGCGAGGAGGGCGAGGTCAGCGTACCCGCGGG
CTCCTCCGTCGCTGTCCACTCCCGCAACGAGAAGAAGGCTCGCAAGGCCATCGCCAAGCTC
GGCCTGAAGCACATCGACGGCATCACACGCGTCACCCTCCGCCGACCCAAGAACATCCTCT
TTGTCATCAACCAGCCCGACGTCTACAAGTCCCCTTCAAGCAACACCTGGATCATCTTCGG
TGAGGCCAAGATCGAGGACCTCAACTCCCAGGCTCAGGCTTCCGCCGCCCAGCAGCTTGCT
CAGGCCGAGGCCGCATCCCACGACCACGCCGGCCACGACCACGGCGACGAGGCCAGCAAGG
GCAAGGGCAAGGCTGTCGAGGACAAGAAGGACGAGGAGGAGGAGGATGACGATGAGGAGAT
TGACGACTCTGGCCTTGAGGCCAAGGACATCGAGCTCGTCATGCAGCAGGCCAGCGTTTCG
CGGAAGAAGGCCGTCAAGGCCCTCAAGGAGAACGATAACGATATAGTCAACTCCATCATGG
CGCTGAGCATATAG
Protein:207aa
MANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVAVHSRNEKKARKAIAKL
GLKHIDGITRVTLRRPKNILFVINQPDVYKSPSSNTWIIFGEAKIEDLNSQAQASAAQQLA
QAEAASHDHAGHDHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQQASVS
RKKAVKALKENDNDIVNSIMALSI

37. Structure of protein PeaT1
Doman analysis of PeaT1
207Amino acid
MANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVAVHSRNEKKARKAIAKLGL
KHIDGITRVTLRRPKNILFVINQPDVYKSPSSNTWIIFGEAKIEDLNSQAQASAAQQLAQAEAA
SHDHAGHDHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQQASVSRKKAV
KALKENDNDIVNSIMALSI
NAC Domain:49-108aa

38. Genes and patents
GenBank
Genes Fungi Patens
Accession No
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

39. Expression of protein elicitor
PeaT1
PeaT1
SDS-PAGE
1. PET-28a/PeaT1 Pichia pastoris
2. PET-28a
3. Marker
E.coli

40. Crystal and X-ray
PeaT1的X-射线晶体学 diffraction
Crystallization X-ray diffraction
Conditions：20% PEG 3350，0.2M MgCl2， 分辨率：2.4Å
0.1M Mes pH5.5 20℃

41. Structure of protein elicitor （Peat1）
a-Helix b-Sheet
0.161 0.307

43. Structure of NAC domain in PeaT1
NAC domain was constructed. NAC domain formed a homodimer
from reverse direction. This structure outline was similar with a
barrel, with a hole at the center and 6 intercrossed β strands
surrounded

44. TCTP2
TCTP1
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

45. Expression of protein elicitor
PeaT1
PeaT1
SDS-PAGE
1. PET-28a/PeaT1 Pichia pastoris
2. PET-28a
3. Marker
E.coli

46. SAR against TMV induced by protein elicitor PeaT1
C
P K
e
a
T
1

47. Protein elicitor products
Certificate for Bio-fertilizer

48. Certificate for pesticide

49. Control of pepper virus disease
Before Treated 3 times Treated 4 times
treated 7d 15d 7d 15d
Treatment
Disease Disease Effect Disease Effect Disease Effect Disease Effect
index index （%） index （%） index （%） index （%）
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
9.82 20.95 62.08 19.21 68.07 17.71 71.91 15.84 76.29
+Bingdu A
Protein+
10.63 55.24 65.11 17.75 70.49 13.24 79.00 11.60 82.64
99 zhibao
ck 10.79 ＼ 60.16 ＼ 63.04 ＼ 66.81 ＼

50. Increase pepper yield

51. Control of virus disease in tomato
Treated 3 times Treated 4 times
7d 15d 7d 15d
Treatment
Disease Effect Disease Effeact Disease Effect Disease Effect
index （%） index （%） index （%） index （%）
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+
31.39 60.83 23.61 73.20 16.82 81.36 11.36 87.92
Bingdu A
Protein+
17.20 78.54 12.41 85.91 6.16 93.18 3.56 96.21
99 Zhibao
CK 80.15 ＼ 88.09 ＼ 90.26 ＼ 94.05 ＼
Hengyang, Hunan

52. Control of pepper virus disease
Before Treated 3 times Treated 4 times
treated 7d 15d 7d 15d
Treatment
Disease Disease Effect Disease Effect Disease Effect Disease Effect
index index （%） index （%） index （%） index （%）
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
9.82 20.95 62.08 19.21 68.07 17.71 71.91 15.84 76.29
+Bingdu A
Protein+
10.63 55.24 65.11 17.75 70.49 13.24 79.00 11.60 82.64
99 zhibao
ck 10.79 ＼ 60.16 ＼ 63.04 ＼ 66.81 ＼

53. Control of virus disease in tomato
Treated 3 times Treated 4 times
7d 15d 7d 15d
Treatment
Disease Effect Disease Effeact Disease Effect Disease Effect
index （%） index （%） index （%） index （%）
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+
31.39 60.83 23.61 73.20 16.82 81.36 11.36 87.92
Bingdu A
Protein+
17.20 78.54 12.41 85.91 6.16 93.18 3.56 96.21
99 Zhibao
CK 80.15 ＼ 88.09 ＼ 90.26 ＼ 94.05 ＼
Hengyang, Hunan

54. Control of tobacco virus disease
Treatment 20 days after treatment 45 days after treatment
Morbidity Disease Effect Morbidity Disease Effect
(%) index （%） (%) index （%）
Activator 8.07 3.95 72.87 9.1 4.32 73.93
WP
bingdubike 8.35 401 71.84 9.56 4.95 70.13
shengwujuns 11.65 5.72 60.71 14.75 7.23 56.37
u
Water 27.11 14.56 — 32.25 16.57 —

55. CK 处理 65% 40%
60%

56. Increase disease resistance and improve yields
of strawberry and grape
Treatment Control Treatment Control
Treatment Control

57. Control Treatment
Chinese cabbage, Cucumber, Pepper, Celery, Rice, Peach

58. Protein gene expressed in
trangenical rice and cotton
水稻
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）

59. 棉
花
A:转化 B:棉铃 C:卡那筛选 D:抗性棉苗
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
500
取食面积/平方毫米
400
2号 300
取食量
200
100
对照 0
对照 2号
棉铃虫取食量差异 卷叶螟取食量差异

60. 农杆菌介导法转化棉花下胚轴： Coker312和CCRI24
获得再生棉苗
无菌 胚状体
苗
成苗
侵染 胚性愈伤
共培 继代
养

63. 转昆虫蜕皮基因烟草饲喂棉铃虫1天后的生测结果

64. 转昆虫蜕皮基因烟草饲喂棉铃虫1天后的生测结果

65. 转昆虫蜕皮基因烟草饲喂棉铃虫5天后的生测结果

66. 昆虫取食后5天脱皮体式显微镜图

67. 昆虫免疫抑制蛋白
昆虫病原线虫：
多种害虫寄生天敌
表皮蛋白
抑制昆虫免疫
Steinernema glaseri
surface coat protein suppresses the
immune response of Popillia
japonica larvae
Cuticle protein on gel
一种什么蛋白，编码基因，到目前为止还没有报道

68. 抗昆虫免疫蛋白
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

69. 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

70. Wash residue culture
medium using the “The Big
funnel”
Wash the nematodes
out from sponge

71. Store in distilled
0.5% sodium hypochlorite for 15min water with bubbled
air for 2 days
Infective stage
Extraction in ice-cold ethanol
for 30 mins and lyophilized
The “sandwich structure” Centrifuge at 13000 rpm, 4 .c
Supernatant: Dissolve the
freeze-dry
Lipid layer
powder in
Protein solution millQ water and
incubate on ice
Pellet: for 30 mins with
Surface coat debris gently vortex
Lyophilize the extraction

72. Marker
97kd,66kd,44kd,22kd,14kd
Dissolve in 1% oNG
12% SDS-PAGE 8% native PAGE
How do they correspond?

73. IEF with ampholine
pH6
pH4
SDS-PAGE
pH4 pH7
2-D results of 35% EtOH extraction proteins

74. 新昆虫免疫抑制蛋白：分离纯化、基因克隆及表达
Xbecotin：483bp
ATGAAAAAGTATCTACTTCCTTTAGCCGCT
72kDa ATGATGGTTTCGGTTTCTACTTTTGCTCAG
GCTGATAAGAAACTTGAAGATGTCGCTCCT
72kDa TATCCTAAAGCTTCAGAGGGAATGGTACGC
55kDa AATGTCATTGACCTTGCACCAGAAGAGGAT
GAAGGCGATTACATGGTTGAACTGATGATT
GGCAAGGATATAAAAGTAGACTGTAATCAT
CACTGGTTTGGTGGGCAACTTGAAACAAAA
ACCTTGGAGGGGTGGGGATACGATTATTAT
GTATTGAATAATGTCACCGGCCCAGCGTCA
ACCCACATGGGTTGTTCAGGACAAAAAGAA
ACCGTGCGCTTTGTTCAAGTGCAGTTAGGC
AAAGATGCGCTTATACGCTATAACAGCAAA
TTACCTATCGTGGTATATACGCCAAAATCA
ATGACAGTCAAATATCGTATTTGGCAGGCA
TTGGATGATGTCGATAATGCAGTAATCAAA
TAA
15% sds page 2-DE
 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

75. In vivo phagocytosis test
1.0 *107 coupled Microspherses
(3ul)
Fluorescent microscope imaging
DAPI treatment
(cell nucleolus
stain)
Grace insect medium
Microinjection
Cherry red microspheres in larva body
Bleeding
12 hours

76. Engulfment of microspheres
Keratin and Enolase protein coupled microspheres
Transparent blue: Cell nucleolus
Bright red: Microspheres
BSA coupled microspheres

77. Supernatant:
Lipid layer
Protein solution
Pellet:
Surface coat debris
Bioassay
抑制昆虫
血细胞免
Phagocytosis 疫
BSA SCP2
2D electrophoresis
蛋白斑点

78. MAIDI-TOF-MS/MS YGLDATAVGDEGGFAPNIQDNK
AAVPSGASTGIHEALELR
LC-MS/MS
ACNCLLLK
GenBank: 秀丽隐杆线虫、单一异尖线虫、旋毛线虫、
enolase 刺尾蝎、拟南芥、番茄、油菜
1 MPITRIHARQIYDSRGNPTVEVDLHTEKGVFRAAVPSGAS
eno1: 1311bp 41 TGIHEALELRDQDKAVHHGKGVEKAVANVIEKIAPALIAK
81 NFDVTDQVAIDKFMIELDGTENKSSLGANAILGVSLAVAK
Eno1: 436aa 121 AGAVHKGVPLYKHLADLAGVSKVVLPVPAFNVINGGSHAG
GenBank: EU333864 161 NKLAMQEFMILPVGAKSFKEAMRMGSEIYHHLKAEIKKRY
201 GLDATAVGDEGGFAPNIQDNKEGLDLLNTAIKLAGYTGLV
241 SIGMDVAASEFYKENEKKYDLDFKNPNSDPSKWINGDELA
281 ALYQSFIKDYPVVSIEDAFDQDDWANWSKLMGNTSIQLVG
专利申请 321 DDLTVTNPKRIQMAVDQKACNCLLLKVNQIGSITESIEAA
200910119907.9 361 KLSRANGWGVMVSHRSGETEDCFIADLVVGLATGQIKTGA
401 PCRSERLAKYNQLLRIEEELGADAVYAGENFRNPQI
表达蛋白：具有抑制昆虫免疫的活性

79.  格式线虫enolase基因序列：
 5’UTR
 GACTCACGCGGCAACCCAACTGTTGAAGTCGACCTCCACACCGAGAAGGGTGAGTCTTTGC
TTGTCTTTGCAGTAGAATAACTGAATCGATTTAGGTGTGTTCCGCGCTGCTGTCCCCAGCG
GAGCCTCCACTGGCATCCACGAGGCTCTGGAACTCCGCGACCAGGACAAGGCTGTCCACCA
CGGAAAGGGAGTCGAGAAGGCCGTCGCCAACGTCATCGAGAAGATCGCGCCGGCCCTCAT
CGCCAAGAACTTCGATGTGACCGACCAGGTCGCCATCGACAAGTTC
 ORF:1032bp:
 ATGATCGAGCTTGACGGAACCGAGAACAAGTCGAGCCTCGGAGCCAACGCCATCCTCGGC
GTGTCGCTCGCCGTCGCCAAGGCCGGTGCCGTGCACAAGGGAGTGCCCCTCTACAAGCACT
TGGCCGATCTCGCCGGAGTGAGCAAGGTTGTCCTCCCCGTTCCTGCCTTCAACGTCATCAAC
GGAGGCTCGCACGCCGGAAACAAGCTCGCCATGCAGGAGTTCATGATCCTCCCCGTCGGA
GCCAAGAGCTTCAAGGAGGCCATGCGCATGGGATCCGAGATCTACCACCACCTCAAGGCC
GAGATCAAGAAGCGCTACGGACTCGACGCCACCGCCGTCGGAGATGAGGGAGGATTCGCC
CCCAACATCCAGGACAACAAGGAGGGTCTTGACCTCCTCAACACCGCTATCAAGCTTGCCG
GATATACCGGACTGGTGTCCATCGGCATGGACGTCGCCGCCTCCGAGTTCTACAAGGAGAA
CGAGAAGAAGTACGACTTGGACTTCAAGAACCCCAACTCCGACCCCAGCAAGTGGATCAAC
GGAGACGAGCTCGCCGCGCTCTACCAGTCGTTCATCAAGGACTATCCCGTCGTCTCCATCG
AGGACGCCTTCGACCAGGACGACTGGGCGAACTGGAGCAAGCTGATGGGCAACACCTCCA
TCCAGCTCGTCGGAGATGATCTCACCGTCACCAACCCCAAGCGCATCCAGATGGCCGTCGA
CCAGAAGGCGTGCAACTGCCTTCTCCTCAAGGTCAACCAGATCGGATCCATCACCGAGTCC
ATCGAGGCCGCCAAGCTGTCCCGCGCCAACGGATGGGGCGTCATGGTTTCCCACCGTTCTG
GAGAAACCGAAGACTGCTTCATCGCTGATCTTGTTGTTGGCCTCGCTACTGGACAGATCAA
GACCGGAGCCCCTTGCCGATCTGAGCGTCTCGCCAAGTACAACCAGCTTCTTCGCATCGAG
GAGGAGCTCGGAGCCGATGCCGTGTACGCCGGAGAGAACTTCCGCAACCCCCAGATCTAA
 3’UTR-98bp:
 ATGACCACCGTACTTTAATCTATCGTGTAGTTTGTAGTTTGACATTGTGATGAGTGGTTAAT
TGTATAATACGTAATTGTTGGTCAAAAAAAAAAAAA

80.  蛋白序列：
 MIELDGTENKSSLGANAILGVSLAVAKAGAVHK
GVPLYKHLADLAGVSKVVLPVPAFNVINGGSHA
GNKLAMQEFMILPVGAKSFKEAMRMGSEIYHHL
KAEIKKRYGLDATAVGDEGGFAPNIQDNKEGLD
LLNTAIKLAGYTGLVSIGMDVAASEFYKENEKKY
DLDFKNPNSDPSKWINGDELAALYQSFIKDYPV
VSIEDAFDQDDWANWSKLMGNTSIQLVGDDLT
VTNPKRIQMAVDQKACNCLLLKVNQIGSITESIE
AAKLSRANGWGVMVSHRSGETEDCFIADLVVGL
ATGQIKTGAPCRSERLAKYNQLLRIEEELGADAV
YAGENFRNPQI

81. 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

83. 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

84. Acknowledgement