Research and application of plant immunity

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Research and application of plant immunity
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  • 1. Research and Application of Plant Immunity Dewen Qiu Institute of Plant ProtectionChinese Academy of Agricultural Sciences
  • 2. 主要内容Concept of plant immunity Mechanism of plant immunityApplication 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 12 12 10 8 6 4 4 2 1 1 0 1991-1995 1996-2000 2001-2005 2006-2010 Nature Cell 1625 23 16 1420 12 1015 8 710 6 7 4 4 5 2 2 0 0 0 0 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 immunityApplication 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 immunitySA→PR →induced plant immune (SAR)?JA/ET→LOX →PAL → induced plant immune(ISR)?JA →MAPK?
  • 12. 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)
  • 13. 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。
  • 14. 主要内容Concept of plant immunity Mechanism of plant immunityApplication of plant immunity
  • 15. Products of Elicitor Eden Bioscienes, “Messenger® ” Redox Chemicals, “Oxycom” Morse, “ KeyPlex” Syngenta , “Actigard” Nippon Kayaku Co Ltd, “NCI” “Chitosan”
  • 16. 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.
  • 17. Protein elicitor from fungi HR –inducing protein elicitor PevD1;Hrip1--- Extracellular Non HR protein elicitor PeaT1;PebC1;PemG1- Intracellular
  • 18. Disease resistance and growth were enhanced by protein elicitor 75.5% Treatment Control 11.09 % 11.9%
  • 19. SAR against TMV induced by protein elicitor PeaT1 C P K e a T 1
  • 20. protein application1 Purification of protein2 Gene clone3 Protein expression and function4 Mechanism5 Application
  • 21. Fungi species Alternaria tenuissima Botrytis cinerea Magnaporthe grisea Penicillium spp Aspergillus Flarres Rhizoctonia solani Trichoderma spp Fusarium spp
  • 22. 一、Protein purification PeaT1 PeaT1 PemG1 PebC1 PebC1
  • 23. Purification of PevD1 HP Q HiTrap™ Ion exchange chromatography SDS-PAGE HR 21, 2, 3, 4, 5and 6representspH 2, 4, 6, 8,11 and 13 hydrophobic chromatography
  • 24. Fungi elicitor protein HR –inducing protein elicitor PevD1;Hrip1--- Extracellular Non HR protein elicitor PeaT1;PebC1;PemG1- Intracellular
  • 25. Purification of Protein elicitor PeaT1 PeaT1 PemG1 PebC1 PebC1
  • 26. Purification of Protein elicitor HP Q HiTrap™ Ion exchange chromatography SDS-PAGE HR 21, 2, 3, 4, 5and 6representspH 2, 4, 6, 8,11 and 13 hydrophobic chromatography
  • 27. 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
  • 28. peptides fingerprintTrypsin TChromtrypsin CTGlu-C V8
  • 29. Gene cloning of protein elicitor The mass-spectrometry/proteomic experiment 1. NILFVINKPDVYKSPSSNTWIIFGEAK 2. DIELVMQQASVSR 3. ALKENDNDIVNSIMALSI 4. RPKNILFVINQPDVYK
  • 30. PeaT1 protein sequence1. NILFVINKPDVYKSPSSNTWIIFGEAK2. DIELVMQQASVSR3. ALKENDNDIVNSIMALSI4. RPKNILFVINQPDVYK
  • 31. 3‘RACE获得该基因的3’全长
  • 32. Alternaria tenuissima NAC protein sequence 交链孢菌激活蛋白的全基因序列207 amono acidsMANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVAVHSRNEKKARKAIAKLGLKHIDGITRVTLRRPKNILFVINQPDVYKSPSSNTWIIFGEAKIEDLNSQAQASAAQQLAQAEAASHDHAGHDHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQQASVSRKKAVKALKENDNDIVNSIMALSI NAC Domain:49-108aa
  • 33. Botrytis cinerea protein sequence1 MSNPRIEELP DNEEPTKQQV TAEDEGSDSS DSEAEGEEVA GIPAGSQVAF SRNEKKARKS61 IAKLGLTRVP GITRVTLRRP KNILFVINQP EVYKSPTSNT YIVFGEAKIE DLNSQAQASA121 AAQLAAQESH DHAGHDHSGH DHSHDHGKGK AVDTEEKKEE EEDDTEEVDA TGLEDKDIEL181 VMTQASVSRN KAVKALKEND NDIVNSIMAL SI
  • 34. Fusarium spp αNAC和βNACαNAC MSNPRVEELPDEEPKKTTVQEHEDDSSDDSEVEEVGEGQLPAGSTVIHNRNE KKARKALEKLHLTRIPGITRVTLRRPKNILFVINTPEVYKSPNSNTYIVFGEAKIE DVNAAAQQAAAAQLASQNAEDHSGHNHGEPSKAVEADEKKEDKEDDEDEEE EEEEEVDASGLEDKDIELVMTQANVSRNKAVKALKENDNDIVNSIMALSI (210aa)βNAC MSDVQERLKKLGLGARTGESNKLIYNTGGKGTPRRKVKRAPARSGADDKK LQLALKKLNTQPIQAIEEVNMFKQDGNVIHFAAPKVHAAVPSNTFAIYGNGED KELTELVPGILNQLGPDSLASLRKLAESYQNLQKEKGEDDDEIPDLVEGENF EGEPKVE (162aa)
  • 35. PeaT1 (Alternaria tenuissima )Gene:624bp ATGGCCAACCCCCGCATTGAAGAGCTCCCCGACGAGCCCGAGAAGAAGAACGTCCAGATCG AGGAGGATGAGTCCAGCGACGAGTCTGAGGGCGAGGAGGGCGAGGTCAGCGTACCCGCGGG CTCCTCCGTCGCTGTCCACTCCCGCAACGAGAAGAAGGCTCGCAAGGCCATCGCCAAGCTC GGCCTGAAGCACATCGACGGCATCACACGCGTCACCCTCCGCCGACCCAAGAACATCCTCT TTGTCATCAACCAGCCCGACGTCTACAAGTCCCCTTCAAGCAACACCTGGATCATCTTCGG TGAGGCCAAGATCGAGGACCTCAACTCCCAGGCTCAGGCTTCCGCCGCCCAGCAGCTTGCT CAGGCCGAGGCCGCATCCCACGACCACGCCGGCCACGACCACGGCGACGAGGCCAGCAAGG GCAAGGGCAAGGCTGTCGAGGACAAGAAGGACGAGGAGGAGGAGGATGACGATGAGGAGAT TGACGACTCTGGCCTTGAGGCCAAGGACATCGAGCTCGTCATGCAGCAGGCCAGCGTTTCG CGGAAGAAGGCCGTCAAGGCCCTCAAGGAGAACGATAACGATATAGTCAACTCCATCATGG CGCTGAGCATATAGProtein:207aa MANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVAVHSRNEKKARKAIAKL GLKHIDGITRVTLRRPKNILFVINQPDVYKSPSSNTWIIFGEAKIEDLNSQAQASAAQQLA QAEAASHDHAGHDHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQQASVS RKKAVKALKENDNDIVNSIMALSI
  • 36. Structure of protein PeaT1 Doman analysis of PeaT1207Amino acidMANPRIEELPDEPEKKNVQIEEDESSDESEGEEGEVSVPAGSSVAVHSRNEKKARKAIAKLGLKHIDGITRVTLRRPKNILFVINQPDVYKSPSSNTWIIFGEAKIEDLNSQAQASAAQQLAQAEAASHDHAGHDHGDEASKGKGKAVEDKKDEEEEDDDEEIDDSGLEAKDIELVMQQASVSRKKAVKALKENDNDIVNSIMALSINAC Domain:49-108aa
  • 37. Genes and patents GenBankGenes Fungi Patens Accession NopemG1 Magnaporthe spp EF062504 ZL200510011580.5peaT1 Alternaria tenuissima EF030819 ZL200610152700.8pebC1 Botrytis cinerea FJ748868 --PevD1 Verticillium dahliae HQ540585 201010593488.5Hrip1 Alternaria tenuissima HQ713431 201110144389.3
  • 38. Expression of protein elicitor PeaT1 PeaT1 SDS-PAGE1. PET-28a/PeaT1 Pichia pastoris2. PET-28a3. Marker E.coli
  • 39. Crystal and X-ray PeaT1的X-射线晶体学 diffraction Crystallization X-ray diffractionConditions:20% PEG 3350,0.2M MgCl2, 分辨率:2.4Å 0.1M Mes pH5.5 20℃
  • 40. Structure of protein elicitor (Peat1)a-Helix b-Sheet 0.161 0.307
  • 41. Structure of NAC domain in PeaT1NAC domain was constructed. NAC domain formed a homodimerfrom reverse direction. This structure outline was similar with abarrel, with a hole at the center and 6 intercrossed β strandssurrounded
  • 42. 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
  • 43. Expression of protein elicitor PeaT1 PeaT1 SDS-PAGE1. PET-28a/PeaT1 Pichia pastoris2. PET-28a3. Marker E.coli
  • 44. SAR against TMV induced by protein elicitor PeaT1 C P K e a T 1
  • 45. Protein elicitor products Certificate for Bio-fertilizer
  • 46. Certificate for pesticide
  • 47. Control of pepper virus disease Before Treated 3 times Treated 4 times treated 7d 15d 7d 15dTreatment 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 44.8799zhibao 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 AProtein+ 10.63 55.24 65.11 17.75 70.49 13.24 79.00 11.60 82.6499 zhibao ck 10.79 \ 60.16 \ 63.04 \ 66.81 \
  • 48. Increase pepper yield
  • 49. Control of virus disease in tomato Treated 3 times Treated 4 times 7d 15d 7d 15dTreatment 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 58.1699 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 84.65Protein+ 31.39 60.83 23.61 73.20 16.82 81.36 11.36 87.92Bingdu AProtein+ 17.20 78.54 12.41 85.91 6.16 93.18 3.56 96.2199 Zhibao CK 80.15 \ 88.09 \ 90.26 \ 94.05 \ Hengyang, Hunan
  • 50. Control of pepper virus disease Before Treated 3 times Treated 4 times treated 7d 15d 7d 15dTreatment 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 44.8799zhibao 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 AProtein+ 10.63 55.24 65.11 17.75 70.49 13.24 79.00 11.60 82.6499 zhibao ck 10.79 \ 60.16 \ 63.04 \ 66.81 \
  • 51. Control of virus disease in tomato Treated 3 times Treated 4 times 7d 15d 7d 15dTreatment 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 58.1699 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 84.65Protein+ 31.39 60.83 23.61 73.20 16.82 81.36 11.36 87.92Bingdu AProtein+ 17.20 78.54 12.41 85.91 6.16 93.18 3.56 96.2199 Zhibao CK 80.15 \ 88.09 \ 90.26 \ 94.05 \ Hengyang, Hunan
  • 52. 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 WPbingdubike 8.35 401 71.84 9.56 4.95 70.13shengwujuns 11.65 5.72 60.71 14.75 7.23 56.37 u Water 27.11 14.56 — 32.25 16.57 —
  • 53. CK 处理 65% 40% 60%
  • 54. Increase disease resistance and improve yields of strawberry and grapeTreatment Control Treatment ControlTreatment Control
  • 55. Control TreatmentChinese cabbage, Cucumber, Pepper, Celery, Rice, Peach
  • 56. 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)
  • 57. 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 4002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
  • 58. 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
  • 59. 转昆虫蜕皮基因烟草饲喂棉铃虫1天后的生测结果
  • 60. 转昆虫蜕皮基因烟草饲喂棉铃虫1天后的生测结果
  • 61. 转昆虫蜕皮基因烟草饲喂棉铃虫5天后的生测结果
  • 62. 昆虫取食后5天脱皮体式显微镜图
  • 63. 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
  • 64. 抗昆虫免疫蛋白Small-scale laboratory entomopathogenic nematode cultureThe 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
  • 65. 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
  • 66. Wash residue culture medium using the “The Big funnel”Wash the nematodes out from sponge
  • 67. 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 lyophilizedThe “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
  • 68. Marker 97kd,66kd,44kd,22kd,14kd Dissolve in 1% oNG12% SDS-PAGE 8% native PAGE How do they correspond?
  • 69. IEF with ampholine pH6 pH4 SDS-PAGE pH4 pH7 2-D results of 35% EtOH extraction proteins
  • 70. 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 TAA15% 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
  • 71. 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
  • 72. Engulfment of microspheres Keratin and Enolase protein coupled microspheres Transparent blue: Cell nucleolus Bright red: Microspheres BSA coupled microspheres
  • 73. 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
  • 74. MAIDI-TOF-MS/MS YGLDATAVGDEGGFAPNIQDNK AAVPSGASTGIHEALELR LC-MS/MS ACNCLLLKGenBank: 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 AGAVHKGVPLYKHLADLAGVSKVVLPVPAFNVINGGSHAGGenBank: 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
  • 75.  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
  • 76.  蛋白序列: MIELDGTENKSSLGANAILGVSLAVAKAGAVHK GVPLYKHLADLAGVSKVVLPVPAFNVINGGSHA GNKLAMQEFMILPVGAKSFKEAMRMGSEIYHHL KAEIKKRYGLDATAVGDEGGFAPNIQDNKEGLD LLNTAIKLAGYTGLVSIGMDVAASEFYKENEKKY DLDFKNPNSDPSKWINGDELAALYQSFIKDYPV VSIEDAFDQDDWANWSKLMGNTSIQLVGDDLT VTNPKRIQMAVDQKACNCLLLKVNQIGSITESIE AAKLSRANGWGVMVSHRSGETEDCFIADLVVGL ATGQIKTGAPCRSERLAKYNQLLRIEEELGADAV YAGENFRNPQI
  • 77. 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
  • 78. 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
  • 79. Acknowledgement