Differential Protein Expression in Sugarcane during Sugarcane-Sporisorium scitamineum Interaction Re
Hindawi Publishing Corporation
Comparative and Functional Genomics
Volume2011,Article ID989016,10pages
doi:10.1155/2011/989016
Research Article
Differential Protein Expression in Sugarcane during Sugarcane-Sporisorium scitamineum Interaction Revealed by
2-DE and MALDI-TOF-TOF/MS
Youxiong Que,Liping Xu,Jianwei Lin,Miaohong Ruan,Muqing Zhang,and Rukai Chen
Key Lab of Sugarcane Genetic Improvement,Ministry of Agriculture,Fujian Agriculture and Forestry University,Fuzhou,
Fujian350002,China
Correspondence should be addressed to Y ouxiong Que,queyouxiong@https://www.wendangku.net/doc/bc8490920.html, and Liping Xu,xlpmail@https://www.wendangku.net/doc/bc8490920.html,
Received24March2011;Accepted17May2011
Academic Editor:G.Pesole
Copyright?2011Y ouxiong Que et al.This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use,distribution,and reproduction in any medium,provided the original work is properly cited.
To understand the molecular basis of a speci?c plant-pathogen interaction,it is important to identify plant proteins that respond to the pathogen attack.Two sugarcane varieties,NCo376and Ya71-374,were used in this study.By applying2-dimensional electrophoresis(2-DE),the protein expression pro?le of sugarcane after inoculating with Sporisorium scitamineum was analyzed.
In total,23di?erentially expressed proteins were identi?ed by MALDI-TOF-TOF/MS.Bioinformatics analysis revealed that the functions of these20di?erential proteins were associated with such functions as photosynthesis,signal transduction,and disease resistance,while the function of the remaining three proteins was not determined.From above,we can assume that the protein regulatory network during the interaction between sugarcane and S.scitamineum is complicated.This represents the ?rst proteomic investigation focused on highlighting the alterations of the protein expression pro?le in sugarcane exposed to S.scitamineum,and it provides reference information on sugarcane response to S.scitamineum stress at the protein level.
1.Introduction
Sugarcane smut,which causes signi?cant losses in cane and sugar yield as well as lowering sucrose content,is a fungal disease caused by Sporisorium scitamineum.Following infec-tion,sugarcane plants often tiller profusely with the shoots being more spindly and the leaves being more upright and narrow(“grass-like”in appearance)and“buggy-like”whips emerging from the shoots.Less common symptoms are leaf and stem galls and proliferating buds.The development of sugarcane smut depends on environment,cultivar,and pathogen interactions.
Plant disease resistance is complex,involving a complex network of recognition of avirulence determinants by plant receptors,triggering of speci?c signal transduction pathways, oxidative bursts,accumulation of pathogenesis-related(PR-) proteins and phytoalexins,and localized cell death[1]. Understanding the basis of pathogens causing a certain disease in one host plant but not in another has long intrigued and motivated plant pathologists.Until recently,increasing attention has been paid to the study of the interaction between sugarcane and S.scitamineum.The research reports have mainly concentrated on the physio-logical and biochemical changes during interaction between sugarcane and S.scitamineum,which should be helpful in accelerating the formulation of short-and long-term strategies of smut disease management[2–4].Additionally, the study of sugarcane-S.scitamineum interaction at the molecular level has also identi?ed an inventory of candidate genes that are preferentially expressed during the process[5–7],suggesting an active role for the host plant.
Despite what we have already learned,little is known about the proteomic background of the interaction between pathogen and host in this pathosystem.Di?erential pro-teomics,whose essence is to discover the di?erential protein expression among di?erent samples caused by a speci?c factor,is an important part of the overall proteomics study. The identi?cation of di?erentially expressed proteins under various exogenous stresses can most probably give clues as to what kind of defensive mechanisms and biochemical
pathways are regulated in speci?c circumstances.Theoreti-cally,once enough information about protein di?erences is obtained,the basis for these changes can be deduced;thus, the study of di?erential proteomics provides a powerful tool for basic life science research.Recently,methods to study proteins that show di?erences in abundance under di?erent conditions,such as2-DE and MALDI-TOF-TOF/MS,have been developed.Moreover,the protein expression pro?les of many plants under various biotic stresses have been studied by the application of di?erential proteomics technology, and the relevant information of the response mechanism of di?erential protein under various stresses has been obtained [8–12].Thus,this technology has been demonstrated to be an excellent tool to identify novel proteins related with plant resistance to certain pathogen.
In the present study,sugarcane varieties NCo376and Ya71-374,which were highly resistant and highly susceptible to S.scitamineum,respectively,were used as plant materials, and two kinds of di?erential proteomics technology,2-DE and MALDI-TOF-TOF/MS(Matrix Assisted Laser Desorp-tion Ionization,tandem Time of Flight Mass Spectrome-ter),were applied in the di?erentially expressed proteins analysis during sugarcane-S.scitamineum interaction.It is anticipated that the results obtained in this study will be helpful toward understanding the molecular response of sugarcane varieties with di?erent smut resistance levels to S. scitamineum,and it will thus provide some information on sugarcane smut resistance basis at the protein level.
2.Materials and Methods
2.1.Plant Materials and Inoculation.Sugarcane varieties, NCo376(highly resistant to S.scitamineum)and Ya71-374 (highly susceptible to S.scitamineum),were used and the source of S.scitamineum Race2inoculum was collected from sugarcane variety F134.Thirty stalks each of NCo376and Ya71-374with robust and uniform growth were selected, cut into two-bud stalk,and divided into two groups of equal sections.The stalks were treated in hot water at50?C for2hours for disinfection.One group of the disinfected stalks was immersed in S.scitamineum spore suspension for 10min(treatment group),and the other group was treated with sterilized double-distilled water(control group).The concentration of the S.scitamineum spore suspension used for inoculation was5×106spores/mL.After inoculation, stalks were kept under humid condition at25?C for24hours and then planted in the?eld.For the control group,the stalks were treated the same as the treatment group except that the S.scitamineum spore suspension was replaced by distilled water.When symptoms of S.scitamineum infection appeared,the+1leaves(the1st leaf under the?eshy band)in the treatment and control groups were collected by freezing in liquid nitrogen and stored at?80?C until later use for protein extraction.Three biological repeats were applied in the experiment.
2.2.Extraction of Whole Protein from Sugarcane Leaves. TCA-acetone(trichloroacetic acid and acetone)precipitation method was adopted for the extraction of whole protein with minor modi?cation[13].Firstly,1g of fresh sugarcane leaves was grinded into powder in liquid nitrogen(PVP:sample was 1:10)with the addition of10mL of10%TCA(prepared by acetone,containing0.07%β-mercaptoethanol).Secondly, the mixed liquid was precipitated for1h at?20?C and cen-trifuged for15min with30000g at4?C,and then the super-natant was discarded and the pellet was re-suspended with cold acetone containing0.07%β-mercaptoethanol and kept overnight at?20?C;after that,the pellet was washed twice with cold acetone containing0.07%β-mercaptoethanol. Thirdly,after centrifugation for15min with30000g,the pellet was resuspended with80%acetone and kept for1h at ?20?C,and then it was centrifuged again.Finally,the pellet was dried into powder by vacuum drying at low temperature and stored at?80?C until use.
2.3.Protein Lysis and Content Determination.50mg of pro-tein dry powder was added into600μL lysate(7mol/L urea, 2mol/L TCA,4%(m/v)3-((3-cholamidopropyl)dimeth-ylammonio-1-propanesulfonate(CHAPS),1%(m/v)am-pholyte with pH of3.5–10,and40mmol/L dithiothreitol (DTT)),and lysed in water bath for2h at30?C,and then 20,000g centrifuged was for20min.The supernatant was used for2-DE analysis.Total protein content was measured according to the method developed by Bradford(1976)using bovine serum albumin as the standard[14].
2.4.First-Dimension Isoelectric Focusing(IEF).Immobilized pH gradient(IPG)strip(24cm,pH4–7,linear gradient; Amersham Biosciences Company)was applied with?rst-dimensional isoelectric focusing(IEF)on Amersham Ettan IPGphor II.Before use,the protein extracts were allowed to thaw at room temperature.Active rehydration was performed,and the rehydration solution contained6mol/L of urea,2mol/L of thiourea,2%CHAPS,0.5%IPG bu?er, 0.4%DTT,and0.002%bromphenol blue.The total sample volume was450μL,containing1,000μg protein.When the sample was added into the gel-holding tank,the plastic protective?lm on the strip was taken o?and the strip surface faced downward and both ends of the strip clung to the electrodes at the bottom of the tank.Attention should be paid that the positive and negative electrodes not be reversed and the generation of bubble between strip and sample?uid be avoided.1.5mL of mineral oil was covered on the strip to prevent volatilization of the sample solution.The working parameters for IEF were as follows:20?C,50μA/strip,30V, 12h;500V,1h;1,000V,2h;3,000V,1h;8,000V,7.5h,of which a total of65,860V hours were achieved.
2.5.Second-Dimension SDS-PAGE.When IEF was complete, the strips were incubated for15min in the equilibration bu?er(6mol/L of urea,50mmol/L Tris-HCl(pH8.8), 2%(m/v)SDS,30%(m/v)glycerol,0.002%bromphenol blue)containing1%(m/v)DTT,and then the strips were reequilibrated for20min in the equilibration bu?er which contained2.5%iodoacetamide to remove the surplus DTT.
After equilibrating twice,the second-dimensional SDS-PAGE was adopted with the gel concentration of12.5% T and 2.6% C.After the solidi?cation of the gel,the strips were placed into the glass plate carefully with good contact with the gel surface(avoiding the generation of bubble),and0.5%agarose containing a trace amount of bromphenol blue(prepared by electrophoresis bu?er)was used for gel sealing,electrophoresis was started,and the parameter was set as15mA/plate.When the front edge of bromphenol blue reached the gel,the electric current was increased to30mA/plate and the electrophoresis was stopped when the front edge of the bromphenol blue reached the position0.5cm away from the bottom of gel.Once the SDS-PAGE?nished,the gel was taken out and stained with the Neuho?colloid staining method[15].Gel image was acquired by a Gel Doc2000(Bio-Rad)image scanner and spot detection,spot matching,and quantitative intensity analysis were performed using PD-Quest7.20software(Bio-Rad).Images of treatment and control gels were normalized through intensity normalization,and only those proteins with obvious upregulated expression or signi?cant down-regulated expression and new proteins after infection were considered to be di?erentially expressed.
2.6.In-Gel Digestion of Di?erentially Expressed Proteins. Proteins of interest were excised from the gels and placed into a96-well microtitre plate.Gel pieces were detained with a solution of15mmol/L potassium ferricyanide and 50mmol/L sodium thiosulfate(1:1),for20min at room temperature.After that,they were washed twice with deion-ized water,and they shrank by dehydration in acetonitrile (ACN).The samples were then swollen in a digestion bu?er containing20mmol/L ammonium bicarbonate and 12.5ng/μL trypsin at4?C.After30min incubation,the gels were digested more than12h at37?C.Peptides were then extracted twice using0.1%TFA in50%ACN.The extracts were dried under the protection of N2.For MALDI-TOF-TOF/MS,the peptides were eluted onto the target plate with 0.7μL matrix solution(α-cyano-4-hydroxy-cinnamic acid in 0.1%TFA and50%ACN).
2.7.MALDI-TOF-TOF/MS and Data Analysis.Samples were analyzed by a4700Proteomics Analyzer(MALDI-TOF/TOF TM)(Applied Biosystems,USA),and the operations were as follows.The completely dry peptide segments were dissolved in0.7μL of0.5g/L CHCA solution(0.1%TFA+50%ACN solvent).The entire solution was spot onto the stainless steel MALDI target plate with air drying at room temperature. Then mass spectrometric analysis was performed on the samples,and the laser light was from Nd:YAG laser with a wavelength of355nm with an accelerating voltage of 20kV.The data was collected by positive ion and automated acquisition mode.The scope of PMF mass scan was from 700D to3500D,and series mass spectrometric analysis was made on5peaks with the maximum intensity.External standard adjustment was made by myoglobin enzymolysis peptide segment on the spectrogram.
The data were searched by GPS Explorer(GPS Explorer TM software,Applied Biosystems,USA)using MASCOT (Matrix Science,London,UK)as a search engine.Setting of parameters was as follows:database was NCBI(nr);retrieval genera was set as all;data acquisition method was set as combined;maximum missing cut site allowed was set as1; enzyme was set as trypsin.Setting of quality error scope was PMF0.3D;MS/MS0.4D;pancreatin self-degradation peak and pollutant peak were rejected manually during the database retrieval.
3.Results and Analysis
3.1.DE Analysis of Di?erentially Expressed Proteins in Sug-arcane under S.scitamineum Stress.By the application of IPG strip(pH3–10),the result showed that the whole protein from sugarcane mainly concentrated between sub-acidity isoelectric points and neutrality,while there were only few extremely acidic proteins with isoelectric points below4and basic proteins with isoelectric points above 8.From the comparison of protein2-DE atlas of highly resistant variety NCo376and highly susceptible variety Ya71-374before and after inoculation,we could see that the pro-tein expression between resistant and susceptible sugarcane varieties presented some di?erence,which was the protein expression of the same variety before and after inoculation and the protein expression between resistant and susceptible varieties were di?erent.While the PI(isoelectric point)of sugarcane proteins mainly concentrated between subacidity and neutrality,IPG strip with pH3–10could not well separate the sugarcane proteins;thus IPG strip with a narrow pH range with pH4–7was further applied for2-DE analysis. Through2-DE and silver staining,protein2-DE gel atlas was obtained and conducted with spectrum analysis by protein discrimination software.In order to assure reproducibility, 2-DE for the control and S.scitamineum-stressed sugarcane was repeated three times.Image analysis showed that these2-DE images were highly reproducible,and2-DE patterns for control and treatment groups are shown in Figures1and2.A total of about500to700visible protein spots were observed on each2-DE gel.
From Figures1and2,the separation e?ect of IPG strip(pH4–7)on sugarcane protein was comparatively better,and we could see that no matter if in high-resistance variety NCo376or in high-susceptibility variety Ya71-374, the protein expression presented signi?cant di?erence before and after inoculation,and the di?erence appeared not only in the expression capacity of the same protein but also in some newly induced proteins(present)or totally inhibited (absent)proteins.At the same time,the quantity and spatial distribution of di?erential proteins in resistant and susceptible varieties were di?erent,which indicated di?erent molecular responses to S.scitamineum challenge.It indicated that the expression of proteins was regulated by di?erent function modes,which resulted in the resistance of resistant variety or the susceptibility of susceptible variety to smut.
In the present study,it was observed that the expression of23proteins,16in NCo376and seven in Ya71-374,had
12
311
1521
19
14
9
6
13
M (KD)
14.4
18.425354566.2116Treatment
NCO376
pH 4–7.24cm (a)
45
8
18
22
M (KD)
14.4
18.425354566.2116Control NCO376
pH 4–7.24cm (b)
Figure 1:2-D SDS-PAGE
proteins in NCo376from treatment (a)and control (b)sugarcane.
Ya71-374
14.4
18.4253454566.2116M (KD)
pH 4–7.24cm (a)
Ya71-374
14.4
18.4254566.2116M (KD)
Control
345pH 4–7.24cm (b)
Figure 2:2-D SDS-PAGE proteins in Ya71-374from treatment (a)and control (b)sugarcane.
changed obviously before and after the S.scitamineum in-oculation.Among these proteins,the expression amount of eight proteins (no.2,no.3,no.6,no.9,no.11,no.13,no.14,and no.19)increased and that of six proteins (no.4,no.5,no.8,no.18,no.21,and no.22)decreased plus two proteins newly induced after infection (no.1,and no.15)in NCo376,while in Ya71-374,the expression of three proteins (no.7,no.16,and no.17)increased,the expression of three proteins (no.12,no.20,and no.23)decreased,and one protein was newly induced after infection (no.10)as shown in Figures
1,2,3,and Table 1.These di ?erentially expressed proteins were separately excised by a clean penknife for subsequent MALDI-TOF-TOF/MS analysis.
3.2.MALDI-TOF-TOF/MS Analysis of Di ?erentially Ex-pressed Proteins.In the MALDI-TOF-TOF/MS analysis of 23di ?erential proteins,the peptide mass ?ngerprinting and tandem mass spectra of 20proteins were successfully obtained.The utility peak was not detected in three proteins numbered as 21,22,and 23,which may be caused by their low abundance or some unknown factors during sample preparation,or because the peak value was too low to obtain the peptide mass ?ngerprinting.For the other 20di ?erential proteins,they were successfully identi?ed in MALDI-TOF-TOF/MS analysis.Bioinformatics analysis demonstrated that the functions of these proteins were associated with such functions as photosynthesis,signal transduction,and disease resistance.Among them,a total of nine photosynthesis-related proteins constituted the largest proportion and the proportion was 45%;?ve were disease-resistance-related proteins that accounted for 25%;one signal-transduction-related protein accounted for 5%,while there were also three function-unknown proteins accounting for 15%of the total 20identi?ed proteins.Figures 4and 5showed the peptide mass ?nger printings,tandem mass spectra,and homologous amino acid sequences of two proteins (no.9and no.10).
4.Discussion
During the process of plant development,sugarcane is fre-quently infested by pathogens (including bacteria,fungi,and viruses)which are the major biotic stresses.Regardless of whether the interaction between plant and pathogen is a disease-resistant (non-a ?nity interaction)or susceptible reaction (a ?nity interaction),it is due to the interaction between the disease-resistance gene of host plant and corre-sponding avirulence gene of pathogen,which could induce the result of the coordination expression of a series of defense genes in host plant.However,in the reactions of a ?nity interaction or non-a ?nity interaction,the di ?erential genes had signi?cant di ?erences in spatial distribution,expression rate,and expression intensity [16].Therefore,these di ?er-ences at the level of gene expression directly appeared in the various generation rate,intensity and spatial distribution of proteins in disease-resistant and susceptible varieties after the interaction between plant and pathogen [8,17].
Until now,the studies on plant proteomics mainly con-centrated on several plant species with completed genomic sequencing,especially on the model plants such as O.sativa and A.thaliana .Up to now,identi?cation of protein functions continues to depend on functional genomics studies at the gene level,especially the study of ETS function.With the implementation and successful completion of sugarcane EST project,about 290,000expressed sequence tag (EST)sequences in sugarcane have been released,but these ETSs could not yet fully cover the large genome of sugarcane,and the functions of most ESTs obtained have not yet been elucidated.Therefore,these data are far away
Treatment Control Treatment Control Treatment Control 11
66
99
1313
1919
22
88
1414
2222
33
1010
1515
2323
Figure3:Enlarged images of partial di?erentially expressed proteins derived from2-DE gel image for treatment and control sugarcane. Notes:Boxed regions show the protein,which is di?erentially expressed,and the number is consistent in Figures1and2.
Table1:Identi?cation of di?erentially expressed proteins in sugarcane under S.scitamineum stress.
Spot Accession no.Homologous protein pI Mr(Da)Species Expression NO.1gi|73970162Echinoderm-microtubule-associated protein8.46233439.8Z.mays+ NO.2gi|3914465Photosystem I reaction center subunit VI10.114920.1Z.mays↑NO.3gi|11134057Oxygen-evolving enhancer protein3-19.7723118.5P.sativum↑NO.4gi|46109064Unknown 4.784190.8O.sativa↓NO.5gi|8996050Protective antigen 6.0262682 A.thaliana↓NO.6gi|66821923Ribulose-1,5-bisphosphate carboxylase 5.9548907.6O.sativa↑NO.7gi|6578770Rubisco large subunit 6.348203.2L.cuneifolia↑NO.8gi|893471492-oxo-acid dehydrogenase E1component 5.7995280.7S.bicolor↓NO.9gi|37780998Chlorophyll-a/b-binding protein7.0318027.3V.vinifera↑NO.10gi|78777782Cytochrome-c peroxidase8.3945113Z.mays+ NO.11gi|27883935Ribulose-1,5-bisphosphate carboxylase 5.9820501.5 C.erectus↑NO.12gi|75817913Predicted oxidoreductases 6.7311799 A.thaliana↓NO.13gi|62734371Abscisic acid and stress-induced protein 6.215455.5O.sativa↑NO.14gi|548603Photosystem I reaction center subunit II9.8121919.4H.vulgare↑NO.15gi|51459711DnaK-like chaperone protein HscA 5.5665281.9Z.mays+ NO.16gi|6691487Oxygen-evolving complex of photosystem II8.6128121.2 C.sativus↑NO.17gi|50508582Rieske Fe-S precursor protein8.5423869.2O.sativa↑NO.18gi|35211352Unknown11.4243495.3G.violaceus↓NO.19gi|30408003NBS-type resistance protein9.7319422.6M.esculenta↑NO.20gi|67004037Unknown 5.48155305.5O.sativa↓Notes:Expression ratio was calculated relative to protein level in control sample.The“↑”indicated that the amount of protein expressed in treatment sample was greater than that in control sample;conversely,“↓”indicated that the protein expression was downregulated under S.scitamineum stress.The“+”indicated it was a newly induced protein after S.scitamineum stress.
701.4937
832.3627
997.5668
1147.6448
1260.68551300.75851395.6792
1503.80941581.95371613.9569
1745.0211
1854.0529
1994.03822086.136
2211.13652295.2114
2429.26072533.384
2641.5503
2786.4255
2908.59253016.6985
1277.7184
699
1261.61824.2
2386.8
2949.4
3512
100806040200I n t e n s i t y (%)
4700re?ector spec #1MC [BP =1277.7,21903]
2.2E +4
Mass (m/z)
(a)
86.071232.1133
170.0433
421.1749558.3256744.4099
1092.5829
74325.4
576.8828.2
1079.61331
4700MS/MS precursor 1260.69spec #1MC [BP =170,3694]
3694.1
Mass (m/z)
100806040200
I n t e n s i t y (%)
(b)
159.0822284.1822
324.1572
496.2647
690.4052
892.5305
1005.63911088.5425
1185.6764
1298.7719
86.0738
609.335
74393.2
712.41031.6
1350.81670
4700MS/MS precursor 1581.95spec #1MC [BP =86.1,2125]
2124.8
Mass (m/z)
100806040200
I n t e n s i t y (%)
(c)
86.0736
120.0547
219.1089328.1759
591.3149
747.4055790.4054
988.5594
1300.707
676.3725
74407.6
741.2
1074.81408.41742
4700MS/MS precursor 1649.93spec #1MC [BP =676.4,759]
759.2
Mass (m/z)
100806040200
I n t e n s i t y (%)
(d)
136.0116
394.1628648.1982
618.2538
1030.3943
1089.48651463.6921
1647.7247
2034.8832
1300.5916
74543.6
1013.21482.8
1952.42422
4700MS/MS precursor 2295.21spec #1MC [BP =1300.6,1941]
1941.4
Mass (m/z)
100806040200
I n t e n s i t y (%)
(e)
lgav g a i ape i l gk l g l ipp a l f vf emalmgf aeh
rrfq dwanpg smgkqyf lg l g eks l k d lk l kevkn
gr lamla i l g y f aqgpvtg v
e e g t k p a y l l l q p g n w g l
f s l k
g d t d
h g p l
v a a f y d p p p p g v a g n g p n t l n y f i n n i y p t
w l s l d f k p g f y k h t d n
161121(f)
Figure 4:Peptide mass ?ngerprinting (a),tandem mass spectra (b,c,d,e),and homologous amino acid sequence (f)of chlorophyll-a/b-binding protein.
701.4985
727.4735829.2804915.5117
1032.5233
1156.5748
1405.71111441.91191540.8815
1686.9198
1804.0122
1932.0215
2086.08742102.0864
2211.12332295.2112
2411.3162
2533.3596
2640.4463
2776.4946
2921.6877
3061.5303
3231.519
3378.574
1275.7759
1397.8446
4700re?ector spec #1MC [BP =1397.8,10651]
1.1E +4
6991261.6
1824.2
2386.8
2949.4
3512
100806040200I n t e n s i t y (%)
Mass (m/z)
(a)
86.0748
120.0613175.0975
342.2183
401.2712554.3119731.389981.5592
74354.4634.8
915.21195.61476
4700MS/MS precursor 1397.84spec #1MC [BP =86.1,3254]
3254.3
Mass (m/z)
100806040200
I n t e n s i t y (%)
(b)
100.9954
355.1365492.131
607.1684833.3389
1076.41871518.5939
1914.6393
74499.4
924.81350.2
1775.62201
4700MS/MS precursor 2085.09spec #1MC [BP =492.1,5882]
5882
Mass (m/z)
100
806040200
I n t e n s i t y (%)
(c)
86.0724
175.1066191.1096
276.1414
375.1886
488.2818
667.4345
841.4847
1008.7354
1279.5925
74363.6
653.2942.8
1232.41522
4700MS/MS precursor 1441.91spec #1MC [BP =1441.9,8899]
723.5
Mass (m/z)
100806040200
I n t e n s i t y (%)
(d)
100.9897
213.0063
492.1209
590.1424833.28251163.4347
1491.5685
1921.8459
74493.6913.2
1332.81752.42172
4700MS/MS precursor 2058.08spec #1MC [BP =492.1,4004]
4004.5
Mass (m/z)
100806040200
I n t e n s i t y (%)
(e)
1mk r l l f y t l i i a imv l sv i l s ayl t ppk v i pqysd k l r e lal f kg l k s t p n t yek l l e 61l v d d a s n pms a ski v lgekl f dvn l skn r qt scs t h sf drdlknrg am s kminspke q 121t nn c v a c h l r dqsg vdr f tf s gdnq eph p nl lntq i ln taf skhf tws ge v s s lr qq s 181e n s f l f l a y h km n i ta d e l v lr v ks e k yr am f s taf q d i t f eninka i ev yv k t lvt r g a 241y d r d gd ns a i s s d akrgl a f i g f g ck g chsdi s ggq s iqrf p l r df aq v ydlkpn f 301e l f p e f k r f d g ef p f ensgg f gknk eh l f rvpi l r v tk t spyf hng av pk i r eavnvm
361a k h q l g r h l t l eqi d e i vaf l i f q g n l r t l e gd i i e
ye i ren d c s d l n r
(f)
Figure 5:Peptide mass ?ngerprinting (a),tandem mass spectra (b,c,d,e),and homologous amino acid sequence (f)of cytochrome-c peroxidase protein.
from the requirement of protein mass spectra identi?cation, while application of protein database of related species under the same or similar stress(such as fungi)can improve the corresponding success rate[18].However,there are still three proteins in this study whose function has not been annotated.In recent years,with the increasing protein expression pro?le study of di?erent plants under various physiological and ecological conditions,though many new proteins that related with plant-microorganism interaction were dug out,there was little research aimed at studying the function and the model of these proteins.We found that only Lee et al.(2004)used proteomics technology and identi?ed one calcium-dependent membrane protein in salt response of root microsomes of A.thaliana,and through the application of reverse-genetic approach,they con?rmed that this protein could mediate osmosis stress and ABA signal transduction by calcium-dependent mode[19].
To our knowledge,this is the?rst report focused on highlighting the alterations of the protein expression pro?le in sugarcane exposed to S.scitamineum.Investigation on proteomic aspect of interaction between sugarcane and S.scitamineum has the ultimate aim of providing infor-mation that may be useful for the development of the e?ective sugarcane smut disease management system.In the present study,the proteomic pro?ling techniques,2-DE coupled with MALDI-TOF-TOF/MS,were used to analyze the di?erentially expressed proteins in sugarcane during sugarcane-S.scitamineum interaction.This approach enables direct qualitative and quantitative analysis of di?erentially expressed proteins during the period of disease develop-ment.In total,23di?erentially expressed proteins were obtained,among which20were successfully identi?ed in MALDI-TOF-TOF/MS analysis.According to mass spectra identi?cation and bioinformatics analysis,these proteins participated in several kinds of metabolism pathways,such as protein synthesis,signal transduction,and photosynthesis. In addition,there were also?ve proteins that played other functions in the interaction between sugarcane and S. scitamineum,and the function of the remaining three was not determined.These proteins can be divided into the following groups.
4.1.Protein-Synthesis-Related Proteins.HSP is a group of speci?c proteins of organism(or isolated culture cells)which can be induced by high salt,ABA stress,and heavy metal contamination and play a role of molecular chaperones[20]. Osmotins,whose expression is related to drought resistance, salt tolerance,and plant disease resistance,is a kind of newly synthesized or increased protein when plant faces osmosis stress[21].Besides,the accumulation of osmotins may be a kind of elementary immune reaction generated by plants for original immune response,and osmotins may even act as the dehydration storage protein which also possesses the antifungal activity[22].From above,the expression increase of two proteins,HSP(No.15)and osmotin(No.13), may protect cellular structure and play a role in the repair of cellular dysfunction during sugarcane-S.scitamineum interaction.4.2.Signal Transduction Proteins.Nucleotide-binding-site (NBS)type resistance proteins are the encoding prod-ucts of NBS disease-resistance genes in plants.They have kinase activity and play a similar role of transcription signal factor and activate the expression of downstream disease-resistance genes[23].They can also activate kinase or G protein,participate in protein phosphorylation,and magnify disease-resistance response signal,and then the plant generates hypersensitive response to pathogen,which plays an important role in disease-resistance response[24, 25].In this study,the generous expression of NBS protein termed No.19in high-smut-resistance sugarcane may just be the basis for its high-disease-resistance.On one hand,it may play a role of smut-resistance directly;on the other hand,it may also function as kinase,and activate the expression of downstream disease-resistance genes and thus the sugarcane smut-resistance.
4.3.Photosynthesis-Related Proteins.Photosynthesis is one of the most important physiologic processes of plant,which plays a decisive role in the growth rate of plant,especially for C4crop.Rubisco is a key enzyme in photosynthesis. It can regulate photosynthesis and light respiration and decide net photosynthesis[26].Previous studies showed that RubisCO not only had organ speci?city but could also be induced by many exogenous factors such as salicylic acid treatment and salt and drought stress[27].CAB gene is an important gene in plant photosynthetic system,whose encoding chlorophyll-a/b-binding protein can bind with pig-ment and form pigment protein complex that can catch light energy,transmit the energy to reaction center quickly,drive photochemical reaction,and thus play an important role in light protection and adaption to various environments[28]. Moreover,Rieske Fe-S precursor protein is an indispensable constituent of chlorophyll body photosynthesis transfer chain.The up-regulated expression of this protein may relate to accumulated H2O2in chlorophyll body and change the redox state of chlorophyll body,which may be a kind of response of Ya71-374to S.scitamineum stress at the protein level.When smut-susceptible sugarcane variety,Ya71-374, was infected with S.scitamineum,the leaves turned from green to black and the main stems and branch stems grew out in the form of dust-brand lash,which resulted in plant death.This indicated that the infection of S.scitamineum a?ected the normal function of plant chlorophyll.However, when high-smut-resistance sugarcane variety,NCo376,was infected with S.scitamineum,the leaf surface had no disease symptom,which indicated that its photosynthesis was not a?ected signi?cantly.In this study,the expression of nine photosynthesis-related proteins(No.2,No.3,No.6,No.7, No.9,No.11,No.14,No.16,and No.17)was upregulated, which most probably suggested that during the interaction between sugarcane and S.scitamineum,in order to defend the pathogen challenge,the expression of photosynthesis-related proteins,which was favorable for the maintenance and repair of the photosynthetic system,was upregulated.Further,the growth potential and thus the growth of sugarcane plants
were improved,and the health status of the plants was maintained,which in turn increased smut resistance.
4.4.Other Functional Proteins.They include?ve proteins
(No.1,NO.5,NO.8,NO.10,and NO.12).MAPs stands for
tubulin,nonspeci?c binding nucleic acid and the proteins
which play an assistant e?ect on microtubule function
during protein-protein interaction.He et al.(2006)found
that MAPs played an important role in the formation of
?ber primary wall[29].It was supposed that the newly
induced expression of MAPs in sugarcane(No.1)was helpful
for resisting the challenge of S.scitamineum.Cytochrome
c peroxidase is a key enzyme during the synthesis of
phytoalexin which has some inhibitory e?ect on disease
[30].In this study,cytochrome c peroxidase termed No.10
was newly induced after infection,and the author believed
that hydrogen-peroxide-redox-type cytochrome c reaction(2
cytochrome c(Fe2+)+H2O2+2H+→2cytochrome c (Fe3+)+2H2O)was catalyzed by the upregulated expression
of cytochrome c peroxidase,which improved the increasing
synthesis of phytoalexin and inhibited the growth of S.
scitamineum and thus reduced the harm of S.scitamineum.
The functions of the other three proteins(NO.5,No.8,and
No.12)still need to be con?rmed.
4.5.Function-Unknown Proteins.They include a total of
three proteins which termed as No.4,No.18,and No.20.
From all the above,it is presumed that there is a com-plicated protein regulatory network during the interaction of sugarcane-S.scitamineum.The regulatory network of these proteins could be just as follows:when challenged by S.scitamineum,sugarcane NBS-type proteins receive and transmit the stress signal,activate the expression of defense response proteins,and thus promote the synthesis of HSP and osmotins;the di?erential expression of photosynthesis related proteins accelerates the growth velocity of sugarcane, along with the expression of other functional resistance proteins.In all,a series of di?erentially expressed proteins form a closely associated regulatory network by the coupling of various endogenous signaling molecules and correlated metabolic pathways,which increase the resistance of sugar-cane to S.scitamineum.
5.Conclusions
The present study reports the di?erential protein expression in sugarcane in response to S.scitamineum infection revealed by2-DE and MALDI-TOF-TOF/MS.The results showed that there were signi?cant di?erences in protein2-DE atlas between resistant and susceptible variety,and also between the inoculated and the control sugarcane.In total,23 proteins,including11upregulated,nine downregulated, and three newly induced after infection,were identi?ed by MALDI-TOF-TOF/MS.The corresponding protein peptide mass?nger printing and tandem mass spectra of20out of these proteins were successfully obtained.Bioinformatics analysis revealed that the functions of these20di?erential proteins were related with photosynthesis,signal transduc-tion,disease resistance,and so on,while the function of the remaining three proteins was not determined.From above,it is assumed to be a complicated protein regulatory network during the interaction between sugarcane and S.scitamineum.This is the?rst proteomic investigation report focused on highlighting the alterations of the protein expression pro?le in sugarcane exposed to S.scitamineum. This study enriches the protein basis for sugarcane response to the infection of S.scitamineum and thus provides reference information for sugarcane response to S.scitamineum stress at the protein level,but the interrelation within each func-tional protein group and among functional protein groups still needs further study.Furthermore,time-consuming e?orts need to be made so that more di?erential proteins can be identi?ed and individual protein be investigated over the duration of the interaction,from initiation to termination. Acknowledgments
This research was funded by the earmarked fund for Mod-ern Agro-industry Technology Research System(CARS-20) and National High Technology Research and Development Program of China(863Program)Project(F2007AA100701). The authors appreciate all ideas and constructive criticism from the reviewers,especially Dr.Thomas L.Tew,who is a research geneticist in Sugarcane Research Unit,USDA-ARS, Houma,LA,USA.
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蛋白质组学研究方法选择及比较
蛋白质组学研究方法选择及比较 目前研究蛋白组学的主要方法有蛋白质芯片及质谱法，本文将从多方面对两种研究方法进行了解与比较； 蛋白质芯片（Protein Array） 将大量不同的蛋白质有序地排列、固定于固相载体表面，形成微阵列。利用蛋白质分子间特异性结合的原理，实现对生物蛋白质分子精准、快速、高通量的检测。 主要类型： ●夹心法芯片(Sandwich-based Array) ●标记法芯片(Label-based Array) ●定量芯片(Quantitative Array) ●半定量芯片(Semi-Quantitative Array) 质谱（Mass Spectrometry） 用电场和磁场将运动的离子按它们的质荷比分离后进行检测，测出离子准确质量并确定离子的化合物组成，即通过对样品离子质荷比的分析而实现对样品进行定性和定量的一种方法。 主要类型：
●二维电泳+质谱(2D/Mass Spectrometry, MS) ●表面增强激光解吸电离飞行时间质谱(Surface-enhanced laser desorption/ionization- time of flight, SELDI) ●同位素标记相对和绝对定量(Isobaric tags for relative and absolute quantitation, iTRAQ) Protein Array or Mass Spectrometry？ 如何选择合适的研究方法？以下将从六个方面进行比较与推荐： 1.筛查蛋白组学表达差异 建议选择：RayBiotech（1000个因子的芯片）+质谱 a)不同的方法学有不同的特点：对于质谱，可以筛查到未知的蛋白，但是对于分子量大、 低丰度的蛋白质，质谱的灵敏度和准确性有一定的限制。 b)不同的方法能筛查到的目标不同：根据Proteome Analysis of Human Aqueous Humor 一文中报道，质谱筛查到的差异蛋白集中在小分子与代谢物。而用RayBiotech芯片筛查到的结果，多是集中在细胞因子、趋化、血管、生长等等。 c)质谱筛查到355个蛋白，而RayBiotech抗体芯片也筛查到328个蛋白，且用定量芯片 验证25个蛋白有差异，这些蛋白是质谱找不到的。目前RayBiotech夹心法抗体芯片已经可以检测到1000个蛋白，采用双抗夹心法，尤其是对于低丰度蛋白，有很好的灵敏度和特异性，很多的低丰度蛋白是抗体芯片可以检测出来，而质谱检测不到的，且样品不经过变性和前处理，保持天然状态的样品直接检测，对于蛋白的检测准确度高。 d)质谱的重复性一直是质谱工作者纠结的问题，不同操作者的结果，不同样品处理条件， 峰值的偏移等影响因素都会产生大的影响；RayBiotech的夹心法芯片重复性高。
实验绿色荧光蛋白
生物技术实验报告 姓名：张龙龙 学号：2011506066 班级：11级生技02班
前言：绿色荧光蛋白（green fluorescent protein，GFP）是一类存在于包括水 母、水螅和珊瑚等腔肠动物体内的生物发光蛋白。当受到紫外或蓝光激发时，GFP 发射绿色荧光。它产生荧光无需底物或辅因子发色团是其蛋白质一级序列固有的。GFP 由3 个外显子组成，长2.6kb；GFP 是由238 个氨基酸所组成的单体蛋白,相对分子质量为27. 0kMr，其蛋白性质十分稳定，能耐受60℃处理。1996 年GFP 的晶体结构被解出，蛋白质中央是一个圆柱形水桶样结构，长420 nm，宽240 nm，由11 个围绕中心α螺旋的反平行β折叠组成，荧光基团的形成就是从这个螺旋开始的，桶的顶部由 3 个短的垂直片段覆盖，底部由一个短的垂直片段覆盖，对荧光活性很重要的生色团则位于大空腔内。发色团是由其蛋白质内部第65-67位的Ser-Tyr-Gly自身环化和氧化形成. 一.实验目的 1、了解表达用基因克隆引物设计的原理和方法。 2、了解利用原核表达系统表达外源基因的原理、流程及方法。 3、掌握PCR、DNA片段的酶切与连接、细菌转化、阳性克隆筛选、质粒提取、DNA样品的纯化、核酸电泳等分子生物学基本技术。 二．实验原理 基因工程一般包括四个步骤：一是取得符合人们要求的DNA片段，这种DNA片段被称为“目的基因”；二是将目的基因与质粒或病毒DNA连接成重组DNA；三是把重组DNA引入某种细胞；四是把目的基因能表达的受体细胞挑选出来。 本实验根据绿色荧光蛋白（GFP）的基因序列设计一对引物，用该引物将GFP基因从含GFP基因的质粒中扩增出来。再利用双酶切切开表达载体pET23b 和目的基因的两端接头，通过T4连接酶GFP基因与表达载体重组。将含GFP 基因的重组表达载体导入宿主菌BL21(DE3),在IPTG的诱导下，使GFP基因表达 三.实验材料及仪器 1、实验材料：含有GFP的质粒；DNA Marker；DH5α；BL21； 2、仪器：恒温培养箱、超净工作台、恒温摇床、制冰机、台式离心机、涡旋振荡器、冰箱、电泳仪、透射仪、PCR仪、PCR管、刀片、玻璃涂棒、酒精灯、无菌牙签、吸水纸、微型离心管、台式冷冻离心机、塑料手套、1.5ml离心管。 四.实验内容 4.1 质粒的提取、酶切及电泳鉴定： 1)实验试剂：LB培养基；溶液Ⅰ；Tris-HCl（pH=8）；溶液Ⅱ；溶液Ⅲ； 酚/氯仿抽提液；无水乙醇；电泳缓冲液；加样缓冲液；GoldView核酸 DNA 染色剂；1%的琼脂糖凝胶；XhoⅠ(10U/μl)；NdeⅠ（10U/μl）；T 4 lisase。 2)实验步骤： 质粒的提取与鉴定
基因组学与蛋白质组学
《基因组学与蛋白质组学》课程教学大纲 学时： 40 学分：2.5 理论学时： 40 实验学时：0 面向专业：生物科学、生物技 术课程代码：B7700005先开课程：生物化学、分子生物 学课程性质：必修/选修执笔人：朱新 产审定人： 第一部分：理论教学部分 一、课程的性质、目的和任务 《基因组学与蛋白质组学》是随着生物化学、分子生物学、结构生物学、晶体学和计算机技术等的迅猛发展而诞生的，是融合了生物信息学、计算机辅助设计等多学科而发展起来的新兴研究领域。是当今生命科学研究的热点与前沿领域。由于基因组学与蛋白质组学学科的边缘性，所以本课程在介绍基因组学与蛋白质组学基本基本技术和原理的同时，兼顾学科发展动向，讲授基因组与蛋白组学中的热点和最新进展，旨在使学生了解现代基因组学与蛋白质组学理论的新进展并为相关学科提供知识和技术。 二、课程的目的与教学要求 通过本课程的学习，使学生掌握基因组学与蛋白质组学的基本理论、基础知识、主要研究方法和技术以及生物信息学和现代生物技术在基因组学与蛋白质组学上的应用及典型研究实例，熟悉从事基因组学与蛋白质组学的重要方法和途
径。努力培养学生具有科学思维方式、启发学生科学思维能力和勇于探索，善于思考、分析问题的能力，激发学生的学习热情，并通过学习提高自学能力、独立思考能力以及科研实践能力，为将来从事蛋白质的研究奠定坚实的理论和实践基础。 三、教学内容与课时分配 第一篇基因组学
第一章绪论（1学时） 第一节基因组学的研究对象与任务； 第二节基因组学发展的历程； 第三节基因组学的分子基础； 第四节基因组学的应用前景。 本章重点： 1. 基因组学的概念及主要任务； 2. 基因组学的研究对象。 本章难点： 1.基因组学的应用及发展趋势； 2.基因组学与生物的遗传改良、人类健康及生物进化。建议教学方法：课堂讲授和讨论 思考题： 查阅有关资料，了解基因组学的应用发展。 第二章人类基因组计划（1学时） 第一节人类基因组计划的诞生； 第二节人类基因组研究的竞赛； 第三节人类基因组测序存在的缺口； 第四节人类基因组中的非编码成分； 第五节人类基因组的概观； 第六节人类基因组多样性计划。 本章重点： 1. 人类基因组的研究； 2. 人类基因组多样性。 本章难点： 人类基因组序列的诠释。 建议教学方法：课堂讲授和讨论 思考题：
基因组学和蛋白质组学对新药研发的影响
通过校园网进入数据库例如维普期刊数据库、CNKI、超星电子图书等。完成 A、任选一题，检索相关资料，截取检索过程图片，做成一个ppt文件（50分）。 B、写综述形式的学术论文(学术论文格式，字数不限，正文字体小四)，做成word文件（50分）。要求：按照自己的思路组织成文件，严禁抄袭。 写明班级学号，打印纸质版交给老师。 1、对检索课题“磷酸对草莓生长和开花的影响”检索中文信息。提示：磷酸的化学物质名称是“Phosphonic acid ”普通商业名称是“ethephon”， 2、基因组学和蛋白质组学对新药研发的影响 3、红霉素衍生物的设计、合成与抗菌活性研究 4、HPLC法测定复方谷氨酰胺肠溶胶囊中L-谷氨酰胺的释放度 姓名：朱艳红 班级： 11生科师范 学号： 11223074 学科教师：张来军
基因组学和蛋白质组学对新药研发的影响琼州学院生物科学与技术学院 11生科师范2班朱艳红 11223074 摘要 20世纪末伴随着人类基因组计划的实施，相继产生了基因组学和蛋白质组学，基因组学和蛋白质组学的迅速发展，对药学科学产生着深远的影响。文章在简介蛋白质组学基本概念、核心技术的基础上，综述了基因组学和蛋白质组学对新药研发带来的影响。 关键词：基因组学；蛋白质组学；药物研发 The impact of genomics and proteomics on the research and development of innovative drug abstract With the implementation of the 20th century,Genomics and proteomics had emerged one after the other. Driven by Soaring development of the omits，pharmaceutical industry presents a new vision，all human life faces a promising future. On the basis of proteomics Introduction to basic concepts, core technology, reviewed the genomics and proteomics research on the impact of new drugs. Keywords:Genomics; proteomics; drug development
蛋白质结构预测在线软件
蛋白质预测在线分析常用软件推荐 蛋白质预测分析网址集锦 物理性质预测： Compute PI/MW http://expaxy.hcuge.ch/ch2d/pi-tool.html Peptidemasshttp://expaxy.hcuge.ch/sprot/peptide-mass.html TGREASE ftp://https://www.wendangku.net/doc/bc8490920.html,/pub/fasta/ SAPS http://ulrec3.unil.ch/software/SAPS_form.html 基于组成的蛋白质识别预测 AACompIdent http://expaxy.hcuge.ch ... htmlAACompSim http://expaxy.hcuge.ch/ch2d/aacsim.html PROPSEARCH http://www.e mbl-heidelberg.de/prs.html 二级结构和折叠类预测 nnpredict https://www.wendangku.net/doc/bc8490920.html,/~nomi/nnpredict Predictprotein http://www.embl-heidel ... protein/SOPMA http://www.ibcp.fr/predict.html SSPRED http://www.embl-heidel ... prd_info.html 特殊结构或结构预测 COILS http://ulrec3.unil.ch/ ... ILS_form.html MacStripe https://www.wendangku.net/doc/bc8490920.html,/ ... acstripe.html 与核酸序列一样，蛋白质序列的检索往往是进行相关分析的第一步，由于数据库和网络技校术的发展，蛋白序列的检索是十分方便，将蛋白质序列数据库下载到本地检索和通过国际互联网进行检索均是可行的。 由NCBI检索蛋白质序列 可联网到：“http://www.ncbi.nlm.ni ... gi?db=protein”进行检索。 利用SRS系统从EMBL检索蛋白质序列 联网到：https://www.wendangku.net/doc/bc8490920.html,/”，可利用EMBL的SRS系统进行蛋白质序列的检索。 通过EMAIL进行序列检索 当网络不是很畅通时或并不急于得到较多数量的蛋白质序列时，可采用EMAIL方式进行序列检索。 蛋白质基本性质分析 蛋白质序列的基本性质分析是蛋白质序列分析的基本方面，一般包括蛋白质的氨基酸组成，分子质量，等电点，亲水性，和疏水性、信号肽，跨膜区及结构功能域的分析等到。蛋白质的很多功能特征可直接由分析其序列而获得。例如，疏水性图谱可通知来预测跨膜螺旋。同时，也有很多短片段被细胞用来将目的蛋白质向特定细胞器进行转移的靶标（其中最典型的
基因组学(结构基因组学和功能基因组学).
问:基因组学、转录组学、蛋白质组学、结构基因组学、功能基因组学、比较基因组学研究有哪些特点? 答:人类基因组计划完成后生物科学进入了人类后基因组时代,即大规模开展基因组生物学功能研究和应用研究的时代。在这个时代,生命科学的主要研究对象是功能基因组学,包括结构基因组研究和蛋白质组研究等。以功能基因组学为代表的后基因组时代主要为利用基因组学提供的信息。 基因组研究应该包括两方面的内容:以全基因组测序为目标的结构基因组学(struc tural genomics和以基因功能鉴定为目标的功能基因组学(functional genomics。结构基因组学代表基因组分析的早期阶段,以建立生物体高分辨率遗传、物理和转录图谱为主。功能基因组学代表基因分析的新阶段,是利用结构基因组学提供的信息系统地研究基因功能,它以高通量、大规模实验方法以及统计与计算机分析为特征。 功能基因组学(functional genomics又往往被称为后基因组学(postgenomics,它利用结构基因组所提供的信息和产物,发展和应用新的实验手段,通过在基因组或系统水平上全面分析基因的功能,使得生物学研究从对单一基因或蛋白质的研究转向多个基因或蛋白质同时进行系统的研究。这是在基因组静态的碱基序列弄清楚之后转入基因组动态的生物学功能学研究。研究内容包括基因功能发现、基因表达分析及突变检测。 基因的功能包括:生物学功能,如作为蛋白质激酶对特异蛋白质进行磷酸化修饰;细胞学功能,如参与细胞间和细胞内信号传递途径;发育上功能,如参与形态建成等采用的手段包括经典的减法杂交,差示筛选,cDNA代表差异分析以及mRNA差异显示等,但这些技术不能对基因进行全面系统的分析。新的技术应运而生,包括基因表达的系统分析,cDNA微阵列,DNA芯片等。鉴定基因功能最有效的方法是观察基因表达被阻断或增加后在细胞和整体水平所产生的表型变异,因此需要建立模式生物体。 功能基因组学
蛋白质结构预测和序列分析软件
蛋白质结构预测和序列分析软件蛋白质数据库及蛋白质序列分析 第一节、蛋白质数据库介绍 一、蛋白质一级数据库 1、 SWISS-PROT 数据库 SWISS-PROT和PIR是国际上二个主要的蛋白质序列数据 库，目前这二个数据库在EMBL和GenBank数据库上均建 立了镜像 (mirror) 站点。 SWISS-PROT数据库包括了从EMBL翻译而来的蛋白质序 列，这些序列经过检验和注释。该数据库主要由日内瓦大 学医学生物化学系和欧洲生物信息学研究所(EBI)合作维 护。SWISS-PROT的序列数量呈直线增长。 2、TrEMBL数据库： SWISS-PROT的数据存在一个滞后问题，即 进行注释需要时间。一大批含有开放阅读 了解决这一问题，TrEMBL(Translated E 白质数据库，它包括了所有EMBL库中的 质序列数据源，但这势必导致其注释质量 3、PIR数据库： PIR数据库的数据最初是由美国国家生物医学研究基金 会（National Biomedical Research Foundation, NBRF） 收集的蛋白质序列，主要翻译自GenBank的DNA序列。 1988年，美国的NBRF、日本的JIPID（the Japanese International Protein Sequence Database日本国家蛋 白质信息数据库）、德国的MIPS（Munich Information Centre for Protein Sequences摹尼黑蛋白质序列信息 中心）合作，共同收集和维护PIR数据库。PIR根据注释 程度(质量)分为4个等级。 4、 ExPASy数据库： 目前，瑞士生物信息学研究所（Swiss I 质分析专家系统（Expert protein anal 据库。 网址：https://www.wendangku.net/doc/bc8490920.html, 我国的北京大学生物信息中心(www.cbi.
荧光免疫技术—试题
第八章荧光免疫技术 一、A1 1、目前应用最广泛的荧光素为（）。 A、异硫氰酸荧光素FITC B、四乙基罗丹明RB200 C、四甲基异硫氰酸罗丹明 D、镧系螯合物 E、藻红蛋白R-BE 2、免疫荧光显微技术中，特异性最高，非特异性荧光染色因素最少的方法是（）。 A、直接法 B、间接法 C、补体结合法 D、双标记法 E、多标记法 3、作为荧光抗体标记的荧光素必须具备的条件中，可以提高观察效果的是（）。 A、必须具有化学上的活性基团能与蛋白稳定结合 B、性质稳定不会影响抗体的活性 C、荧光效率高，荧光与背景组织色泽对比鲜明 D、与蛋白质结合的方法简便快速 E、与蛋白质的结合物稳定 4、荧光抗体试验所没有的类型是（）。 A、直接法 B、间接法 C、补体结合法 D、间接抑制法 E、双标记法 5、要使荧光强度与荧光物质的浓度成正比，应使（）。 A、激发光必须很强 B、样品浓度应适中 C、待测物吸光系数必须很大 D、光源与检测器应与样品在同一线上 E、液槽厚度要足够厚 6、下列有关直接法荧光抗体染色技术的叙述，错误的是（）。 A、简单易行，特异性好 B、敏感性较间接法差 C、可对抗原或抗体作检测 D、检测一种抗原需要制备一种荧光抗体 E、结果直观，易于判断 7、荧光抗体染色技术中，只制备一种标记抗体，却可检测几乎所有的抗原抗体的方法是（）。 A、直接法 B、间接法 C、补体结合法 D、双标记法 E、混合法
8、荧光效率是指（）。 A、荧光色素将吸收的光能转变为荧光的百分率 B、荧光色素产生荧光的强度 C、接受激发光后，荧光物质所产生的荧光的色调 D、特异性荧光和非特异性荧光的强度比 E、物质产生荧光的效率 答案部分 一、A1 1、 【正确答案】A 2、 【正确答案】A 【答案解析】荧光抗体染色技术直接法：用将特异荧光抗体直接滴加于标本上，使之与抗原发生特异性结合，本法优点是操作简便，特异性高，非特异性荧光染色因素最少，缺点是敏感度偏低，每检查一种抗原需制备相应的特异荧光抗体。不可对抗原或抗体作检测。 3、 【正确答案】C 【答案解析】作为标记的荧光素应符合以下要求：①应具有能与蛋白质分子形成共价健的化学基团，与蛋白质结合后不易解离，而未结合的色素及其降解产物易于清除。②荧光效率高，与蛋白质结合后，仍能保持较高的荧光效率。③荧光色泽与背景组织的色泽对比鲜明。④与蛋白质结合后不影响蛋白质原有的生化与免疫性质。⑤标记方法简单、安全无毒。 ⑥与蛋白质的结合物稳定，易于保存。其中，②③与提高观察效果有关。 4、 【正确答案】D 【答案解析】荧光抗体试验的类型包括：直接法、间接法、补体结合法和双标记法。 5、 【正确答案】B 【答案解析】要使荧光强度与荧光物质的浓度成正比，应使样品的浓度适中。 6、 【正确答案】C 【答案解析】荧光抗体染色技术直接法是用特异荧光抗体直接滴加于标本上，使之与抗原发生特异性结合。本法的优点是操作简便，特异性高，结果直观，易于判断，非特异性荧光染色因素最少，缺点是敏感度偏低，每检查一种抗原需制备相应的特异荧光抗体。不可对抗体作检测。间接法可用于检测抗原和抗体。 7、 【正确答案】B 【答案解析】荧光抗体染色法中，间接法可用于检测抗原和抗体。本法有两种抗体相继作用，第一抗体为针对抗原的特异抗体，第二抗体（荧光抗体）为针对第一抗体的抗抗体。本法灵敏度高，而且只制备一种标记抗体，却可检测几乎所有的抗原抗体系统。 8、 【正确答案】A 【答案解析】荧光分子不能将全部吸收的光能都转变成荧光，总或多或少地以其他形式释放。
质谱技术在蛋白质组学研究中的应用_甄艳
第35卷 第1期2011年1月 南京林业大学学报(自然科学版) J o u r n a l o f N a n j i n g F o r e s t r y U n i v e r s i t y (N a t u r a l S c i e n c e E d i t i o n ) V o l .35,N o .1 J a n .,2011 h t t p ://w w w .n l d x b .c o m [d o i :10.3969/j .i s s n .1000-2006.2011.01.024] 收稿日期:2009-12-31 修回日期:2010-10-26 基金项目:国家自然科学基金项目(31000287);江苏省高校自然科学基础研究项目(10K J B 220002) 作者简介:甄艳(1976—),副教授,博士。＊施季森(通信作者),教授。E -m a i l :j s h i @n j f u .e d u .c n 。 引文格式:甄艳,施季森.质谱技术在蛋白质组学研究中的应用[J ].南京林业大学学报:自然科学版,2011,35(1):103-108. 质谱技术在蛋白质组学研究中的应用 甄 艳,施季森 ＊ (南京林业大学,林木遗传与生物技术省部共建教育部重点实验室,江苏 南京 210037) 摘要:随着蛋白质组学研究的迅速发展,质谱技术已成为应用于蛋白质组学研究中的强有力工具和核心技术。质谱技术的先进性在于为蛋白质组学研究提供的通量和分子信息。笔者重点概述了基于质谱路线的蛋白质组学研究,介绍了基于质谱的定量蛋白质组学﹑翻译后修饰蛋白质组学、定向蛋白质组学、功能蛋白质组学以及基于串联质谱技术的蛋白质组学数据解析的研究 进展。 关键词:质谱;蛋白质组学;定量蛋白质组学;翻译后修饰;定向蛋白质组学;功能蛋白质组学中图分类号:Q 81 文献标志码:A 文章编号:1000-2006(2011)01-0103-06 A p p l i c a t i o n o f m a s s s p e c t r o m e t r y i n p r o t e o m i c s s t u d i e s Z H E NY a n ,S H I J i s e n ＊ (K e y L a b o r a t o r y o f F o r e s t G e n e t i c s a n d B i o t e c h n o l o g y M i n i s t r y o f E d u c a t i o n , N a n j i n g F o r e s t r y U n i v e r s i t y ,N a n j i n g 210037,C h i n a ) A b s t r a c t :W i t ht h e r a p i d d e v e l o p m e n t o f p r o t e o m i c s ,m a s s s p e c t r o m e t r y i s m a t u r i n g t o b e a p o w e r f u l t o o l a n dc o r e t e c h -n o l o g y f o r p r o t e o m i c s s t u d i e s d u r i n g t h e r e c e n t y e a r s .T h e s u p e r i o r i t y o f m a s s s p e c t r o m e t r y l i e s i n p r o v i d i n g t h e t h r o u g h -p u t a n d t h e m o l e c u l a r i n f o r m a t i o n ,w h i c hn o o t h e r t e c h n o l o g y c a n b e m a t c h e di np r o t e o m i c s .I nt h i s r e v i e w ,w e m a d e a g l a n c e o n t h e o u t l i n e o f m a s s s p e c t r o m e t r y -b a s e d p r o t e o m i c s .A n dt h e nw e a d d r e s s e d o n t h e a d v a n c e s o f d a t a a n a l y s i s o f m a s s s p e c t r o m e t r y -b a s e dp r o t e o m i c s ,q u a n t i t a t i v em a s ss p e c t r o m e t r y -b a s e dp r o t e o m i c s ,p o s t -t r a n s l a t i o n a l m o d i f i c a t i o n s b a s e d m a s s s p e c t r o m e t r y ,t a r g e t e d p r o t e o m i c s a n df u n c t i o n a l p r o t e o m i c s b a s e d -m a s s s p e c t r o m e t r y . K e yw o r d s :m a s ss p e c t r o m e t r y ;p r o t e o m i c s ;q u a n t i t a t i v ep r o t e o m i c s ;p o s t -t r a n s l a t i o n m o d i f i c a t i o n ;t a r g e t e d p r o -t e o m i c s ;f u n c t i o n a l p r o t e o m i c s 蛋白质组学(P r o t e o m i c s )是从整体水平上研究细胞内蛋白质的组成、活动规律及蛋白质与蛋白质的相互作用,是功能基因组学时代一门新的学科。目前蛋白质组学的研究主要有两条路线:一是基于双向电泳的蛋白质组学;二是基于质谱的蛋白质组学,其中基于双向电泳的蛋白质组学研究路线最终也离不开质谱技术的应用。自20世纪80年代末,两种质谱软电离方式即电喷雾电离(e l e c t r o s p r a y i o n i z a t i o n ,E S I )和基质辅助激光解析离子化(m a -t r i x a s s i s t e d l a s e r d e s o r p t i o n i o n i z a t i o n ,M A L D I )的发明和发展解决了极性大、热不稳定蛋白质和多肽分 析的离子化和分子质量大的测定问题[1] ,蛋白质组学研究中常用的质谱分析仪包括离子阱(i o n t r a p ,I T ),飞行时间(t i m e o f f l i g h t ,T O F ),串联飞行时间(T O F -T O F ),四级杆/飞行时间(q u a d r u p o l e /T O F h y b r i d s ),离子阱/轨道阱(I T /o r b i t r a ph y b r i d ) 和离子阱/傅里叶变换串联质谱分析仪(I T /F o u r i e r t r a n s f o r m i o n c y c l o t r o nr e s o n a n c em a s s s p e c t r o m e t e r s h y b r i d s ,I T /F T M S ),这些质谱仪具有不同的灵敏度、分辨率、质量精确度和产生不同质量的M S /M S 谱[2] 。质谱作为蛋白质组学研究的一项强有力的工具日趋成熟,并作为样品制备及数据分析的信息学工具被广泛地应用。因此,有学者指出质谱技术 已在蛋白质组学研究中处于核心地位[3] 。目前在通量及所包含的分子信息内容上,基于质谱的蛋白质组学技术在细胞生物学研究中可以鉴定和量化
免疫荧光染色
荧光免疫染色和DAPI染色实验 1．实验原理 免疫染色的实验原理类似于Western Blotting，两者都是运用抗体的特异性识别作用来显示目的蛋白，但是由于免疫染色需要在原位进行，而且蛋白没有经过富集，因此其实验难度较高。 实验的基本原理是：利用固定剂（通常是甲醛或多聚甲醛）将细胞固定，使得细胞膜的通透性大大增加，并且利用Triton-X-100使得一部分膜蛋白变性，从而使通透性进一步加强。利用正常羊血清封闭，可以令许多蛋白先与血清内的非特异性抗体结合，而特异性的抗体由于动力学的关系可以通过竞争性的反应与目的蛋白结合，这一过程可以保证抗体识别的特异性。二抗可以特异性识别一抗的Fc区域，利用二抗连接不同的荧光基团，就可以在荧光显微镜下观察到不同的荧光，从而显示目的基因的表达情况。 另外，免疫荧光实验由于其较高的敏感性可以显示出基因表达的亚细胞情况(核内，核外，膜上以及一些较大的细胞器上)，所以通常被用来作为基因定位的方法。 DAPI的中文名称是4，6-联脒-2-苯基吲哚，是一种常用的荧光染料，其作用机理与溴化乙锭（EB）等染色剂的机理类似：它们与DNA双螺旋的凹槽部分可以发生相互作用，从而与DNA 的双链紧密结合。结合后产生的荧光基团的吸收峰是358nm而散射峰是461nm，正好UV （紫外光）的激发波长是356nm，使得DAPI成为了一种常用的荧光检测信号。 Jagielski M. et. Al在1976年首次运用该技术检测细胞培养中的支原体感染。后来随着技术的进步，该技术被运用于各种微生物的检测、生长监测，胚胎发育过程的检测，细胞周期的检测和各种核定位的实验。 本实验就是利用DAPI染色标记细胞核的位置。 免疫染色实验方法和步骤 免疫染色(immunol staining)包括免疫荧光(immunol fluorescence)、免疫组化(immunol histochemistry)、免疫细胞化学(immunol cytochemistry)等，可以参考如下步骤进行操作。 1. 样品准备(Sample preparation) 对于贴壁细胞： 可以直接用多孔板，例如6孔板、24孔板等，培养细胞，然后到预定时间时进行固定等后续操作。 也可以用洁净的盖玻片，70%乙醇中浸泡后，用无菌的镊子放置到6孔板内，然后用无菌的生理盐水、PBS或培养液洗去残留的乙醇。这时就可以种入细胞进行培养，待细胞贴在盖玻片上生长良好后，即可进行固定等后续操作。 对于悬浮细胞： 把细胞先在固定液中固定，然后把细胞滴加在载玻片上，干燥后细胞会紧贴在载玻片上。然后就可以进行后续操作。如果细胞的粘附能力不佳，可以在载玻片上用PDL等物质进行处理，以增强载玻片的粘附能力。 对于冷冻切片： 切片放置在载玻片上后，可以直接进行固定等后续操作。 对于石蜡切片：
蛋白质结构预测在线软件
蛋白质预测分析网址集锦? 物理性质预测：? Compute PI/MW?? ?? SAPS?? 基于组成的蛋白质识别预测? AACompIdent???PROPSEARCH?? 二级结构和折叠类预测? nnpredict?? Predictprotein??? SSPRED?? 特殊结构或结构预测? COILS?? MacStripe?? 与核酸序列一样，蛋白质序列的检索往往是进行相关分析的第一步，由于数据库和网络技校术的发展，蛋白序列的检索是十分方便，将蛋白质序列数据库下载到本地检索和通过国际互联网进行检索均是可行的。? 由NCBI检索蛋白质序列? 可联网到：“”进行检索。? 利用SRS系统从EMBL检索蛋白质序列? 联网到：”，可利用EMBL的SRS系统进行蛋白质序列的检索。? 通过EMAIL进行序列检索?
当网络不是很畅通时或并不急于得到较多数量的蛋白质序列时，可采用EMAIL方式进行序列检索。? 蛋白质基本性质分析? 蛋白质序列的基本性质分析是蛋白质序列分析的基本方面，一般包括蛋白质的氨基酸组成，分子质量，等电点，亲水性，和疏水性、信号肽，跨膜区及结构功能域的分析等到。蛋白质的很多功能特征可直接由分析其序列而获得。例如，疏水性图谱可通知来预测跨膜螺旋。同时，也有很多短片段被细胞用来将目的蛋白质向特定细胞器进行转移的靶标（其中最典型的例子是在羧基端含有KDEL序列特征的蛋白质将被引向内质网。WEB中有很多此类资源用于帮助预测蛋白质的功能。? 疏水性分析? 位于ExPASy的ProtScale程序（?）可被用来计算蛋白质的疏水性图谱。该网站充许用户计算蛋白质的50余种不同属性，并为每一种氨基酸输出相应的分值。输入的数据可为蛋白质序列或SWISSPROT数据库的序列接受号。需要调整的只是计算窗口的大小（n）该参数用于估计每种氨基酸残基的平均显示尺度。? 进行蛋白质的亲/疏水性分析时，也可用一些windows下的软件如，bioedit,dnamana等。? 跨膜区分析? 有多种预测跨膜螺旋的方法，最简单的是直接，观察以20个氨基酸为单位的疏水性氨基酸残基的分布区域，但同时还有多种更加复杂的、精确的算法能够预测跨膜螺旋的具体位置和它们的膜向性。这些技术主要是基于对已知
蛋白质结构预测方法综述
蛋白质结构预测方法综述 卜东波陈翔王志勇 《计算机不能做什么？》是一本好书，其中文版序言也堪称佳构。在这篇十余页的短文中，马希文教授总结了使用计算机解决实际问题的三步曲，即首先进行形式化，将领域相关的实际问题抽象转化成一个数学问题；然后分析问题的可计算性；最后进行算法设计，分析算法的时间和空间复杂度，寻找最优算法。 蛋白质空间结构预测是很有生物学意义的问题，迄今亦有很多的工作。有意思的是，其中一些典型工作恰恰是上述三步曲的绝好示例，本文即沿着这一路线作一总结，介绍于后。 1 背景知识 生物细胞种有许多蛋白质（由20余种氨基酸所形成的长链），这些大分子对于完成生物功能是至关重要的。蛋白质的空间结构往往决定了其功能，因此，如何揭示蛋白质的结构是非常重要的工作。 生物学界常常将蛋白质的结构分为4个层次：一级结构，也就是组成蛋白质的氨基酸序列；二级结构，即骨架原子间的相互作用形成的局部结构，比如alpha螺旋,beta片层和loop区等；三级结构，即二级结构在更大范围内的堆积形成的空间结构；四级结构主要描述不同亚基之间的相互作用。 经过多年努力，结构测定的实验方法得到了很好的发展，比较常用的有核磁共振和X光晶体衍射两种。然而由于实验测定比较耗时和昂贵，对于某些不易结晶的蛋白质来说不适用。相比之下，测定蛋白质氨基酸序列则比较容易。因此如果能够从一级序列推断出空间结构则是非常有意义的工作。这也就是下面的蛋白质折叠问题： 1蛋白质折叠问题（Protein Folding Problem） 输入: 蛋白质的氨基酸序列
输出: 蛋白质的空间结构 蛋白质结构预测的可行性是有坚实依据的。因为一般而言，蛋白质的空间结构是由其一级结构确定的。生化实验表明：如果在体外无任何其他物质存在的条件下，使得蛋白质去折叠，然后复性，蛋白质将立刻重新折叠回原来的空间结构，整个过程在不到1秒种内即可完成。因此有理由认为对于大部分蛋白质而言，其空间结构信息已经完全蕴涵于氨基酸序列中。从物理学的角度讲，系统的稳定状态通常是能量最小的状态，这也是蛋白质预测工作的理论基础。 2 蛋白质结构预测方法 蛋白质结构预测的方法可以分为三种： 同源性（Homology ）方法：这类方法的理论依据是如果两个蛋白质的序列比较相似，则其结构也有很大可能比较相似。有工作表明，如果序列相似性高于75％，则可以使用这种方法进行粗略的预测。这类方法的优点是准确度高，缺点是只能处理和模板库中蛋白质序列相似性较高的情况。 从头计算（Ab initio ） 方法：这类方法的依据是热力学理论，即求蛋白质能量最小的状态。生物学家和物理学家等认为从原理上讲这是影响蛋白质结构的本质因素。然而由于巨大的计算量，这种方法并不实用，目前只能计算几个氨基酸形成的结构。IBM 开发的Blue Gene 超级计算机，就是要解决这个问题。 穿线法(Threading )方法：由于Ab Initio 方法目前只有理论上的意义，Homology 方法受限于待求蛋白质必需和已知模板库中某个蛋白质有较高的序列相似性，对于其他大部分蛋白质来说，有必要寻求新的方法。Threading 就此应运而生。 以上三种方法中，Ab Initio 方法不依赖于已知结构，其余两种则需要已知结构的协助。通常将蛋白质序列和其真实三级结构组织成模板库，待预测三级结构的蛋白质序列，则称之为查询序列(query sequence)。 3 蛋白质结构预测的Threading 方法 Threading 方法有三个代表性的工作：Eisenburg 基于环境串的工作、Xu Ying 的Prospetor 和Xu Jinbo 、Li Ming 的RAPTOR 。 Threading 的方法：首先取出一条模版和查询序列作序列比对(Alignment)，并将模版蛋白质与查询序列匹配上的残基的空间坐标赋给查询序列上相应的残基。比对的过程是在我们设计的一个能量函数指导下进行的。根据比对结果和得到的查询序列的空间坐标，通过我们设计的能量函数，得到一个能量值。将这个操作应用到所有的模版上，取能量值最低的那条模版产生的查询序列的空间坐标为我们的预测结果。 需要指出的是，此处的能量函数却不再是热力学意义上的能量函数。它实质上是概率的负对数，即 ，我们用统计意义上的能量来代替真实的分子能量，这两者有大致相同的形式。 p E log ?=如果沿着马希文教授的观点看上述工作 ，则更有意思：Eisenburg 指出如果仅仅停留在简单地使用每个原子的空间坐标(x,y,z)来形式化表示蛋白质空间结构，则难以进一步深入研究。Eisenburg 创造性地使用环境串表示结构，从而将结构预测问题转化成序列串和环境串之间的比对问题；其后，Xu Ying 作了进一步发展，将蛋白质序列表示成一系列核（core ）组成的序列，Core 和Core 之间存在相互作用。因此结构就表示成Core 的空间坐标，以及Core 之间的相互作用。在这种表示方法的基础上，Xu Ying 开发了一种求最优匹配的动态规划算法，得到了很好的结果。但是由于其较高的复杂度，在Prospetor2上不得不作了一些简化；Xu Jinbo 和Li Ming 很漂亮地解决了这个问题，将求最优匹配的过程表示成一个整数规划问题，并且证明了一些常用
浅析功能基因组学和蛋白质组学的概念及应用
【摘要】基因组相对较稳定，而且各种细胞或生物体的基因组结构有许多基本相似的特征；蛋白质组是动态的，随内外界刺激而变化。对蛋白质组的研究可以使我们更容易接近对生命过程的认识。蛋白质组学是在细胞的整体蛋白质水平上进行研究、从蛋白质整体活动的角度来认识生命活动规律的一门新学科，简要介绍功能基因组学和蛋白质组学的科学背景、概念及其应用。 【关键词】基因组；功能基因组学；蛋白质组学； 一、基因组及基因组学的概念 基因组(genome)一词系由德国汉堡大学H．威克勒教授于1920年首创，用以表示真核生物从其亲代所继承的单套染色体，或称染色体组。更准确地说，基因组是指生物的整套染色体所含有的全部DNA序列。由于在真核细胞的线粒体和植物的叶绿体中也发现存在遗传物质，因此又将线粒体或叶绿体所携带的遗传物质称为线粒体基因组或叶绿体基因组。原核生物基因组则包括细胞内的染色体和质粒DNA。此外非独立生命形态的病毒颗粒也携带遗传物质，称为病毒基因组。所有生命都具有指令其生长与发育，维持其结构与功能所必需的遗传信息，本书中将生物所具有的携带遗传信息的遗传物质总和称为基因组。[1] 基因组学(genomic)一词系由T．罗德里克(T．Roderick)于1986年首创，用于概括涉及基因组作图、测序和整个基因组功能分析的遗传学学科分支，并已用来命名一个学术刊物Genomics。基因组学是伴随人类基因组计划的实施而形成的一个全新的生命科学领域。[1] 基因组学与传统遗传学其他学科的差别在于，基因组学是在全基因组范围研究基因的结构、组成、功能及其进化，因而涉及大范围高通量收集和分析有关基因组DNA的序列组成，染色体分子水平的结构特征，全基因组的基因数目、功能和分类，基因组水平的基因表达与调控以及不同物种之间基因组的进化关系。基因组学的研究方法、技术和路线有许多不同于传统遗传学的特点，各相关领域的研究仍处于迅速发展和不断完善的过程中。 基因组学的主要工具和方法包括：生物信息学，遗传分析，基因表达测量和基因功能鉴定。 二、功能基因组学的概念及应用
绿色萤光蛋白
绿色萤光蛋白(green fluorescent protein)，简称GFP，这种蛋白质最早在一种学名Aequorea victoria的水母中发现。其基因所产生的蛋白质，在蓝色波长范围的光线激发下，会发出绿色萤光。这个发光的过程中还需要冷光蛋白质Aequorin的帮助，且这个冷光蛋白质与钙离子(Ca+2)可产生交互作用。 由水母Aequorea victoria中发现的野生型绿色萤光蛋白，395nm和475nm分别是最大和次大的激发波长，它的发射波长的峰点是在509nm，在可见光绿光的范围下是较弱的位置。由海肾(sea pansy)所得的绿色萤光蛋白，仅有在498nm有一个较高的激发峰点。 在细胞生物学与分子生物学领域中，绿色萤光蛋白基因常被用作为一个报导基因(reporter gene)。一些经修饰过的型式可作为生物探针，绿色萤光蛋白基因也可以克隆到脊椎动物(例如:兔子上进行表现，并拿来映证某种假设的实验方法。 我们这边细胞组的基本上都在用这个东东。标记细胞 GFP的分子结构和发光机制 绿色荧光蛋白为一个由238个氨基酸残基组成的单链，GFP有两个吸收峰，主峰在395nm，次峰在470nm，其荧光发射峰在509nm。GFP 的化学性质相当稳定，其变性需要在90℃或pH<4或pH>12的条件下用6mollL盐酸胍处理，这一性质与GFP的结构特性相关。 Yang等的研究表明，GFP是由两个相当规则的内含一个α-螺旋和外面包围l1个β-折叠的β-桶状结构组成的二聚体，β-桶状结构直径约3nm，高约4nm。β折叠彼此紧密结合，象桶板一样形成桶状结构的外围，并且形成了一个规则的氢键带。桶状结构和位于其末端的短α螺旋以及环状结构一起组成一个单独的致密结构域，没有可供扩散的配体进入缝隙。这种坚实的结构保证了其稳定和抗热、抗变性的特点。 GFP的生色基团附着于α-螺旋上，几乎完美的包被于桶状结构中心。位于圆桶中央的α-螺旋含有一个由六肽组成的发光中心，而发光团是由其中的三肽Ser65-Tyr66-Gly67经过环化形成了对羟基苯咪唑啉酮。GFP的生色基团是蛋白质自身催化环化的结果，环化是一个有氧过程，在严格厌氧条件下GFP不能形成荧光，因为GFP的生色团形成需要O2使Tyr66脱氢氧化。生色基团通过Tyr66的脱质子(酚盐)和质子化状态(羟酚基)的转换决定荧光发射，此模型为Yang等的晶体学证据所支持。 GFP在生物技术中的应用研究 1．分子标记 作为一种新型的报告基因，GFP已在生物学的许多研究领域得到应用。利用绿色荧光蛋白独特的发光机制，可将GFP作为蛋白质标签(protein tagging)，即利用DNA重组技术，将目的基因与GFP基因构成融合基因，转染合适的细胞进行表达，然后借助荧光显微镜便可对标记的蛋白质进行细胞内活体观察。由于GFP相对较小，只有238个氨基酸，将其与其他蛋白融合后不影响自身的发光功能，利用GFP的这一特性已经加深了我们对细胞内一些过程的了解，如细胞分裂、染色体复制和分裂，发育和信号转导等。1996年，Ehrdardt等人首次报道了利用GFP的特性研究细胞分化蛋白FtsZ的定位。研究显示FtsZ在细胞分裂位点形成了一个环状物，且至少有9种蛋白在细胞分裂中起重要作用，尽管对这些蛋白功能仍然不是很清楚，但是利用GFP融合蛋白已经搞清楚了它们聚合的顺序以及在蛋白定位中的一些特征。利用GFP来检测目标蛋白的定位已为我们提供了一种对细胞内的一些基本的生理过程进行更详尽观察的新方法。 除用于特定蛋白的标记定位外，GFP亦大量用于各种细胞器的标记如细胞骨架、质膜、细胞核等等。Shi等人曾报道将GFP融合到大肠杆菌细胞膜表面用作标记蛋白，这一技术将有助于提高多肽库的筛选效率、疫苗的研制、构建细胞生物传感器用作环境检测以及探测信号转导过程等等。这些都为传统生物学研究提供了新思路和新方法，成为交叉学科研究的热点。 2．药物筛选 许多新发展的光学分析方法已经开始利用活体细胞来进行药物筛选，这一技术能从数量众多的化合物中快速筛选出我们所感兴趣的药物。基于细胞的荧光分析可分为三类：即根据荧光的密度变化、能量转移或荧光探针的分布来研究目标蛋白如受体、离子通道或酶的状态的变化。荧光探针分布是利用信号传导中信号分子的迁移功能，将一荧光蛋白与信号分子相偶联，根据荧光蛋白的分布情况即可推断信号分子的迁移状况，并推断该分子在迁移中的功能。由于GFP分子量小，在活细胞内可溶且对细胞毒性较小，因而常用作荧光探针。 在细胞体内分子之间的相互作用非常复杂，其中很多涉及到信号分子在细胞器之间的迁移。例如当信号分子和某一特殊受体结合后常会导致配体-受体复合物从某一细胞区域迁移到另一区域，而这一迁移过程通常会介导一重要的生理功能。因而，这些受体常常被用作药物筛选的目标，若某一药物具有与信号分子类似的功能，那么该药物即具有潜在的医药价值。利用GFP荧光探针，将很容易从数量众多的化合物中判断出那些化合物具有与信号分子相似的能引起配体一受体复合物迁移并介导生理反应的功能，且这一筛选过程简单方便，所需成本也很低。利用这一原理，已经成功构建了一个筛选模型用于研究药物介导的糖皮质激素受体(hGR)的迁移过程。在一96孔板中培养细胞，并以一编码hGR GFP蛋白的质粒转染该细胞。当细胞用待筛选的药物处理后，hGR-GFP从细胞质迁移人细胞核的过程可实时或在某一时段