CN102372781B - Insect antibacterial peptide thanatin (s) fusion protein and application thereof to plant disease resistance - Google Patents
Insect antibacterial peptide thanatin (s) fusion protein and application thereof to plant disease resistance Download PDFInfo
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Abstract
The invention discloses insect antibacterial peptide thanatin (s) fusion protein and an application thereof to the plant disease resistance. The insect antibacterial peptide thanatin (s) fusion protein provided by the invention is protein shown as 1) or 2): 1) protein which consists of amino acid sequences and is shown by sequences 2 in a sequence table; 2) protein which is derived from 1), is formed through substituting and/or deleting and/or adding amino acid sequences shown by the sequences 2 in the sequence table through one or a plurality of amino acid residues and is relevant to the plant disease resistance. Disease resistance detection results show that ChtSP-thanatin(s) transgenic plants have the obvious resistance on golovinomyces cichoracearum, erysiphe graminis, botrytis cinerea and pseudomonas syringae pv.tomato DC3000. The obvious effect of thanatin (s) on an aspect of controlling the plant fungus and bacterial disease is proved, and certain application prospects are realized.
Description
Technical field
The present invention relates to insect antimicrobial peptide Thanatin (S) fusion rotein and the application in plant disease-resistant thereof.
Background technology
Plant is faced with the threat of 20th-century disease pathogenic microorganism at any time in completing the process of its life history.Loss every year of the crop yield that Plant diseases causes that is caused by bacterium, fungi and virus in the world, is up to hundred million dollars of 300-500.Agro-chemicals control is still a Critical policies controlling corps diseases, but this strategy brings obvious threat for human health and environment protection.Traditional breeding way enantiopathy breed breeding has been made of paramount importance contribution, but the disease resistance that mediates due to the single disease-resistant gene of the disease-resistant gene finite sum of plant own has the height specialization, disease-resistant spectrum is narrower, can only resist limited microspecies, so that when pathogenic bacterium colony changes, just face the risk of resistant lose, thereby can't satisfy the needs of crop disease control.How with the affluent resources of current various biological genomes and gene manipulation techniques efficiently, systematically being applied to the improvement of cropper resistance, is focus and the focal issue of current transgenic engineering.What is more important, plant genetic engineering can provide more powerful means for obtaining new disease-resistant varieties not changing introducing disease resistance phenotype in the existing excellent genotypic situation of plant, becomes one of Critical policies of Agricul tural Sustain able Development.
Plant itself can produce multiple antibacterial peptide, but research is found the expression of the antibacterial peptide gene of plant origin and only pathogenic bacteria is produced moderate resistance, this is due to through long-term cause of disease and host's interaction and evolution, and phytopathogen has produced tolerance to these Antimicrobial Peptides From Plants.Therefore, the antibacterial peptide gene in non-plant source more and more comes into one's own in the application in plant disease-resistant field.
Insect antimicrobial peptide is to performance resistance of wide spectrum such as bacterium, fungi, viruses, and germ resistance is strong, antibacterial effect is fast.According to the Shai-Matsuzaki-Huanf model, the mechanism of action of most antibacterial peptides all is based on polypeptide and the phosphatide interaction causes membranolysis and causes cell injury.Therefore the non-enzyme because antibacterial peptide acts on phospholipid, seldom has cause of disease antagonism bacterium peptide to produce resistance.These characteristics make antibacterial peptide have tempting potentiality aspect the cultivation resistance crop.
Found that at present insect antimicrobial peptide can suppress very important plant pathogenic fungi and bacterium in many agriculture productions, as bacteriums such as the fungies such as Powdery Mildew, Pyricularia oryzae, Botrytis cinerea and Pseudomonas syringae, Erwinia Carotovora Pv. carotovora, rice leaf spot bacterias.By transgenic technology, antibacterial peptide is imported in plant and express, can make crop produce effective resistance to plant pathogenetic bacteria and fungi.In China, existing with antibacterial peptide gene successful expression and obtain the report of disease-resistant strain in the plants such as potato, tobacco, paddy rice, capsicum, tomato.Yet the insect antimicrobial peptide that is applied at present in transgenic plant is mainly the cecropin gene, and the also not test of the disease-resistant function of other most antibacterial peptides in transgenic plant.
Thanatin is the antibacterial peptide of the present minimum of finding, only contains 21 amino-acid residues, and it comes from hemipteran thorn shoulder stinkbug (Podisus maculiventris).Thanatin has the anti-microbial activity of wide spectrum, to fungi, G+ and G-all show anti-microbial activity (Fehlbaum P P, Bulet et al. (1994). " Insect immunity.Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. " J Biol Chem 269 (52): 33159-63.).Its aminoacid sequence is GSKKPVPIIYCNRRTGKCQRM, and first three amino acid is little to antibiotic activity influence, and then six amino acid (C holds hydrophobic region) form a ring, and are larger to antibiotic activity influence; Different bacterium had concentration effect.May act on the lipopolysaccharides of bacterium, form precipitation.Change T into S or removal, increased activity (Wu, G.Q., J.X.Ding, et al., 2009).There is no hemolytic, humans and animals is not had toxicity.At present, less what study aspect plant disease control for Thanatin.
Arabidopis thaliana (Arabidopsis thaliana) belongs to Cruciferae, and genome is very little, but its 2.5 ten thousand gene is roughly similar with other flowering plants on functional category; The Arabidopis thaliana life cycle is very short, only needs for 6～8 weeks from being seeded into the seed results; The Arabidopis thaliana individuality is less, is suitable for plantation in the laboratory; These characteristics make Arabidopis thaliana become a kind of desirable plant genetics and molecular biology research material.In addition, Arabidopis thaliana can be infected by multiple different plant pathogenetic bacteria, fungi and virus etc. under inoculation condition, and this is very beneficial for studying plant and pathogenic bacteria does mutually.Wheat is important food crop, it is carried out genetically engineered operate to improve its resistance against diseases and will play to the breeding for disease resistance of wheat larger pushing effect.
Summary of the invention
An object of the present invention is to provide a kind of insect antimicrobial peptide thanatin (S) fusion rotein.
Fusion rotein provided by the present invention, the name be called ChtSP-Thanatin (S), be following 1) or 2) protein:
1) protein that is formed by the aminoacid sequence shown in sequence in sequence table 2;
2) with the amino acid residue sequence of sequence in sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant to disease resistance of plant by 1) protein that derives.
Another object of the present invention is to provide the encoding gene of described fusion rotein.
The encoding gene of described fusion rotein provided by the present invention (called after ChtSP-Thanatin (S)) is following 1)-3) in arbitrary described gene:
1) its nucleotide sequence is the sequence 1 in sequence table;
2) under stringent condition with 1) gene recombination and the gene of encoding said fusion protein;
3) with 1) or 2) gene have homology more than 90% and the gene of encoding said fusion protein.
Above-mentioned stringent condition can be with 6 * SSC, the solution of 0.5%SDS, and hybridization, then use 2 * SSC under 65 ℃, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Sequence 1 in sequence table by 123 based compositions, is the encoding sequence of the signal peptide of paddy rice chitinase Cht-1 from 5 ' the 1st-60, end, and the 61st-123 is the encoding sequence of insect antimicrobial peptide thanatin (S).
The expression cassette, recombinant expression vector, transgenic cell line or the recombinant bacterium that contain described encoding gene also belong to protection scope of the present invention.
Described recombinant expression vector has inserted for the attR site at the pGWB11 carrier recombinant expression vector that described encoding gene obtains.
Described recombinant expression vector is to have inserted the recombinant expression vector that described encoding gene obtains between the SmaI of pAHC25 carrier and SacI restriction enzyme site.
Another purpose of the present invention is to provide a kind of method of cultivating transgenic plant.
The method of cultivation transgenic plant provided by the present invention is that described encoding gene is changed in the purpose plant, obtains comparing with described purpose plant the transgenic plant that the Anti-bacterium ability improves.
Described encoding gene imports in the purpose plant by weighing described recombinant expression vector.
Described purpose plant is monocotyledons or dicotyledons; Described dicotyledons is specially Arabidopis thaliana; Described monocotyledons is wheat.
Described germ is Arabidopis thaliana powdery mildew (Golovinomyces cichoracearum), botrytis cinerea (Botrytis cinerea), the pathogenic mutation (Pseudomonas syrmgae pv.tomato) of pseudomonas syringae tomato and/or wheat powdery mildew (Erysiphe grammis).
The signal peptide of paddy rice chitinase Cht-1 is connected to the N end of thanatin (S), and carry out codon optimized to this fusion structure, make it be conducive to high expression level in plant, subsequently, it is building up in the pGWB11 (the C end connects the FLAG label) and wheat conversion carrier pAHC25 of Gateway carrier series.The result demonstration that disease resistance detects, ChtSP-Thanatin (S) transgenic plant all have obvious resistance to Powdery Mildew (Golovinomyces cichoracearum and Erysiphe grammis), botrytis cinerea (Botrytis cinerea) and pseudomonas syringae (Pseudomonas syrmgae).Proof thanatin (S) effect aspect control plant epiphyte and Micobial Disease is remarkable, has certain application prospect.
The present invention adopts transgenic technology to change the fusion gene shown in sequence in sequence table 1 over to disease resistance that plant is improved plant, and this is that traditional breeding technology can't be accomplished.The present invention utilizes the outer gene of plant-sourced to improve the disease resistance of plant, for plant disease control provides a new way, will hold out broad prospects if it is dissolved into the crop breeding program.
Description of drawings
Fig. 1 is the expression analysis result of ChtSP-Thanatin (S) fusion gene in transgenic arabidopsis; Wherein, in Fig. 1, A is the part-structure schematic diagram of recombinant vectors pGWB11:ChtSP-Thanatin (S); In Fig. 1, B is for detecting the result of Thanatin (S) gene integration to the genome to 15 strains of transgenic arabidopsis plant T1 with PCR; In Fig. 1, C uses real-time PCR to detect the result of Thanatin (S) gene transcription level to 7 strains of transgenic arabidopsis plant T2; In Fig. 1, D is for selecting 3 higher strains of transcriptional level protein expression result of western blot technology for detection ChtSP-Thanatin (S)-FLAG.
Fig. 2 is that ChtSP-Thanatin (S) transgenic arabidopsis is to the detected result of powdery mildew resistance; Wherein, the phenotype result that in Fig. 2, A infects powdery mildew for inoculation rear 12 days transgenic arabidopsis line 1, line 6 and line 7 and wild-type Col-0; In Fig. 2, B is for inoculating the rear 6 days original monospore on Arabidopsis leaf; In Fig. 2, C is the spore count statistics that on Arabidopsis leaf, monospore produces.
Fig. 3 is that ChtSP-Thanatin (S) transgenic arabidopsis is to the resistance detected result of botrytis cinerea; Wherein, in Fig. 3, A is the phenotype of transgenic arabidopsis line 1, line 6 and line 7 and wild-type contrast Col-0, and in Fig. 3, B is the statistic analysis result of the lesion area of transgenic arabidopsis line 1, line 6 and line 7 and wild-type contrast Col-0.
The resistance detected result of Fig. 4 ChtSP-Thanatin (S) transgenic arabidopsis to the pathogenic mutation DC3000 of pseudomonas syringae tomato; Wherein, in Fig. 4, A is the phenotype of 2 days after the blade inoculation of transgenic arabidopsis line 1, line 6 and line 7 and wild-type contrast Col-0, and in Fig. 4, B is the measurement result of 2 days chemoluminescence number of photons after transgenic arabidopsis line 1, line 6 and line 7 and wild-type contrast Col-0 blade inoculation.
Fig. 5 is the part-structure schematic diagram of recombinant vectors p AHC25:ChtSP-Thanatin (S)-FLAG.
Fig. 6 turns the disease-resistant phenotype of wheat inoculation wheat powdery mildew (Erysiphe grammis) after 7 days of ChtSP-Thanatin (S)-FLAG fusion gene.
Embodiment
The experimental technique that uses in following embodiment is ordinary method if no special instructions.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The acquisition of embodiment one, transgenic arabidopsis and detection thereof
One, the expression analysis of ChtSP-Thanatin (S) fusion gene in transgenic arabidopsis
1, genetic transformation Vector construction
the Agrobacterium-mediated Transformation carrier that adopts is that (public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity for pGWB11 carrier in gateway series, the non-patent literature of putting down in writing this material is: Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka, K., Matsuoka, K., Jinbo, T.and Kimura, T. (2007) Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation.J.Biosci.Bioeng.104, 34-41.), this carrier carry can constitutive expression cauliflower mosaic virus promoter (CaM35S promoter), can the constitutive expression target gene.(public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity to transform Agrobacterium used and be the GV3101 bacterial strain, the non-patent literature of putting down in writing this material is: Van Larebeke N, Engler G, Holsters M, Van den Elsacker S, Zaenen I, Schilperoort RA, Schell J (1974) Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability.Nature 252:169-170.).
First with the online software of codon
Http:// www.jcat.de/With
Http:// miracle.igib.res.in/dynavac/The ChtSP-Thanatin that optimized (S) gene fusion sequence trust genescript company is synthetic, and this sequence is connected on pUC57 carrier (available from Nanjing Genscript Biotechnology Co., Ltd., catalog number is SD1176).Take pUC57:ChtSP-Thanatin (S) as template, with following primer process pcr amplification, upstream primer: 5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTGCTCTAGAGCCACCATGAGAGCGC TC-3 ' and downstream primer: 5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTCGGCGGCTGTACACATTCTTTGACATT TTC-3 ', amplified production checks order in the large genome company of China.Sequencing result shows, the nucleotide sequence of the fusion gene of acquisition is as shown in sequence in sequence table 1, and coding has the fusion rotein of the amino acid residue sequence shown in sequence 2 in sequence table, and in sequence table, sequence 2 is comprised of 41 amino-acid residues.In sequence table, sequence 1 is the encoding sequence of the signal peptide of paddy rice chitinase Cht-1 from 5 ' the 1st-60, end, and the 61st-123 is the encoding sequence of insect antimicrobial peptide thanatin (S).With this fusion gene called after ChtSP-Thanatin (S), with the fusion rotein called after ChtSP-Thanatin (S) of its coding.
The PCR product that order-checking is correct and pDONR207 carrier are (available from Invitrogen, catalog number is 12213013) and Gateway BP Clonase enzyme mixture (available from Invitrogen, catalog number is 11789-021) jointly hatch in 25 ℃, obtain recombinant vectors pDONR207:ChtSP-Thanatin (S).With this recombinant vectors transformed competence colibacillus intestinal bacteria DH10B (available from Beijing hundred Tyke Bioisystech Co., Ltd, catalog number is DP77), card is received the mycin plate screening and is obtained positive colony, expands numerous, the extraction plasmid.Again with pDONR207:ChtSP-Thanatin (S), pGWB 11 and Gateway LR Clonase enzyme mixture (available from Invitrogen, catalog number is 11791-043) jointly hatch in 25 ℃, obtain recombinant expression vector pGWB11:ChtSP-Thanatin (S).This recombinant expression vector is transformed intestinal bacteria DH10B (available from Beijing hundred Tyke Bioisystech Co., Ltd, catalog number is DP77), and Totomycin plate screening positive colony expands numerously, extracts plasmid and also send the large genome company of China to check order.Sequencing result shows, inserted the encoding gene shown in sequence 1 in the sequence table in the attR site of pGWB11 carrier, proves construction of recombinant plasmid correct (A in Fig. 1).
PGWB11:ChtSP-Thanatin (S) is transformed Agrobacterium GV3101 competence, and the Totomycin plate screening is also identified (primer is with above described) positive colony by bacterium colony PCR reaction, expands numerously, is used for conversion wild-type Arabidopis thaliana Col-0.
2, genetic transformation and T1 are for the analysis of transformed plant
The environmental Col-0 (available from the Biological resources center (ABRC) of Ohio State Univ-Columbus USA, catalog number is CS39005) of pGWB11:ChtSP-Thanatin (S) arabidopsis thaliana transformation that adopts the inflorescence infusion method that GV3101 is carried.
Inflorescence infusion method concrete steps are: with Agrobacterium suspension (0.5% sucrose of inflorescence in OD600=0.6～0.8,0.02 soaked about 30 seconds～0.05%Sliwet L-77), plant after conversion was placed 16-24 hour with the moisturizing of black plastic bag lucifuge, then between being the plant-growth of 22 ℃, 9 hours illumination/15 hour dark, temperature cultivate, until seed maturity.The planting seed of results is on the flat board that contains Totomycin, and screening obtains positive transformant.
Simultaneously, obtain turning empty carrier pGWB 11 contrast Arabidopis thaliana plant with aforesaid method.If unconverted Arabidopis thaliana Col-0 is wild-type contrast Arabidopis thaliana plant.
The genomic dna that the pGWB11:ChtSP-Thanatin (S) that obtains is transformed 15 strains (Line1-15) of Arabidopis thaliana T1 in generation that obtain carries out the PCR detection of Thanatin (S).PCR detects primer sequence used: 5 '-ATCTAAAAAACCTGTTC-3 ' and 5 '-CATTCTTTGACATTTTCCAG-3 '.
Result: in the independent transformed plant of detected pGWB11:ChtSP-Thanatin (S), except strain 5 (L5), all the other 14 strains all can amplify the purpose band; Wild-type contrast Arabidopis thaliana Col-0 plant does not amplify purpose band (B in Fig. 1).
For understanding Thanatin (S) transcriptional expression whether in transgenic arabidopsis, Real-time PCR has been carried out in 7 strains in the independent transformed plant T2 of PGWB11:ChtSP-Thanatin (S) detected, primer sequence used is as follows: 5 '-ATCTAAAAAACCTGTTC-3 ' and 5 '-CATTCTTTGACATTTTCCAG-3 '.
Result shows goal gene equal energy transcriptional expression in transgenic plant L1-L8, and can not transcriptional expression (C in Fig. 1) in wild-type contrast Arabidopis thaliana plant Col-0.
In order further to study the accurate translation situation of ChtSP-Thanatin (S) fusion rotein in transgenic arabidopsis, the higher strain 1 (L1) of transcriptional level, strain 6 (L6) and strain 7 (L7) have been carried out western blot to be detected, antibody is that mouse-anti FLAG antibody is (available from sigma company, F1804).
Result demonstration, ChtSP-Thanatin (S) fusion rotein be equal energy accurate translation in transgenic arabidopsis L1, L6 and L7, and can not accurate translation (D in Fig. 1) in wild-type contrast Arabidopis thaliana plant Col-0.
Two, the Analysis of Resistance of ChtSP-Thanatin (S) transgenic arabidopsis to powdery mildew
the strain 1 (line 1) higher to ChtSP-Thanatin (S) fusion rotein accurate translation in transgenic arabidopsis), (public can obtain with developmental biology institute from the Chinese Academy of Sciences's heredity strain 6 (line 6) and strain 7 (line 7) inoculation Arabidopis thaliana powdery mildew (Golovinomyces cichoracearum), the non-patent literature of putting down in writing this material is: Yiping Wang, Marc T.Nishimura, Ting Zhao, Dingzhong Tang.2011.ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis.The Plant Journal, 68, 10:74-87).Inoculation method is: with blower, the powdery mildew spore is blown in the encloses container of high 1 meter left and right, standing more than 1 hour, spore can evenly become scattered about on the interior Arabidopsis leaf of encloses container.Inoculate rear 6 days and get blade trypan blue dyeing (B in Fig. 2, red arrow refers to original monospore), examine under a microscope the spore count that monospore produces, count and carry out statistical study, the results are shown in Figure C in 2, result shows that ChtSP-Thanatin (S) transgenic arabidopsis line 1, line 6 and line 7 compare powdery mildew with wild-type Col-0 and have significant resistance.Inoculate rear 12 days, take the phenotype photo that Col-0, line 1, line 6 and line 7 infect powdery mildew, see A in Fig. 2, as seen from the figure, ChtSP-Thanatin (S) transgenic arabidopsis line 1, line 6 and line 7 have obvious resistance than wild-type contrast Col-0.Turn the empty carrier adjoining tree consistent with the phenotype of wild-type adjoining tree.
Three, the Analysis of Resistance of ChtSP-Thanatin (S) transgenic arabidopsis to botrytis cinerea
the strain 1 (line1) higher to ChtSP-Thanatin (S) fusion rotein accurate translation in transgenic arabidopsis, (public can obtain with developmental biology institute from the Chinese Academy of Sciences's heredity strain 6 (line6) and strain 7 (line7) inoculation botrytis cinerea (Botrytis cinerea), the non-patent literature of putting down in writing this material is: Yiping Wang, Marc T.Nishimura, Ting Zhao, Dingzhong Tang.2011.ATG2, an autophagy-related protein, negaively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis.The Plant Journal, 68, (10): 74-87).Inoculation method is: getting blade and be tiled on 0.8% agar plate, is 5 * 10 with concentration
5The botrytis cinerea spore suspension 10 μ L of spores/ml drop to blade central authorities.22 ℃ of moisturizings were observed phenotype after 3 days, took pictures and measured the scab diameter and carry out statistical study.The results are shown in Figure 3, in Fig. 3, A is the leaf spot lesion phenotype of transgenic arabidopsis line 1, line 6 and line 7 and wild-type contrast Col-0, and in Fig. 3, B is the statistic analysis result of the scab diameter of transgenic arabidopsis line 1, line 6 and line 7 and wild-type contrast Col-0.Result shows that three strain line 1, the line 6 of Thanatin (S) transgenic arabidopsis and line 7 have significant resistance than wild-type contrast Col-0 to botrytis cinerea.Turn the empty carrier adjoining tree consistent with the phenotype of wild-type adjoining tree.
Four, the Analysis of Resistance of ChtSP-Thanatin (S) transgenic arabidopsis to the pathogenic mutation of pseudomonas syringae tomato
bacterial strain uses therefor is to cause a disease with the pseudomonas syringae tomato of chemoluminescence reporter gene that (public can obtain with developmental biology institute from the Chinese Academy of Sciences's heredity mutation bacterial strain DC3000 (luxCDABE-tagged Pseudomonas syrmgae pv.tomato DC3000), the non-patent literature of putting down in writing this material is: Jun Fan, Casey Crooks, Chris Lamb.2008.High-throughput quantitative luminescence assay of the growth in planta of Pseudomonas syrmgae chromosomally tagged with Photorhabdus luminescens luxCDABE.The Plant Journal, 53, (2): 393-399, January 2008).Be bacteria suspension injection inoculation Thanatin (S) the transgenic arabidopsis strain of OD600=0.002 and the blade of wild-type with the 1ml needleless injector with concentration, get blade after 2 days, take pictures and measure the bacterium amount.The measuring method of bacterium amount is as follows: be placed in the 1.5ml centrifuge tube with the punch tool blade that to get 3 pieces of areas be 0.6cm2, add 200ul 10mM MgCl
2Solution grinds to form uniform suspension with blade, directly put into FB12 luminometer (Berthold Detection Systems,
Http:// www.berthold-ds.com/) the mensuration number of photons.result as shown in Figure 4, in Fig. 4, A is transgenic arabidopsis line 1, the phenotype of 2 days after the blade inoculation of line 6 and line 7 and wild-type contrast Col-0, in Fig. 4, B is transgenic arabidopsis line 1, the mensuration (asking the calculation formula of the fluorescence volume shown in the B ordinate zou in explanatory view 4) of 2 days chemoluminescence number of photons after line 6 and line 7 and wild-type contrast Col-0 blade inoculation, result shows, three strain line 1 of ChtSP-Thanatin (S) transgenic arabidopsis, line 6 and line 7 have obvious resistance than wild-type contrast Col-0 to the pathogenic mutation of pseudomonas syringae tomato.Turn the empty carrier adjoining tree consistent with the phenotype of wild-type adjoining tree.
The acquisition of embodiment 2, Thanatin (S) transgenic wheat and to the Analysis of Resistance of wheat powdery mildew
One, genetic transformation Vector construction
The carrier that transformed wheat adopts is that (public can obtain with developmental biology institute from the Chinese Academy of Sciences's heredity pAHC25, the non-patent literature of putting down in writing this material is: Christensen AH, Quail PH.1996.Ubiquitine promoter-based vectors for highlevel expression of selectable and/or screenable marker genes in monocotyledonous plants.Transgenic Research 5:213-218.).At first adopt Restriction enzyme Sma I and SacI double digestion, the GUS sequence in pAHC25 is removed, reclaim the carrier large fragment; Take pGWB11:ChtSP-Thanatin (S) as template, pcr amplification is ChtSP-Thanatin (S)-FLAG fusion gene sequence, amplified production is also used SmaI and SacI double digestion, and the pcr amplification the primer is: 5 '-TATCCCCCGGGAACAAGTTTG-3 ' and 5 '-TACGAGCTCCTAAGCCTTGTC-3 '.Then the PCR product after enzyme being cut and the carrier large fragment of recovery links with the T4 ligase enzyme, i.e. acquisition contains the recombinant conversion carrier pAHC25:ChtSP-Thanatin (S) of goal gene-FLAG.This recombinant vectors is transformed intestinal bacteria DH10B (available from Beijing hundred Tyke Bioisystech Co., Ltd, catalog number is DP77), and ammonia benzyl plate screening positive colony expands numerously, extracts plasmid and also send the large genome company of China to check order.Sequencing result shows, inserted the encoding gene (A in Fig. 5) shown in sequence 1 in the correct sequence table of sequence between the SmaI of pAHC25 carrier and SacI restriction enzyme site.
(state examines wheat 2006017 with Bombardment-Mediated Transformation wheat breed agriculture 199 (KN199) of section with carrier pAHC25:ChtSP-Thanatin (S)-FLAG, the stock breeding stage) (public can obtain with developmental biology institute from Chinese Academy of Sciences heredity, the non-patent literature of putting down in writing this material is: Li Junming, Zhang Xiangqi, Zhang Aimin, Wang Zhiguo, peace was transferred, discipline army, Wang Jing, 2007, high yield is suitable new variety of wheat--section's agriculture 199. wheat crops journals extensively, 27 (2): 368), 14 transgenic lines of final acquisition, PCR method detects proof 8 strains positive (B in Fig. 5), primer sequence used is: 5 '-ATCTAAAAAACCTGTTC-3 ' and 5 '-CATTCTTTGACATTTTCCAG-3 '.
Simultaneously, obtain turning empty carrier pAHC25 contrast wheat plant with aforesaid method.If the agriculture 199 of unconverted wheat section is wild-type contrast wheat plant.
Two, the Analysis of Resistance of ChtSP-Thanatin (S) transgenic wheat T0 to wheat powdery mildew
The transgenic wheat seedling of 5 strains (L5, L8, L10, L11, L13) in T0 generation and contrast KN199 are adopted the method with wheat leaves in vitro inoculation wheat powdery mildew (Erysiphe grammis), and (public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity, the non-patent literature of putting down in writing this material is: ten thousand is flat, make sharp army, Zhou Wenjuan, Zhang Wenjun, Ling Hongqing, Zhu Lihuang, Zhang Xiangqi.2004. the clone of wheat zinc finger protein gene, sequence and expression analysis.Acta Genetica Sinica, Vol 31, (9): 895-900).At first preparation contains the 0.5% wide part of agar plate excision middle part 3cm of 50mg/L benzoglyoxaline, the blade to be inoculated of clip 4cm left and right, the agar tangent plane is inserted at two ends, the powdery mildew spore is evenly shaken off to blade, cultivate in 17 ℃, 85% relative humidity, 2000lx intensity of illumination, after 7 days, observe disease-resistant phenotype and take pictures.The blade that 5 strains (L5, L8, L10, L11, L13) arranged is significantly higher than contrast KN199 to the resistance of wheat powdery mildew, and result as shown in Figure 6.Turn the empty carrier adjoining tree consistent with the phenotype of contrast KN199 plant.
Claims (11)
1. fusion rotein, the protein that is formed by the aminoacid sequence shown in sequence in sequence table 2.
2. the encoding gene of the described albumen of claim 1.
3. encoding gene according to claim 2, it is characterized in that: the nucleotide sequence of described encoding gene is the sequence 1 in sequence table.
4. the expression cassette that contains claim 2 or 3 described encoding genes.
5. the recombinant bacterium that contains claim 2 or 3 described encoding genes.
6. the transgenic cell line that contains claim 2 or 3 described encoding genes.
7. the recombinant expression vector that contains claim 2 or 3 described encoding genes.
8. recombinant expression vector according to claim 7 is characterized in that: described recombinant expression vector is for having inserted in the att of pGWB11 carrier R site the recombinant expression vector that the described encoding gene of claim 2 or 3 obtains.
9. recombinant expression vector according to claim 7 is characterized in that: described recombinant expression vector is for having inserted the recombinant expression vector that the described encoding gene of claim 2 or 3 obtains between the SmaI of pAHC25 carrier and SacI restriction enzyme site.
10. a method of cultivating transgenic plant, be that the described encoding gene of claim 2 or 3 is changed in the purpose plant, obtains comparing with described purpose plant the transgenic plant that the Anti-bacterium ability improves; Described purpose plant is Arabidopis thaliana or wheat; Described Anti-bacterium ability is the resistibility to Arabidopis thaliana powdery mildew (Golovinomyces cichoracearum), botrytis cinerea (Botrytis cinerea), the pathogenic mutation (Pseudomonas syringae pv.tomato) of pseudomonas syringae tomato and/or wheat powdery mildew (Erysiphe graminis).
11. method according to claim 10 is characterized in that: the described encoding gene of claim 2 or 3 imports in the purpose plant by arbitrary described recombinant expression vector in claim 7-9.
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| Title |
|---|
| Solution structure of thanatin, a potent bactericidal and fungicidal insect peptide, determined from proton two-dimensional nuclear magnetic resonance data.;Mandard N.et al.,;《Eur J Biochem.》;19981231;第256卷(第2期);404-410 * |
| structure-activity analysis of thanatin,a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides;PASCALE FEHLBAUM et al.,;《PNAS》;19961231;第93卷;1221-1225 * |
| UniProtKB/TrEMBL.UniProtKB/TrEMBL Accession Number Q42993.《Signal Peptide Database- Bacteria》.2008,全文. * |
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