CN101200727B - Farm crop fungus resistant polypeptides and method for making same - Google Patents
Farm crop fungus resistant polypeptides and method for making same Download PDFInfo
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- CN101200727B CN101200727B CN 200710050926 CN200710050926A CN101200727B CN 101200727 B CN101200727 B CN 101200727B CN 200710050926 CN200710050926 CN 200710050926 CN 200710050926 A CN200710050926 A CN 200710050926A CN 101200727 B CN101200727 B CN 101200727B
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Abstract
The present invention discloses a novel anti-crop-epiphyte polypeptide and a preparation method thereof. A recombined anti-crop-epiphyte polypeptide is formed by linking gene which codes white candida pheromone and the gene which codes colicin. Compared with the prior anti-crop-epiphyte medicine, the novel anti-crop-epiphyte polypeptide of the present invention has the advantages that the novel anti-crop-epiphyte polypeptide directly forms ionic passage at epiphytic cell membrane to kill the epiphyte, so the killing efficiency is thousands times stronger than the prior anti-crop-epiphyte medicine; the epiphyte can hardly amend the geometrical damage at the cell membrane caused by the polypeptide by the mutation-expression form, so the polypeptide can not induce the epiphyte producing the traditional drug resistance; the polypeptide is a biological preparation and has target killing towards the epiphyte, so the novel anti-crop-epiphyte polypeptide does not have the toxic and side effects of the prior anti-crop-epiphyte medicine.
Description
Technical field
The present invention relates to gene, recombinant plasmid, polypeptide of a kind of farm crop fungus resistant polypeptides of restructuring and preparation method thereof.
Background technology
Fungi infestation is the common harm of farm crop, causes huge financial loss for the rice production of China such as the fungi Magnaporthe grisea that causes rice blast is annual, and hazardness is very big.Along with the vast agricultural district of China accelerates urbanization, ecological environmental pollution and deterioration increasingly sharpen, and corps diseases sickness rate straight line rises, and the bionomic control of corps diseases has become the significant problem that we must face and solve.
Now use antibiotic agricultural chemicals undesirable to farm crop fungal infection curative effect; The fungi eukaryotic cell, the similarity of it and human body cell makes agricultural antifungal drug such as tricyclazole class also kill and wound human body cell when killing and wounding the fungal cell.The chemicalses such as life-time service ring azole lure that fungi has produced resistance to it into, and the peasant infects and often do not stint repeatedly repeated drug taking of overdose for suppressing farm crop fungus, and this just causes the farm crop fungus resistance further to increase the weight of; Simultaneously also cause the residual quantity of agricultural chemicals in environment and agricultural-food increasing, so the mankind are being devoted to develop efficient and free of contamination fungicide always.
Fungi fundamental research in recent years obtains very large progress, determines the aminoacid sequence of some fungi such as Candida albicans pheromone, is the polypeptide that 14 amino-acid residues form.It can freely move about in life medium, has the biologic activity of corresponding acceptor on Automatic-searching fungal cell's after birth of the same race.Therefore can utilize this Automatic-searching activity of fungi pheromone to induce this bacterioid extracellular toxin of colicin to arrive to form ionic channel on the cytolemma of these fungies and kill and wound these fungies, thereby construct a class novel biopesticide.
Summary of the invention
An object of the present invention is to provide gene, recombinant plasmid, the polypeptide of novel restructuring farm crop fungus resistant polypeptides, this peptide species can be special kills the farm crop fungus cell and can not injure the human normal cell, and prevention effect greatly is better than and now uses agricultural chemicals.A further object of the present invention is to provide the preparation method of above-mentioned restructuring farm crop fungus resistant polypeptides.
Technical scheme of the present invention is as follows:
The gene of farm crop fungus resistant polypeptides of the present invention contains the gene of the colicin of encoding and the gene of coding Candida albicans pheromone.The gene of coding Candida albicans pheromone is operably connected with the colicin gene, thereby the nucleotide sequence of restructuring farm crop fungus resistant polypeptides is expressed in acquisition.The gene of coding Candida albicans pheromone is the described nucleotide sequence of SEQ ID NO.2 in sequence table.The optional self energy of colicin forms colicin E1 or Ia or Ib or A or B or the N of ionic channel.In a preferred embodiment of the invention, above-mentioned Candida albicans pheromone gene is formed as the described nucleotide sequence of SEQ ID NO.4 in sequence table because being connected with the carboxyl end groups of colicin Ia (SEQ ID NO.1).In another embodiment of the present invention, the aminoterminal gene of above-mentioned Candida albicans pheromone gene with colicin Ia (SEQ ID NO.1) is connected and forms as the described nucleotide sequence of SEQ IDNO.6 in sequence table.
Recombinant plasmid of the present invention is that carboxyl terminal or aminoterminal that the nucleotide sequence of coding Candida albicans pheromone as above inserts colicin through double-stranded oligonucleotide point mutation technology are formed recombinant plasmid of the present invention.If select colicin Ia, the nucleotide sequence of the Candida albicans pheromone of encoding inserts after the 626th amino acids of Ia gene before (carboxyl terminal) or the 1st amino acids (aminoterminal) and forms all recombinant plasmids of the present invention.The original commercial plasmid pET-15b of construction recombination plasmid comes from Novagen company, has wherein loaded colicin Ia and corresponding Immunity protein gene, for Candida albicans pheromone gene design several to primer, its sequence is seen embodiment 1.Utilize double-stranded oligonucleotide point mutation technology, obtain recombinant plasmid according to the medicine-chest operation of Strategene company.
Another aspect of the present invention, the preparation method of farm crop fungus resistant polypeptides of the present invention is provided, the gene of coding Candida albicans pheromone operationally is connected the recombinant plasmid that obtains coding restructuring farm crop fungus resistant polypeptides gene with the colicin gene, the Transfected Recombinant Plasmid that obtains is entered in colibacillus engineering and obtains to produce the engineering bacteria cell of farm crop fungus resistant polypeptides of recombinating.Namely obtain farm crop fungus resistant polypeptides of the present invention with Sepharose High Performance column separating purification.
Farm crop fungus resistant polypeptides of the present invention can be used in the agricultural chemicals of preparation treatment or the infection of prevention farm crop fungus.Can by polypeptide of the present invention is added pharmaceutically acceptable carrier or vehicle or optionally other composition make and be suitable for the pesticide composition that farm crop are used.Its mechanism is: the Candida albicans pheromone that can be combined with target fungal cell surface receptor in polypeptide of the present invention induces colicin to arrive near target farm crop fungus cytolemma, then the colicin passage forms in the hydrophobic section insertion target fungal cell membrane of structural domain, and then colicin passage formation structural domain forms ionic channel on target farm crop fungus cytolemma, target farm crop fungus entocyte leaked and cause fungal cell's death, thereby reaching the purpose of killing the farm crop fungus cell.
Farm crop fungus resistant polypeptides of the present invention is compared to the advantage of traditional fungicide, and it has special targeting, thereby in use can not attack zooblast, and its toxicity and untoward reaction are far below traditional fungicide.Because it is directly to form ionic channel to kill and wound the farm crop fungus cell on the farm crop fungus cytolemma, therefore compare with traditional fungicide, the farm crop fungus cell is much lower to the probability that it produces resistance again; Farm crop fungus resistant polypeptides-ionic channel cause leak and cause the farm crop fungus necrocytosis this than short duration in, the farm crop fungus cell is more difficult, and to utilize mutator gene-protein expression mode to repair the geometry that farm crop fungus resistant polypeptides-ionic channel causes on fungal cell membrane damaged, so the farm crop fungus cell is lower to the probability that farm crop fungus resistant polypeptides of the present invention produces resistance.Of particular note, in an embodiment of the present invention, farm crop fungus resistant polypeptides is thousands of times of existing agricultural chemicals tricyclazole result for the treatment of at least to the results for the treatment of of the farm crop of the infection such as fungi Magnaporthe grisea and Aspergillus flavus, has proved that farm crop fungus resistant polypeptides of the present invention has a good application prospect.
The method of the invention can obtain the farm crop fungus resistant polypeptides (greater than 95%) of desired purity and farthest preserve its biological activity, and the farm crop fungus resistant polypeptides purity lower (approximately 80-90%) of general CM column separating purification.
Description of drawings
Fig. 1 is the structural representation that contains the recombinant plasmid pCHCEBCH1 of Candida albicans pheromone gene and colicin Ia gene.
Fig. 2 is the structural representation that contains the recombinant plasmid pCHCEBCH2 of Candida albicans pheromone gene and colicin Ia gene.
Fig. 3 is farm crop fungus resistant polypeptides of the present invention to the inhibition test result of the fungi Magnaporthe grisea that grows in solid medium, shows that farm crop fungus resistant polypeptides is stronger thousands of times than tricyclazole to the growth inhibitory effect of fungi Magnaporthe grisea; A tricyclazole 5mg/ml in figure, B tricyclazole 50 μ g/ml, C farm crop fungus resistant polypeptides 2,5 μ g/ml, D farm crop fungus resistant polypeptides 1,5 μ g/ml.
Fig. 4 is farm crop fungus resistant polypeptides of the present invention to the inhibition test result of the fungi Magnaporthe grisea that grows in solid medium, shows that farm crop fungus resistant polypeptides is stronger thousands of times than tricyclazole to the growth inhibitory effect of fungi Magnaporthe grisea; A tricyclazole 5mg/ml in figure, B tricyclazole 500 μ g/ml, C tricyclazole 50 μ g/ml, D farm crop fungus resistant polypeptides 1,5 μ g/ml.
Fig. 5 is farm crop fungus resistant polypeptides of the present invention to the inhibition test result of the Aspergillus flavus that grows in solid medium, shows that farm crop fungus resistant polypeptides is stronger thousands of times than tricyclazole to the growth inhibitory effect of Aspergillus flavus; A tricyclazole 5mg/ml in figure, B tricyclazole 500 μ g/ml, C tricyclazole 50 μ g/ml, D farm crop fungus resistant polypeptides 1,5 μ g/ml.
Fig. 6 is that farm crop fungus resistant polypeptides of the present invention is in the situation of field experiment; Medication zone situation is divided in the A farmland, and wherein, region intermediate Control is the blank zone, and the right Ph-CA zone be that farm crop fungus resistant polypeptides 1 of the present invention uses the zone, and Tricyc zone, the left side is that the tricyclazole use is regional; B field investigation situation; The C situation of spraying insecticide.
Embodiment
The structure of the plasmid of embodiment 1 expression farm crop fungus resistant polypeptides and the preparation of restructuring farm crop fungus resistant polypeptides
Original plasmid is the commercial plasmid of pET-15b (plasmid size 6.3 kb that loaded colicin Ia and Immunity protein gene, available from Novagen company, said gene is loaded by this laboratory), through double-stranded oligonucleotide point mutation technology (QuickChange
TMKit, Strategene company) will encode after the gene (the described nucleotide sequence of SEQ ID NO.2 in sequence table) of Candida albicans pheromone is inserted into the I626 site of colicin Ia gene, prepare a kind of recombinant plasmid pCHCCACH1 (as shown in Figure 1) of farm crop fungus resistant polypeptides.Transfected Recombinant Plasmid enters in E.coli BL21 (DE3) plyS engineering bacteria to prepare polypeptide, obtain the described farm crop fungus resistant polypeptides of SEQ ID NO.5 in sequence table, continue after embodiment in be called " farm crop fungus resistant polypeptides 1 ", its molecular weight is 70,000.
The sudden change program is undertaken by Strategene QuickChange Site-Directed Mutagenesis Kit (catalog#200518) medicine-chest handbook:
1, prepare the point mutation reactant
5ul 10xbuffer
2ul (10ng) wild-type colicin Ia plasmid
5 '-3 ' oligonucleotide primer of 1ul (125ng) design
3 '-5 ' oligonucleotide primer of 1ul (125ng) design
1ul dNTP
Distilled water 50ul
1ul pfu
(except plasmid, outside primer and distilled water, being the standby reagent of medicine-chest institute)
2, carry out pcr amplification, amplification condition: 95 ℃ of sex change, 35 seconds, anneal 53 ℃, 70 seconds, extend 68 ℃, totally 20 circulations in 17 minutes;
3, add (37 ℃, 1 hour) after Dpnl restriction endonuclease 1ul digestion mother body D NA chain, get 1ul reactant and HMS174 competent cell 50ul ice and incubated 30 minutes, 42 ℃ of capable thermal shockings, 45 seconds, then inserted in ice 2 minutes;
4, add 220rpm after the basic 0.5ml of NZY training, 37 ℃ were shaken bacterium after 1 hour, got 50-100ul reactant bed board (LB training base adds 1% agar and adds the 50ug/ml penbritin, and 37 ℃ are spent the night);
5, choose bacterium after 18 hours, follow Qiagene, the companies such as Gibco various commercial extract the plasmid medicine-chests extract plasmids all can, order-checking is determined to suddenly change successfully;
6, E.coliBL21 (DE3) the ply engineering bacteria competent cell 50ul ice of plasmid 50 ng and preparation was incubated 30 minutes, 42 ℃, thermal shocking in 90 seconds, get the 50-100ul reactant and add the basic 0.5ml of LB training, 220rpm, 37 ℃ are shaken 37 ℃, bacterium bed board after 1 hour (LB training base adds 1% agar and adds the 50ug/ml penbritin) and hatch, picking colony after 18 hours;
7, increase in a large number bacterium, 8-16 rises LB training base, 250rpm, 37 ℃, 6-8 hour, the centrifugation thalline, 4 ℃, 6000g, 20 minutes, get 4 ℃, 20mMPBS, 0.15MNaCl, 2mMEDTA, pH8.0) 50-80ml suspension thalline, add after the 0.2MPMSF250 microlitre (4 ℃ of row ultrasonications, 400W, 2 minutes), (4 ℃ of the broken thalline of high speed centrifugation precipitation, 75000g, 1.5 hour), get the supernatant molecular weight 15 of packing into, 000 dialysis tubing is in 4 ℃, 20mMPBS, 0.15MNaCl, after 5mMimidazole10 rises dialysed overnight, supernatant is splined on HisTrap post (Amersham Biosciences), through 20mM PBS, 1Mimidazole, pH8.0, 4 ℃) can obtain the farm crop fungus resistant polypeptides of recombinating after wash-out, through 20mM sodium phosphate, 0.15MNaCl, 2mM EDTA, standby after the pH8.0 dialysis.
The below is listed as the designed concrete double-stranded oligonucleotide sequence of Candida albicans pheromone gene of preparation pCHCCACH1 plasmid:
pCHCCACH1
1、5’-3’
gcg aat aag ttc tgg ggtatt GGG TTT CGT CTC ACA AAC TTC taa ata aaa tat aag
aca ggc
3’-5’
gcctgtctt ata ttt tat tta GAA GTT TGT GAG ACG AAA CCC aat acc cca gaa ctt att
cgc
2、5’-3’
att ggg ttt cgt ctc aca aac ttc GGA TAC TTT GAG CCC GGC AAA taa ata aaa tat
aag aca ggc
3’-5’
gcc tgt ctt ata ttt tat tta TTT GCC GGG CTC AAA GTA TCC gaa gtt tgt gag acg aaa
ccc aat
The structure of the plasmid of embodiment 2 expression farm crop fungus resistant polypeptides and the preparation of restructuring farm crop fungus resistant polypeptides
Original plasmid is the commercial plasmid of pET-15b (the plasmid size 6.3kb that has loaded colicin Ia and Immunity protein gene, available from Novagen company, said gene is loaded by this laboratory), through double-stranded oligonucleotide point mutation technology (QuickChange
TMKit, Strategene company) will encode before the gene (the described nucleotide sequence of SEQ ID NO.2 in sequence table) of Candida albicans pheromone is inserted into the S1 site of colicin Ia, prepare another recombinant plasmid pCHCCACH2 (as shown in Figure 2) of farm crop fungus resistant polypeptides.Transfected Recombinant Plasmid enters in E.coli BL21 (DE3) plyS engineering bacteria to prepare polypeptide, obtain the described farm crop fungus resistant polypeptides of SEQ ID NO.7 in sequence table, continue after embodiment in be called " farm crop fungus resistant polypeptides 2 ", its molecular weight is 70,000.
The sudden change program is undertaken by Strategene QuickChange Site-Directed Mutagenesis Kit (catalog#200518) medicine-chest handbook:
1, prepare the point mutation reactant
5ul 10xbuffer
2ul (10ng) wild-type colicin Ia plasmid
5 '-3 ' oligonucleotide primer of 1ul (l25ng) design
3 '-5 ' oligonucleotide primer of 1ul (125ng) design
1ul dNTP
Distilled water 50ul
1ulpfu
(except plasmid, outside primer and distilled water, being the standby reagent of medicine-chest institute)
2, carry out pcr amplification, amplification condition: 95 ℃ of sex change, 35 seconds, anneal 53 ℃, 70 seconds, extend 68 ℃, totally 20 circulations in 17 minutes;
3, add (37 ℃, 1 hour) after Dpn1 restriction endonuclease 1ul digestion mother body D NA chain, get 1ul reactant and HMS174 competent cell 50ul ice and incubated 30 minutes, 42 ℃ of capable thermal shockings, 45 seconds, then inserted in ice 2 minutes;
4, add 220rpm after the basic 0.5ml of NZY training, 37 ℃ were shaken bacterium after 1 hour, got 50-100ul reactant bed board (LB training base adds 1% agar and adds the 50ug/ml penbritin, and 37 ℃ are spent the night);
5, choose bacterium after 18 hours, follow Qiagene, the companies such as Gibco various commercial extract the plasmid medicine-chests extract plasmids all can, order-checking is determined to suddenly change successfully;
6, E.coli BL21 (DE3) the ply engineering bacteria competent cell 50ul ice of plasmid 50ng and preparation was incubated 30 minutes, 42 ℃, thermal shocking in 90 seconds, get the 50-100ul reactant and add the basic 0.5ml of LB training, 220rpm, 37 ℃ are shaken 37 ℃, bacterium bed board after 1 hour (LB training base adds 1% agar and adds the 50ug/ml penbritin) and hatch, picking colony after 18 hours;
7, increase in a large number bacterium, 8-16 rises LB training base, 250rpm, 37 ℃, 6-8 hour, the centrifugation thalline, 4 ℃, 6000g, 20 minutes, get 4 ℃, 20mMPBS, 0.15M NaCl, 5mMimidazole, 2mMEDTA, pH 8.0) 50-80ml suspension thalline, add after 0.2M PMSF250 microlitre (4 ℃ of row ultrasonications, 400W, 2 minutes), (4 ℃ of the broken thalline of high speed centrifugation precipitation, 75000g, 1.5 hour), get the supernatant molecular weight 15 of packing into, 000 dialysis tubing is in 4 ℃, 20mM PBS, 0.15M NaCl, after 5mM imidazole10 rises dialysed overnight, supernatant is splined on HisTrap post (Amersham Biosciences), through 20mM PBS, 1Mimidazole, pH8.0, 4 ℃) can obtain the farm crop fungus resistant polypeptides of recombinating after wash-out, through 20mMsodium phosphate, 0.15MNaCl, 2mM EDTA, standby after the pH8.0 dialysis.
The double-stranded oligonucleotide sequence of the Candida albicans pheromone gene of preparation pCHCCACH2 plasmid is pressed the method design of embodiment 1.
The body outer suppressioning test of embodiment 3 farm crop fungus resistant polypeptides to farm crop fungus
Test 1: fungi is the fungi Magnaporthe grisea bacterial strain (Pyricularia oryzae) that Ya'an, Sichuan Yucheng District bureau of agriculture plant protection unit separates from the field, gets bacterium liquid 5 microlitres (10
8CFU/ml level bacterium amount) evenly coat husky Bo Shi solid and train basic (Hangzhou microorganism reagent factory) surface, place again the plastics cuvette of absorption 50 each treatment solutions of μ l in the training primary surface, each treatment solution is respectively tricyclazole concentration 5mg/ml, tricyclazole concentration 50 μ g/ml, farm crop fungus resistant polypeptides 2 concentration 5 μ g/ml, farm crop fungus resistant polypeptides 1 concentration 5 μ g/ml.To train base and be placed in 35 ℃ of cultivations two days, result as shown in Figure 3.Result shows to only have the treatment solution of dress tricyclazole concentration 5mg/ml and the treatment solution cuvette of farm crop fungus resistant polypeptides 1 concentration 5 μ g/ml to occur complete bacteriolyze ring on every side.This test shows, the growth of fungi Magnaporthe grisea can only be suppressed by farm crop fungus resistant polypeptides 1 and heavy dose of tricyclazole.
Test 2: fungi is the fungi Magnaporthe grisea bacterial strain from the center purchase of the Chinese Academy of Agricultural Sciences agricultural microorganism, bacterium liquid 5 microlitres (10
6CFU/ml level bacterium amount) evenly coat husky Bo Shi solid and train basic (Hangzhou microorganism reagent factory) surface, place again the plastics cuvette of absorption 50 each treatment solutions of μ l in the training primary surface, each treatment solution is respectively tricyclazole concentration 5mg/ml, tricyclazole concentration 500 μ g/ml, tricyclazole concentration 50 μ g/ml, farm crop fungus resistant polypeptides 1 concentration 5 μ g/ml.To train base and be placed in 35 ℃ of cultivations two days, result as shown in Figure 4.Result shows, the bacteriolyze ring that occurs around farm crop fungus resistant polypeptides 1 cuvette is greater than the bacteriolyze ring around tricyclazole concentration 5mg/ml cuvette, and this test shows, strong thousands of times at least of the tricyclazoles of crossing of the effect of farm crop fungus resistant polypeptides 1 Antifungi Magnaporthe grisea growth.
Test 3: fungi is the Aspergillus flavus bacterial strain (Asperigillus flavus) that buy back at the Chinese Academy of Agricultural Sciences agricultural microorganism center, bacterium liquid 5 microlitres (10
6CFU/ml level bacterium amount) evenly coat husky Bo Shi solid and train basic (Hangzhou microorganism reagent factory) surface, place again the plastics cuvette of absorption 50 each treatment solutions of μ l in the training primary surface, each treatment solution is respectively concentration 5mg/ml, tricyclazole concentration 500 μ g/ml, tricyclazole concentration 50 μ g/ml, farm crop fungus resistant polypeptides 1 concentration 5 μ g/ml.To train base and be placed in 35 ℃ of cultivations two days, result as shown in Figure 5.Result demonstration, the bacteriolyze ring that occurs around farm crop fungus resistant polypeptides 1 cuvette are greater than the bacteriolyze ring around tricyclazole concentration 5mg/ml cuvette, and this test shows, strong thousands of times at least of the tricyclazoles of crossing of effect that farm crop fungus resistant polypeptides 1 inhibition Aspergillus flavus grows.
The field control test of the anti-agriculture fungi polypeptide of embodiment 4 to blast resisting
1, experiment material
(1) medicine
Farm crop fungus resistant polypeptides 1 and tricyclazole (contrast).
(2) farm crop and tested fungi
The fungi Magnaporthe grisea that field paddy rice and rice plant infect
2, experimental technique
the heavier rice field of random selection rice blast morbidity, by testing standard, the rice field is divided into and uses farm crop fungus resistant polypeptides 1 (Ph-CA), tricyclazole (Tricyc) and blank (Control) Three regions, with farm crop fungus resistant polypeptides 1 (liquid agent), tricyclazole (pulvis) is diluted to respectively the working concentration of 10 μ g/ml farm crop fungus resistant polypeptides 1 and 2mg/ml tricyclazole in proportion with paddy field water either, respectively spray once in rice blast leaf pest phase and stem pest phase, check respectively and record farm crop fungus resistant polypeptides 1 after 10-14 days by the rice blast examination criteria, leaf pest phase protection ratio and the stem pest phase damaging factor in tricyclazole and blank zone.Gather spike of rice in the Three regions sampling after the paddy maturation, take relatively prevention effect of thousand seed weight.Situation as shown in Figure 6 on the spot.
3, result
The field experiment result of 2005 and 2006 2 years shows; under above-mentioned working concentration; farm crop fungus resistant polypeptides 1 and tricyclazole have obtained comparatively close prevention effect: farm crop fungus resistant polypeptides 1 leaf pest phase protection ratio 71%; stem pest phase damaging factor 0.53%; tricyclazole leaf pest phase protection ratio 75%, stem pest phase damaging factor 1.6%.
Molecular weight (the MW70 of farm crop fungus resistant polypeptides 1,000) be 370 times of tricyclazole molecular weight (MW189), and the working concentration of farm crop fungus resistant polypeptides 1 and tricyclazole differs 200 times, therefore, if by comparing after same medicine molecular amounts mark, the present embodiment shows, the prevention effect that farm crop fungus resistant polypeptides of the present invention infects blast resisting should be 74,000 times of tricyclazole prevention effect.
The paddy thousand seed weight that tricyclazole zone (Tricyc) sampling gathers is 17.65mg and 20.5mg, the paddy thousand seed weight that blank zone (Control) sampling gathers is 18.6mg and 20.95mg, and the paddy thousand seed weight that farm crop fungus resistant polypeptides 1 zone (Ph-CA) gathers is 24.05mg and 24.2mg.Calculate by these data, the paddy thousand seed weight of farm crop fungus resistant polypeptides 1 area sampling collection has increased 13.4-29.3% than the paddy thousand seed weight of blank area sampling collection.
Because the damage that rice blast causes is only added up in field examination, and the fungal disease in the rice field also has other fungal disease except rice blast, as fungal diseases such as Aspergillus flavus, black-koji moulds.What thousand seed weight was added up is the comprehensive accumulation of farm crop fungus resistant polypeptides 1 all fungal diseases of control.
Claims (6)
- The coding farm crop fungus resistant polypeptides gene, its nucleotide sequence such as SEQ ID NO.4.
- 2. the recombinant plasmid of farm crop fungus resistant polypeptides, is characterized in that containing gene claimed in claim 1.
- 3. farm crop fungus resistant polypeptides, is characterized in that it is formed by expression of recombinant plasmid claimed in claim 2.
- 4. farm crop fungus resistant polypeptides according to claim 3, its aminoacid sequence is as shown in SEQ ID NO.5, and its molecular weight is 70,000.
- 5. the application of the described farm crop fungus resistant polypeptides of claim 3 or 4 in the agricultural chemicals of preparation treatment or the infection of prevention farm crop fungus.
- 6. the preparation method of claim 3 or 4 described farm crop fungus resistant polypeptides is characterized in that comprising the following steps:The gene of Candida albicans pheromone is connected with the gene of coding colicin Ia the gene that obtains coding restructuring farm crop fungus resistant polypeptides, the gene that obtains is imported in expression system express, separate the polypeptide of expressing and obtain farm crop fungus resistant polypeptides.
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