CN103937816A - Method of efficiently expressing Bt protein Cry30Fal in rice - Google Patents
Method of efficiently expressing Bt protein Cry30Fal in rice Download PDFInfo
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Abstract
The invention provides a method for efficiently expressing Bt protein Cry30Fal in rice. The method is characterized in that on the basis of the original protein Cry30Fal (GenBank:Aci22625.1), a gene coding the protein is modified into codon that the rice prefers to, and then the modified gene (Seq ID No.1) is introduced into rice; the expression amount of the protein Cry30Fal in rice is higher than that of unmodified gene; the transgenic rice has remarkable resistance to brown planthopper so that the dosage of a pesticide can be reduced and the environmental pollution can be reduced; the method has important economic value and application prospect.
Description
Technical field
The present invention relates to biological technical field, specifically, relate to a kind of in paddy rice the method for high efficient expression Bt PROTEIN C ry30Fa1.
Background technology
Brown paddy plant hopper (Nilaparvata lugens
) claim again brown back rice plant hopper, belong to Homoptera (Homoptera), Delphacidae (Delphacide), it is a kind of migratory pest that spreads all over the world, it is one of insect that harm Asia rice workspace is the most serious, have a strong impact on output and the quality thereof of paddy rice, the year of China brown paddy plant hopper, area occurred is 1330-2000 ten thousand hm
2, account for greatly 1/2 of Monitoring of Paddy Rice Plant Area.Brown paddy plant hopper is following several respects to the harm main manifestations of paddy rice: 1) adult and nymph are clustered in rice clump base portion, and thorn is inhaled juice in stems and leaves of rice phloem, consumes rice strain nutrition; Be injured while weight, rice strain bottom blackening, rotten smelly and shape of paralysis.2) stab rice strain cauline leaf tissue when Adult worms producting eggs, form a large amount of wounds, impel moisture outwards to scatter and disappear by stabbing a little, transfusion tissue destroys simultaneously, and assimilation weakens, and accelerates rice strain lodging.3) brown paddy plant hopper is still propagated the entomophila of Virus Diseases of Rice, can propagate or bring out rice disease.In addition, a large amount of wounds that cause when brown paddy plant hopper takes food or lays eggs, are conducive to infecting of the germs such as rice sheath blight disease; While taking food harm, " honeydew " of excretion, is rich in various carbohydrates and amino acid, covers in rice strain, easily causes multiplying of germ.
For a long time, mainly depend on chemical pesticide and prevent and treat brown paddy plant hopper, but traditional single medicament control method has been difficult to meet the demand that reality is produced, and abuse chemical insecticide can cause the problem such as environmental pollution and environmental destruction.In addition, the change of Biotypes of The Brown Planthopper Nilaparvata Lugens Stal, food chain abundant and some factors that cure mainly the generation resistances such as medicament Provado have been impelled to repeatedly breaking out of brown paddy plant hopper.Therefore, many countries are all constantly excavating and are utilizing brown planthopper resistant gene in the world, and then seed selection has lasting insect-resistance rice varieties.
Investigator has located a series of anti-plant hopper key-genes successively from Rice Resistance brown paddy plant hopper kind, so far, has identified 35 Brown Planthopper Resistance sites in paddy rice, and wherein 4 are cloned, and cultivate a series of brown planthopper resistant rice varieties.In addition, homoptera pest is there is to the gene of control action kou, also comprise that cholesterol oxidase gene from rhodococcus equi, soybean trypsin suppress (SKTI) gene and Snowdrop lectin (GNA) gene.Because GNA albumen is extremely low to people's toxicity, but brown paddy plant hopper, rice green leafhopper are had to extremely strong toxic action, can also suppress the growth of aphid, thereby be applied in transgenic paddy rice simultaneously.The harm of brown paddy plant hopper has been alleviated in the popularization of these resistant varieties at certain phase, obtained larger economic benefit, is also that the friendly type of development environment and resource-conserving agricultural have been made significant contribution.
But, brown paddy plant hopper has quick differentiation and forms new biotype to adapt to the ability of new pest-resistant cultivar, the rice varieties of a series of single resistances is often endangered by new biotype soon, within 2 years, just lost resistance as IR26 only promotes, and the resistance of IR36 has also only maintained 8 years.Therefore, screening has the novel gene of unique insecticidal mechanism to brown paddy plant hopper, the multiple anti insect genes of binding molecule assisted Selection technology aggregation or QTL, become the harm of control brown paddy plant hopper and alleviated the selective pressure of pest-resistant cultivar to brown paddy plant hopper, extends the kind top priority of working life.
Tribactur (Bacillus thuringiensis, Bt) be a kind of gram positive bacterium, in forming brood cell, can produce insecticidal crystal protein (the Insecticidal Crystal Proteins various pests such as lepidopteran, Diptera, Coleoptera, Homoptera, nematode to special insecticidal activity, ICPs), the encode gene of these albumen is called cry or cyt gene.These insecticidal crystal proteins have person poultry harmless, eco-friendly feature.Therefore, be widely used in the pest control in the fields such as agricultural, forestry, health, become the maximum microbial pesticide of most widely used, output in the world; Its killing gene is successfully applied in transgenic anti-insect plants simultaneously, is the main gene source of transgenic anti-insect plants.
The gene of coded insect-killing crystallin is called cry or cyt gene, by the definite designation of international Bt killing gene NK, is mainly to classify according to the homology of the aminoacid sequence of killing gene coding; Because aminoacid sequence difference and desinsection specificity and virulence have close dependency, NK specifies to be new pattern gene (http://www.biols.susx.ac.uk/Home/Neil-Crickmore/Bt/index.html) with known albumen homology lower than 95%.At present, investigator is separated to tens thousand of strain Bt bacterial strains from insect all over the world, soil and plant body surface equal samples, and therefrom excavates from Cry1～Cry73 class insecticidal crystal protein, but the high virulence Bt bacterial strain of insect and Cry albumen are but rarely had to report.
The application of Bt and insecticidal crystal protein thereof mainly concentrates on three aspects: 1) direct suitability for industrialized production; 2) build engineering bacteria, and then transform production; 3) cultivate transgenic anti-insect plants.The Cry albumen of commercial applications mainly contains Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1D, Cry1E, Cry1F, Cry2Aa, Cry2Ab, Cry3A, Cry3B, Cry8A, Cry8B and Cry34/Cry35 at present.Bt and insecticidal crystal protein thereof have been made huge contribution to the green control of plant insect, sanitary insect pest.
But the insecticidal spectrum of Cry toxin protein is only confined to the particular target insects such as lepidopteran, Coleoptera, Diptera, only has few effectively report of Cry toxin for insects such as the aphid of Hemiptera piercing-sucking mouthparts, plant hopper, leafhopper.Meanwhile, derive from the gene of different plant species, the expression efficiency in heterologous receptor can be affected, and expression amount, virulence and the wild strain of insecticidal crystalline gene in plant exists significant difference.For the loss of avoiding resistant insects to cause, improve the resistance of paddy rice to brown paddy plant hopper, finding new high virulence gene resource is the effective way addressing this problem, and the biological control of China is had to very important meaning.
Within 2009, the inventor separates the new Bt bacterial strain BtMC28 obtaining from the virgin forest Soils In The Region of Muchuan, Sichuan Province.Show by the virulence test to BtMC28 bacterial strain, BtMC28, to lepidoptera pest, Diptera pest etc., all has high virulence.
Later stage is tested pest species by expansion, finds that this bacterial strain pure protein has very strong insecticidal activity to brown paddy plant hopper, is 95% at the desinsection corrected mortality of 48h.By all Cry protein biological activities of BtMC28 bacterial strain are measured, only insecticidal crystal protein Cry30Fa1(GenBank:ACI22625.1) albumen has insecticidal activity (48h, LC to brown paddy plant hopper
50be 1 μ M).Under same background, its insecticidal activity is compared the higher (LC of GNA (GNA) that Powell etc. reports
50be 4 μ M), be the Cry proteinoid of finding first brown paddy plant hopper to have at present special insecticidal activity both at home and abroad.This albumen can be used for the wide spectrum Bt sterilant of preparation mainly for brown paddy plant hopper and other insect pests, by its gene order rice transformation, make it possess corresponding anti-insect activity, thereby reduce the usage quantity of agricultural chemicals, reduce environmental pollution, there is important economic worth and application prospect.
Summary of the invention
The object of this invention is to provide a kind of in paddy rice the method for high efficient expression Bt PROTEIN C ry30Fa1.
In order to realize the object of the invention, first the present invention provides the gene of a kind of Bt of coding PROTEIN C ry30Fa1, i.e. Cry30Fa1 gene, and its nucleotides sequence is classified as:
I) Seq ID No.1; Or
Ii) the nucleotide sequence shown in Seq ID No.1 is substituted, lacks and/or increase the nucleotide sequence of one or more Nucleotide and expression identical function protein; Or
Iii) under stringent condition with the nucleotide sequence of sequence hybridization shown in Seq ID No.1;
Described stringent condition is containing 0.1 × SSPE of 0.1%SDS or containing in 0.1 × SSC solution of 0.1%SDS, hybridization at 65 DEG C, and wash film with this solution.
The present invention also provides the carrier, host cell and the engineering bacteria that contain Cry30Fa1 gene.
The application of the carrier that the present invention also provides Cry30Fa1 gene or contains this gene in preparation transgenic plant (as paddy rice).
The present invention's be also provided for increasing Auele Specific Primer pair of Cry30Fa1 gene, comprises forward primer F5'-GCGCATATGATGAAGCCGTACCAGAGCGAGAAT-3' and reverse primer R5'-CGGAATTCTTAGTTCACTGGACAAGCAAACGCA-3'.
The present invention also provide a kind of in paddy rice the method for high efficient expression Bt PROTEIN C ry30Fa1, adopt agriculture bacillus mediated method, the carrier that contains Cry30Fa1 gene is proceeded in paddy rice (as extensive in rice varieties another name for Sichuan Province 818) callus, the material transforming through cultivating altogether-screen-break up-take root-exercise and the transplanting of transgenic seedling, screen the transgenic rice plant of high efficient expression Bt PROTEIN C ry30Fa1.
Preferably, described carrier is plant binary expression vector.
More preferably, the carrier that sets out of described carrier is pCDMAR-Hyg.
The amino acid composition of Cry30Fa1 albumen is as shown in table 1.
The amino acid composition of table 1Cry30Fa1 albumen
| Amino acid | Per-cent % | Amino acid | Per-cent % |
| Ala(A): | 6.7 | Met(M): | 1.60 |
| Cys(C): | 1.02 | Asn(N): | 9.46 |
| Asp(D): | 4.95 | Pro(P): | 6.11 |
| Glu(E): | 3.06 | Gln(Q): | 5.39 |
| Phe(F): | 4.22 | Arg(R): | 3.64 |
| Gly(G): | 5.53 | Ser(S): | 6.70 |
| His(H): | 1.02 | Thr(T): | 8.01 |
| Ile(I): | 9.46 | Val(V): | 3.64 |
| Lys(K): | 4.51 | Trp(W): | 1.16 |
| Leu(L): | 8.30 | Tyr(Y): | 5.53 |
According to the principle of BT transformation, at original PROTEIN C ry30Fa1(GenBank:ACI22625.1) basis on, the codon that is paddy rice preference by new Bt genetic modification, remove special construction, increase restriction enzyme site etc., improved cry gene is connected into (pUPROK) between P-ubi and T-nos, after structure completes, utilize Hind III and EcoR V to cut element, after filling, Klenow is connected into pCDMAR-Hyg carrier to obtain the high efficient expression binary vector of final marker-free, Cry30Fa1 gene (Seq ID No.1) the Introduced into Rice kind another name for Sichuan Province extensive 818 of transformation will be modified, its expression amount in paddy rice, Cry30Fa1 gene than not transformation is high, render transgenic paddy rice has the resistance to brown paddy plant hopper.
The present invention is based on the insecticidal activity of Cry30Fa1 albumen to brown paddy plant hopper itself, by improved Cry30Fa1 gene high efficient expression in paddy rice, render transgenic paddy rice is obtained has extremely strong insecticidal activity to brown paddy plant hopper.
Brief description of the drawings
Fig. 1 is the pSTK carrier conversion Bacillus thuringiensis with crystal negative mutant strain HD-73 that shuttles back and forth in the embodiment of the present invention 1
-the scanning electron microscope (SEM) photograph of expressed albumen.
Fig. 2 is the schematic flow sheet that builds high efficient expression binary vector PCDMARcry30Fa1 in the embodiment of the present invention 3.
Fig. 3 is the 327 strain transgenosis monoclonal anti Totomycin rice plants that obtain in the embodiment of the present invention 3.
Fig. 4 is the electrophoresis result that in the embodiment of the present invention 3, PCR detects positive transgenic paddy rice (partial results).
Fig. 5 is the result that in the embodiment of the present invention 3, Totomycin solution detects positive transgenic paddy rice.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is all according to normal experiment condition, as Sambrook equimolecular cloning experimentation handbook (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001), or according to the condition of manufacturer specification sheets suggestion.
Embodiment 1 is according to paddy rice codon preference transformation Cry30Fa1 protein coding gene
In the case of not changing the aminoacid sequence of original Cry30Fa1 coded by said gene, use paddy rice preference codon to be optimized (table 1) to codon, carry G+C rich, avoid some may reduce the sequence of expression: to remove original PPSS(AATAAA, AATAAT, AATTAA and AACCAA), remove original ATTTA sequence, remove original cutting sequence C ATTG that includes, give up continuous AT enrichment region (>=4A/T), some conventional restriction enzyme sites that exist in coding region are transformed to (table 2), improved situation is as shown in table 3, improved Cry30Fa1 gene order is as shown in SEQ ID No.1.
Table 1CG and TA are respectively in the situation of codon 2-3 position
Table 2 is transformed (removal) to some conventional restriction enzyme sites
Situation before and after table 3 genetic modification
Expression and the insecticidal activity assay of Cry30Fa1 gene after embodiment 2 transformations
1, the structure of shuttle expression carrier, expression and purifying
Design a pair of Auele Specific Primer cryF:5'-GCGCATATG (NdeI) ATGAAGCCGTACCAGAGCGAGAAT-3'; CryR:5'-CGGAATTC (EcoR I) TTAGTTCACTGGACAAGCAAACGCA-3', obtains gene fragment by pcr amplification.Gene fragment clone is upper to intestinal bacteria-Bt shuttle expression carrier pSTK, and after demethylation, electricity transforms Tribactur Bt without crystal mutant strain HD-73
-in; Then be PB(0.5% peptone and 0.3% beef extract of 50 μ g/ml in kalamycin concentration) in substratum, cultivate 50h in 30 DEG C.Result shows, improved gene is without crystal mutant strain HD-73
-middle successful expression, and form coccoid crystal (Fig. 1).
The 1M NaCl solution washing of precooling for the bacterial sediment of collecting, the centrifugal rear washing of the sterilized water with precooling; Precipitation is suspended in lysate (containing 3% beta-mercaptoethanol, pH9.6), and pH value is adjusted to 9.5-10, shakes 4h, soluble protein on ice; In 10,000rpm, 4 DEG C of centrifugal 20min, get supernatant, and with 4M sodium acetate-acetic acid solution (pH4.5), adjust pH is to 4.5-5, and 4 DEG C precipitate 4h; 12,000rpm, 4 DEG C of centrifugal 15min, sterilized water washing several for precipitation.
2, biological activity determination
Brown paddy plant hopper artificial breeding: raising device is long 15cm, the bottomless cylinder glass vessel of diameter 2.5cm, feed chamber fitted draws thin Parafilm film folder nutrition drop to form by bilayer.Every pipe connects after 25 of 2 age in days nymphs with gauze sealing, keeps flat, and with black cloth shading moisturizing, only exposes Parafilm film sealing end, is placed in the culturing room or incubator of 27 ± 1 DEG C of envrionment temperatures, within every 2 days, changes feed (artificial brown paddy plant hopper nutritive medium).
Determination of activity: the Cry30Fa1 albumen after artificial brown paddy plant hopper nutritive medium and purifying is fully mixed, dilute 6 different concns gradients, add in feed chamber fitted.Double-way pipe one side is sealed one section of double-way pipe with sealed membrane, then add 100 μ l artificial diet, then artificial diet are enclosed between two membranes with sealed membrane, i.e. access 30 of worms of examination (adult) after sealing, finally seal the other end with nylon wire.The double-way pipe that examination worm is housed is placed in artificial culture case, and the one end covering with nylon wire encases with wet black cloth and towel.Observation experiment result after cultivation 24-48h, adds up dead borer population and the borer population of living, statistics mortality ratio, calculation correction mortality ratio and LC
50.Each concentration arranges and independently repeats to test three times.
Result shows that Cry30Fa1 albumen has insecticidal activity to brown paddy plant hopper, LC
50be 1 μ M.
Embodiment 3 Cry30Fa1 genes high efficient expression in paddy rice
Improved cry gene is connected into (pUPROK) between P-ubi and T-nos, after structure completes, utilize Hind III and EcoR V to cut element, after filling, Klenow is connected into pCDMAR-Hyg carrier to obtain high efficient expression binary vector PCDMARcry30Fa1(Seq ID No.5, Fig. 2 of final marker-free).With agrobacterium-mediated transformation, the Cry30Fa1 gene (Seq ID No.1) of modifying transformation is proceeded to rice varieties another name for Sichuan Province extensive 818, obtain 327 strain transgenosis monoclonal anti Totomycin rice plants (Fig. 3), detected and found that there is 233 strains containing hygromycin gene (Fig. 4) by PCR, 46 strains are containing Cry30Fa1 and hygromycin gene (Fig. 5).
Utilize Q-PCR and protein detection technology, the Cry30Fa1 gene (Seq ID No.1) of modifying transformation is imported to another name for Sichuan Province extensive 818, its expression amount in paddy rice, than Cry30Fa1 gene (the Seq ID No.2) height of transformation not, remarkable to the resistance of Brown Planthopper.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Reference
[1] Cheng Xianian, Wu Jincai, Ma Fei.Brown paddy plant hopper research and control.Beijing: Chinese agriculture press, 2003.
[2]Cheng,H.P.,Deng,W.,Fu,Y.,et?al.Research?progress?in?genetics?and?breeding?for?rice?resistance?to?Brown?Planthopper[J].Acta?Agriculturae?Jiang?Xi,2013,25(10)：60－64.
[3]Tong,X.H.,Qi,J.F.,Zhu,X.D.,et?al.The?rice?hydroperoxidelyase?OsHPL3functions?in?defense?responses?by?modulating?the?oxylipin?pathway[J].The?Plant?J.,2012,71(5):763-775.
[4] Shi Zhenying, Zhu Lili, He Guangcun.The cloning and analysis [J] of rice protein kinase gene Ptil.Wuhan phytology research, 2004,22 (3): 187-192.
[5]Hu,J.,Zhou,J.B,and?Peng,X.X.,et?a1.The?BphiOO8a?gene?interacts?with?the?ethylene?pathway?and?transcriptionally?regulates?MAPK?genes?in?the?response?of?rice?to?brown?planthopper?feeding[J].Plant?Physiol.J.,201l,56(2):856—872.
[6]Du,B.,Zhang,W.L,and?Liu,B.F.,et?al.Identification?and?characterizationof?Bph14,a?gene?conferring?resistance?to?brown?planthopper?in?rice[J].Proc.Natl.Acad.Sci.USA,2009,106(52):22163-22168.
[7]Jongsma,M.A,Bakke,P.L,and?Peters,J.,et?al.Adaptation?of?Spodoptera?exigua?larvae?to?plant?proteinase?inhibitors?by?induction?of?gut?proteinase?activity?insensitive?to?inhibitors[J].Proc.Natl.Acad.Sci.USA,1995,92:8041-8045.
[8]Powell,K?S.,atehouse,A.M.R.,and?Hilder,V.A.,et?al.Different?antimetabolic?effects?of?related?lectins?towards?nymphal?stages?of?Nilaparvata?lugens[J].Entomol.Exp.Appl.,1995,75:61-65.
[9]Lee,S.I.,Lee,S.H.,and?Koo,J.C.,et?al.Soybean?Kunitz?trypsin?inhibitor?(SKTI)confers?resistance?to?the?brown?planthoper(Nilaparvata?lugens?Stal)in?transgenic?rice[J].Mol.Breeding.,1999,5:1-9.
[10]Nagadhara,D.,Ramesh,S.,and?Pasalu,I.C.,et?al.Transgenic?indica?rice?resistant?to?sap-sucking?insects[J].Plant?Biotechnol.J.,2003:231-240.
[11] He Jun.The Fine Mapping of Rice Resistance brown paddy plant hopper key-gene bph22 (t), Agricultural University Of Nanjing, doctorate paper, 2011.
[12]Huang,D.,Qiu,Y.,and?Zhang,Y.,et?al.Fine?mapping?and?characterization?of?BPH27,a?brown?planthopper?resistance?gene?from?wild?rice(Oryza?rufipogon?Griff.)[J].Theor.Appl.Genet.,2013,126:219–229.
[13]Gurdev,S.，and?Khush,G.S.Plant?breeding[M]//Pimentel?D.Encyclopedia?of?pest?management.New?York:Blackwell?Publishing.2003:1-5.
[14]Khush,G.S.,and?Brar,D.S.Genetics?of?resistance?to?insect?in?crop?plants?[J].Adv.Agron.,1991,45:223-228.
[15] Lv Zhongxian, Yu Xiaoping, Tao Linyong etc.The evaluation of new rice variety (being) to Brown Planthopper Resistance.Scientia Agricultura Sinica, 2002,35 (2): 225-229.
Claims (10)
1. the gene of coding Bt PROTEIN C ry30Fa1, is characterized in that, its nucleotides sequence is classified as:
I) Seq ID No.1; Or
Ii) the nucleotide sequence shown in Seq ID No.1 is substituted, lacks and/or increase the nucleotide sequence of one or more Nucleotide and expression identical function protein; Or
Iii) under stringent condition with the nucleotide sequence of sequence hybridization shown in Seq ID No.1;
Described stringent condition is containing 0.1 × SSPE of 0.1%SDS or containing in 0.1 × SSC solution of 0.1%SDS, hybridization at 65 DEG C, and wash film with this solution.
2. contain the carrier of gene described in claim 1.
3. contain the engineering bacteria of gene described in claim 1.
4. the application of carrier in preparation transgenic plant described in gene or claim 2 described in claim 1.
5. application according to claim 4, is characterized in that, described plant is paddy rice.
6. for the Auele Specific Primer pair of gene described in the claim 1 that increases, it is characterized in that, comprise forward primer F5'-GCGCATATGATGAAGCCGTACCAGAGCGAGAAT-3' and reverse primer R5'-CGGAATTCTTAGTTCACTGGACAAGCAAACGCA-3'.
7. the method for a high efficient expression Bt PROTEIN C ry30Fa1 in paddy rice, it is characterized in that, adopt agriculture bacillus mediated method, carrier claimed in claim 2 is proceeded in Rice Callus, the material transforming through cultivating altogether-screen-break up-take root-exercise and the transplanting of transgenic seedling, screen the transgenic rice plant of high efficient expression Bt PROTEIN C ry30Fa1.
8. method according to claim 7, is characterized in that, described carrier is plant binary expression vector.
9. method according to claim 8, is characterized in that, the carrier that sets out of described carrier is pCDMAR-Hyg.
10. according to the method described in claim 7-9 any one, it is characterized in that, the rice varieties of use is another name for Sichuan Province extensive 818.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105017391A (en) * | 2015-06-30 | 2015-11-04 | 杭州瑞丰生物科技有限公司 | Insect-resistant protein, insect-resistant fusion protein, coding gene, carrier and application |
| CN107759702A (en) * | 2017-10-30 | 2018-03-06 | 中国科学院遗传与发育生物学研究所 | A kind of insecticidal proteins HY131c of anti-brown plant-hopper and its encoding gene and application |
| CN110904143A (en) * | 2019-09-12 | 2020-03-24 | 黑龙江省农业科学院耕作栽培研究所 | Multifunctional glyphosate-resistant rice transformation vector pCDMAR-epsps and construction method and application thereof |
-
2014
- 2014-03-27 CN CN201410119119.0A patent/CN103937816B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| TAN F.R.等: "Cloning and characterization of two novel crystal protein genes , Cry54AaJ and Cry30Fal, from Bacillus thuringiensis Strain BtMC28", 《CURR MICROBIOL》, vol. 58, no. 6, 31 December 2009 (2009-12-31), pages 654 - 659, XP019708389 * |
| TAN,F等: "登录号：EU751609.1", 《GENBANK》, 1 June 2009 (2009-06-01), pages 1 * |
| TU J等: "Field performance of transgenic rice plants with a synthetic crylAb gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest species", 《MOLECULAR BREADING》, no. 6, 31 December 2000 (2000-12-31), pages 433 - 439 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105017391A (en) * | 2015-06-30 | 2015-11-04 | 杭州瑞丰生物科技有限公司 | Insect-resistant protein, insect-resistant fusion protein, coding gene, carrier and application |
| CN107759702A (en) * | 2017-10-30 | 2018-03-06 | 中国科学院遗传与发育生物学研究所 | A kind of insecticidal proteins HY131c of anti-brown plant-hopper and its encoding gene and application |
| CN107759702B (en) * | 2017-10-30 | 2020-10-27 | 中国科学院遗传与发育生物学研究所 | Brown planthopper resistant insecticidal protein HY131c as well as coding gene and application thereof |
| CN110904143A (en) * | 2019-09-12 | 2020-03-24 | 黑龙江省农业科学院耕作栽培研究所 | Multifunctional glyphosate-resistant rice transformation vector pCDMAR-epsps and construction method and application thereof |
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