CN102146126A - Protein related to insect resistance and encoding gene and application thereof - Google Patents
Protein related to insect resistance and encoding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a protein related to insect resistance and an encoding gene and application thereof. The protein is one of the following proteins in (1) and (2): (1) a protein shown in SEQ ID NO: 2; and (2) a protein which is obtained by carrying out substitution and/or deletion and/or addition of one or more amino acid residues on an amino acid residue sequence shown in SEQ ID NO:2, is related to insect resistance of plants and is derivative from the protein in (1). A method for cultivating transgenic plants has important theoretical and practical significances and can be used for cultivating and identifying varieties of the insect resistance plants required by agriculture and animal husbandry and treatment of the ecological environment.
Description
Technical field
The present invention relates to a kind of and pest-resistant associated protein and encoding gene and application.
Background technology
Worldwide current, because the production loss that insect pest infestation causes farm crop is very huge.Controlling the approach that insect pest infestation the most generally adopts traditionally is to use chemical insecticide in a large number.The use of chemical insecticide has also brought residual toxicity, environmental pollution, ecological damage, pest resistance to insecticide and serious problems such as wildness again when having obtained certain economic benefit.For adapting to the needs of agricultural sustainable development, environment protection and human security, strengthen the sustainable Control Study of agro-ecology property disaster energetically, be the human problem that faces jointly, also be the severe challenge that this field scientific research faces.Therefore, thus study and utilize self resistant gene of plant effectively to control agricultural insect pest and become the target that the mankind seek assiduously.
In the evolution of long period of time process, plant has been set up complicated and diversified resistance reaction mechanism in order to resist the predation from insect.And the plant hormone jasmonic plays an important role in the defensive reaction that the regulation and control plant is biological to insect pest infestation, pathogen invasion and mechanical wounding etc. and inanimate is coerced.Jasmonic is found in the beginning of the nineties in last century the regulation and control plant to the effect aspect the sick worm resistance, and after this people have obtained impressive progress aspect the biosynthesizing of research jasmonic and the signal transduction through the effort of more than ten years in model plant Arabidopis thaliana and tomato.The jasmonate acid signal approach that present people recognize comprises 3 signal of interests transduction element: COI1 that all guard, JAZ albumen and transcription factor MYC2 in Arabidopis thaliana and tomato.The COI1 F-box albumen of encoding forms SCF in vivo
COI1The ubiquitin ligase complex body, the nearest COI1 that studies show that is the acceptor of active jasmonic (JA-Ile).Hereditary and biochemical evidence shows that the JAZ family protein is SCF
COI1The substrate of ubiquitin ligase complex body plays the effect of supressor in the genetic expression of jasmonic inductive.The genetic expression of jasmonic inductive is subjected to the regulation and control of MYC2 and other transcription factor.Under normal circumstances, JAZ albumen suppresses the transcriptional activation activity of MYC2, and the jasmonate acid signal approach is in closing condition; And under the situation of pathogenic bacteria or insect pest infestation, active jasmonic promotes COI1 and the proteic interaction of JAZ and the latter degraded, and then discharges the function of MYC2 and induce Expression of Related Genes.
Summary of the invention
The purpose of this invention is to provide a kind of albumen relevant and encoding gene thereof with plant anti-insect.
The proteic encoding gene relevant with plant anti-insect provided by the present invention, name is called SlNAC072, derives from tomato and belongs to tomato (Solanum lycopersicon).
The albumen relevant with plant anti-insect provided by the present invention is the albumen with one of following amino acid residue sequences:
1) protein shown in the SEQ ID NO:2;
2) with the amino acid residue sequence shown in the SEQ ID NO:2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant resistance to insect by 1) deutero-protein.
The proteic encoding gene relevant with plant anti-insect provided by the present invention is following 1)-4) in arbitrary described gene:
1) dna molecular shown in the SEQ ID NO:1;
2) dna molecular shown in the Nucleotide of 28-1077 position among the SEQ ID NO:1;
3) under stringent condition with 1) or 2) dna molecular of the dna molecule hybridize that limits;
4) with 1) or 2) dna molecular that limits has the homology more than 90% and the gene of encoding said proteins.
Described stringent condition is: (or 0.1 * SSC), the solution of 0.1 * SDS is hybridized under 65 ℃ and is washed film with 0.1 * SSPE.
In order to make Protein S lNAC072 be convenient to purifying, can connect label as shown in table 1 at proteinic N-terminal or the C-terminal that the aminoacid sequence shown in the SEQ ID NO:2 is formed.
The sequence of table 1. label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag?II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned 2) but in SlNAC072 gene synthetic.Above-mentioned 2) encoding gene of the SlNAC072 in can be by lacking SEQ ID NO:1 the codon of one or several amino-acid residue in the dna sequence dna shown in the 5 ' terminal 28-1077 bit base, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
Contain above-mentioned and expression cassette, recombinant vectors, transgenic cell line and reorganization bacterium tomato anti insect associated protein encoding gene and also belong to protection scope of the present invention.
Described recombinant vectors is for inserting the recombinant expression vector that above-mentioned encoding gene obtains between the multiple clone site of pCAMBIA2300-35S-OCS.
Arbitrary segmental primer also belongs to protection scope of the present invention in amplification and above-mentioned and the anti insect related gene.
Last purpose of the present invention provides a kind of method of cultivating insect-resistance enhanced transgenic plant.
The method of cultivation insect-resistance enhanced transgenic plant provided by the present invention is that above-mentioned arbitrary described encoding gene is changed in the plant that sets out, and obtains the transgenic plant that insect-resistance is better than the described plant that sets out.
In the aforesaid method, described encoding gene is to import in the described plant that sets out by above-mentioned arbitrary described recombinant expression vector.
The described plant that sets out can be dicotyledons, and described dicotyledons specifically can be tomato.
Described worm is an insect; Described insect specifically can be bollworm.
Experiment showed, that Protein S lNAC072 of the present invention is a transcription factor, it regulates and control the resistance of plant to insect by the expression of regulating some downstream resistance related genes.The transgenic Fructus Lycopersici esculenti of overexpression SlNAC072 can accumulate more resistance-associated protein PIs than wild-type tomatoes when being subjected to insect pest infestation, its resistance to insect is improved.Utilize the transgenic line of SlNAC072 overexpression can obtain plant transgene strain or the kind stronger to insect-resistant.Therefore, Protein S lNAC072 and encoding gene thereof can be used for the cultivation and the evaluation of the required zoophobous kind of husbandry and ecological environment treatment, have vital role in plant anti-insect breeding field.
Description of drawings
Fig. 1 is the transcriptional activation activity check of SlNAC072 as transcription factor.
Fig. 2 is the pest-resistant phenotype result of overexpression SlNAC072 tomato.
Fig. 3 be overexpression SlNAC072 tomato by insect bite after the mRNA expression amount of PI-II detect.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Tomato (Solanum lycopersicon) kind M82, the public can (TGRC, http://tgrc.ucdavis.edu/) obtain from U.S. tomato genetic resources center.
The acquisition of the clone of embodiment 1, goal gene and overexpression SlNAC072 transgenic Fructus Lycopersici esculenti
One, the clone of goal gene
Extracting total RNA of tomato (Solanum lycopersicon) kind M82, is template with its reverse transcription product, under the guiding of primer 1 and primer 2, carries out pcr amplification.
Primer 1:5 '-TCT
GGTACCATGGGTGTTCAAGAAAAAGATCCAC-3 ' (upstream primer, underscore part base are Kpn I recognition site)
Primer 2: 5 '-CAG
CTGCAGCTACTGCTTGAACCCGAGATTTAAC-3 ' (downstream primer, underscore part base are Pst I recognition site)
Reaction finishes the back PCR product is carried out purifying and is connected into the pMD18-T carrier, transformed into escherichia coli DH5 α, select positive colony, positive colony is carried out liquid culture, extracting positive plasmid checks order, the result has inserted gene shown in the Nucleotide of 28-1077 position among the SEQ ID NO:1 that two ends have Kpn I recognition site and Pst I recognition site in pMD18-T, show that the recombinant vectors of structure is correct, and note is made pMD18-SlNAC072.The proteic aminoacid sequence of genes encoding shown in the Nucleotide of 28-1077 position is shown in SEQ ID NO:2 among the SEQ ID NO:1.This proteinic molecular weight is 39.47KD, and iso-electric point is 8.43.
Two, the acquisition of overexpression SlNAC072 transgenic Fructus Lycopersici esculenti
Carrier pCAMBIA2300-35S-OCS disclosed in document " Wu S; Yu Z; Wang F.2007.Cloning; characterization; and transformation of the phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) in maize (Zea mays L.) .Mol Biotechnol36:102-112 ", and the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity.
Agrobacterium tumefaciens AGL0 is at document " Li C; Liu G; Xu C; Lee G; Bauer P; Ganal M, Ling H and Howe GA. (2003) .The tomato Suppressor of prosystemin-mediated response2 gene encodes a fatty acid desaturase required for the biosynthesis of jasmonic acid and the production of a systemic wound signal for defense gene expression.The Plant Cell.15,1646-1661 " in disclosed, the public can obtain with developmental biology institute from Chinese Academy of Sciences heredity.
With restriction enzyme Kpn I and Pst I double digestion pMD18-SlNAC072, reclaim gene SlNAC072 fragment; With restriction enzyme Kpn I and Pst I double digestion carrier pCAMBIA2300-35S-OCS, reclaim the big fragment of carrier; The gene SlNAC072 fragment of recovery and the big fragment of carrier of recovery are connected, obtain recombinant expression vector.With the evaluation of checking order of this recombinant expression vector, the result, between the Kpn of carrier pCAMBIA2300-35S-OCS I and Pst I restriction enzyme site, inserted gene shown in the Nucleotide of 28-1077 position among the SEQ ID NO:1, the recombinant expression vector that shows structure is correct, should positive recombinant expression vector note make pCAMBIA2300-35S-SlNAC072-OCS.
Recombinant expression vector pCAMBIA2300-35S-SlNAC072-OCS and empty carrier pCAMBIA2300-35S-OCS are transformed agrobacterium tumefaciens AGL0 respectively, on the LB solid medium that contains 50mg/L kantlex and 50mg/L Rifampin, screen, obtain positive recombinant, note is made AGL0-pCAMBIA2300-35S-SlNAC072-OCS and AGL0-pCAMBIA2300-35S-OCS respectively.
Transform tomato conventional variety M82 with AGL0-pCAMBIA2300-35S-SlNAC072-OCS and AGL0-pCAMBIA2300-35S-OCS respectively, concrete steps are as follows:
1, vegetable material: tomato conventional variety M82.
2, medium component:
Liquid MS medium: 4.4g MS, 30g sucrose and water are mixed, and water is settled to 1L, obtains liquid MS medium.
Seed growth substratum (1/2MS substratum): 2.2g MS, 30g sucrose and water are mixed, and water is settled to 1L, adds 0.8% agar, obtains the 1/2MS substratum.
(be total to) culture medium (D1) in advance: 4.4g MS, 1.0mg zeatin (Zeatin) and 30g sucrose is soluble in water, and water is settled to 1L, adds 0.8% agar;
Screening division culture medium (2Z): 4.4g MS, 2.0mg zeatin (Zeatin), 50mg kantlex (Kanamycin), 100mg inositol (Inositol), 0.5mg folic acid (Folic acid) and 20g sucrose is soluble in water, water is settled to 1L, adds 0.8% agar;
Root media: 4.4g MS, 50mg Kanamycin, 0.5mg Folic acid, 0.5mg indolebutyric acid (IBA) and 30g sucrose is soluble in water, and water is settled to 1L, adds 0.8% agar.
Above medicine is all available from Sigma company, and article No. is respectively MS (M0404), Zeatin (Z3541), Folic acid (F8758), Inositol (I7508).
3. the preparation of vegetable material
Get tomato conventional variety M82, select full, the big seed of grain, soak 15-20min, use aseptic water washing again 5 times, be seeded on the seed growth substratum, in 25 ℃ of illumination cultivation with 10%NaClO.After 7-8d sprouts, under aseptic condition, cotyledon is diced, the cotyledon dice is inoculated in the pre-culture medium, in 25 ℃, 16 hours illumination/8 hour dark, light intensity 70microeinsteins m
-2s
-2Condition under cultivate, behind the 1-2d, can be used for transforming.
4. agrobacterium tumefaciens bacterial strain and cultivation
The preparation of Agrobacterium bacterium liquid: positive single reorganization bacterium colony AGL0-pCAMBIA2300-35S-SlNAC072-OCS of picking or AGL0-pCAMBIA2300-35S-OCS are inoculated in respectively in the LB liquid nutrient medium (additional 50mg/L Kan) and in 28 ℃, 200rpm incubated overnight, to OD600=0.6-0.7.Bacterium liquid is abandoned supernatant with the centrifugal 10min of 5000r/min, collects thalline.With the liquid MS medium thalline that suspends again, be diluted to OD600=0.3-0.4 after, in order to the usefulness of conversion.
5. the conversion of explant, screen and take root
Adopt " Ye Panfa ".The cotyledon piece that step 3 is obtained immersed the Agrobacterium bacterium liquid AGL0-pCAMBIA2300-35S-SlNAC072-OCS for preparing and AGL0-pCAMBIA2300-35S-OCS respectively 10 minutes.Be inoculated in then and (put filter paper on the substratum) on the D1 substratum and cultivate 2d altogether, be transferred to screening and culturing in the screening division culture medium (2Z), per 2 all subcultures are once cultivated 5-8 and are produced resistant buds after week.When indefinite bud is stretched to 2-3cm, with scalper resistant buds is downcut, and be forwarded on the root media, carry out root culture.The T0 that to take root moves into Routine Management in the soil for transfer-gen plant, and results T0 is for the seed of transfer-gen plant.
The condition of altogether cultivation, screening and culturing, root culture is: temperature is 25 ℃, 16 hours illumination/8 hour dark, light intensity 50-60microeinsteins m
-2s
-2
Design the evaluation that primer is used for transgenic plant according to 35S promoter sequence before the pCAMBIA2300-35S-OCS carrier multiple clone site and SlNAC072 encoding sequence, primer sequence is as follows:
Primer 3:5 '-AAGACTGGCGAACAGTTCATACAGA-3 '
Primer 4:5 '-GTTGTCGCTGTGAATGTGGT-3 '
Utilize the method for PCR, under the guiding of primer 3 and primer 4, the transfer-gen plant that obtains is identified, obtain PCR and identify correct commentaries on classics pCAMBIA2300-35S-SlNAC072-OCS plant 7 strains, detect through fluorescence quantitative RT-RCR, the mRNA abundance of SlNAC072 significantly improves in the transgenic plant, and conclusive evidence has obtained the transgenic Fructus Lycopersici esculenti of overexpression SlNAC072.
Three, SlNAC072 is to the genetic regulation of insect-resistant
Plantation T0 obtains T1 for transfer-gen plant for the seed of transfer-gen plant, therefrom identifies transgenosis homozygote and numerous kind, is used for the experiment of feeding of follow-up insect.After homozygote authentication method: T1 plants for transfer-gen plant individual plant receipts, (wild-type M82 growth on this substratum can be subjected to severe inhibition containing on the 1/2MS substratum of 100mg/L kantlex screening with broadcasting after the seed disinfection of 30-40 grain respectively, it is shorter to show as main root, lateral root does not take place substantially, and transgenic line is not suppressed substantially owing to having the kalamycin resistance growth, the generation of taproot and lateral root is all very normal), if some T1 show as kalamycin resistance for the seed of transfer-gen plant results, then this T1 is a homozygote for transfer-gen plant, the seed of its results also all is a homozygote, can be used for the experiment of feeding of follow-up insect.
The insect experiment of feeding:
Bollworm (Helicoverpa armigera) disclosed in document " Wu K.; Y.Lu; H.Feng; Y.Jiang; and J.Zhao.2008.Suppression of cotton bollworm in multiple crops in China in areas with Bt toxin-containing cotton.Science.321 (5896): 1676-1678. ", and the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity.
Before insect was fed, transgenosis was cultivated under normal condition by the plant of isozygotying: illumination 16 hours, dark 8 hours, intensity of illumination 70microeinsteins m
-2s
-2, 25 ℃ of temperature.When treating that plant grows to 4-6 sheet true leaf and launches (approximately need 30 days) fully, get the growing way unanimity 2 the length of time bollworm carefully be put on the plant, let alone freely to get food.Respectively get the transfer-gen plant that 20 strains change the adjoining tree of pCAMBIA2300-35S-OCS over to and change pCAMBIA2300-35S-SlNAC072-OCS over to and test, every plant is put 3 bollworms.By the time fed the 9th day, plant is carried out Taking Pictures recording, and collection blade extraction RNA is used for subsequent analysis.Simultaneously with the same terms cultivate down but do not connect worm commentaries on classics pCAMBIA2300-35S-SlNAC072-OCS plant and with the same terms cultivate down but the commentaries on classics pCAMBIA2300-35S-OCS plant that do not connect worm in contrast.
The result shows, the transgenic Fructus Lycopersici esculenti that isozygotys can accumulate more resistance-associated protein PIs after being subjected to physical abuse or insect to chew food, compare with the M82 that changes empty carrier pCAMBIA2300-35S-OCS over to, the transgenic Fructus Lycopersici esculenti of overexpression SlNAC072 (35S::SlNAC072) significantly improves the resistance of insect, is embodied in:
1) the control plant blade that changes empty carrier pCAMBIA2300-35S-OCS in the overwhelming majority is chewed food to the greatest extent the time by insect, and the blade of transgenic plant is kept perfectly substantially; Wild-type tomatoes is identical with the control plant performance that changes empty carrier pCAMBIA2300-35S-OCS over to.
2) fed 9 days after, it is clean that the vegetative point that changes the control plant of empty carrier pCAMBIA2300-35S-OCS over to is chewed food substantially, and the vegetative point of transgenic plant is not damaged substantially, (Fig. 2, A is for before feeding; B is for after feeding 9 days); Wild-type tomatoes is identical with the control plant performance that changes empty carrier pCAMBIA2300-35S-OCS over to.
3) PI-II is used to weigh the resistance to insect for a long time as an important marker gene in the tomato jasmonate acid signal transduction pathway always.Transfer-gen plant after detection was fed the 9th day and change the mRNA expression amount of PI-II in the adjoining tree of empty carrier pCAMBIA2300-35S-OCS over to.
The RT-PCR primer that detects PI-II is
Primers F: 5 '-AATTATCCATCATGGCTGTTCAC-3 '
Primer R:5 '-CCTTTTTGGATCAGATTCTCCTT-3 '
The result is as shown in Figure 3 (among Fig. 3, A is not for connecing the commentaries on classics pCAMBIA2300-35S-OCS plant of worm, B is the commentaries on classics pCAMBIA2300-35S-OCS plant that connects behind the worm, C is not for connecing the commentaries on classics pCAMBIA2300-35S-SlNAC072-OCS plant of worm, and D is the commentaries on classics pCAMBIA2300-35S-SlNAC072-OCS plant that connects behind the worm).Compare with changeing the pCAMBIA2300-35S-OCS control plant, the transgenic Fructus Lycopersici esculenti of overexpression SlNAC072 is being subjected to having accumulated more PI-II after insect is chewed food, and the transgenic Fructus Lycopersici esculenti that shows overexpression SlNAC072 significantly improves the resistance of insect.
Experiment repeats 3 times, as a result unanimity.
The transcriptional activation activity checking of embodiment 2, SlNAC072
One, the clone of goal gene
Carrier pGBKT7 is available from U.S. Clontech company, and catalog number is 630442.PGADT7 is available from U.S. Clontech company, and catalog number is 630443.
According to the complete encoding sequence of the suitable restriction enzyme site design primer amplification SlNAC072 of the cDNA sequence of SlNAC072 and carrier pGBKT7, N-terminal encoding sequence and C-terminal encoding sequence, primer sequence is as follows:
Primer 5:5 '-
CATATGGGTGTTCAAGAAAAAGA-3 ' (complete encoding sequence upstream primer, underscore part base are Nde I recognition site)
Primer 6:5 '-
CTGCAGCTACTGCTTGAACCCGAGAT-3 ' (complete encoding sequence downstream primer, underscore part base are Pst I recognition site)
Primer 7:5 '-
CATATGGGTGTTCAAGAAAAAGA-3 ' (N-terminal encoding sequence upstream primer, underscore part base are Nde I recognition site)
Primer 8:5 '-
CTGCAGCTATGGTCCACTTGAATTCTTCT-3 ' (N-terminal encoding sequence downstream primer, underscore part base are Pst I recognition site)
Primer 9:5 '-
CATATGAAACCTCTGATGTCTGGTTT-3 ' (C-terminal encoding sequence upstream primer, underscore part base are Nde I recognition site)
Primer 10:5 '-
CTGCAGCTACTGCTTGAACCCGAGAT-3 ' (C-terminal encoding sequence downstream primer, underscore part base are Pst I recognition site)
PMD18-SlNAC072 with acquisition among the embodiment 1 is a template, and respectively at primer 5 and primer 6, primer 7 and primer 8 under the guiding of primer 9 and primer 10, carry out pcr amplification, obtain 1050bp respectively, the fragment of 504bp and 546bp size.To checking order behind the PCR product purification, show that three bar segment have in the sequence table SEQ IDNO:1 respectively from 5 ' end 28-1077, the nucleotide sequence of 28-531 and 532-1077 position.
Two, the structure of recombinant vectors
3 dna fragmentations that amplification in the above-mentioned steps one is obtained carry out double digestion with restriction enzyme Nde I and Pst I, then purified enzyme is cut product and be connected with carrier pGBKT7 behind the PstI double digestion through Nde I, to connect product and carry out the enzyme evaluation of cutting and check order, respectively evaluation is shown the complete encoding sequence (SEQID NO:1 is from 5 ' end 28-1077) that contains SlNAC072, correct recombinant expression vector called after pGBKT7-F, pGBKT7-N and the pGBKT7-C of N-terminal encoding sequence (SEQ ID NO:1 is from 5 ' end 28-531) and C-terminal encoding sequence (SEQ ID NO:1 is from 5 ' end 532-1077).
Three, transcriptional activation activity check
Yeast AH109 is available from U.S. Clontech company.Contain AD on the pGADT7 carrier, contain BD on the pGBKT7 carrier.
With three recombinant expression vector pGBKT7-F that obtain in the step 2, pGBKT7-N and pGBKT7-C advance the yeast AH109 competence of prepared fresh respectively with the pGADT7 cotransformation, separate application lacks on (SD/Trp-Leu-) and four scarce (SD/Trp-Leu-His-Ade-) plate culture mediums two then, is inverted for 30 ℃ and cultivates 4～6 days.The result shows (Fig. 1), contain respectively three recombinant expression vectors and negative control (BD, yeast pGBKT7) can both lack on (SD/Trp-Leu-) flat boards two grow bacterium colony, shows to transform successfully; But have only the yeast that transforms pGBKT7-F and pGBKT7-C to lack on (SD/Trp-Leu-His-Ade-) flat board and grow bacterium colony four.Four lack the bacterium colony that grows on (SD/Trp-Leu-His-Ade-) flat boards through X-GAL (available from U.S. Amresco company, article No. 0428) dyeing can be dyed blueness, show that further the yeast that transforms pGBKT7-F and pGBKT7-C has all successfully activated the sweet enzymic activity of beta galactose (β-gal activity), cause because the total length of SlNAC072 and C-end have transcriptional activation activity and this activation must be.
Above-mentioned experimental result shows that SlNAC072 has transcriptional activation activity, and this transcriptional activation activity depends on its C-terminal sequence.
Among Fig. 1,1 expression AD+BD-SlNAC072,2 expression AD+BD-SlNAC072-N, 3 expression AD+BD-SlNAC072-C, 4 expression AD+BD.
Claims (10)
1. an albumen is following 1) or 2) protein:
1) protein of forming by aminoacid sequence shown in the SEQ ID NO:2;
2) with the amino acid residue sequence shown in the SEQ ID NO:2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant resistance to insect by 1) deutero-protein.
2. the described proteic encoding gene of claim 1.
3. encoding gene according to claim 2 is characterized in that: described encoding gene is following 1)-4) in arbitrary described:
1) dna molecular shown in the SEQ ID NO:1;
2) dna molecular shown in the Nucleotide of 28-1077 position among the SEQ ID NO:1;
3) under stringent condition with 1) or 2) dna molecular of the dna molecule hybridize that limits;
4) with 1) or 2) dna molecular that limits has the homology more than 90% and the gene of encoding said proteins.
4. the recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 described encoding genes.
5. recombinant vectors according to claim 4 is characterized in that: described recombinant vectors is for inserting the recombinant expression vector that claim 2 or 3 described encoding genes obtain between the multiple clone site of pCAMBIA2300-35S-OCS.
6. the primer of arbitrary fragment or its total length in amplification claim 2 or the 3 described encoding genes.
7. a method of cultivating insect-resistance enhanced transgenic plant is that claim 2 or 3 described encoding genes are changed in the plant that sets out, and obtains the transgenic plant that insect-resistance is better than the described plant that sets out.
8. method according to claim 7 is characterized in that: claim 2 or 3 described encoding genes are to import in the described plant that sets out by claim 4 or 5 described recombinant expression vectors.
9. according to claim 7 or 8 described methods, it is characterized in that: the described plant that sets out is a dicotyledons; Described dicotyledons is a tomato.
10. according to claim 7 or 8 or 9 described methods, it is characterized in that: described worm is an insect; Described insect is a bollworm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667265A (en) * | 2012-09-06 | 2014-03-26 | 中国科学院遗传与发育生物学研究所 | Application of Lox (lipoxygenase) D gene of tomato in regulating and controlling plant resistance |
| CN104628836A (en) * | 2015-01-29 | 2015-05-20 | 中国农业科学院油料作物研究所 | Protein GmIFR associated with insect tolerance of plants as well as coding gene and application thereof |
| CN111363020A (en) * | 2020-04-22 | 2020-07-03 | 浙江省农业科学院 | MYC2 transcription factor of tea tree and application thereof |
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| EP2090662A2 (en) * | 2006-04-05 | 2009-08-19 | Metanomics GmbH | Process for the production of a fine chemical |
| WO2009073069A2 (en) * | 2007-10-31 | 2009-06-11 | Monsanto Technology, Llc | Genes and uses for plant enhancement |
| WO2010075143A1 (en) * | 2008-12-22 | 2010-07-01 | Monsanto Technology Llc | Genes and uses for plant enhancement |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667265A (en) * | 2012-09-06 | 2014-03-26 | 中国科学院遗传与发育生物学研究所 | Application of Lox (lipoxygenase) D gene of tomato in regulating and controlling plant resistance |
| CN103667265B (en) * | 2012-09-06 | 2015-10-28 | 中国科学院遗传与发育生物学研究所 | The application of tomato LoxD gene in regulating plant resistance |
| CN104628836A (en) * | 2015-01-29 | 2015-05-20 | 中国农业科学院油料作物研究所 | Protein GmIFR associated with insect tolerance of plants as well as coding gene and application thereof |
| CN111363020A (en) * | 2020-04-22 | 2020-07-03 | 浙江省农业科学院 | MYC2 transcription factor of tea tree and application thereof |
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| CN102146126B (en) | 2013-04-03 |
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