CN102533820B - Peanut beta-1,3-glucanase gene and application thereof in improvement of disease resistance of peanut - Google Patents
Peanut beta-1,3-glucanase gene and application thereof in improvement of disease resistance of peanut Download PDFInfo
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- CN102533820B CN102533820B CN 201210065250 CN201210065250A CN102533820B CN 102533820 B CN102533820 B CN 102533820B CN 201210065250 CN201210065250 CN 201210065250 CN 201210065250 A CN201210065250 A CN 201210065250A CN 102533820 B CN102533820 B CN 102533820B
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Abstract
The invention provides a peanut beta-1,3-glucanase gene and application thereof in improvement of the disease resistance of peanut. The gene contains an intron of 434bp, an open reading frame has the size of 1,047bp, and a translation product contains 348 amino acids, wherein signal peptide has the length of 27 amino acids and protein has the molecular weight of 38,806.55 daltons. The gene is connected to a pCAMBIA1301 plasmid to construct a plant overexpression vector pCAMBIA1301-Glu, and the peanut is transformed through agrobacterium-mediated transformation. Reverse transcription-polymerase chain reaction (RT-PCR) results show that the expression of Ah-Glu gene in a transgenic plant is obviously higher than that in a non-transgenic plant. Excised leaves of the transgenic plant are inoculated with cercospora personata, and through field observation, the disease resistance is higher than that of the non-transgenic plant. By the technical scheme, a new way for breeding for disease resistance in peanut genic engineering and avoiding potential safety hazards is opened up, and has important theoretical significance and wide application prospect.
Description
Technical field
The present invention relates to biological technical field, relate in particular to peanut beta-1,3-glucanase gene and the application in improving the peanut disease resistance thereof.
Background technology
Peanut is the important oil crops of China and cash crop, all has critical role in agricultural and even whole national economy.There are the multiple fungus diseases such as leaf spot, rust, bacterial wilt, Aspergillosis Huang in the peanut production midium or long term, is having a strong impact on the yield and quality of peanut.Along with the development of biotechnology, utilize the genetic engineering technique resistant gene of will being correlated with to import peanut, make plant resist the invasion and attack of pathogenic fungi by the overexpression foreign gene, be the available strategy that improves now peanut disease resistance and productivity.
Plant is subjected to pathogen infection can produce various lytic enzymes and pathogenesis-related proteins (PR albumen), β-1,3-dextranase (β-1,3-glucanas, Glu) be lytic enzyme important in the PR albumen, the main component β-1 in the degradable pathogenic fungi cell walls, 3-dextran, thereby the deposition of broken garland cells wall novel substance causes pathogenic fungi dead, plays an important role in plant is resisted the defense response of fungal disease.The beta-1,3-glucanase expression amount is few in the normal plants, although but induce lower abduction delivering at pathogenic fungi and exciton, but still can not effectively resist infecting of pathogenic fungi.Therefore people attempt clone Glu gene from plant, and improve the disease resistance of transgenic plant by genetic transformation, but at home and abroad are not reported about peanut Glu gene cloning.
Summary of the invention
For β-1 in the normal peanut in the prior art; 3-dextranase expression amount seldom is difficult to effectively resist the present situation that pathogenic fungi infects; the invention provides peanut β-1; 3-glucanase gene and the application in improving the peanut disease resistance thereof; the present invention clones the full length cDNA sequence of Glu gene in peanut; and make up Overexpression vector conversion peanut, and study the expression pattern of Glu gene in transgenic peanuts, filter out the transgenic peanuts strain of overexpression high resistance fungal disease.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
Peanut beta-1,3-glucanase gene, its sequence table is shown in SEQ ID No:1.
This gene the 134bp～567bp is an intron, and the open reading frame size is 1047bp, and translation product contains 348 amino acid, 27 amino acid of signal peptide length wherein, protein molecular weight 38806.55 dalton.
The PCR primer of cloning described Ah-Glu gene is:
Forward primer: 5 '-AGCTTCAGCTTCACCTCTCG-3 ',
Reverse primer: 5 '-CAACAGCACCTCTTCAAGTG-3 '.
The pcr amplification system of cloning described Ah-Glu gene is: cumulative volume 25 μ L, dNTPs 0.4mmol/L wherein, each 0.4mmol/L of upstream and downstream primer, 10 * PCR buffer, 2.5 μ L, Mg
2+3.0mmol/L, genomic dna 50ng, Taq polysaccharase 2.5U, distilled water 13.5 μ L.
The pcr amplification program is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 1min, 58 ℃ of renaturation 1min, 72 ℃ are extended 1min, 35 circulations; 72 ℃ are extended 10min.
The present invention also provides the plant expression vector of described peanut beta-1,3-glucanase gene.
Described plant vector is pCAMBIA1301-Glu.
The present invention also provides the application of described peanut beta-1,3-glucanase gene in improving the peanut disease resistance.Transfer-gen plant strengthens the resistance of the cercospora black spot of peanut and brown spot.
Change peanut beta-1,3-glucanase gene over to peanut, the primer that the peanut transfer-gen plant is carried out the PCR detection is:
Upstream primer: 5 '-CACACCGATACCATCAGAGAT-3 ',
Downstream primer: 5 '-TCACCGAAGGGCATGCCAGTC-3 '.
It is 25 μ L that the peanut transfer-gen plant is carried out the reaction system that PCR detects, dNTPs 0.25mmol/L wherein, each 0.4mmol/L of upstream and downstream primer, 10 * PCR buffer, 2.5 μ L, genomic dna 50ng, Taq polysaccharase 1.0U, distilled water 13.5 μ L.
The PCR response procedures is: 94 ℃ of sex change 4min; 94 ℃ of sex change 50s, 56 ℃ of annealing 50s, 72 ℃ are extended 40s, 30 circulations; 72 ℃ are extended 10min.
Compared with prior art, advantage of the present invention and positively effect are:
1, the present invention has cloned β-1 from peanut, 3-glucanase gene (Ah-Glu), sequencing result show that this gene open reading frame size is 1047bp, and translation product contains 348 amino acid, 27 amino acid of signal peptide length wherein, protein molecular weight 38806.55 dalton.
2, this gene is connected to structure plant Overexpression vector pCAMBIA1301-Glu on the pCAMBIA1301 plasmid, and transforms peanut by agrobacterium-mediated transformation.RT-PCR result shows that transfer-gen plant Ah-Glu gene expression amount is apparently higher than the non-transgenic plant.Transfer-gen plant Isolated leaf inoculation cercospora black spot of peanut bacterium and land for growing field crops are observed, and disease resistance all is higher than the non-transgenic plant.Technical scheme of the present invention will and avoid security hidden danger to open up new approach for peanut genetically engineered breeding for disease resistance, have important theory significance and wide application prospect.
After reading the specific embodiment of the present invention by reference to the accompanying drawings, other characteristics of the present invention and advantage will become clearer.
Description of drawings
Fig. 1 is Ah-Glu gene PCR and RT-PCR result among the present invention.M:Maker G; 1: the water contrast; The 2:DNA amplification; 3: without the RT-PCR of Induced by Salicylic Acid; 4: through the RT-PCR of Induced by Salicylic Acid.
Fig. 2 is Ah-Glu protein blast comparison result figure.
Fig. 3 is plant expression vector figure pCAMBIA1301-Glu constructed among the present invention.
Fig. 4 is pcr amplification and the double digestion checking of plant expression vector pCAMBIA1301-Glu among the present invention.M1:DL 2000maker; 1: the water contrast; The 2:PCR amplification; 3:XbaI and PstI double digestion result; M2:DL15000maker.
Fig. 5 is peanut genetic transformation process agriculture bacillus mediated among the present invention.5-1: body embryonal induction; 5-2: body embryo germination; 5-3: regeneration plant.
Fig. 6 is that the PCR of figure transgenic peanuts among the present invention detects.M:DL2000Maker; 1: the water contrast; 2: plasmid; 3: the non-transgenic plant; 4-13: transfer-gen plant.
Fig. 7 is the GUS staining examine result of transfer-gen plant among the present invention.The left side is the non-transgenic plant, and the right side is transfer-gen plant.
Fig. 8 is transfer-gen plant Ah-Glu gene RT-PCR amplification among the present invention.M:Marker G; 1: plasmid; 2: the water contrast; 3: the non-transgenic plant; 4-8: transfer-gen plant.
Fig. 9 is transfer-gen plant Ah-Actin reference gene RT-PCR amplification among the present invention.M:Marker DL2000; 1: plasmid; 2: the water contrast; 3: the non-transgenic plant; 4-8: transfer-gen plant.
Figure 10 is that transgenic peanuts Isolated leaf inoculation cercospora black spot of peanut bacterium detects the disease resistance result among the present invention.The left side is the non-transgenic plant leaf, and the right side is rotaring gene plant blade.
Figure 11 be non-transgenic and transgenic peanuts among the present invention field resistance relatively.The left side is the non-transgenic plant, and the right side is transfer-gen plant.
Embodiment
Below in conjunction with the drawings and specific embodiments technical scheme of the present invention is described in further detail.
One, experiment material
1. bacterial classification and peanut varieties
Bacillus coli DH 5 alpha, agrobacterium tumefaciens bacterial strain EHA105 are preserved by Qingdao Agricultural University genetic research chamber, and the gene clone material is peanut varieties " flower is educated No. 20 ", and transgenic acceptor is peanut varieties " flower is educated No. 22 ".
2. plant culture
The substratum that adopts in the peanut genetic transformation has 3 kinds:
(1) body embryonal induction substratum (MS-B
5, add 10mg/L 2,4-D);
(2) body embryo germination substratum (MS-B
5, add 4mg/L BAP);
(3) screening culture medium: (MS-B
5, add the 15mg/L Totomycin).
Two, experimental technique
The present invention includes following concrete experimental procedure:
1, the acquisition of the extraction of peanut genome, mRNA and cDNA sequence
Utilize the CTAB method to extract the Peanut Leaflet genomic dna.The Trizol method is adopted in the extraction of mRNA: spray peanut leaf with the 1.5mmol/L salicylic acid solution and induce processing 48 hours, get 0.3g through the peanut leaf of Induced by Salicylic Acid and the corresponding blank without Induced by Salicylic Acid, add 1mL Trizol ice bath and grind, place 5min under the room temperature.Add the 2mL chloroform, acutely mix 3min, 4 ℃ of centrifugal 15min of lower 12000r/min.Supernatant moves in the centrifuge tube of aseptic and RNase free, adds 2 times of volume ethanol, places 10min, 4 ℃ of centrifugal 5min of lower 12000r/min for-20 ℃.Abandon supernatant, precipitate twice with 75% washing with alcohol.After slightly dried, use the aqua sterilisa dissolution precipitation without RNase.The synthetic of cDNA undertaken by reverse transcription test kit specification sheets step.
2, peanut beta-1,3-glucanase gene cloning
Conservative amino acid sequence (GeneBank:AAA32864.1) according to the Arabidopis thaliana beta-1,3-glucanase exists
Http:// www.ncbi.nlm.nih.govBLAST/Website carries out the TBLASTN retrieval of peanut est database, obtains the with it similar peanut EST group of part, utilizes
Http:// pbil.univ-lyon1.fr/cap3.phpOn CAP3 software, carry out est sequence splicing and be assembled into contig (contig), be the peanut beta-1,3-glucanase gene cDNA sequence of prediction, total length 1326bp.
Design Ah-Glu gene amplification primer according to this forecasting sequence:
Forward primer: 5 '-AGCTTCAGCTTCACCTCTCG-3 ' (SEQ ID No:3);
Reverse primer: 5 '-CAACAGCACCTCTTCAAGTG-3 ' (SEQ ID No:4).
With this primer DNA and cDNA are increased, DNA cloning product 1.5kb, amplified production 1.1kb after Induced by Salicylic Acid, without Induced by Salicylic Acid without amplified production (as shown in Figure 1).Amplified production recovery, purifying rear clone to the pMD18-T carrier, are obtained the T-Glu recombinant plasmid, send living worker Shanghai company to check order.Shown in the following SEQ ID of the sequencing result No:1.
SEQ ID No:1 sequence total length 1563bp, ORF:1481bp, intron: 434bp (is that 134bp～567bp), exon: 1047bp meets GT......AG montage rule.
The cDNA sequence is shown in SEQ ID No:2, and the cDNA sequence is 1129bp altogether; Encoding sequence is 1047bp, and coding produces 348 amino acid; The aminoacid sequence that coding produces is shown in SEQ ID NO:5.
3, the structural analysis of peanut beta-1,3-glucanase gene and function prediction and homology analysis
Utilize NCBI network resource and DNAMAN software that clone's Ah-Glu gene coded protein formation and physic-chemical property is carried out analyses and prediction.With the protein sequence (SEQ ID NO:5) of prediction, usefulness BLASTP (
Http:// blast.ncbi.nlm.nih.gov/) the search Protein Data Bank, carry out homology search and comparison, make up the protein evolution tree, as shown in Figure 2.
The aminoacid sequence of Ah-Glu proteins encoded and other plant beta-1,3-glucanase genes encoding is compared, and homology is 41%～90%, reaches 90% with the homology of soybean.
4, the structure of plant expression vector
Use XbaI, PstI is double digestion pCAMBIA1301 and T-Glu carrier respectively, reclaims the purpose fragment, with the T4DNA ligase enzyme Glu fragment is connected to Actinp promotor back among the expression vector pCAMBIA1301, obtain recombinant plasmid pCAMBIA1301-Glu, as shown in Figure 3.Recombinant plasmid is through pcr amplification and XbaI, and the PstI double digestion all obtains the 1.1kb fragment, as shown in Figure 4.
5, peanut Study on Genetic Transformation
Utilize agrobacterium-mediated transformation that recombinant plasmid pCAMBIA1301-Glu is transformed the little leaf explant of peanut embryo.At first the peanut embryo leaflet is downcut together with the plumular axis radicle and carry out surface sterilization, sterilized water soaked overnight; The embryo leaflet was separated out preculture 3 days from the plumular axis radicle; Use OD
600Be that 0.6 Agrobacterium is infected, infected 10 to 15 minutes that dark was cultivated 3 days altogether; The embryo leaflet that common cultivation is finished moves to 4 weeks of inductor embryo in the interpolation 15mg/L hygromycin selection agent body embryonal induction substratum; The body embryo that induces is moved to the body embryo germination culture medium culturing 4 months of adding the agent of 15mg/L hygromycin selection, can obtain the seedling of regenerating, as shown in Figure 5.
6, transgenic plant Molecular Identification and GUS dyeing
Extract the transfer-gen plant genomic dna, utilize gus gene to carry out pcr amplification and detect, obtain the purpose fragment of about 410bp, as shown in Figure 6.The GUS dyeing process: the clip blade is put in the sky centrifuge tube, adds the GUS staining fluid in the centrifuge tube, covers the centrifuge tube lid, be put into 37 ℃ 3 hours～spend the night, blade is changed over to decolours in 70% alcohol 2～3 times, to negative control material be yellow or white.Coloration result as shown in Figure 7.
Gus gene detects primer:
Upstream primer: 5 '-CACACCGATACCATCAGAGAT-3 ' (SEQ ID No:6),
Downstream primer: 5 '-TCACCGAAGGGCATGCCAGTC-3 ' (SEQ ID No:7).
7, transfer-gen plant RT-PCR analyzes
Extract the total RNA of transgenosis, reverse transcription is cDNA, utilizes the Ah-Glu gene primer to carry out the RT-PCR augmentation detection, and amplification primers is:
Forward primer: 5 '-AGCTTCAGCTTCACCTCTCG-3 ',
Reverse primer: 5 '-CAACAGCACCTCTTCAAGTG-3 '.
Transfer-gen plant all amplifies the fragment of 1.1kb, and the non-transgenic plant does not amplify corresponding purpose fragment, as shown in Figure 8.Illustrate that the foreign gene that imports obtains expressing in the mRNA level.
Carry out the RT-PCR augmentation detection take peanut Actin gene as confidential reference items, non-transgenic and transfer-gen plant all amplify corresponding 380bp fragment (such as Fig. 9).The Ah-Actin amplimer is:
Forward primer: 5 '-GTGGCCGTACAACTGGTATTGT-3 ',
Reverse primer: 5 '-ATGGATGGCTGGAAGAGAACT-3 '.
8, the disease resistance of transgenic plant detects
With being immersed in the cercospora black spot of peanut bacterium spore liquid behind the wet cloth wiping excised leaf that speckles with tween, the excised leaf that speckles with spore and mycelia is put into moistening sterilization sandy soil, after moisturizing cultivated for 3 weeks, observe rotaring gene plant blade and the non-transgenic plant leaf situation of catching an illness.Grafting is observed its under field conditions (factors) disease-resistant situation in field to the transfer-gen plant in field.The result shows, connects in bacterium and the field self-sow situation stripped, and the occurring degree of transfer-gen plant is all than a little less than the non-transgenic plant.Such as Figure 10, shown in Figure 11.
Above embodiment is only in order to illustrating technical scheme of the present invention, but not limits it; Although with reference to previous embodiment the present invention is had been described in detail, for the person of ordinary skill of the art, still can make amendment to the technical scheme that previous embodiment is put down in writing, perhaps part technical characterictic wherein is equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution break away from the spirit and scope of the present invention's technical scheme required for protection.
Claims (10)
1. peanut beta-1,3-glucanase gene
Ah-Glu, its sequence is shown in SEQ ID No:1.
2. peanut β-1 according to claim 1, the 3-glucanase gene, it is characterized in that this gene the 134bp ~ 567bp is an intron, the open reading frame size is 1047bp, translation product contains 348 amino acid, 27 amino acid of signal peptide length wherein, protein molecular weight 38806.55 dalton.
3. peanut beta-1,3-glucanase gene according to claim 1 is characterized in that cloning described
Ah-GluThe PCR primer of gene is:
Forward primer: 5'-AGCTTCAGCTTCACCTCTCG-3',
Reverse primer: 5'-CAACAGCACCTCTTCAAGTG-3'.
4. peanut beta-1,3-glucanase gene according to claim 1 is characterized in that cloning described
Ah-GluThe pcr amplification system of gene is: cumulative volume 25mL, dNTPs 0.4 mmol/L wherein, each 0.4 mmol/L of upstream and downstream primer, 10 * PCR buffer 2.5mL, Mg
2+3.0 mmol/L, genomic dna 50ng, Taq polysaccharase 2.5U, distilled water 13.5mL;
The pcr amplification program is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 1min, 58 ℃ of renaturation 1min, 72 ℃ are extended 1 min, 35 circulations; 72 ℃ are extended l 0min.
5. the plant expression vector that contains peanut beta-1,3-glucanase gene claimed in claim 1.
6. the plant expression vector of peanut beta-1,3-glucanase gene according to claim 5 is characterized in that described plant vector is pCAMBIA1301-Glu.
7. the application of peanut beta-1,3-glucanase gene according to claim 1 in improving the peanut disease resistance.
8. the application of peanut beta-1,3-glucanase gene according to claim 7 in improving the peanut disease resistance is characterized in that transfer-gen plant strengthens the resistance of the cercospora black spot of peanut and brown spot.
9. the application of peanut beta-1,3-glucanase gene according to claim 7 in improving the peanut disease resistance is characterized in that changing peanut beta-1,3-glucanase gene over to peanut, and the primer that the peanut transfer-gen plant is carried out the PCR detection is:
Upstream primer: 5-CACACCGATACCATCAGAGAT-3,
Downstream primer: 5-TCACCGAAGGGCATGCCAGTC-3.
10. peanut β-1 according to claim 9, the application of 3-glucanase gene in improving the peanut disease resistance, it is characterized in that it is 25 μ L that peanut transfer-gen plant gus gene is carried out the reaction system that PCR detects, dNTPs 0.25 mmol/L wherein, each 0.4 mmol/L of upstream and downstream primer, 10 * PCR buffer, 2.5 mL, genomic dna 50ng, Taq polysaccharase 1.0 U, distilled water 13.5mL; The PCR response procedures is: 94 ℃ of sex change 4 min; 94 ℃ of sex change 50s, 56 ℃ of annealing 50s, 72 ℃ are extended 40s, 30 circulations; 72 ℃ are extended 10 min.
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| CN104561060A (en) * | 2014-05-30 | 2015-04-29 | 河北省科学院生物研究所 | Beta-1, 3-1, 4-dextranase gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101134949A (en) * | 2006-08-30 | 2008-03-05 | 中国农业科学院饲料研究所 | Beta-glucanase, encoding gene thereof, recombinant plasmid and bacterial strain and uses thereof |
| CN101182527A (en) * | 2007-10-10 | 2008-05-21 | 邢苗 | Alkaline endo glucanase gene, recombinase and applications thereof |
| CN101280290A (en) * | 2008-05-13 | 2008-10-08 | 江南大学 | Genetic engineering bacteria for producing high-thermal stability recombinant beta-glucanase and construction thereof |
| CN101285071A (en) * | 2008-05-13 | 2008-10-15 | 江南大学 | Recombination bacillus coli of high producing beta-glucanase and construction thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101134949A (en) * | 2006-08-30 | 2008-03-05 | 中国农业科学院饲料研究所 | Beta-glucanase, encoding gene thereof, recombinant plasmid and bacterial strain and uses thereof |
| CN101182527A (en) * | 2007-10-10 | 2008-05-21 | 邢苗 | Alkaline endo glucanase gene, recombinase and applications thereof |
| CN101280290A (en) * | 2008-05-13 | 2008-10-08 | 江南大学 | Genetic engineering bacteria for producing high-thermal stability recombinant beta-glucanase and construction thereof |
| CN101285071A (en) * | 2008-05-13 | 2008-10-15 | 江南大学 | Recombination bacillus coli of high producing beta-glucanase and construction thereof |
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