CN103088035B - Application of soybean agglutinin gene lec-s - Google Patents
Application of soybean agglutinin gene lec-s Download PDFInfo
- Publication number
- CN103088035B CN103088035B CN201310008139.6A CN201310008139A CN103088035B CN 103088035 B CN103088035 B CN 103088035B CN 201310008139 A CN201310008139 A CN 201310008139A CN 103088035 B CN103088035 B CN 103088035B
- Authority
- CN
- China
- Prior art keywords
- tobacco
- lec
- plant
- resistant
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108010048090 soybean lectin Proteins 0.000 title claims abstract description 24
- 241000196324 Embryophyta Species 0.000 claims abstract description 55
- 241000723873 Tobacco mosaic virus Species 0.000 claims abstract description 31
- 201000010099 disease Diseases 0.000 claims abstract description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 14
- 230000009261 transgenic effect Effects 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 45
- 239000013612 plasmid Substances 0.000 claims description 34
- 241000607479 Yersinia pestis Species 0.000 claims description 11
- 241001477931 Mythimna unipuncta Species 0.000 claims description 4
- 235000002637 Nicotiana tabacum Nutrition 0.000 abstract description 88
- 241000208125 Nicotiana Species 0.000 abstract description 85
- 238000011160 research Methods 0.000 abstract description 6
- 208000035240 Disease Resistance Diseases 0.000 abstract description 4
- 241000238631 Hexapoda Species 0.000 abstract description 4
- 238000009395 breeding Methods 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 244000003416 Asparagus officinalis Species 0.000 abstract 1
- 235000005340 Asparagus officinalis Nutrition 0.000 abstract 1
- 108700019146 Transgenes Proteins 0.000 description 36
- 239000003550 marker Substances 0.000 description 17
- 238000011081 inoculation Methods 0.000 description 16
- 206010039509 Scab Diseases 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 229940088598 enzyme Drugs 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 9
- 239000013641 positive control Substances 0.000 description 8
- 230000019617 pupation Effects 0.000 description 8
- 241000256247 Spodoptera exigua Species 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 239000013642 negative control Substances 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 102100036816 Eukaryotic peptide chain release factor GTP-binding subunit ERF3A Human genes 0.000 description 6
- 101000906005 Fasciola hepatica Glutathione S-transferase class-mu 26 kDa isozyme 1 Proteins 0.000 description 6
- 101000851788 Homo sapiens Eukaryotic peptide chain release factor GTP-binding subunit ERF3A Proteins 0.000 description 6
- 101001071694 Homo sapiens Glutathione S-transferase Mu 1 Proteins 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 241000589158 Agrobacterium Species 0.000 description 5
- 235000016068 Berberis vulgaris Nutrition 0.000 description 5
- 241000335053 Beta vulgaris Species 0.000 description 5
- 235000010469 Glycine max Nutrition 0.000 description 5
- 244000068988 Glycine max Species 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 4
- 108090001090 Lectins Proteins 0.000 description 4
- 102000004856 Lectins Human genes 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 239000002523 lectin Substances 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 3
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 3
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 3
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 3
- 244000061176 Nicotiana tabacum Species 0.000 description 3
- 108010047620 Phytohemagglutinins Proteins 0.000 description 3
- 238000002105 Southern blotting Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000004665 defense response Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 230000001885 phytohemagglutinin Effects 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- -1 beta galactose aldehyde Chemical class 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000013043 chemical agent Substances 0.000 description 2
- 235000019504 cigarettes Nutrition 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 108010011619 6-Phytase Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 101001009070 Homo sapiens HBS1-like protein Proteins 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108020005089 Plant RNA Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000002507 anti-phytoviral effect Effects 0.000 description 1
- 208000027697 autoimmune lymphoproliferative syndrome due to CTLA4 haploinsuffiency Diseases 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 1
- 229960003669 carbenicillin Drugs 0.000 description 1
- RTYJTGSCYUUYAL-YCAHSCEMSA-L carbenicillin disodium Chemical compound [Na+].[Na+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)C(C([O-])=O)C1=CC=CC=C1 RTYJTGSCYUUYAL-YCAHSCEMSA-L 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 1
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000007852 inverse PCR Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000001418 larval effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000024241 parasitism Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000010496 root system development Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the field of genetic engineering and relates to an application of a soybean agglutinin gene lec-s. The soybean agglutinin gene lec-s (accession number in GenBank is DQ235094) is subjected to transgenic tobacco research and disease resistance and insect resistance research of transgenic tobacco, and the obtained transgenic tobacco expresses obvious resistance to TMV (Tobacco Mosaic Virus), and also expresses good resistance to asparagus caterpillar. The facts prove that the soybean agglutinin gene lec-s can be applied to breeding disease resistant and/or insect resistant transgenic plant varieties.
Description
Technical field
The invention belongs to genetically engineered field, relate to the application of soybean lectin plain gene lec-s.
Background technology
Tobacco mosaic virus (TMV) (Tobacco mosaic virus, TMV) is a kind of plant positive chain RNA virus, can infect 38 section's 268 kind of plant, causes the multiple important viroses of plant (Scholthof, 2004).Because TMV resistance is extremely strong, to host's obligatory parasitism, add that plant lacks complete immune metabolic system, make this disease be difficult to control.Beet armyworm results from South East Asia at first, but is also worldwide a kind of important insect pest, can endanger multiple garden crop and vegetables (Smagghe et al, 2003).The use of chemical agent is a kind of important measures that prevent and treat at present plant pest, but the abuse of chemical agent has caused serious impact to environment and agricultural product security.The engineered development of plant disease-resistant worm is in recent years for control plant pest provides a new way, main thought is that resistance related gene is proceeded in recipient plant by genetic engineering means, thereby obtain plant disease-resistant worm new variety, and there is the features (Kang et al, 2005) such as genetic resources is many, breeding cycle is short compared with traditional breeding method.
Lectin is the glycoprotein in the nonimmune source of a kind of energy aggegation red corpuscle, polysaccharide or saccharide complex.So far, people have isolated lectin (Tateno et al, 1998 from various plants, animal, bacterium, virus and fungi; Wang et al, 1996; Inamori and Saito, 1999; Leteux and Chai, 2000).In many lectins of having found up to now, phytohemagglutinin is maximum Yi Ge family.In these plants, especially abundant with lectin content in fabaceous seed.Phytohemagglutinin has important defense function in plant.Many researchs report in the past shows, phytohemagglutinin can improve resistance (Ye et al, 2001 of plant to the multiple pathogens such as fungi, bacterium, nematode and harmful insect; Ooi et al, 2000; Machuka and Oladapo, 2000).But the research about soybean agglutinin Antiphytoviral have not been reported.In contriver's previous work, utilize RACE from soybean varieties, to close a separated new soybean lectin plain gene, the called after lec-s(DQ235094 of obtaining rich No. 29 with inverse PCR technology).
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, the application of a kind of new soybean lectin plain gene lec-s is provided.
Object of the present invention is achieved through the following technical solutions:
The application of soybean lectin plain gene lec-s in cultivating disease-resistant and/or pest-resistant transgenic plant kind, wherein, the GenBank accession number of described soybean lectin plain gene lec-s is DQ235094.
Wherein, described disease-resistant be resisting tobacco mosaic virus, the described anti-beet armyworm of pest-resistant finger.
Wherein, the GenBank accession number of described soybean lectin plain gene lec-s is DQ235094 in the application of the recombinant plasmid that contains soybean lectin plain gene lec-s in cultivating disease-resistant and/or pest-resistant transgenic plant kind.
Wherein, described disease-resistant be resisting tobacco mosaic virus, the described anti-beet armyworm of pest-resistant finger.
Beneficial effect:
Transgene tobacco is because its gene transformation technology is ripe, from infecting to obtaining the cycle of regrowth shorter, be used as a work pattern system, be often used to carry out plant disease-resistant worm Journal of Sex Research.The present invention has carried out the research of transgene tobacco and disease-resistant insect-resistance thereof to soybean lectin plain gene lec-s, and the transgene tobacco of acquisition shows significant resistance to TMV, and beet armyworm is also showed to good resistance.The detected result of qRT-PCR also shows, with turn empty carrier tobacco and compare, 12h and 24h after transgene tobacco inoculation TMV, the equal remarkable up-regulated expression of Analysis of Defence Genes Involved PR-1a, Pa1 and GST1 and HR marker gene hsr515 in tobacco, and finally show as transgene tobacco the resistance of TMV is strengthened.So we infer, lec-s gene, at tobacco expression in vivo, may activate disease resistance SA signal path and hypersensitive cell death (hypersensitive cell death, the HCD) signal path in tobacco simultaneously, has given the resistance of tobacco to TMV.Visible soybean lectin plain gene lec-s is proceeded to farm crop by genetic engineering means, can obtain the transgenic plant new variety with disease resistance and insect-resistance.
Accompanying drawing explanation
Fig. 1 lec-s gene RT-PCR amplification figure (A) and pMD18-T Simple::lec-s(B) enzyme cut proof diagram A:M. molecular weight marker (DNA Marker DL2000); 1 ~ lec-s 2. increases from soybean varieties 29; 3. clear water contrasts B:M. molecular weight marker (DNA Marker DL2000); 1.Sma I and Xba I enzyme are cut pMD18-T Simple::lec-s
The amino acid of Fig. 2 soybean agglutinin lec-s genes encoding is with the homology comparison of other soybean agglutinin
The site that has * is the position that similarity is high
Fig. 3 plant recombinant expression plasmid pBI121::lec-s builds collection of illustrative plates
LB.T-DNA left margin; RB.T-DNA right margin; Npt II. neomycin phosphotransferase gene; Pro. promotor; Ter. terminator; Gus. beta galactose aldehyde phytase gene
Fig. 4 plant expression vector pBI121::lec-s builds cleavage map
M1. molecular weight marker (DNA Marker DL2000); M2. molecular weight marker (DNA Marker DL15000); 1 ~ 2. Sma I and Xba I double digestion for plasmid pBI121::lec-s; 3. Sma I and Xba I double digestion for plasmid pBI121.
Fig. 5 recombinant vectors pBI121::lec-s transforms the PCR checking of Agrobacterium
M. molecular weight marker (DNA Marker DL2000); W. negative control, clear water; P. positive control, plasmid pBI121::lec-s; 1～3. different transformant.
Fig. 6 transgene tobacco T
0the PCR in generation detects
A, B:M. molecular weight marker (DNA Marker DL2000); P. positive control, plasmid pBI121::lec-s; W. negative control, clear water; CK. negative control, unconverted plant; 1 ~ 45. transfer-gen plant;
C:M. molecular weight marker (DNA Marker DL2000); P. positive control, plasmid pBI121; W. negative control, clear water;
CK. negative control, unconverted plant; 1 ~ 24. turns empty carrier plant.
Fig. 7 transfer-gen plant T
0the Southern hybridization analysis in generation
M. molecular weight marker (DNA Marker DL2000, λ-Hind III digest); 1. positive control, lec-s pcr amplification product; 2. unconverted plant; 3. turn empty carrier plant; 4～8. transfer-gen plant
Fig. 8 T
1resistance for transgene tobacco to TMV
WT. unconverted tobacco; VEC. turn empty carrier tobacco; T1-11, T1-20. transgene tobacco
Fig. 9 T
1for scab after transgene tobacco inoculation TMV, count diversity ratio
WT. unconverted tobacco; VEC. turn empty carrier tobacco; T1-11, T1-20. transgene tobacco; Data are expressed as mean+SD; * represent and turn empty carrier tobacco and compare significant difference in 0.05 level
Figure 10 qRT-PCR measures defense response related gene expression
A: 12h after inoculation TMV; B. inoculate 24h after TMV; WT. unconverted tobacco; VEC. turn empty carrier tobacco;
B:T1-11, T1-20. transgene tobacco
Data are expressed as mean+SD; * represent and turn empty carrier tobacco and compare significant difference in 0.05 level
Figure 11 T
1impact for transgene tobacco on beet exigua larvae body weight
WT. unconverted tobacco; VEC. turn empty carrier tobacco; T1-11, T1-20. transgene tobacco
Figure 12 T
1impact for transgene tobacco on beet exigua larvae percentage of pupation
WT. unconverted tobacco; VEC. turn empty carrier tobacco; T1-11, T1-20. transgene tobacco
Embodiment
Related material and reagent in following examples:
Tobacco bred is three lives cigarette (Nicotiana tabacum cv.Samsun NN), gets tobacco leaf as converting material.Agrobacterium tumefaciens (Agrobacterium tumefaciens) bacterial strain EHA105, plant expression vector pBI121 are purchased from Invitrogen.
Tissues of Tobacco substratum be take MS substratum as basis, pH5.8,121 ℃ of sterilizing 15min.Preculture substratum: MS+6BA0.5mg/L; Be total to culture medium: MS+6-BA1mg/L; Screening culture medium: MS+6-BA1mg/L+Km100mg/L+Cb500mg/L; Root media: 1/2MS+Km100mg/L+Cb200mg/L+IAA0.2mg/L; Agrobacterium is cultivated and uses YEB substratum.
Taq archaeal dna polymerase, restriction enzyme, T4 ligase enzyme, dNTPs, nucleic acid molecule quality standard, DNase I, PrimeScript
tMreverse Transcriptase and SYBR Premix ExTaq
tMkit is all purchased from TaKaRa company.Plasmid extraction and DNA gel reclaim test kit purchased from Axygen company.Plant RNA extraction test kit Plant RNAKit is purchased from Omega company.Kantlex (Kanamycin), Pyocianil (Carbenicillin) are purchased from Suo Laibao bio tech ltd, Shanghai.
The clone of the structure 1.1lec-s of embodiment 1pBI121::lec-s plasmid
Get soybean varieties and close rich No. 29 blades, use the Plant RNAKit plant RNA extraction test kit of Omega company to extract the total RNA of soybean, use the DNase I without RNase to process purifying.Use PrimeScript
tMreverse Transcriptase(Takara) carry out the synthetic cDNA of RNA reverse transcription.It is template that the total RNA of 1 μ g of take obtains cDNA the first chain through ThermoScript II, and lec-s-F/lec-s-R is primer, reacts the lec-s gene fragment that contains Xba I/Sma I restriction enzyme site to two ends of amplification by PCR.Adopt the AxyPrep DNA gel recovery test kit of Axygen company to reclaim PCR electrophoretic band.The lec-s fragment of recovery is connected to sequencing vector pMD18-T Simple Vector, obtains plasmid pMD-18T Simple vector::lec-s.Plasmid pMD-18T Simple vector::lec-s transforms DH5 α competent cell, use the plasmid of the AxyPrep plasmid DNA small volume of reagent box extraction primary dcreening operation positive colony of Axygen company, through PCR screening positive clone, the plasmid extracting from the positive colony through PCR screening, use TaKaRa company restriction enzyme to carry out enzyme and cut evaluation, enzyme is cut to the correct plasmid of checking and send order-checking.
PCR reaction system is:
Wherein, upstream primer lec-s-F:5 '-
tCTAGAaTGGCCACCTCCAACTTCTC-3 ' (containing XbaI, SEQ ID NO.1)
Downstream primer lec-s-R:5 '-
cCCGGGtTAGATGGCCTCATTGAGCAC-3 ' (Sma I, SEQ ID NO.2) PCR response procedures is:
95 ℃ of denaturation 5min; 95 ℃ of sex change 45sec, 58.6 ℃ of renaturation 45sec, 72 ℃ of extension 40sec, 30 circulations; Last 72 ℃ are extended 10min.
Utilize RT-PCR method to close and rich 29, amplify lec-s gene (Figure 1A) from soybean varieties, be connected to (Figure 1B) after pMD18-T Simple carrier, order-checking, result shows that this gene is 849bp, with the homology of DQ235094 sequence in database (GenBank) be 100%.The aminoacid sequence that soybean agglutinin lec-s gene is inferred is with upper listed other soybean agglutinin le1 of GenBank and le2 compare (Fig. 2), result shows: the aminoacid sequence that this full-length cDNA is coded and le1 homology 60%, with le2 homology be 34%.
The structure of 1.2pBI121::lec-s plasmid
Extract plasmid pMD-18T Simple vector::lec-s and pBI121, with Sma I and Xba I enzyme, cut pMD-18T Simple vector::lec-s carrier and pBI121 plasmid, lec-s is connected on pBI121 carrier, obtains pBI121::lec-s plasmid (Fig. 3).PBI121::lec-s plasmid transforms DH5 α competent cell, use the AxyPrep plasmid DNA small volume of reagent box of Axygen company to extract pBI121::lec-s plasmid, with Sma I and Xba I enzyme, cut pBI121::lec-s plasmid, enzyme is cut to the correct plasmid of checking and send order-checking.Method with reference to Yu Yunzhou (2003) etc., imports edaphic bacillus EHA105 by recombinant plasmid.Use the AxyPrep plasmid DNA small volume of reagent box of Axygen company to extract EHA105/pBI121::lec-s plasmid, with Sma I and Xba I enzyme, cut EHA105/pBI121::lec-s plasmid, enzyme is cut to the correct plasmid of checking and send order-checking.
As can be seen from Figure 4, after recombinant plasmid pBI121::lec-s enzyme being cut with Sma I and Xba I, obtained the fragment of expection 849bp.Sequence in the sequencing result of lec-s gene and database is compared, and homology is 100%.This explanation lec-s gene has successfully been linked pBI121 plasmid, and vivoexpression carrier pBI121::lec-s successfully constructs.The restructuring transgene carrier freeze-thaw method that checking is correct transforms Agrobacterium tumefaciens EHA105.After resistance list bacterium colony PCR after transforming is detected, select positive colony and send order-checking, sequencing result and lec-s gene order are compared, homology is 100%.The result transgene carrier that shows to recombinate successfully proceeds to Agrobacterium tumefaciens EHA105(Fig. 5).
The tobacco genetic transformation that embodiment 2 is agriculture bacillus mediated
The conversion of tobacco adopts agriculture bacillus mediated Ye Panfa.Get tobacco leaf disk (diameter 0.5cm) as transformation receptor, in pre-culture medium, after preculture 1～2d, immerse the Agrobacterium bacterium liquid inductance of getting ready and dye 20min, tobacco leaf disc and Agrobacterium dark are cultivated after 2d altogether, proceed in the screening training base containing carboxylic Bian mycin (500mg/L) and kantlex (100mg/L).25 ℃, L: D=16h: 8h cultivation, within 1 week, subculture is 1 time.Within 1 week, left and right leaf dish starts to break up resistant calli, then differentiates gradually resistant buds.Within 3 ~ 6 weeks, start to have bud differentiation.After green bud grows to 2～3cm height, cut regeneration bud and be placed in root media and cultivate.Seedling plant height to be transformed 6～8cm, the good rear uncork hardening 2d of root system development, then intermediate house is cultivated.
Wherein, Tissues of Tobacco substratum be take MS substratum as basis (Murashige and Skoog, 1962), pH5.8,121 ℃ of sterilizing 15min.Preculture substratum: MS+6-BA0.5mg/L; Be total to culture medium: MS+6-BA1mg/L; Screening culture medium: MS+6-BA1mg/L+Km100mg/L+Cb500mg/L; Root media: 1/2MS+Km100mg/L+Cb200mg/L+IAA0.2mg/L; Agrobacterium is cultivated and uses YEB substratum.
Previous step obtains altogether transgene tobacco resistance seedling and amounts to 45 strains, and plant genomic DNA extracts the CTAB method that adopts.Use transgene tobacco T
0for the total DNA of blade, be template, empty carrier transformed plant and the total DNA of unconverted plant make negative control, and plasmid pBI121::lec-s does positive control and carries out pcr amplification.According to goal gene primers lec-s-F/lec-s-R(SEQ ID NO.1/SEQ ID NO.2), amplifying target genes (amplified production 849bp), detects 31 strains that have that are positive.Detected result shows, transgenic tobacco plant is the same with positive control plasmid, all can amplify the specific fragment 849bp of corresponding goal gene, and unconverted plant and turn sky body plant and do not have amplified band to occur (Fig. 6 A).Therefore the result proof foreign gene that, PCR detects has been transferred in these regeneration plants.In order to ensure the accuracy of pcr amplification, also use part carrier primers pBI-vector-F/pBI-vector-R(SEQ ID NO.13/SEQ ID NO.14) increase.Detected result shows, transgenic tobacco plant is the same with positive control plasmid, all can amplify the specific fragment 1303bp of corresponding goal gene, and unconverted plant and clear water contrast do not have amplified band to occur (Fig. 6 B) turning specific fragment 454bp(Fig. 6 C that all can amplify corresponding goal gene in empty carrier plant).Therefore the result preliminary proof foreign gene that, PCR detects has been transferred in these regeneration plants.
The transfer-gen plant of the selected part PCR positive carries out Southern hybridization analysis, and with unconverted plant with turn empty carrier plant and make negative control, lec-s segment pcr amplification product is done positive control.Use 1.2% agarose gel electrophoresis, voltage 2V/cm, electrophoresis finishes with siphon blotting, PCR product to be transferred on nylon membrane afterwards.Digoxigenin labeled for probe gene (part for 1ec-s gene, about 400bp), mark and hybridizing method carry out according to test kit specification sheets.Results of hybridization (Fig. 7) shows, in 5 strain transfer-gen plants, all occur hybridization signal, there are 1 hybridization signal bands of a spectrum in 3 strains wherein, and 2 hybridization signal bands of a spectrum appear in 1 strain, there are 3 hybridization signal bands of a spectrum in 1 strain, illustrates and in corresponding transfer-gen plant, have 1,2 and 3 copy; Unconverted plant and turn empty carrier plant and do not occur hybridization signal.Molecular hybridization further proved foreign gene stable integration arrived in the genome of tobacco.
Embodiment 5T
1anti-TMV effect assessment for transgene tobacco
With the tobacco with batch hot-house culture, 10 times of TMV virus juice dilutions, inoculate respectively after 50 times and 100 times.Choose the even and more concentration of 48-72 hour sequela for formal inoculation.Tobacco T
1in generation, is transplanted the tobacco of latter 6 weeks.Adopt in frictional inoculation method inoculation TMV(side and reach, 1998).Concrete operation step is as follows:
(1) 1g of QuTMVBing leaf texture adds aqua sterilisa 1ml, in clean mortar, is ground into homogenate;
(2) homogenate is added in 2ml ep pipe, low-speed centrifugal is got supernatant and is placed in frozen water standby;
(3) a small amount of silicon carbide (600 order) is sprinkling upon on blade to be inoculated, with sample injector, draw TMV suspension after the dilution of 100ul evenly point drop on blade face, with finger frictional inoculation gently on blade face;
(4) postvaccinal blade water rinses gently, test plant is placed in to 22-25 ℃ of greenhouse and cultivates.
Each processes inoculation 10 strains, 3 blades that leaf age is consistent in every strain inoculation middle part, and test repeats 3 times, compares to turn empty carrier plant at every turn, and a situation arises for the withered spot of 48h " Invest, Then Investigate " blade.Disease resistance is expressed as transfer-gen plant compared with the control, the percentage ratio that scab number reduces, that is: and scab is counted decrement (%)=[(transgene tobacco scab number-turn empty carrier tobacco scab number)/turn empty carrier tobacco scab number] * 100.
After inoculation TMV, on 48h inoculation blade, there is necrotic plaque, for TMV infects the classical symptom of three lives cigarette sequela.The scab of inoculation blade is counted statistics and is shown, compare with the tobacco that turns empty carrier with unconverted, the T1-11 and the T1-20 strain scab digital display work that after inoculation TMV, turn lec-s genetic tobacco reduce, TMV is all shown to obvious resistance (Fig. 8), wherein T1-11 strain scab number approximately reduces 41.72%, T1-20 strain scab and approximately reduces 43.79%(Fig. 9).After unconverted tobacco inoculates TMV with the tobacco that turns empty carrier, scab number does not have notable difference.
Get respectively the transgene tobacco blade of 12h and 24h after inoculation TMV and grind with liquid nitrogen freezing, use test kit to extract the total RNA of blade.After the DNase I without RNase is processed, get respectively 2 μ g RNA and make template, adopt Prime Script
tMreverse Transcriptase carries out reverse transcription.After 1 times of reverse transcription product dilution, get 2 μ L as template, use SYBR Premix Ex Taq
tMkit, adopts Two-step-qRT-PCR on ABI PRISM7500 quantitative real time PCR Instrument, to carry out the mensuration of PR-1a, GST1, Pal and hsr515 gene expression amount, and EF-1a gene is as internal reference, and primer sequence is in Table 1.3 repetitions are all established in test, with empty carrier transformed plant and unconverted plant, compare, and adopt 2
-Δ Δ CTmethod is carried out Data Processing in Experiment.
PCR reaction system:
Pcr amplification reaction condition:
95 ℃, 10sec; 95 ℃, 5sec, 40 circulations; 60 ℃, 34sec; 95 ℃, 15sec; 60 ℃, 1min; 95 ℃, 15sec.
Table 1 primer
After inoculation TMV, in 12h and 24h transgene tobacco blade, the expression of Defense response gene PR-1a, GST1, Pa1 and hsr515 is shown in Figure 10.Result shows, after inoculation TMV12h and 24h, turn in the blade of two strains of lec-s genetic tobacco all remarkable up-regulated expressions of PR-1a, GST1, Pa1 and hsr515 gene, illustrate that TMV infects the mRNA Rapid Accumulation of rear transgene tobacco leaf portion said gene, and in unconverted tobacco and the leaf portion that turns empty carrier tobacco, TMV infects the up-regulated expression that does not cause PR-1a, GST1, Pa1 and hsr515 gene.Infer thus, it may be that PR-1a in transgene tobacco, GST1, Pal and hsr515 gene up-regulated expression are necessary that the soybean agglutinin of lec-s genes encoding is expressed.
Embodiment 7T
1resistance Identification for transgene tobacco to beet armyworm
With reference to the method for (1997) such as Van de Veire, carry out the resistance experiment of transgene tobacco to beet armyworm.Doing a bottom surface diameter is 9cm, the cylindrical plastic cage of high 3cm.With gauze, seal above, prevent that larva from climbing out of, can breathe freely simultaneously.Tobacco excised leaf is placed in cage, with cotton, encases petiole moisturizing.Select beet armyworm 2 instar larvaes to start to test.Each cage is put 3 larvas, 10 cages of each strain.Change fresh blade every day.Death toll and the individual fresh weight of a larva of investigation in every two days, until larvae pupation.Finally according to statistics calculate the survival rate of larvae of beet armyworm, to time used, the percentage of pupation etc. of pupating.
Choose beet armyworm 2 instar larvaes of same batch, respectively feeding wild-type tobacco, turn the transgene tobacco of empty carrier tobacco and two strains, until larvae pupation, the whole cycle continues average approximately 18 days.
Statistics shows, compare with the larva that turns empty carrier tobacco with feeding wild-type tobacco, the beet exigua larvae of feeding transgene tobacco strain T1-11 is (rate of losing weight is 33.83%) after taking food 2 days, and beet exigua larvae body weight after taking food 4 days of feeding transgene tobacco strain T1-20 starts obvious decline (rate of losing weight is 17.34%), reach conspicuous level, and the situation of this weight loss is continued until that larval feeding is after 12 days (Figure 11).
In addition, compare with the larva that turns empty carrier tobacco with feeding wild-type tobacco, the beet exigua larvae of feeding transgene tobacco shows the delay of growing, and percentage of pupation also obviously declines.Feeding wild-type tobacco starts to pupate at the 15th day with the larva that turns empty carrier tobacco, and the larvae pupation of feeding transgene tobacco is postponed till the 16th day.During by the 18th day, feeding wild-type tobacco and the larvae pupation rate that turns empty carrier tobacco reach 100% and 96%, and the larvae pupation rate that takes food transgene tobacco is down to respectively 60%(T1-11) and 73%(T1-20) (Figure 12).
Claims (1)
1. soybean lectin plain gene
lec-sapplication in cultivating disease-resistant and/or pest-resistant transgenic plant kind, wherein, described soybean lectin plain gene
lec-sgenBank accession number be DQ235094; Described disease-resistant be resisting tobacco mosaic virus, the described anti-beet armyworm of pest-resistant finger.
2, contain soybean lectin plain gene
lec-sthe application of recombinant plasmid in cultivating disease-resistant and/or pest-resistant transgenic plant kind, wherein, described soybean lectin plain gene
lec-sgenBank accession number be DQ235094; Described disease-resistant be resisting tobacco mosaic virus, the described anti-beet armyworm of pest-resistant finger.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310008139.6A CN103088035B (en) | 2013-01-10 | 2013-01-10 | Application of soybean agglutinin gene lec-s |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310008139.6A CN103088035B (en) | 2013-01-10 | 2013-01-10 | Application of soybean agglutinin gene lec-s |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103088035A CN103088035A (en) | 2013-05-08 |
| CN103088035B true CN103088035B (en) | 2014-04-16 |
Family
ID=48201165
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310008139.6A Expired - Fee Related CN103088035B (en) | 2013-01-10 | 2013-01-10 | Application of soybean agglutinin gene lec-s |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103088035B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305525B (en) * | 2012-03-14 | 2015-02-18 | 林忠平 | Lectin gene of locust tree as well as coded protein and application thereof |
| CN111254148B (en) * | 2018-11-30 | 2023-03-24 | 东北农业大学 | Cultivation method and application of soybean mosaic virus resistant gene GmST1 and GmST1 transgenic soybeans |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1222578A (en) * | 1998-10-06 | 1999-07-14 | 中国农业科学院生物技术研究中心 | Composite coded homoptera pest-resisting gene of galanthus agglutinin and its application |
| CN1357553A (en) * | 2001-12-11 | 2002-07-10 | 中国科学院遗传研究所 | Heliangine and its encoding gene and application in insect-resisting plant gene engineering |
| CN1386859A (en) * | 2002-06-20 | 2002-12-25 | 上海复旦迪恩生物技术有限公司 | Coding sequence of arisaema agglutinant protein and its application |
| CN103305525A (en) * | 2012-03-14 | 2013-09-18 | 林忠平 | Lectin gene of locust tree as well as coded protein and application thereof |
-
2013
- 2013-01-10 CN CN201310008139.6A patent/CN103088035B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1222578A (en) * | 1998-10-06 | 1999-07-14 | 中国农业科学院生物技术研究中心 | Composite coded homoptera pest-resisting gene of galanthus agglutinin and its application |
| CN1357553A (en) * | 2001-12-11 | 2002-07-10 | 中国科学院遗传研究所 | Heliangine and its encoding gene and application in insect-resisting plant gene engineering |
| CN1386859A (en) * | 2002-06-20 | 2002-12-25 | 上海复旦迪恩生物技术有限公司 | Coding sequence of arisaema agglutinant protein and its application |
| CN103305525A (en) * | 2012-03-14 | 2013-09-18 | 林忠平 | Lectin gene of locust tree as well as coded protein and application thereof |
Non-Patent Citations (10)
| Title |
|---|
| A jacalin-related lectin-like gene in wheat is a component of the plant defence system;Xiang et al;《Journal of Experimental Botany》;20110823;第62卷(第15期);全文 * |
| Chrispeels and Raikhel.Lectins, Lectin Genes, and Their Role in Plant Defense.《The Plant Cell》.1991,第3卷(第1期),全文. |
| Hwang and Hwang.The Pepper Mannose-Binding Lectin Gene CaMBL1 Is Required to Regulate Cell Death and Defense Responses to Microbial Pathogens.《Plant Physiology》.2011,第155卷(第1期),全文. |
| Lectins, Lectin Genes, and Their Role in Plant Defense;Chrispeels and Raikhel;《The Plant Cell》;19910131;第3卷(第1期);全文 * |
| Plant lectins as defense proteins against phytophagous insects;Vandenborre et al;《Phytochemistry》;20110321;第72卷(第13期);全文 * |
| The Pepper Mannose-Binding Lectin Gene CaMBL1 Is Required to Regulate Cell Death and Defense Responses to Microbial Pathogens;Hwang and Hwang;《Plant Physiology》;20110131;第155卷(第1期);全文 * |
| Vandenborre et al.Plant lectins as defense proteins against phytophagous insects.《Phytochemistry》.2011,第72卷(第13期),全文. |
| Xiang et al.A jacalin-related lectin-like gene in wheat is a component of the plant defence system.《Journal of Experimental Botany》.2011,第62卷(第15期),全文. |
| 孙慧等.杨树菇凝集素AAVP具有抗病毒和促进菌丝分化功能.《中国生物化学与分子生物学报》.2003,第19卷(第2期),全文. |
| 杨树菇凝集素AAVP具有抗病毒和促进菌丝分化功能;孙慧等;《中国生物化学与分子生物学报》;20030430;第19卷(第2期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103088035A (en) | 2013-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102732554B (en) | A kind of method improving plant anti-insect ability | |
| CN102659934B (en) | Zn-finger protein transcriptional factor of plant, encoding gene and application thereof | |
| CN105543269B (en) | Plant is improved to the method for resistance to verticillium wilt using beauveria bassiana BbP4-ATPase gene | |
| US20130007916A1 (en) | Modulation of low carbon dioxide inducible proteins (lci) for increased biomass production and photosynthesis | |
| WO2017076306A1 (en) | Method for improving plant resistance to verticillium wilt using verticillium-wilt bacteria vdp4-atpase gene | |
| CN107267526B (en) | Radix Notoginseng myb transcription factor gene PnMYB2 and its application | |
| CN103710357B (en) | Alfalfa stress response gene M sNAC2 and application thereof | |
| CN105838723B (en) | A kind of alfalfa Cold resistant genes MsZFP and its coding albumen and application | |
| CN103088035B (en) | Application of soybean agglutinin gene lec-s | |
| CN103627716B (en) | Obtaining method and application for gene used for modifying plant type of maize and improving maize yield | |
| CN112359049B (en) | Lilium regale chitinase gene LrCHI2 and application thereof | |
| CN107253980B (en) | Application of OsGRF7 gene in rice plant type regulation | |
| CN109837291A (en) | A kind of method and its special DNA fragment improving plant resistance to insect using RNA perturbation technique | |
| CN103819549B (en) | A kind of rice protein and encoding gene application in regulation and control plant drought resistance thereof | |
| Zhu et al. | Enhancing disease resistances of Super Hybrid Rice with four antifungal genes | |
| CN107406844A (en) | The transgenic rice plant of improvement | |
| CN103451225B (en) | Application of rice WRKY transcription factor gene OsWRKY21 in cultivation of resistant varieties of plants | |
| CN109628475B (en) | Application of brassinolide synthetic gene PaCYP724B1 in regulation and control of plant branches | |
| CN103409432B (en) | Phytophthora resistance application utilizing soybean agglutinin gene lec-s | |
| CN101712718B (en) | Protein relevant to plant drought resistance, coding gene and application thereof | |
| US20130174299A1 (en) | Method for production of stolon-forming plant having improved tuber production ability or stolon production ability compared with wild type, and stolon-forming plant produced by the method | |
| CN110656118A (en) | Geranium strictipes inulin degrading enzyme gene Tk1-FEH and application thereof | |
| CN107267525B (en) | Application of panax notoginseng polygalacturonase inhibitor protein gene PnPGIP | |
| CN103320448B (en) | Lilium regle bZIP transcription factor LrbZIP1 and application | |
| CN115976052A (en) | Wheat stem basal rot resistance gene TaHSP18.6, expression product, recombinant vector and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140416 |