CN114317591A - Construction method and application of transgenic plant with interaction of cucumber root system and meloidogyne incognita - Google Patents
Construction method and application of transgenic plant with interaction of cucumber root system and meloidogyne incognita Download PDFInfo
- Publication number
- CN114317591A CN114317591A CN202111093736.4A CN202111093736A CN114317591A CN 114317591 A CN114317591 A CN 114317591A CN 202111093736 A CN202111093736 A CN 202111093736A CN 114317591 A CN114317591 A CN 114317591A
- Authority
- CN
- China
- Prior art keywords
- root system
- cucumber
- transgenic
- target gene
- meloidogyne incognita
- 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.)
- Pending
Links
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 48
- 241000243786 Meloidogyne incognita Species 0.000 title claims abstract description 40
- 241000196324 Embryophyta Species 0.000 title claims abstract description 39
- 230000003993 interaction Effects 0.000 title claims abstract description 26
- 244000305550 Streptopus amplexifolius Species 0.000 title claims abstract description 19
- 235000001231 Streptopus amplexifolius Nutrition 0.000 title claims abstract description 19
- 238000010276 construction Methods 0.000 title claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 47
- 230000001580 bacterial effect Effects 0.000 claims abstract description 42
- 240000008067 Cucumis sativus Species 0.000 claims abstract description 37
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 32
- 239000013598 vector Substances 0.000 claims abstract description 26
- 241000589156 Agrobacterium rhizogenes Species 0.000 claims abstract description 21
- 239000000243 solution Substances 0.000 claims description 32
- 239000007788 liquid Substances 0.000 claims description 27
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 claims description 24
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000012258 culturing Methods 0.000 claims description 12
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 11
- 239000001110 calcium chloride Substances 0.000 claims description 11
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 11
- 238000011534 incubation Methods 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 239000003337 fertilizer Substances 0.000 claims description 9
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.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 OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 claims description 9
- 229960002064 kanamycin sulfate Drugs 0.000 claims description 9
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 9
- 239000008223 sterile water Substances 0.000 claims description 9
- 229960005322 streptomycin Drugs 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 239000002689 soil Substances 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 238000005507 spraying Methods 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 239000003125 aqueous solvent Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 241000244206 Nematoda Species 0.000 abstract description 14
- 230000008859 change Effects 0.000 abstract description 8
- 208000000291 Nematode infections Diseases 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 5
- 230000004044 response Effects 0.000 abstract description 4
- 230000004069 differentiation Effects 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000002347 injection Methods 0.000 description 12
- 239000007924 injection Substances 0.000 description 12
- 238000011081 inoculation Methods 0.000 description 8
- 230000001954 sterilising effect Effects 0.000 description 8
- 239000013604 expression vector Substances 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- 108091033409 CRISPR Proteins 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000010354 CRISPR gene editing Methods 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 238000009630 liquid culture Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 230000001131 transforming effect Effects 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 101150054900 gus gene Proteins 0.000 description 3
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 241000219104 Cucurbitaceae Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 230000001133 acceleration Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- 241000219112 Cucumis Species 0.000 description 1
- 235000010071 Cucumis prophetarum Nutrition 0.000 description 1
- 235000009849 Cucumis sativus Nutrition 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 108020004687 Malate Synthase Proteins 0.000 description 1
- 241000243785 Meloidogyne javanica Species 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- 241000212749 Zesius chrysomallus Species 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000004790 biotic stress Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000009335 monocropping Methods 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
Images
Abstract
The invention provides a construction method and application of a transgenic plant with interaction between a cucumber root system and meloidogyne incognita, and belongs to the technical field of plant genetic engineering and plant protection. In the period that the division and differentiation of cucumber seedlings with undeployed cucumber cotyledons are vigorous, the method inoculates a bacterial solution of agrobacterium rhizogenes which is transformed with a target gene vector to be tested or knocked out to generate a hairy root system, and establishes a high-efficiency transgenic root system for nematode infection and research on interaction with the root system. The method can solve the problem that the change of the aerial part target gene caused by the whole transgenic system can influence the change of the root system, so that the phenotype and the response of the root system to nematode infection are biased, and more accurately screen and functionally verify the root system related nematode-resistant gene.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering and plant protection, in particular to a construction method and application of a transgenic plant with interaction of a cucumber root system and meloidogyne incognita.
Background
Cucumber is an annual vine plant of the cucumis genus of the cucurbitaceae family. In the cultivation system of solar greenhouses in northern China, cucumbers occupy an important position. However, due to the closed soil environment of the sunlight greenhouse, and the continuous cropping of the same kind or same family crops in most greenhouses all year round, the accumulation of soil pathogenic bacteria and the occurrence of diseases are caused. Plant parasitic nematodes are the most harmful pathogens in sunlight greenhouses, while northern sunlight greenhouses are mainly dominated by southern root-knot nematodes.
Southern root knot nematodes cause serious losses in agricultural production worldwide and can cause as much as 40% of yield loss, even outcrop, in vegetable crops, especially in solanaceae and cucurbitaceae, due to their extremely broad host range. Therefore, the method has important significance for preventing and treating the meloidogyne incognita. There are several methods for the agricultural control of meloidogyne incognita, and nematode-resistant varieties are one of the most efficient means, but there are no nematode-resistant varieties in cucumber cultivars. Therefore, the research on the interaction mechanism of the root system of the cucumber and the meloidogyne incognita is a basis for breeding and cultivation production of the cucumber to further explore the prevention and treatment of meloidogyne incognita diseases.
At present, the interaction between the root system of cucumber and meloidogyne incognita is generally researched by stably transforming a target gene into a target cucumber to form a transgenic plant, so as to research the function of the target gene and the interaction between the target gene and the biological and non-biological adversities. However, in plant tissue systems, expression of genes is tissue specific and in the interaction with biotic and abiotic stress, the target gene localization and high expression of the tissue is specific. Since the overground part of the plant and the underground part are mutually related to each other in growth and development, the overground part growth can generate signals to be transmitted to the root system to influence the development and the function of the root system. In the process of researching the target gene, the whole plant transgenic system enables the target gene to be over-expressed or interfered at the specific part of the original tissue, and simultaneously, the target gene changes the original expression mode in the cells within the whole plant tissue range. Therefore, in the interaction research of cucumber root systems and meloidogyne incognita, the change of the aerial part target genes caused by the whole plant transgenic system can influence the change of the root systems, so that the phenotype and the response of the root systems to nematode infection are biased.
Disclosure of Invention
The invention aims to provide a construction method and application of a transgenic plant with interaction between a cucumber root system and meloidogyne incognita, and the method can solve the problem that the change of an overground part target gene caused by a whole plant transgenic system can influence the change of the root system, so that the phenotype and the response of the root system to nematode infection are biased, and can more accurately screen and functionally verify the root system related nematode-resistant gene.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for constructing a transgenic plant with interaction between a cucumber root system and meloidogyne incognita, which comprises the following steps:
infecting cucumber seedlings with a bacterial solution of agrobacterium rhizogenes transformed with a target gene vector to be tested or a target gene vector knocked out, and culturing to obtain transgenic plants;
the infected position is a cotyledon node below an undeployed cotyledon of the cucumber seedling.
Preferably, the agrobacterium rhizogenes comprises agrobacterium rhizogenes K599.
Preferably, the preparation method of the bacterial liquid comprises the following steps:
inoculating agrobacterium rhizogenes transformed with a target gene vector to be detected or knocked out of the target gene vector to a resistance culture medium, culturing until monoclonal bacterial plaques grow out, and collecting the monoclonal bacterial plaques by using sterile water to obtain monoclonal bacterial plaque resuspension;
and mixing the monoclonal plaque resuspension and acetosyringone, and incubating to obtain a bacterial liquid.
Preferably, the resistant medium is in aqueous solvent, comprising the following components in the following concentrations: 5-10 g/L of peptone, 3-5 g/L of yeast extract, 10ml/L of calcium chloride aqueous solution, 10-15 g/L of agar, 50-80 mg/L of streptomycin and 50-80 mg/L of kanamycin sulfate; the pH value of the resistant culture medium is 7.0-7.2; the concentration of calcium chloride in the calcium chloride aqueous solution is 1M.
Preferably, the incubation temperature is 24-28 ℃; the incubation time is 0.8-1 h.
Preferably, the humidity of the cultivation is 80-100%; the cultivation temperature is 24-28 ℃;
the lighting conditions of the incubation include: culturing for 0-24 h in the dark; after 24h, the light was 16h and the dark was 8 h.
Preferably, in the cultivation process, the method further comprises the step of irrigating a nitrogen-containing nutrient solution and/or spraying a nitrogen-containing foliar fertilizer.
The invention also provides application of the transgenic plant constructed by the construction method in the scheme in constructing an interaction model of cucumber root systems and meloidogyne incognita.
Preferably, the application comprises the following steps:
and after the transgenic plant grows out of hairy roots, removing the non-transgenic root system, cutting off the transgenic root system from the lower part of the cotyledonary node, burying the cut transgenic root system in soil, and inoculating the meloidogyne incognita to obtain the interaction model of the cucumber root system and the meloidogyne incognita.
The invention provides a method for constructing a transgenic plant with interaction between a cucumber root system and meloidogyne incognita, which comprises the following steps: infecting cucumber seedlings with a bacterial solution of agrobacterium rhizogenes transformed with a target gene vector to be tested or a target gene vector knocked out, and culturing to obtain transgenic plants; the infected position is a cotyledon node below an undeployed cotyledon of the cucumber seedling. In the period that the division and differentiation of cucumber seedlings with undeployed cucumber cotyledons are vigorous, the method inoculates a bacterial solution of agrobacterium rhizogenes which is transformed with a target gene vector to be tested or knocked out to generate a hairy root system, and establishes a high-efficiency transgenic root system for nematode infection and research on interaction with the root system. The method can solve the problem that the change of the aerial part target gene caused by the whole transgenic system can influence the change of the root system, so that the phenotype and the response of the root system to nematode infection are biased, and more accurately screen and functionally verify the root system related nematode-resistant gene.
Drawings
FIG. 1 is a schematic representation of the bacterial solution used for Agrobacterium rhizogenes infection in example 1;
FIG. 2 is a pictorial representation of a seedling to be inoculated;
FIG. 3 is a diagram showing callus and root primordia grown after injection of the bacterial suspension in example 1;
FIG. 4 shows the growth of hairy roots of different treatments in example 1;
FIG. 5 shows the result of identifying the CUS gene in example 1, wherein A is the result of amplification of the GUS gene, B is a hairy root system produced after injection of Agrobacterium rhizogenes, C is the result of dyeing the hairy root, the root system of the right plant in C appears bluish, which is a transgenic root system, and the root system of the left plant does not appear bluish, which is a non-transgenic root system;
fig. 6 shows the fluorescence verification result of gfp, the transformed CRISPR expression vector in example 2, wherein a is the hairy root system after injection of agrobacterium rhizogenes solution, B, C and D are the transgenic root system photographed under a fluorescence microscope;
fig. 7 is the root morphology of the positive root system after nematode inoculation and control infection in example 2, where a is CK, i.e., the root knot generated after non-transgenic root system infection of nematode, B shows that the root knot in a picture is non-fluorescent under a fluorescent microscope, C is the root knot generated after transgenic root system infection of nematode after malic acid synthase gene in cucumber is knocked out by CRISPR, and D is the fluorescence generated by the positive root system transformed with CRISPR expression vector under a fluorescent microscope; root knots can be formed after the root-knot nematodes are infected, the root system develops in the root knots, normal physiological activities are damaged, and damage is caused to plants;
FIG. 8 shows the difference between the number of transgenic positive roots and the number of control root knots and the number of egg masses and the statistical result obtained 28 days after inoculation of Meloidogyne incognita, wherein the red spots are egg masses, the expanded part of the roots is root knots, and the staining enables the nematode and the nematode to produce red egg masses, thereby indicating the growth rate of the nematode and observing the effect of the target gene on the nematode.
Detailed Description
The invention provides a method for constructing a transgenic plant with interaction between a cucumber root system and meloidogyne incognita, which comprises the following steps:
infecting cucumber seedlings with a bacterial solution of agrobacterium rhizogenes transformed with a target gene vector to be tested or a target gene vector knocked out, and culturing to obtain transgenic plants;
the infected position is a cotyledon node below an undeployed cotyledon of the cucumber seedling.
In the present invention, the original vector transformed with the target gene vector to be tested is preferably a PBI121 expression vector; the target gene knockout vector is preferably a CRISPR knockout vector. The method for transforming the target gene vector to be tested or knocking out the target gene vector is not particularly limited, and the conventional method in the field can be adopted. In the present invention, the Agrobacterium rhizogenes preferably includes Agrobacterium rhizogenes K599, available from Shanghai Diego Biotechnology Ltd.
In the present invention, the method for preparing the bacterial liquid preferably comprises the following steps:
inoculating agrobacterium rhizogenes transformed with a target gene vector to be detected or knocked out of the target gene vector to a resistance culture medium, culturing until monoclonal bacterial plaques grow out, and collecting the monoclonal bacterial plaques by using sterile water to obtain monoclonal bacterial plaque resuspension; and mixing the monoclonal plaque resuspension and acetosyringone, and incubating to obtain a bacterial liquid.
In the invention, the culture time is preferably 48-72 h.
In the present invention, the resistant medium is in aqueous solvent, preferably comprising the following components in the following concentrations: 5-10 g/L of peptone, 3-5 g/L of yeast extract, 10ml/L of calcium chloride aqueous solution, 10-15 g/L of agar, 50-80 mg/L of streptomycin and 50-80 mg/L of kanamycin sulfate; the pH value of the resistant culture medium is 7.0-7.2; the concentration of calcium chloride in the calcium chloride aqueous solution is 1M.
In the present invention, the acetosyringone is mixed in the form of an acetosyringone solution, and the solvent of the acetosyringone solution is preferably dimethyl sulfoxide (DMSO); the concentration of the acetosyringone in the acetosyringone solution is preferably 2 mg/L; the acetosyringone has the function of activating the infection toxicity of agrobacterium rhizogenes and helping to infect plant tissues. In the present invention, the monoclonal plaque resuspension is light brownish red in color; the volume ratio of the monoclonal plaque resuspension to the acetosyringone solution is preferably 1: 1000. in the invention, the incubation temperature is preferably 24-28 ℃, and more preferably 25-26 ℃; the incubation time is preferably 0.8-1 h.
The invention has no special limitation on the acquisition mode of the cucumber seedlings, and the conventional cucumber seedling raising method in the field can be adopted.
In the present invention, the time of infection is preferably when the cotyledons are exposed to turn green and the cotyledons are not yet spread; the mode of infection is preferably injection; the number of times of injection is preferably 3-5 times, and more preferably 4 times; the dosage of each injection is preferably 3-6 mu l, and more preferably 4-5 mu l; during the injection process, mechanical damage is made to the infected site without breaking the seedlings to accelerate the spread of the bacterial solution.
In the invention, the cultivation humidity is preferably 80-100%, more preferably 90%, and the cultivation temperature is preferably 24-28 ℃, more preferably 25-26 ℃; the illumination conditions for the incubation preferably include: culturing for 0-24 h in the dark; after 24h, the lamp is lighted for 16h and is dark for 8 h; during the incubation process, the incubated dishes are preferably covered with a lid to maintain a high humidity.
In the invention, in the cultivation process, preferably, the method further comprises irrigating a nitrogen-containing nutrient solution and/or spraying a nitrogen-containing foliar fertilizer; the nitrogen-containing nutrient solution is counted by 1L, and preferably comprises the following components: 5mL of A solution, 5mL of B solution, 1mL of C solution and the balance of water; the solution A takes water as a solvent, and preferably comprises the following components in molar concentration: ca (NO)3)2·4H2O4 mM and KNO33 mM; the solution B takes water as a solvent, and preferably comprises the following components in molar concentration: KH (Perkin Elmer)2PO41mM、K2SO41.5mM and MgSO4·7H2O2 mM; the solution C preferably comprises the following components in molar concentration by taking water as a solvent: NaFe-EDTA 0.1mM, H3BO30.02mM、MnSO4·H2O 1μM、ZnSO4·7H2O 1μM、CuSO4·5H2O0.2. mu.M and (NH)4)6Mo7O24·4H2O 0.005μM。
In the invention, the nitrogenous foliar fertilizer is based on a foliar fertilizer and also comprises KNO3An aqueous solution; KNO in per liter of foliar fertilizer3The adding amount of the aqueous solution is 1-3 mL, and more preferably 2 mL; the KNO3The molar concentration of the aqueous solution is preferably 1 mM. In the present invention, the foliar fertilizer is preferably a foliar fertilizer that is conventional in the art.
In the present invention, the administration frequency of the nitrogen-containing nutrient solution is preferably 1 time per 3 days; the application frequency of the foliar fertilizer is preferably 1 time of spraying every 7 days.
The invention also provides application of the transgenic plant constructed by the construction method in the scheme in constructing an interaction model of cucumber root systems and meloidogyne incognita.
In the present invention, the application comprises the following steps: and after the transgenic plant grows out of hairy roots, removing the non-transgenic root system, cutting off the transgenic root system from the lower part of the cotyledonary node, burying the cut transgenic root system in soil, and inoculating the meloidogyne incognita to obtain the interaction model of the cucumber root system and the meloidogyne incognita.
In the present invention, the meloidogyne incognita is preferably a meloidogyne incognita second-instar larva; the inoculation amount of the meloidogyne incognita is preferably 400-600, more preferably 500, inoculated to each plant.
In the invention, the southern root-knot nematode invades into a transgenic root system and then carries out interaction, and the interaction is carried out 28 days after inoculation, so that an interaction model of the cucumber root system and the southern root-knot nematode is obtained; in the invention, after the interaction model of the cucumber root system and the meloidogyne incognita is obtained, the number of positive root system knots and the number of egg masses are calculated, so as to identify whether the target gene can influence the life history process of the meloidogyne incognita in the root. If the developmental dynamics of the meloidogyne incognita on the transgenic root system is different compared with the non-transgenic root system (control CK), the target gene influences the developmental dynamics of the meloidogyne incognita, and if the developmental dynamics of the meloidogyne incognita is not different compared with the control CK, the target gene does not influence the developmental dynamics of the meloidogyne incognita.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 GUS Gene cucumber transgenic root System
(1) Transformation of the target gene vector: transforming agrobacterium rhizogenes K599 competent cells by using a PBI121 expression vector carrying GUS genes through a chemical transformation method, incubating for 2h at 28 ℃ of a constant temperature shaking table, coating kanamycin sulfate and streptomycin resistance TY solid plates, taking positive monoclonals, shaking bacteria in 5ml of TY liquid culture medium, 50mg/L streptomycin and 50mg/L kanamycin sulfate at 28 ℃ and 180rpm overnight, and preserving bacteria liquid transformed with the target gene vector.
TY liquid medium: 5g of tryptone and 3g of yeast extract, adding water to 1L of the mixture, and after complete dissolution, sterilizing at the high temperature of 121 ℃ for 20 min. Preparing 1M calcium chloride water solution, and sterilizing at 121 deg.C for 20 min. Adding 10ml of sterile 1M calcium chloride aqueous solution into 1L of sterilized TY liquid nutrient solution.
TY solid plate: on the basis of TY liquid culture medium, 15g of agar powder is added.
(2) Preparation of a bacterial liquid for injection: and diluting 500 mu L of bacterial liquid with 2ml of TY liquid culture medium again, coating kanamycin sulfate and streptomycin resistance TY solid plates, adding 1ml of sterile water into the plates after the monoclonal antibody grows out, collecting the bacterial liquid by using a sterile coating rod, putting the collected bacterial liquid into a 2ml centrifuge tube, adding 1 mu L of acetosyringone (2mg/L), incubating for 1h by using a shaking table at 28 ℃ for 180 revolutions, and referring to the figure 1 of a real object picture of the bacterial liquid for injection.
(3) Cucumber seedling culture: firstly, sterilizing cucumber seeds, sterilizing the cucumber seeds for 30s by using 75% alcohol in an ultra-clean workbench, then cleaning the cucumber seeds for 3-5 times by using sterile water, then sterilizing the cucumber seeds for 5min by using 0.05% sodium hypochlorite, then cleaning the cucumber seeds for 5 times by using the sterile water, and placing the seeds in a sterile culture dish filled with sterilized filter paper for germination acceleration. After the seeds are exposed to white, the seeds are cultivated in a sterilized matrix, cotyledons are broken, injection inoculation is prepared, and a real figure of seedlings to be inoculated is shown in figure 2.
(4) Injecting bacterial liquid: and (3) sucking the bacterial liquid by using a 1ml sterile injector, injecting the bacterial liquid into cotyledon nodes for 3-5 times, creating mechanical damage and not breaking seedlings. Injecting the bacteria liquid, placing in a tray with a cover, and maintaining high humidity.
(5) Seedling management: and (3) placing the seedlings injected with the bacterial liquid in a culture room, covering a cover to keep high humidity, pouring proper amount of high-nitrogen nutrient solution, culturing in the dark for 24 hours, circulating normally day and night, and waiting for hairy roots to grow out.
(6) And (3) detecting the GUS gene hairy root transgenosis: and (4) cutting off the grown hairy roots, placing the cut hairy roots in GUS staining solution, and incubating overnight in a shaking table in a dark place for staining.
(7) After the root system is dyed, the color is easily decolored for 2-3 times by using 70% ethanol until the leaves and other parts are nearly transparent.
Example 2 transgenic root acquisition of cucumber malate synthase gene CsMS induced by nematode infestation
(1) Construction and transformation of gene CRISPR knockout vector: two targets are obtained by analyzing and predicting the gene sequence of the CsMS gene, and the two targets are added to a CRISPR expression vector by adopting a four-primer amplification method and an enzyme digestion connection method for knocking out the CsMS gene of the cucumber. Transforming agrobacterium rhizogenes K599 competent cells by using a chemical transformation method for an expression vector, incubating at 28 ℃ for 2 hours by using a constant temperature shaking table, coating kanamycin sulfate and streptomycin resistance TY solid plates, taking positive monoclonals, shaking the positive monoclonals in a 5ml TY +50mg/L streptomycin +50mg/L kanamycin sulfate liquid culture medium at 28 ℃ and 180rpm overnight, and preserving and transforming a bacterial liquid of a target gene vector.
(2) Preparation of a bacterial liquid for injection: and diluting 500. mu.l of the bacterial solution with 2ml of TY liquid culture medium, plating kanamycin sulfate and streptomycin resistance TY solid plates, adding 1ml of sterile water into the plates after the monoclonal antibody grows out, collecting the bacterial solution by using a sterile coating rod, placing the collected bacterial solution into a 2ml centrifuge tube, adding 1. mu.l of acetosyringone (2mg/L), and incubating for 1h by 180 revolutions in a shaking table at 28 ℃.
(3) Cucumber seedling culture: firstly, sterilizing cucumber seeds, sterilizing the cucumber seeds for 30s by using 75% alcohol in an ultra-clean workbench, then cleaning the cucumber seeds for 3-5 times by using sterile water, then sterilizing the cucumber seeds for 5min by using 0.05% sodium hypochlorite, then cleaning the cucumber seeds for 5 times by using the sterile water, and placing the seeds in a sterile culture dish filled with sterilized filter paper for germination acceleration. After the seeds are exposed to the white, the seeds are cultivated in a sterilized substrate, and the cotyledons are broken to prepare injection inoculation.
(4) Injecting bacterial liquid: and (3) sucking the bacterial liquid by using a 1ml sterile injector, injecting the bacterial liquid into cotyledon nodes for 3-5 times, creating mechanical damage and not breaking seedlings. After injecting the bacteria liquid, placing the bacteria liquid in a plug tray with a cover, and keeping high humidity.
(5) Seedling management: and (3) placing the seedlings injected with the bacterial liquid in a culture room, covering a cover to keep high humidity, pouring proper amount of high-nitrogen nutrient solution, culturing in the dark for 24 hours, circulating normally day and night, and waiting for hairy roots to grow out.
(6) Transgenic root system verification: the resulting hairy roots were placed in a fluorescence microscope for detection of gfp fluorescence. To confirm the transfer of the expression vector. Plants containing positive hairy roots were cut from under the cotyledonary node and the hairy roots were buried in soil for inoculation of second instar larvae of meloidogyne incognita.
(7) 500 second-instar larvae are inoculated to each plant, and the number of positive root knots and the number of egg masses are counted 28 days after inoculation, so that whether knocking out the cucumber root gene can influence the life history process of the meloidogyne incognita in roots or not is identified.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A method for constructing a transgenic plant with interaction between a cucumber root system and Meloidogyne incognita, comprises the following steps:
infecting cucumber seedlings with a bacterial solution of agrobacterium rhizogenes transformed with a target gene vector to be tested or a target gene vector knocked out, and culturing to obtain transgenic plants;
the infected position is a cotyledon node below an undeployed cotyledon of the cucumber seedling.
2. The method of claim 1, wherein the agrobacterium rhizogenes comprises agrobacterium rhizogenes K599.
3. The method for constructing according to claim 1, wherein the method for preparing the bacterial liquid comprises the following steps:
inoculating agrobacterium rhizogenes transformed with a target gene vector to be detected or knocked out of the target gene vector to a resistance culture medium, culturing until monoclonal bacterial plaques grow out, and collecting the monoclonal bacterial plaques by using sterile water to obtain monoclonal bacterial plaque resuspension;
and mixing the monoclonal plaque resuspension and acetosyringone, and incubating to obtain a bacterial liquid.
4. The construction method according to claim 1, wherein the resistant medium is in aqueous solvent and comprises the following components in concentration: 5-10 g/L of peptone, 3-5 g/L of yeast extract, 10ml/L of calcium chloride aqueous solution, 10-15 g/L of agar, 50-80 mg/L of streptomycin and 50-80 mg/L of kanamycin sulfate; the pH value of the resistant culture medium is 7.0-7.2; the concentration of calcium chloride in the calcium chloride aqueous solution is 1M.
5. The construction method according to claim 3, wherein the incubation temperature is 24-28 ℃; the incubation time is 0.8-1 h.
6. The construction method according to claim 1, wherein the humidity of the incubation is 80% to 100%; the cultivation temperature is 24-28 ℃;
the lighting conditions of the incubation include: culturing for 0-24 h in the dark; after 24h, the light was 16h and the dark was 8 h.
7. The construction method according to claim 1, wherein in the cultivation process, the method further comprises irrigating a nitrogen-containing nutrient solution and/or spraying a nitrogen-containing foliar fertilizer.
8. The application of the transgenic plant constructed by the construction method of any one of claims 1 to 7 in constructing a cucumber root system and meloidogyne incognita interaction model.
9. The application according to claim 8, characterized in that it comprises the following steps:
and after the transgenic plant grows out of hairy roots, removing the non-transgenic root system, cutting off the transgenic root system from the lower part of the cotyledonary node, burying the cut transgenic root system in soil, and inoculating the meloidogyne incognita to obtain the interaction model of the cucumber root system and the meloidogyne incognita.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111093736.4A CN114317591A (en) | 2021-09-17 | 2021-09-17 | Construction method and application of transgenic plant with interaction of cucumber root system and meloidogyne incognita |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111093736.4A CN114317591A (en) | 2021-09-17 | 2021-09-17 | Construction method and application of transgenic plant with interaction of cucumber root system and meloidogyne incognita |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114317591A true CN114317591A (en) | 2022-04-12 |
Family
ID=81045650
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111093736.4A Pending CN114317591A (en) | 2021-09-17 | 2021-09-17 | Construction method and application of transgenic plant with interaction of cucumber root system and meloidogyne incognita |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114317591A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1018553A1 (en) * | 1999-01-08 | 2000-07-12 | Korea Kumho Petrochemical Co. Ltd. | Transgenic plants with divergent SCaM4 or SCaM5 gene to achieve multiple disease resistance |
| CN101899470A (en) * | 2010-02-25 | 2010-12-01 | 杭州师范大学 | Method for breeding root-knot nematodes by using transgenic adventitious roots |
| CN102181473A (en) * | 2011-02-17 | 2011-09-14 | 杭州师范大学 | Construction method for plant root related functional gene research model |
| CN102653763A (en) * | 2012-04-26 | 2012-09-05 | 华南农业大学 | Meloidogyne javanica dominant-effect gene (Mj-nulg), related protein and application of Mj-nulg |
| CN103503924A (en) * | 2013-08-29 | 2014-01-15 | 中国农业科学院蔬菜花卉研究所 | Preparation method and applications of fusarium oxysporum inoculant |
| CN106748555A (en) * | 2017-02-27 | 2017-05-31 | 沂南县知识产权局 | A kind of Biological organic fertilizer special for cucumber and preparation method thereof |
-
2021
- 2021-09-17 CN CN202111093736.4A patent/CN114317591A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1018553A1 (en) * | 1999-01-08 | 2000-07-12 | Korea Kumho Petrochemical Co. Ltd. | Transgenic plants with divergent SCaM4 or SCaM5 gene to achieve multiple disease resistance |
| CN101899470A (en) * | 2010-02-25 | 2010-12-01 | 杭州师范大学 | Method for breeding root-knot nematodes by using transgenic adventitious roots |
| CN102181473A (en) * | 2011-02-17 | 2011-09-14 | 杭州师范大学 | Construction method for plant root related functional gene research model |
| CN102653763A (en) * | 2012-04-26 | 2012-09-05 | 华南农业大学 | Meloidogyne javanica dominant-effect gene (Mj-nulg), related protein and application of Mj-nulg |
| CN103503924A (en) * | 2013-08-29 | 2014-01-15 | 中国农业科学院蔬菜花卉研究所 | Preparation method and applications of fusarium oxysporum inoculant |
| CN106748555A (en) * | 2017-02-27 | 2017-05-31 | 沂南县知识产权局 | A kind of Biological organic fertilizer special for cucumber and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 鄢小宁;SIKORA R. A.;郑经武;: "七株黄瓜内生真菌对南方根结线虫二龄幼虫的不同作用", 中国生物防治, no. 02 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110747210B (en) | Application of tea tree glycosyltransferase gene UGT91Q2 in improving cold resistance of plants | |
| CN102246662B (en) | Method for identifying resistance against heterodera glycines | |
| CN101213938B (en) | Method for cultivating detoxification tissue culture bulb of hyacinth | |
| CN112544440A (en) | Method for cultivating new transgenic or gene-edited seedlings of root tuber plants | |
| CN108260470A (en) | A kind of method for improving matsutake mycorrhizal seedling raising and application | |
| CN107667738A (en) | The method of seedling stage assay watermelon blight resistance | |
| CN107593626B (en) | Method for breeding corn aphids by using living corn plants | |
| CN109294930A (en) | A method of obtaining dendrobium candidum plantlet stage mycorrhizal fungi | |
| CN103194524B (en) | Method for rapidly identifying peanut germplasm with bacterial wilt resistance | |
| CN103340180A (en) | Method for identifying resistance function of aphid-resisting genes through transforming hairy roots of soybean | |
| CN108391591B (en) | Tissue culture and rapid propagation method for tabebuia flavedo | |
| CN114350546B (en) | Pseudomonas bacteria and their use in promoting plant growth, flowering and fruit setting | |
| CN114317591A (en) | Construction method and application of transgenic plant with interaction of cucumber root system and meloidogyne incognita | |
| CN113652446B (en) | Agrobacterium rhizogenes mediated one-step transformation method for hairy roots of Caragana carryover | |
| CN115152352A (en) | Industrial hemp sterile seedling breeding method | |
| CN106358661B (en) | Method for cultivating rhizobium mother strain | |
| CN104221648A (en) | Method for cultivating tilletia controversa kuhn disease plants in indoor environments | |
| CN110724640B (en) | Tomato root knot nematode biocontrol bacteria, preparation and application thereof | |
| CN114303955A (en) | Breeding method of high-quality chrysanthemum morifolium ramat | |
| CN112385424A (en) | Cucumber grafting method and application thereof in root-knot nematode prevention and control | |
| CN104630261A (en) | Method for improving genetic transformation transient expression efficiency of neolamarckia cadamba | |
| CN106613287B (en) | Method for rapidly identifying aphid resistance of mung beans | |
| CN108841832A (en) | The application of the transhipment helper factor GmPHF1b of phosphate cotransporter | |
| CN105325196B (en) | Method for infecting wheat by inoculating tilletia foetida at root | |
| CN101250503B (en) | In-vitro propagation method of tuberose virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |