CN118185997A - Application method of melatonin receptor MdCAND-2 in promoting generation of adventitious roots of apple plants - Google Patents
Application method of melatonin receptor MdCAND-2 in promoting generation of adventitious roots of apple plants Download PDFInfo
- Publication number
- CN118185997A CN118185997A CN202410413646.6A CN202410413646A CN118185997A CN 118185997 A CN118185997 A CN 118185997A CN 202410413646 A CN202410413646 A CN 202410413646A CN 118185997 A CN118185997 A CN 118185997A
- Authority
- CN
- China
- Prior art keywords
- mdcand
- liquid
- culture medium
- plants
- microliter
- 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
- 238000000034 method Methods 0.000 title claims abstract description 34
- 102000001419 Melatonin receptor Human genes 0.000 title claims abstract description 25
- 108050009605 Melatonin receptor Proteins 0.000 title claims abstract description 25
- 230000001737 promoting effect Effects 0.000 title claims abstract description 23
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 30
- 230000009261 transgenic effect Effects 0.000 claims abstract description 28
- 241000589158 Agrobacterium Species 0.000 claims abstract description 25
- 230000002018 overexpression Effects 0.000 claims abstract description 16
- 239000013604 expression vector Substances 0.000 claims abstract description 14
- 238000012795 verification Methods 0.000 claims abstract description 9
- 230000001404 mediated effect Effects 0.000 claims abstract description 7
- 230000003321 amplification Effects 0.000 claims abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 59
- 230000001580 bacterial effect Effects 0.000 claims description 55
- 238000006243 chemical reaction Methods 0.000 claims description 30
- 239000013612 plasmid Substances 0.000 claims description 21
- 239000013598 vector Substances 0.000 claims description 21
- 239000012880 LB liquid culture medium Substances 0.000 claims description 18
- 238000007664 blowing Methods 0.000 claims description 15
- 239000001963 growth medium Substances 0.000 claims description 15
- 238000011144 upstream manufacturing Methods 0.000 claims description 15
- 241000233866 Fungi Species 0.000 claims description 12
- 230000015572 biosynthetic process Effects 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 238000011084 recovery Methods 0.000 claims description 12
- 241000588724 Escherichia coli Species 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 230000009466 transformation Effects 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000005520 cutting process Methods 0.000 claims description 8
- 230000012010 growth Effects 0.000 claims description 7
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 6
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 claims description 6
- 239000012881 co-culture medium Substances 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 230000004927 fusion Effects 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 239000013642 negative control Substances 0.000 claims description 6
- 238000005086 pumping Methods 0.000 claims description 6
- 239000012883 rooting culture medium Substances 0.000 claims description 6
- 230000035939 shock Effects 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 4
- 101000942941 Arabidopsis thaliana DNA ligase 6 Proteins 0.000 claims description 3
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 claims description 3
- 239000003242 anti bacterial agent Substances 0.000 claims description 3
- 229940088710 antibiotic agent Drugs 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 230000002068 genetic effect Effects 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 3
- 230000006870 function Effects 0.000 claims description 2
- 239000003292 glue Substances 0.000 claims 2
- 241000196324 Embryophyta Species 0.000 abstract description 56
- 241000220225 Malus Species 0.000 abstract description 27
- 235000013399 edible fruits Nutrition 0.000 abstract description 7
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 25
- YJPIGAIKUZMOQA-UHFFFAOYSA-N Melatonin Natural products COC1=CC=C2N(C(C)=O)C=C(CCN)C2=C1 YJPIGAIKUZMOQA-UHFFFAOYSA-N 0.000 description 12
- 229960003987 melatonin Drugs 0.000 description 12
- DRLFMBDRBRZALE-UHFFFAOYSA-N melatonin Chemical compound COC1=CC=C2NC=C(CCNC(C)=O)C2=C1 DRLFMBDRBRZALE-UHFFFAOYSA-N 0.000 description 12
- 229940088597 hormone Drugs 0.000 description 5
- 239000005556 hormone Substances 0.000 description 5
- 102100024382 Cullin-associated NEDD8-dissociated protein 2 Human genes 0.000 description 4
- 101000910263 Homo sapiens Cullin-associated NEDD8-dissociated protein 2 Proteins 0.000 description 4
- 235000021016 apples Nutrition 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000035882 stress Effects 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- IXVMHGVQKLDRKH-VRESXRICSA-N Brassinolide Natural products O=C1OC[C@@H]2[C@@H]3[C@@](C)([C@H]([C@@H]([C@@H](O)[C@H](O)[C@H](C(C)C)C)C)CC3)CC[C@@H]2[C@]2(C)[C@@H]1C[C@H](O)[C@H](O)C2 IXVMHGVQKLDRKH-VRESXRICSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- IXVMHGVQKLDRKH-KNBKMWSGSA-N brassinolide Chemical compound C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@@H](O)[C@H](O)[C@@H](C)C(C)C)CC[C@H]3[C@@H]21 IXVMHGVQKLDRKH-KNBKMWSGSA-N 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- GEWDNTWNSAZUDX-WQMVXFAESA-N (-)-methyl jasmonate Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-WQMVXFAESA-N 0.000 description 1
- JLIDBLDQVAYHNE-LXGGSRJLSA-N 2-cis-abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\C1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-LXGGSRJLSA-N 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 102000034353 G alpha subunit Human genes 0.000 description 1
- 108091006099 G alpha subunit Proteins 0.000 description 1
- 108091006027 G proteins Proteins 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000004722 NADPH Oxidases Human genes 0.000 description 1
- 108010002998 NADPH Oxidases Proteins 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- 239000003630 growth substance Substances 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- ZNJFBWYDHIGLCU-HWKXXFMVSA-N jasmonic acid Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(O)=O)CCC1=O ZNJFBWYDHIGLCU-HWKXXFMVSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- GEWDNTWNSAZUDX-UHFFFAOYSA-N methyl 7-epi-jasmonate Natural products CCC=CCC1C(CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 108091005720 plant hormone receptors Proteins 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000003823 potassium efflux Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000021749 root development Effects 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- 230000010496 root system development Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000015378 stomatal closure Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides an application method of melatonin receptor MdCAND-2 in promoting the generation of adventitious roots of apple plants, and relates to the technical field of plant genetic engineering. The application method of the melatonin receptor MdCAND-2 in promoting the generation of adventitious roots of apple plants comprises the following steps: step S1. Monoclonal amplification of MdCAND2-2 gene; s2, constructing a plant super-expression vector MdCAND-2-pBI 121; s3, transferring the plant super-expression vector MdCAND-2-pBI 121 into a GALA-3 plant by using an agrobacterium-mediated method; and S4, performing functional verification on the transgenic plant of the over-expression MdCAND 2-2. In the invention, mdCAND < 2 > -2 is over-expressed in the apple plant to be enhanced in the form of transgenic plant, so that the rooting capacity of the apple is enhanced, and the quality and the yield of the fruit tree are improved.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to an application method of melatonin receptor MdCAND-2 in promoting the generation of adventitious roots of apple plants.
Background
Apples are the main cultivated fruit trees in China. In recent years, the apple industry in China develops rapidly, and the yield is in a continuous increasing trend. In the apple seedling breeding practice in China, the dwarf stock with excellent ecological adaptability has poor rooting capability, which becomes an important bottleneck problem for restricting the propagation of high-quality big apple seedlings. Traditional apple breeding has a longer period, and researches show that the application of exogenous hormone substances or the application of genetic engineering methods to perform fine variety breeding are feasible ways for promoting plant rooting. Exogenous hormone substances capable of effectively improving rooting capacity of plants are selected, genes of apple response root system development are mined, and the method has important significance for improving rooting capacity of fruit trees and cultivating new apple germplasm with robust root system.
The plant Melatonin (MT) can fully play the role of a growth regulator to induce cell expansion, root development, participate in flowering regulation and the like. MT also can enhance the tolerance of plants to stresses such as light stress, saline-alkali stress, cold-hot dry-wet stress, insect diseases and the like, and the action mechanism of plant physiology and molecular biology has become a research hot spot. MT can promote the continuous expansion and growth of grape young fruits. MT and IAA have similar concentration distribution in soybean hypocotyl, decreasing from top to bottom. In addition, both have similar modes of action, both of which promote growth by promoting further expansion of cells. MT regulates plant growth by regulating sugar metabolism, and exogenously applies MT to photosynthesis and organic accumulation of corn seedling to inhibit at high concentration and promote at low concentration.
The plant hormone receptor is a specific recognition protein in the plant sensing and receiving hormone signal transduction pathway, and influences the plant hormone to regulate the growth and development of plants and resist environmental stress. The work on melatonin receptor research in 2018 has made a significant advance in the scientific community that is a major breakthrough in the history of melatonin receptor research. The first plant melatonin receptor CAND2 was found in arabidopsis by Wei et al, with the main evidence: CAND2 localizes to the plasma membrane, has a similar topology to the receptor, interacts with the G protein alpha subunit, and highly specifically binds melatonin and CAND2 mutants exhibit insensitivity to melatonin-induced pore closure. Melatonin binding to CAND2 triggers dissociation of the two subunits β, γ and α of the G protein, thereby activating NADPH oxidase-dependent mass production of H 2O2, enhancing Ca 2+ influx and K + efflux, ultimately leading to stomatal closure.
Accordingly, one skilled in the art would provide a method of using melatonin receptor MdCAND-2 in promoting adventitious root formation in apple plants to address the problems set forth in the background above.
Disclosure of Invention
(One) solving the technical problems
Aiming at the defects of the prior art, the invention provides an application method of melatonin receptor MdCAND-2 in promoting the generation of adventitious roots of apple plants, which is presented in the form of transgenic plants, and MdCAND-2 is over-expressed in the apple plants to be enhanced, so that the rooting capacity of apples is enhanced, and the quality and the yield of fruit trees are improved.
(II) technical scheme
In order to achieve the above purpose, the invention is realized by the following technical scheme:
a method of using melatonin receptor MdCAND-2 in promoting adventitious root formation in apple plants, comprising the steps of:
Step S1. Monoclonal amplification of MdCAND2-2 gene;
S2, constructing a plant super-expression vector MdCAND-2-pBI 121;
S3, transferring the plant super-expression vector MdCAND-2-pBI 121 into a GALA-3 plant by using an agrobacterium-mediated method;
and S4, verifying the functions of the transgenic plant of the over-expression MdCAND 2-2.
Preferably, the specific method of step S1 is as follows:
1) PCR amplification MdCAND of the 2-2 Gene
Using specific upstream primer F and downstream primer R as primer pairs, amplifying MdCAND-2 gene with the following 40 microliter reaction system to obtain MdCAND2-2 nucleotide double strand, wherein the 40 microliter reaction system is as follows:
2) The MdCAND-2 nucleotide double strand obtained by the PCR amplification was ligated with a PMD-19-T (Simple) vector, and ligated overnight at 16℃with the following 10. Mu.l reaction system to obtain a ligation product MdCAND-2-T, the 10. Mu.l reaction system being as follows:
PMD-19-T(Simple) 0.5μL
Solution 1 5μL
Recovery of the product (MdCAND-2) 4.5. Mu.L
3) Ligation product transformation competent cells
Taking out DH5 alpha competent cells of escherichia coli from an ultralow temperature refrigerator at the temperature of minus 80 ℃, putting the competent cells on ice to melt, sucking 20 microliter DH5 alpha competent cells in a sterile centrifuge tube of 1.5mL, blowing and mixing the competent cells with 10 microliter of a connecting product, carrying out ice bath for 30min, carrying out water bath heat shock for 90s at the temperature of 42 ℃, carrying out ice bath for 2min, adding 200 mu L of LB liquid culture medium in a sterile environment, putting the culture medium in a shaking table with the rotation speed of 180rpm at the temperature of 37 ℃, shaking for 1h, taking 100 mu L of bacterial liquid to coat an LB plate with antibiotics after shaking, starting to coat the plate until the plate dries, covering a cover seal to make a mark, and putting the culture medium in a constant temperature incubator at the temperature of 37 ℃ for 10-12h until bacterial plaques grow;
4) Spot picking
In a sterile super clean bench, sucking 100 microliters of LB liquid culture medium by using a sterile gun head, pumping into a 0.5mL sterile centrifuge tube, picking 10 bacterial plaques with a regular shape by using a 10 microliter sterile gun head, placing the bacterial plaques in the LB liquid culture medium, blowing for several times, covering a cover, marking the centrifuge tube, placing the centrifuge tube in a shaking table, and obtaining bacterial liquid at 37 ℃ at 180rpm for 4-6 hours;
5) Identification of bacterial liquid
After the shaking, the bacterial liquid is identified by using the following 10 microliter reaction system, the bacterial liquid is used as a template, the selected upstream primer is used as a carrier upstream primer, the selected downstream primer is used as a downstream primer of a gene, the identification is carried out according to a normal verification flow, and water is used as a negative control. And (3) testing the positive rate of the bacterial liquid, and selecting the relatively bright bacterial liquid in positive identification, wherein the 10 microliter reaction system is as follows:
preferably, the specific method of step S2 is as follows:
1) Extracting positive bacterial liquid plasmids, and carrying out a plasmid extraction method according to the steps of a kit to obtain PMD-19-T (Simple) fusion plasmids connected with MdCAND-2;
2) The PMD-19-T (Simple) fusion plasmid with MdCAND-2 and the pBI121 empty vector were subjected to double digestion in the following 40. Mu.l reaction system, which was used for 2-6h at 37℃and was described as follows:
3) Recovery and ligation of the target gene and the target vector: the above-mentioned band for cutting the target gene was subjected to gel recovery (according to the kit), and at the same time, the enzyme-cut band of the target vector was also cut out to perform gel recovery (according to the kit), and then the gel was ligated in a 10-micro-organism reaction system as follows:
T4 ligase 1. Mu.L
10x T4 buffer 1μL
8 Mu L of target gene and target vector
4) The ligation products described above were transformed into E.coli DH 5. Alpha. Competent cells in the following 30. Mu.l reaction system, which was:
competent cells (E.coli DH 5. Alpha.) 20. Mu.L
Ligation product 10. Mu.L
Then taking out the competent cells of the escherichia coli DH5 alpha from a refrigerator at the temperature of minus 80 ℃, putting a horse on ice prepared in advance to melt, sucking 20 mu L of the competent cells of the escherichia coli DH5 alpha into a sterile centrifuge tube with the volume of 1.5mL, sucking 10 mu L of the constructed connection product, blowing and mixing uniformly, carrying out ice bath for 30min, carrying out heat shock at the temperature of 42 ℃ for 90s, carrying out ice bath for 2min, adding 200 mu L of LB liquid culture medium in a sterile environment, putting in a shaking table with the rotation speed of 180rpm, shaking for 1h, taking 100 mu L of bacterial liquid to coat an LB plate, coating the plate until the plate is dried, covering a cover seal to make a mark, and putting in a constant temperature incubator with the temperature of 37 ℃ for culturing for 10-12h;
5) Spot picking: in a sterile super clean bench, sucking 100 mu L of LB liquid culture medium by using a sterile gun head, pumping into a 0.5mL sterile centrifuge tube, picking 10 bacterial plaques by using a 10 mu L sterile gun head, placing in the LB liquid culture medium, blowing for several times, covering a cover, marking the centrifuge tube, placing in a shaking table at 37 ℃ at 180rpm for 4-6 hours;
6) And (3) bacterial liquid identification: after the shaking is finished, carrying out PCR identification by using a 10 mu L system shown in the table 3, taking bacterial liquid as a template, taking a primer as an upstream primer of a carrier, carrying out identification by using a downstream primer of a gene according to a normal verification flow, taking water as a negative control, detecting the positive rate of the bacterial liquid, and selecting a relatively bright bacterial liquid in the positive identification;
7) Extracting positive bacterial liquid plasmids, and carrying out a plasmid extraction method according to the steps of the kit to obtain the plant super-expression vector MdCAND-2-pBI 121.
Preferably, the specific method of step S3 is as follows:
1) Transformation of plant overexpression vector MdCAND2-2-pBI121 into Agrobacterium EHA105
Taking out the agrobacterium EHA105 competent cells from the ultralow temperature refrigerator at the temperature of minus 80 ℃, sucking 20 microliter of the agrobacterium EHA105 competent cells into a 1.5mL sterile centrifuge tube, sucking 5 microliter of the overexpression vector MdCAND2-2-pBI121 plasmid, adding 50 microliter of the agrobacterium EHA105 competent cells, blowing and mixing uniformly, carrying out ice bath for 30min, heating at 37 ℃ for 90s, carrying out ice bath for 2min, adding the obtained product into 200 microliter of LB liquid medium in an ultralow working table, shaking for 4h in a shaking table at the temperature of 28 ℃ and the rotating speed of 180rpm, taking 100 microliter of bacterial liquid to coat an LB plate after shaking, starting to coat the plate until the plate dries, covering a cover seal to make a mark, and culturing in a constant temperature incubator at the temperature of 28 ℃ for 10-12h;
2) Agrobacterium-mediated transfer of expression vectors into GALA-3 plants
Cutting leaves of GALA-3 plants from tissue culture seedlings subjected to secondary culture for about 30 days for genetic transformation, selecting young leaves developed from 3-4 leaves at the top of the tissue culture seedlings, cutting off leaf tips and leaf stalk parts, marking 3 trails on the middle part, immediately placing the young leaves in a preculture medium for preculture, eliminating light, at 24 ℃ for 2 days, culturing the obtained agrobacterium EHA105 in a YEP culture medium culture dish at 180rpm and 28 ℃ for 50mL in a shaking table for 4-6 hours until the OD value is 0.4-0.6, centrifuging at 25 ℃ for 5min by using a centrifuge, collecting thalli by using a glucose liquid culture medium, suspending the thalli, and adding 20mg acetosyringone for standby;
Then, placing the pre-cultured leaves in a liquid co-culture medium with agrobacterium, shaking for several times every 2min, soaking for about 8min, transferring the leaf blocks to sterile filter paper to absorb bacterial liquid, rapidly transferring the leaf blocks without bacterial liquid on the surfaces to a culture dish in which the co-culture medium is split-charged, expanding the buds by using an expanding culture medium after the leaves grow out transgenic buds, and placing the buds into a rooting culture medium for rooting after the buds grow into seedlings to obtain transgenic plants.
Preferably, the specific method of step S4 is as follows:
1) Selecting 20 transgenic GALA-3 plants which grow for one month and have consistent growth vigor and 20 non-transgenic GALA-3 plants of a control group;
2) Transferring the plant to a rooting culture medium, and growing for 40 days to obtain a phenotype;
3) After phenotype appears, root system scanning is carried out, the fresh weight, root length, root tip number, root surface area and root volume of the control group and transgenic group plants are statistically analyzed, and the test is repeated for more than three times.
Preferably, in the step S1, the CDS sequence of MdCAND-2 gene is specifically:
ATGCAAGCTCCCCAAGAAGCTTCCTCGCAATCCGGACTCCCAATTCCACTCACCCAGGCCCCAAATTCAACCATAGACGACGGCGTTTCGGGCTCCGAATTCTACAGCTGGCTCGGCGAATGTCATGGGTTCTTCCACAATGTGGCTCTGATGGTGCCCTCGCTTCTCTTCGTCCTGTATTTGGCGTTTCAATCGAGAAAAAGCTTCTCGAAGCTCTCCCATGGCCGCTCTTATATCATGATTGCCTACTACGGGTGCCTCTGGCTTGTTAGCTTGCTCAATCTTGCTTGGTGCTGTTTTCAGGCATGGGAGTGCAGTCCTGGAAAAGAAGTAACATGGAATATATTATCTTTGTTCACAACCTCCGGGATGCTATTTTTGGAAGTAAGCTTGCTGGCATTTTTGCTTCAGGGGAATTATGCAAGTGGACTGGAAGCTTTGACACGGACTTTTTTTATCTCAGGGATCCTTGTTGTTTTGGATATACTTCTCAAGGTATCGCTCGTTCTTTTTCGTGAAATGGATTTTCACCCTTATGACCTATGTATGCTTGTTAGTTACTTCAAGAATATACTAATGGAAACATATATGTTGTTGGAAGTGCACATTGGTGCATTATTGTGGTGCATAACGGACTTACAATTATACTCTGTTTTCTTCCAGGCAATATATCTATTTGGACTTGGCATTCCATTGTTCATTGACAATAATGACCGTGCACATCGAACAAAGTGGGCCTTGTGGGTTGTCCACAGGCTAGTGATTACTGCAATTTACGGCTTCATATTGTTCATGTACCATTCCAAGTGGAGAGAGAGGTTACCAGCAAGACCTGCATTCTACAAGTATACCGCTATCATGTTTACGTTAAACGTACTAGTACTGTTTGCTTGCTTGCTTACTGGAAATGGGGCTGGATTTGGATTCTGGCTATATAGTGCTACTATTGTCTGTTATCACGCCTTTTATCTTCCACTTCTGTACGTAACATTTCTGGCAGACTTCTTCCAGGAGGAAGATTTAAATTTGGAGAATGTGTACTATTCAGAAATGAAAGATGCCGGTTTCTTTGACGCGGATTGGGAGTGA.
preferably, in the step S1, the nucleotide sequence of the specific upstream primer F is specifically:
ACTTCCCCAACCTTTCTAGTCAT。
preferably, in the step S1, the nucleotide sequence of the specific downstream primer R is specifically:
TAACATCATACAAACACTTAAAAAC。
according to a second aspect of the present invention there is also provided the use of melatonin receptor MdCAND-2 in promoting adventitious root formation in apple plants.
(III) beneficial effects
The invention provides an application method of melatonin receptor MdCAND-2 in promoting the generation of adventitious roots of apple plants. The beneficial effects are as follows:
1. The application method of melatonin receptor MdCAND-2 in promoting the generation of adventitious roots of apple plants is presented in the form of transgenic plants, and MdCAND-2 is overexpressed in the apple plants to be enhanced, so that the rooting capacity of apples is enhanced, and the quality and the yield of fruit trees are improved.
2. The application method of melatonin receptor MdCAND-2 in promoting the generation of adventitious roots of apple plants, which is provided by the invention, can obviously enhance the rooting capability of apple plants, does not need to cultivate new varieties of fruit trees for a long time, improves the quality of plants in a short time, and is simple and easy to implement, simple to operate and good in stability.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Examples:
the embodiment of the invention provides an application method of melatonin receptor MdCAND-2 in promoting the generation of adventitious roots of apple plants, which comprises the following steps:
step S1. Monoclonal amplification of MdCAND2-2 Gene
1) PCR amplification MdCAND of the 2-2 Gene
The specific upstream primer F and the specific downstream primer R are used as primer pairs, the MdCAND-2 gene is amplified by using a 40 microliter reaction system as follows, and MdCAND-2 nucleotide double chains are obtained, wherein the 40 microliter reaction system is as follows:
Wherein, the CDS sequence of MdCAND-2 gene is specifically:
ATGCAAGCTCCCCAAGAAGCTTCCTCGCAATCCGGACTCCCAATTCCACTCACCCAGGCCCCAAATTCAACCATAGACGACGGCGTTTCGGGCTCCGAATTCTACAGCTGGCTCGGCGAATGTCATGGGTTCTTCCACAATGTGGCTCTGATGGTGCCCTCGCTTCTCTTCGTCCTGTATTTGGCGTTTCAATCGAGAAAAAGCTTCTCGAAGCTCTCCCATGGCCGCTCTTATATCATGATTGCCTACTACGGGTGCCTCTGGCTTGTTAGCTTGCTCAATCTTGCTTGGTGCTGTTTTCAGGCATGGGAGTGCAGTCCTGGAAAAGAAGTAACATGGAATATATTATCTTTGTTCACAACCTCCGGGATGCTATTTTTGGAAGTAAGCTTGCTGGCATTTTTGCTTCAGGGGAATTATGCAAGTGGACTGGAAGCTTTGACACGGACTTTTTTTATCTCAGGGATCCTTGTTGTTTTGGATATACTTCTCAAGGTATCGCTCGTTCTTTTTCGTGAAATGGATTTTCACCCTTATGACCTATGTATGCTTGTTAGTTACTTCAAGAATATACTAATGGAAACATATATGTTGTTGGAAGTGCACATTGGTGCATTATTGTGGTGCATAACGGACTTACAATTATACTCTGTTTTCTTCCAGGCAATATATCTATTTGGACTTGGCATTCCATTGTTCATTGACAATAATGACCGTGCACATCGAACAAAGTGGGCCTTGTGGGTTGTCCACAGGCTAGTGATTACTGCAATTTACGGCTTCATATTGTTCATGTACCATTCCAAGTGGAGAGAGAGGTTACCAGCAAGACCTGCATTCTACAAGTATACCGCTATCATGTTTACGTTAAACGTACTAGTACTGTTTGCTTGCTTGCTTACTGGAAATGGGGCTGGATTTGGATTCTGGCTATATAGTGCTACTATTGTCTGTTATCACGCCTTTTATCTTCCACTTCTGTACGTAACATTTCTGGCAGACTTCTTCCAGGAGGAAGATTTAAATTTGGAGAATGTGTACTATTCAGAAATGAAAGATGCCGGTTTCTTTGACGCGGATTGGGAGTGA;
the nucleotide sequence of the specific upstream primer F is specifically:
ACTTCCCCAACCTTTCTAGTCAT；
The nucleotide sequence of the specific downstream primer R is specifically:
TAACATCATACAAACACTTAAAAAC；
2) The MdCAND-2 nucleotide double strand obtained by the PCR amplification was ligated with a PMD-19-T (Simple) vector, and ligated overnight at 16℃with the following 10. Mu.l reaction system to obtain a ligation product MdCAND-2-T, the 10. Mu.l reaction system being as follows:
PMD-19-T(Simple) 0.5μL
Solution 1 5μL
Recovery of the product (MdCAND-2) 4.5. Mu.L
3) Ligation product transformation competent cells
Taking out DH5 alpha competent cells of escherichia coli from an ultralow temperature refrigerator at the temperature of minus 80 ℃, putting the competent cells on ice to melt, sucking 20 microliter DH5 alpha competent cells in a sterile centrifuge tube of 1.5mL, blowing and mixing the competent cells with 10 microliter of a connecting product, carrying out ice bath for 30min, carrying out water bath heat shock for 90s at the temperature of 42 ℃, carrying out ice bath for 2min, adding 200 mu L of LB liquid culture medium in a sterile environment, putting the culture medium in a shaking table with the rotation speed of 180rpm at the temperature of 37 ℃, shaking for 1h, taking 100 mu L of bacterial liquid to coat an LB plate with antibiotics after shaking, starting to coat the plate until the plate dries, covering a cover seal to make a mark, and putting the culture medium in a constant temperature incubator at the temperature of 37 ℃ for 10-12h until bacterial plaques grow;
4) Spot picking
In a sterile super clean bench, sucking 100 microliters of LB liquid culture medium by using a sterile gun head, pumping into a 0.5mL sterile centrifuge tube, picking 10 bacterial plaques with a regular shape by using a 10 microliter sterile gun head, placing the bacterial plaques in the LB liquid culture medium, blowing for several times, covering a cover, marking the centrifuge tube, placing the centrifuge tube in a shaking table, and obtaining bacterial liquid at 37 ℃ at 180rpm for 4-6 hours;
5) Identification of bacterial liquid
After the fungus shaking is finished, the fungus liquid is identified by using the following 10 microliter reaction system, the fungus liquid is used as a template, the selected upstream primer is used as a carrier upstream primer, the selected downstream primer is used as a downstream primer of a gene, the fungus liquid is identified according to a normal verification flow, water is used as a negative control, the positive rate of the fungus liquid is checked, and the fungus liquid with brighter positive identification is selected, wherein the 10 microliter reaction system is as follows:
S2, constructing a plant super-expression vector MdCAND-2-pBI 121
1) Extracting positive bacterial liquid plasmids, and carrying out a plasmid extraction method according to the steps of a kit to obtain PMD-19-T (Simple) fusion plasmids connected with MdCAND-2;
2) The PMD-19-T (Simple) fusion plasmid with MdCAND-2 and the pBI121 empty vector were subjected to double digestion in the following 40. Mu.l reaction system, which was used for 2-6h at 37℃and was described as follows:
3) Recovery and ligation of the target gene and the target vector: the above-mentioned band for cutting the target gene was subjected to gel recovery (according to the kit), and at the same time, the enzyme-cut band of the target vector was also cut out to perform gel recovery (according to the kit), and then the gel was ligated in a 10-micro-organism reaction system as follows:
T4 ligase 1. Mu.L
10x T4 buffer 1μL
8 Mu L of target gene and target vector
4) The ligation products described above were transformed into E.coli DH 5. Alpha. Competent cells in the following 30. Mu.l reaction system, which was:
competent cells (E.coli DH 5. Alpha.) 20. Mu.L
Ligation product 10. Mu.L
Then taking out the competent cells of the escherichia coli DH5 alpha from a refrigerator at the temperature of minus 80 ℃, putting a horse on ice prepared in advance to melt, sucking 20 mu L of the competent cells of the escherichia coli DH5 alpha into a sterile centrifuge tube with the volume of 1.5mL, sucking 10 mu L of the constructed connection product, blowing and mixing uniformly, carrying out ice bath for 30min, carrying out heat shock at the temperature of 42 ℃ for 90s, carrying out ice bath for 2min, adding 200 mu L of LB liquid culture medium in a sterile environment, putting in a shaking table with the rotation speed of 180rpm, shaking for 1h, taking 100 mu L of bacterial liquid to coat an LB plate, coating the plate until the plate is dried, covering a cover seal to make a mark, and putting in a constant temperature incubator with the temperature of 37 ℃ for culturing for 10-12h;
5) Spot picking: in a sterile super clean bench, sucking 100 mu L of LB liquid culture medium by using a sterile gun head, pumping into a 0.5mL sterile centrifuge tube, picking 10 bacterial plaques by using a 10 mu L sterile gun head, placing in the LB liquid culture medium, blowing for several times, covering a cover, marking the centrifuge tube, placing in a shaking table at 37 ℃ at 180rpm for 4-6 hours;
6) And (3) bacterial liquid identification: after the shaking is finished, carrying out PCR identification by using a 10 mu L system shown in the table 3, taking bacterial liquid as a template, taking a primer as an upstream primer of a carrier, carrying out identification by using a downstream primer of a gene according to a normal verification flow, taking water as a negative control, detecting the positive rate of the bacterial liquid, and selecting a relatively bright bacterial liquid in the positive identification;
7) Extracting positive bacterial liquid plasmids, and carrying out a plasmid extraction method according to the steps of a kit to obtain a plant super-expression vector MdCAND-2-pBI 121;
s3, transferring the plant super-expression vector MdCAND-2-pBI 121 into a GALA-3 plant by using an agrobacterium-mediated method
1) Transformation of plant overexpression vector MdCAND2-2-pBI121 into Agrobacterium EHA105
Taking out the agrobacterium EHA105 competent cells from the ultralow temperature refrigerator at the temperature of minus 80 ℃, sucking 20 microliter of the agrobacterium EHA105 competent cells into a 1.5mL sterile centrifuge tube, sucking 5 microliter of the overexpression vector MdCAND2-2-pBI121 plasmid, adding 50 microliter of the agrobacterium EHA105 competent cells, blowing and mixing uniformly, carrying out ice bath for 30min, heating at 37 ℃ for 90s, carrying out ice bath for 2min, adding the obtained product into 200 microliter of LB liquid medium in an ultralow working table, shaking for 4h in a shaking table at the temperature of 28 ℃ and the rotating speed of 180rpm, taking 100 microliter of bacterial liquid to coat an LB plate after shaking, starting to coat the plate until the plate dries, covering a cover seal to make a mark, and culturing in a constant temperature incubator at the temperature of 28 ℃ for 10-12h;
2) Agrobacterium-mediated transfer of expression vectors into GALA-3 plants
Cutting leaves of GALA-3 plants from tissue culture seedlings subjected to secondary culture for about 30 days for genetic transformation, selecting young leaves developed from 3-4 leaves at the top of the tissue culture seedlings, cutting off leaf tips and leaf stalk parts, marking 3 trails on the middle part, immediately placing the young leaves in a preculture medium for preculture, eliminating light, at 24 ℃ for 2 days, culturing the obtained agrobacterium EHA105 in a YEP culture medium culture dish at 180rpm and 28 ℃ for 50mL in a shaking table for 4-6 hours until the OD value is 0.4-0.6, centrifuging at 25 ℃ for 5min by using a centrifuge, collecting thalli by using a glucose liquid culture medium, suspending the thalli, and adding 20mg acetosyringone for standby;
Then, placing the pre-cultured leaves in a liquid co-culture medium with agrobacterium, shaking for several times every 2min, soaking for about 8min, transferring the leaf blocks to sterile filter paper to absorb bacterial liquid, rapidly transferring the leaf blocks without bacterial liquid on the surfaces to a culture dish in which the co-culture medium is split-filled, amplifying the buds by using an expanding culture medium after the leaves grow out transgenic buds, and placing the buds into a rooting culture medium for rooting after the buds grow into seedlings to obtain transgenic plants;
S4, functional verification of over-expression MdCAND2-2 transgenic plant
1) Selecting 20 transgenic GALA-3 plants which grow for one month and have consistent growth vigor and 20 non-transgenic GALA-3 plants of a control group;
2) Transferring the plant to a rooting culture medium, and growing for 40 days to obtain a phenotype;
3) After phenotype appears, root system scanning is carried out, the fresh weight, root length, root tip number, root surface area and root volume of the control group and transgenic group plants are statistically analyzed, and the test is repeated for more than three times.
Verification conclusion:
① . MdCAND2-2 over-expression plants grow for 7 days and are subjected to rooting treatment, the over-expression MdCAND-2 transgenic GALA-3 plants show obvious phenomenon of increasing adventitious roots, and the GALA-3 plants of a control group have sparse root systems and few adventitious roots, which means MdCAND2-2 can obviously root the plants.
② . The fresh weight of the transgenic plant of the over-expression MdCAND-2 is obviously higher than that of the control group by measuring the fresh weight of the root of the two groups of plants, the root length, the root tip number, the root surface area and the root volume of the transgenic plant of the over-expression MdCAND-2 are counted by root system scanning, the root length, the root tip number, the root surface area and the root volume of the transgenic plant of the over-expression MdCAND-2 are obviously higher than those of the control group, and obvious phenotype data show that the transgenic plant of the over-expression MdCAND-2 has stronger root system growth capacity.
③ . The two groups of plants were subjected to the determination of the content of the root hormone, compared with WT, the content of the root auxin (IAA), cytokinin (IPA) and Brassinolide (BR) of MdCAND-2-OE seedlings was significantly increased, the content of abscisic acid (ABA) was significantly decreased, and the content of gibberellin (GA 3) and methyl jasmonate (JA-Me) in the roots were not significantly different. Elevated melatonin levels in MdCAND2-2-OE plants suggest that MdCAND-2 may affect endogenous melatonin synthesis. MdCAND2-2 is over expressed in apples to cause the change of hormone content in seedlings, so that the rooting process of plants is regulated.
In summary, mdCAND2-2 transgenic GALA-3 plants have more excellent root growth ability.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A method of using melatonin receptor MdCAND-2 in promoting adventitious root formation in apple plants, comprising the steps of:
Step S1. Monoclonal amplification of MdCAND2-2 gene;
S2, constructing a plant super-expression vector MdCAND-2-pBI 121;
S3, transferring the plant super-expression vector MdCAND-2-pBI 121 into a GALA-3 plant by using an agrobacterium-mediated method;
and S4, verifying the functions of the transgenic plant of the over-expression MdCAND 2-2.
2. The method of using melatonin receptor MdCAND-2 according to claim 1, wherein the specific method of step S1 is:
1) PCR amplification MdCAND of the 2-2 Gene
Using specific upstream primer F and downstream primer R as primer pairs, amplifying MdCAND-2 gene with the following 40 microliter reaction system to obtain MdCAND2-2 nucleotide double strand, wherein the 40 microliter reaction system is as follows:
2) The MdCAND-2 nucleotide double strand obtained by the PCR amplification was ligated with a PMD-19-T (Simple) vector, and ligated overnight at 16℃with the following 10. Mu.l reaction system to obtain a ligation product MdCAND-2-T, the 10. Mu.l reaction system being as follows:
PMD-19-T(Simple)0.5μL
Solution 1 5μL
Recovery of the product (MdCAND-2) 4.5. Mu.L
3) Ligation product transformation competent cells
Taking out DH5 alpha competent cells of escherichia coli from an ultralow temperature refrigerator at the temperature of minus 80 ℃, putting the competent cells on ice to melt, sucking 20 microliter DH5 alpha competent cells in a sterile centrifuge tube of 1.5mL, blowing and mixing the competent cells with 10 microliter of a connecting product, carrying out ice bath for 30min, carrying out water bath heat shock for 90s at the temperature of 42 ℃, carrying out ice bath for 2min, adding 200 mu L of LB liquid culture medium in a sterile environment, putting the culture medium in a shaking table with the rotation speed of 180rpm at the temperature of 37 ℃, shaking for 1h, taking 100 mu L of bacterial liquid to coat an LB plate with antibiotics after shaking, starting to coat the plate until the plate dries, covering a cover seal to make a mark, and putting the culture medium in a constant temperature incubator at the temperature of 37 ℃ for 10-12h until bacterial plaques grow;
4) Spot picking
In a sterile super clean bench, sucking 100 microliters of LB liquid culture medium by using a sterile gun head, pumping into a 0.5mL sterile centrifuge tube, picking 10 bacterial plaques with a regular shape by using a 10 microliter sterile gun head, placing the bacterial plaques in the LB liquid culture medium, blowing for several times, covering a cover, marking the centrifuge tube, placing the centrifuge tube in a shaking table, and obtaining bacterial liquid at 37 ℃ at 180rpm for 4-6 hours;
5) Identification of bacterial liquid
After the fungus shaking is finished, the fungus liquid is identified by using the following 10 microliter reaction system, the fungus liquid is used as a template, the selected upstream primer is used as a carrier upstream primer, the selected downstream primer is used as a downstream primer of a gene, the fungus liquid is identified according to a normal verification flow, water is used as a negative control, the positive rate of the fungus liquid is checked, and the fungus liquid with brighter positive identification is selected, wherein the 10 microliter reaction system is as follows:
3. The method for promoting adventitious root formation in apple plants using melatonin receptor MdCAND-2 according to claim 1, wherein the specific method of step S2 is:
1) Extracting positive bacterial liquid plasmids, and carrying out a plasmid extraction method according to the steps of a kit to obtain PMD-19-T fusion plasmids connected with MdCAND < 2 > -2;
2) The PMD-19-T fusion plasmid connected with MdCAND-2 and the pBI 121 empty vector are subjected to double digestion according to the following 40 microliter reaction system, and the reaction is carried out for 2-6 hours at 37 ℃, wherein the 40 microliter reaction system is as follows:
3) Recovery and ligation of the target gene and the target vector: the bands of the target genes are cut for glue recovery, and simultaneously enzyme-cut bands of the target vectors are cut for glue recovery, and then are connected in a 10-micro-organism reaction system, wherein the 10-micro-organism reaction system is as follows:
T4 ligase 1. Mu.L
10x T4 buffer 1μL
8 Mu L of target gene and target vector
4) The ligation products described above were transformed into E.coli DH 5. Alpha. Competent cells in the following 30. Mu.l reaction system, which was:
Competent cells (E.coli DH 5. Alpha.) 20. Mu.L
Ligation product 10. Mu.L
Then taking out the competent cells of the escherichia coli DH5 alpha from a refrigerator at the temperature of minus 80 ℃, putting a horse on ice prepared in advance to melt, sucking 20 mu L of the competent cells of the escherichia coli DH5 alpha into a sterile centrifuge tube with the volume of 1.5mL, sucking 10 mu L of the constructed connection product, blowing and mixing uniformly, carrying out ice bath for 30min, carrying out heat shock at the temperature of 42 ℃ for 90s, carrying out ice bath for 2min, adding 200 mu L of LB liquid culture medium in a sterile environment, putting in a shaking table with the rotation speed of 180rpm, shaking for 1h, taking 100 mu L of bacterial liquid to coat an LB plate, coating the plate until the plate is dried, covering a cover seal to make a mark, and putting in a constant temperature incubator with the temperature of 37 ℃ for culturing for 10-12h;
5) Spot picking: in a sterile super clean bench, sucking 100 mu L of LB liquid culture medium by using a sterile gun head, pumping into a 0.5mL sterile centrifuge tube, picking 10 bacterial plaques by using a 10 mu L sterile gun head, placing in the LB liquid culture medium, blowing for several times, covering a cover, marking the centrifuge tube, placing in a shaking table at 37 ℃ at 180rpm for 4-6 hours;
6) And (3) bacterial liquid identification: after the shaking is finished, carrying out PCR identification by using a 10 mu L system shown in the table 3, taking bacterial liquid as a template, taking a primer as an upstream primer of a carrier, carrying out identification by using a downstream primer of a gene according to a normal verification flow, taking water as a negative control, detecting the positive rate of the bacterial liquid, and selecting a relatively bright bacterial liquid in the positive identification;
7) Extracting positive bacterial liquid plasmids, and carrying out a plasmid extraction method according to the steps of the kit to obtain the plant super-expression vector MdCAND-2-pBI 121.
4. The method for promoting adventitious root formation in apple plants using melatonin receptor MdCAND-2 according to claim 1, wherein the specific method of step S3 is:
1) Transformation of plant overexpression vector MdCAND2-2-pBI 121 into Agrobacterium EHA105
Taking out the agrobacterium EHA105 competent cells from the ultralow temperature refrigerator at the temperature of minus 80 ℃, sucking 20 microliter of the agrobacterium EHA105 competent cells into a 1.5mL sterile centrifuge tube, sucking 5 microliter of the overexpression vector MdCAND2-2-pBI 121 plasmid, adding 50 microliter of the agrobacterium EHA105 competent cells, blowing and mixing uniformly, carrying out ice bath for 30min, heating at 37 ℃ for 90s, carrying out ice bath for 2min, adding the obtained product into 200 microliter of LB liquid medium in an ultralow working table, shaking for 4h in a shaking table at the temperature of 28 ℃ and the rotating speed of 180rpm, taking 100 microliter of bacterial liquid to coat an LB plate after shaking, starting to coat the plate until the plate dries, covering a cover seal to make a mark, and culturing in a constant temperature incubator at the temperature of 28 ℃ for 10-12h;
2) Agrobacterium-mediated transfer of expression vectors into GALA-3 plants
Cutting leaves of GALA-3 plants from tissue culture seedlings subjected to secondary culture for about 30 days for genetic transformation, selecting young leaves developed from 3-4 leaves at the top of the tissue culture seedlings, cutting off leaf tips and leaf stalk parts, marking 3 trails on the middle part, immediately placing the young leaves in a preculture medium for preculture, eliminating light, at 24 ℃ for 2 days, culturing the obtained agrobacterium EHA105 in a YEP culture medium culture dish at 180rpm and 28 ℃ for 50mL in a shaking table for 4-6 hours until the OD value is 0.4-0.6, centrifuging at 25 ℃ for 5min by using a centrifuge, collecting thalli by using a glucose liquid culture medium, suspending the thalli, and adding 20mg acetosyringone for standby;
Then, placing the pre-cultured leaves in a liquid co-culture medium with agrobacterium, shaking for several times every 2min, soaking for about 8min, transferring the leaf blocks to sterile filter paper to absorb bacterial liquid, rapidly transferring the leaf blocks without bacterial liquid on the surfaces to a culture dish in which the co-culture medium is split-charged, expanding the buds by using an expanding culture medium after the leaves grow out transgenic buds, and placing the buds into a rooting culture medium for rooting after the buds grow into seedlings to obtain transgenic plants.
5. The method for promoting adventitious root formation in apple plants using melatonin receptor MdCAND-2 according to claim 1, wherein the specific method of step S4 is:
1) Selecting 20 transgenic GALA-3 plants which grow for one month and have consistent growth vigor and 20 non-transgenic GALA-3 plants of a control group;
2) Transferring the plant to a rooting culture medium, and growing for 40 days to obtain a phenotype;
3) After phenotype appears, root system scanning is carried out, the fresh weight, root length, root tip number, root surface area and root volume of the control group and transgenic group plants are statistically analyzed, and the test is repeated for more than three times.
6. The method for promoting adventitious root formation in apple plants using melatonin receptor MdCAND-2 according to claim 1, wherein in step S1, the CDS sequence of MdCAND2-2 gene is specifically:
ATGCAAGCTCCCCAAGAAGCTTCCTCGCAATCCGGACTCCCAATTCCACTCACCCAGGCCCCAAATTCAACCATAGACGACGGCGTTTCGGGCTCCGAATTCTACAGCTGGCTCGGCGAATGTCATGGGTTCTTCCACAATGTGGCTCTGATGGTGCCCTCGCTTCTCTTCGTCCTGTATTTGGCGTTTCAATCGAGAAAAAGCTTCTCGAAGCTCTCCCATGGCCGCTCTTATATCATGATTGCCTACTACGGGTGCCTCTGGCTTGTTAGCTTGCTCAATCTTGCTTGGTGCTGTTTTCAGGCATGGGAGTGCAGTCCTGGAAAAGAAGTAACATGGAATATATTATCTTTGTTCACAACCTCCGGGATGCTATTTTTGGAAGTAAGCTTGCTGGCATTTTTGCTTCAGGGGAATTATGCAAGTGGACTGGAAGCTTTGACACGGACTTTTTTTATCTCAGGGATCCTTGTTGTTTTGGATATACTTCTCAAGGTATCGCTCGTTCTTTTTCGTGAAATGGATTTTCACCCTTATGACCTATGTATGCTTGTTAGTTACTTCAAGAATATACTAATGGAAACATATATGTTGTTGGAAGTGCACATTGGTGCATTATTGTGGTGCATAACGGACTTACAATTATACTCTGTTTTCTTCCAGGCAATATATCTATTTGGACTTGGCATTCCATTGTTCATTGACAATAATGACCGTGCACATCGAACAAAGTGGGCCTTGTGGGTTGTCCACAGGCTAGTGATTACTGCAATTTACGGCTTCATATTGTTCATGTACCATTCCAAGTGGAGAGAGAGGTTACCAGCAAGACCTGCATTCTACAAGTATACCGCTATCATGTTTACGTTAAACGTACTAGTACTGTTTGCTTGCTTGCTTACTGGAAATGGGGCTGGATTTGGATTCTGGCTATATAGTGCTACTATTGTCTGTTATCACGCCTTTTATCTTCCACTTCTGTACGTAACATTTCTGGCAGACTTCTTCCAGGAGGAAGATTTAAATTTGGAGAATGTGTACTATTCAGAAATGAAAGATGCCGGTTTCTTTGACGCGGATTGGGAGTGA.
7. The method for promoting adventitious root formation in apple plants using melatonin receptor MdCAND-2 according to claim 1, wherein in step S1, the nucleotide sequence of specific upstream primer F is specifically:
ACTTCCCCAACCTTTCTAGTCAT。
8. the method for promoting adventitious root formation in apple plants using melatonin receptor MdCAND-2 according to claim 1, wherein in step S1, the nucleotide sequence of specific downstream primer R is specifically:
TAACATCATACAAACACTTAAAAAC。
9. Use of the melatonin receptor MdCAND-2 according to any one of claims 1-8 for promoting adventitious root formation in apple plants.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410413646.6A CN118185997A (en) | 2024-04-08 | 2024-04-08 | Application method of melatonin receptor MdCAND-2 in promoting generation of adventitious roots of apple plants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410413646.6A CN118185997A (en) | 2024-04-08 | 2024-04-08 | Application method of melatonin receptor MdCAND-2 in promoting generation of adventitious roots of apple plants |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118185997A true CN118185997A (en) | 2024-06-14 |
Family
ID=91396631
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410413646.6A Pending CN118185997A (en) | 2024-04-08 | 2024-04-08 | Application method of melatonin receptor MdCAND-2 in promoting generation of adventitious roots of apple plants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118185997A (en) |
-
2024
- 2024-04-08 CN CN202410413646.6A patent/CN118185997A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111607599B (en) | Potato KNOX transcription factor StKNOX1 gene, coding protein and application thereof | |
| CN102787124A (en) | Tomato fruit ripening gene SINAC3 and application thereof | |
| WO2020221029A1 (en) | Zea mays receptor-like kinase gene zmrlk7 and use thereof | |
| Yang et al. | Construction of high efficiency regeneration and transformation systems of Pyrus ussuriensis Maxim | |
| CN110713529A (en) | Application of VvDUF642 gene in causing abortion of plant seeds | |
| CN113846121B (en) | Method for regulating and controlling occurrence of tomato lateral branches | |
| CN105294847A (en) | Stress tolerance-related protein of plants and encoding gene and application of stress tolerance-related protein | |
| CN118185997A (en) | Application method of melatonin receptor MdCAND-2 in promoting generation of adventitious roots of apple plants | |
| Parks et al. | Composite plants for a composite plant: an efficient protocol for root studies in the sunflower using composite plants approach | |
| CN102559676A (en) | Rice root specific promoter and application thereof | |
| CN107488669B (en) | CauliflowerBoTLP1Coding sequence of gene and application thereof in cultivating salt-tolerant drought-resistant transgenic plant | |
| CN113215186A (en) | Application of light respiration branch protein in regulation and control of plant characters | |
| CN113249394B (en) | Application of receptor-like kinase gene MdMRLK2 in improving plant water utilization efficiency | |
| CN104805065B (en) | A kind of paddy rice histone deacetylase and its encoding gene and application | |
| CN108707613A (en) | NtHAK8 genes and application thereof | |
| Jin et al. | Techniques for the regeneration and genetic transformation of Arabidopsis pumila: An ephemeral plant suitable for investigating the mechanisms for adaptation to desert environments | |
| CN114517202B (en) | Application of aquaporin gene SlPIP, 2 in improving resistance of tomatoes to facility continuous cropping soil | |
| CN116479007B (en) | Celery AgDREBA6a gene and application thereof in improving high-temperature stress resistance of plants | |
| KR101238259B1 (en) | ADH gene increasing seed germination of plant at anaerobic condition and uses thereof | |
| CN118165996A (en) | Application of MdNAC gene in improving potassium deficiency stress resistance of apples | |
| CN116064578A (en) | Gene related to stress resistance of Wucai and application thereof | |
| CN116590301A (en) | Hybridized tulip tree LhWUS gene and expression protein and application thereof | |
| Jin et al. | Regeneration and Genetic Transformation of Arabidopsis Pumila: An Ephemeral Plant Suitable for Investigating the Mechanisms for Adaptation to Desert Environments | |
| CN113881686A (en) | Method and sequence for improving branching capability of plant and fruit seedling breeding method | |
| CN116970638A (en) | Application of knockout tomato SlZF3 gene in improving tomato yield |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication |