CN118006675A - Efficient genetic transformation method for grape seeds independent of tissue culture - Google Patents
Efficient genetic transformation method for grape seeds independent of tissue culture Download PDFInfo
- Publication number
- CN118006675A CN118006675A CN202410071900.9A CN202410071900A CN118006675A CN 118006675 A CN118006675 A CN 118006675A CN 202410071900 A CN202410071900 A CN 202410071900A CN 118006675 A CN118006675 A CN 118006675A
- Authority
- CN
- China
- Prior art keywords
- grape
- grape seeds
- genetic transformation
- dip
- independent
- 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
- 235000009754 Vitis X bourquina Nutrition 0.000 title claims abstract description 49
- 235000012333 Vitis X labruscana Nutrition 0.000 title claims abstract description 49
- 235000014787 Vitis vinifera Nutrition 0.000 title claims abstract description 49
- 230000002068 genetic effect Effects 0.000 title claims abstract description 33
- 238000011426 transformation method Methods 0.000 title claims abstract description 13
- 240000006365 Vitis vinifera Species 0.000 title 1
- 241000219095 Vitis Species 0.000 claims abstract description 68
- 230000009466 transformation Effects 0.000 claims abstract description 29
- 239000007788 liquid Substances 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000004043 dyeing Methods 0.000 claims abstract description 19
- 230000009261 transgenic effect Effects 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 210000001015 abdomen Anatomy 0.000 claims abstract description 7
- 230000001954 sterilising effect Effects 0.000 claims abstract description 7
- 238000005520 cutting process Methods 0.000 claims abstract description 6
- 239000001963 growth medium Substances 0.000 claims abstract description 6
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 claims description 14
- 230000001580 bacterial effect Effects 0.000 claims description 11
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- 229940087559 grape seed Drugs 0.000 claims description 5
- 208000015181 infectious disease Diseases 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 229930191978 Gibberellin Natural products 0.000 claims description 3
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 3
- 210000003323 beak Anatomy 0.000 claims description 3
- 230000001419 dependent effect Effects 0.000 claims description 3
- 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 claims description 3
- 239000003448 gibberellin Substances 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 16
- 239000000463 material Substances 0.000 abstract description 14
- 241000589158 Agrobacterium Species 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 5
- 230000001404 mediated effect Effects 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 230000007547 defect Effects 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 239000002609 medium Substances 0.000 description 6
- 241000219094 Vitaceae Species 0.000 description 5
- 235000021021 grapes Nutrition 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000004161 plant tissue culture Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- YLNDNABNWASMFD-UHFFFAOYSA-N 4-[(1,3-dimethylimidazol-1-ium-2-yl)diazenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=[N+](C)C=CN1C YLNDNABNWASMFD-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 241000589176 Agrobacterium vitis Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 229930189077 Rifamycin Natural products 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 235000011869 dried fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 229960003292 rifamycin Drugs 0.000 description 1
- HJYYPODYNSCCOU-ODRIEIDWSA-N rifamycin SV Chemical compound OC1=C(C(O)=C2C)C3=C(O)C=C1NC(=O)\C(C)=C/C=C/[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@H](C)[C@@H](OC)\C=C\O[C@@]1(C)OC2=C3C1=O HJYYPODYNSCCOU-ODRIEIDWSA-N 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 210000000707 wrist Anatomy 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a high-efficiency genetic transformation method of grape seeds independent of tissue culture, and relates to the fields of agricultural biotechnology and plant genetic engineering. The genetic transformation comprises the following steps: sterilizing grape seeds, performing skin cutting treatment, and pre-culturing until belly shells are cracked; placing the grape seeds with split belly shells into a dip dyeing solution for dip dyeing treatment, then planting the grape seeds into a culture medium, and culturing to obtain transgenic grape plants; the dip-dyeing liquid contains recombinant engineering strains capable of expressing heterologous genes. The invention takes grape seeds as test materials, and introduces exogenous genes into the genome of grape successfully by an agrobacterium-mediated method, thereby realizing genetic transformation of grape and obtaining transgenic grape plants. The transformation method effectively overcomes the defects of the traditional genetic transformation technology, can realize genetic transformation on the basis of not depending on the tissue culture technology, and has higher transformation efficiency and operation convenience.
Description
Technical Field
The invention relates to the fields of agricultural biotechnology and plant genetic engineering, in particular to a high-efficiency genetic transformation method for grape seeds without tissue culture dependence.
Background
Grape (grape l.), which belongs to the family of vitiaceae, is one of the economic fruit trees widely planted worldwide, is known as the first of four fruits in the world because of its unique taste and high nutritive value. The grape has rich nutrition, wide application, bright color, strong fragrance and delicious taste, and is suitable for fresh eating and can be used for brewing wine, juice making and dried fruit production. Among them, the variety 'Cabernet Sauvignon' is known to be easy to cultivate, high in juice yield and excellent in sugar-acid ratio, and the brewed wine has ruby red color, mellow taste and unique flavor, is favored by consumers, and has extremely high economic value. However, the traditional cross breeding method has long period and low efficiency, and is difficult to meet the requirement of rapid cultivation of new varieties. In contrast, genetic engineering techniques provide a more efficient way to cultivate new varieties. Although the research of grape transgenic technology is relatively extensive, the research of grape seed transgenic under the condition of non-tissue culture (short non-tissue culture) is still blank.
In agrobacterium-mediated genetic transformation of grapes, commonly used acceptor materials generally include leaves, petioles, anthers, filaments, stem tips, stem segments, and the like. These materials originate from different stages of grape growth and have significant space-time variability. In order to improve the conversion efficiency and create new varieties, the selection of ideal conversion materials is important. The ideal material should meet the following conditions: firstly, the treatment mode is unified, and the repeatability is high; secondly, the differentiation degree is low, the totipotency of cells is strong, and the potential of developing into plants is provided; thirdly, the method is easy to obtain, and the interference of the materials to the test is reduced; and fourthly, the quantity is enough, which is favorable for successful transformation. Grape seeds are excellent in quantity, uniformity, repeatability, availability and cell totipotency, and are ideal genetic transformation materials. However, the traditional agrobacterium vitis genetic transformation needs to rely on tissue culture, and the method has long period, high pollution rate and easy death of plants. Therefore, the development of a grape genetic transformation method which is not dependent on tissue culture is particularly critical to the creation of new varieties.
Disclosure of Invention
The invention aims to provide a high-efficiency genetic transformation method of grape seeds without tissue culture dependence, so as to solve the problems of the prior art, effectively overcome the defects of the traditional genetic transformation technology, realize genetic transformation on the basis of independent tissue culture technology, and have higher transformation efficiency and operation convenience.
In order to achieve the above object, the present invention provides the following solutions:
The invention provides a high-efficiency genetic transformation method of grape seeds independent of tissue culture, which comprises the following steps:
Sterilizing grape seeds, performing skin cutting treatment, and pre-culturing until belly shells are cracked;
Placing the grape seeds with split belly shells into a dip dyeing liquid for dip dyeing treatment, then planting the grape seeds into a culture medium, and culturing to obtain transgenic grape plants;
the dip-dyeing liquid contains recombinant engineering strains capable of expressing heterologous genes.
Further, the disinfection treatment comprises alcohol disinfection and sodium hypochlorite solution disinfection.
Further, the grape seeds are soaked in gibberellin before being disinfected.
Further, the skin-cutting treatment refers to the scarification of the back seed coat and the beak of the grape seed.
Further, the dip dyeing liquid also comprises acetosyringone.
Further, the acetosyringone concentration is 100mM.
Further, the dip dyeing liquid is obtained by adding acetosyringone into bacterial liquid containing the recombinant engineering bacterial strain and performing dark culture.
Further, the time of the dark culture was 2 hours.
Further, the OD 600 =0.6 of the bacterial liquid.
Further, the time of the vacuuming and dyeing treatment is 6min, and the time of the shaking and dyeing treatment is 14min.
The invention discloses the following technical effects:
The invention takes grape seeds as test materials, and introduces exogenous genes into the genome of grape successfully by an agrobacterium-mediated method, thereby realizing genetic transformation of grape and obtaining transgenic grape plants.
The genetic transformation method of the invention directly uses grape seeds to operate without tissue culture, and has the following advantages:
(1) The operation is simple. Avoiding a series of complicated processes of culture medium preparation, explant disinfection, callus induction, subculture, co-culture, screening, regeneration bud induction, rooting and the like which are necessary in the plant tissue culture process.
(2) The growth period is short. The transgenic grape seedlings can be obtained only by 35-40d, and the time required by the method is shorter than that required by the traditional tissue culture method (85 d).
(3) High repeatability and universality. The material treatment mode is unified, simple, convenient and easy to repeat.
(4) The economic benefit is high. The invention adopts a method of directly infecting grape seeds by using agrobacterium tumefaciens bacteria liquid to carry out genetic transformation and uses a nutrient medium to cultivate grape plants, and compared with the traditional plant tissue culture transformation process, the method remarkably reduces the investment of manpower and material resources, thereby greatly reducing the overall cost of genetic transformation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of seed transformation according to the present invention; wherein, A is the engineering bacteria liquid of VvBBM gene carrier to activate and prepare the dyeing liquid; b is to obtain grape seeds, and then to carry out skin cutting after being treated in 2.5g/L GA 3; c, clamping grape seeds pre-cultured until abdominal shell cracks into an infection liquid; d, vacuumizing and manually infecting grape seeds; e, planting grape seeds with the dry bacterial liquid absorbed into a nutrient medium sterilized at high temperature, and culturing to form complete plants;
FIG. 2 is a statistical plot of seed germination for wild type grapes (UT) and transgenic grapes (OE-VvBBM);
FIG. 3 shows plant height, root length, total root number of UT and OE-VvBBM plants grown with 38d seed;
FIG. 4 is a graph of the detection of UT, OE-VvBBM under a fluorescence confocal microscope;
FIG. 5 is a PCR identification map of grape plants; wherein, #1, #3, #5, #7, #12 and #13 show positive bands; NE is ddH 2 O negative control group; PO is VvBBM plasmid positive control group;
FIG. 6 shows the results of detection of the expression level of OE-VvBBM transgenic plants; wherein a is the typical phenotype of the over-expressed plant; b is VvBBM relative expression quantity detection result;
FIG. 7 is a Western blot (A) and PCR amplification (B) detection of VaMIEL 1.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The preparation method of the culture medium used in the following examples was as follows:
LB liquid medium: 10g/L sodium chloride+10 g/L peptone+5 g/L yeast extract.
LB solid medium: 10g/L sodium chloride+10 g/L peptone+5 g/L yeast extract+15 g/L agar.
The invention discloses a high-efficiency rapid tissue-independent agrobacterium-mediated grape seed genetic transformation method, the transformation flow chart is shown in figure 1, and the method is specifically described as follows:
Example 1
1. VvBBM Gene plant expression vector construction
PCR amplification was performed using the 'Chardonnay' grape somatic cDNA as a template using primers 2300-BBM-Bam-F and 2300-BBM-Sal-R to obtain a PCR product (SEQ ID NO. 1). The vector pHZM was digested with SalI and SacI restriction endonucleases at 30℃for 15min,37℃for 30min and 65℃for 20min, respectively. And carrying out homologous recombination reaction on the PCR product obtained by purification and recovery and the enzyme digestion product. The PCR reaction was performed at 55℃for 30min and at 30℃for 20min. The recombinant solution was transformed into LB solid medium resistant to kanamycin (Kan), and the transformed culture was inverted and incubated overnight in a constant temperature incubator at 37 ℃. Single colonies were picked, inoculated into LB medium containing KAN, placed on a 37℃constant temperature shaker at 180rpm for 16h, and plasmids were extracted. The concentration and purity were determined. And (5) enzyme digestion detection is carried out. mu.L of plasmid was taken and sent to the company for sequencing.
SEQ ID NO.1：
ATGGCTTCCATGAACAACTGGTTGGGTTTCTCTTTGTCCCCTCGAGAACTTCCACCACAGCCTGAAAATCACTCACAGAACAGTGTCTCTAGACTTGGTTTCAACTCTGATGAAATCTCTGGGACTGATGTGTCAGGTGAGTGTTTTGATCTCACTTCAGATTCCACTGCTCCCTCTCTCAACCTCCCTCCCCCTTTTGGGATACTTGAAGCATTCAACAGGAATAATCAGCCCCAAGATTGGAACATGAAGGGTTTGGGCATGAATTCAGATACTAACTACAAAACCACCACTTCTGAGCTCTCCATGCTCATGGGTAGTTCATGCAGTAGTCATCATAACCTCGAAAACCAAGAACCCAAACTTGAAAATTTCCTGGGCTGCCGCTCTTTTGCTGATCATGAGCAGAAACTTCAAGGGTGTAACTCCATTGCAGCAGCAGCTTATGATAGCTCTGCAGACTACATGTTCCCCAACTGCTCACTGCAGCTTCCATCTGAGCCGGTAGACACCCCCACTCCCCGCGGTGGCGGCGGTGGAAGCACTACCGTCAACAATAGTTCCATTGGTTTATCCATGATCAAGACATGGCTGCGGAACCAACCTGCACCCACCCATCAGGATAACAACAAGAGTACTGATACTGGGCCTGTCGGTGGAGCCGCCGCTGGGAACCTACCCAATGCACAGACCTTATCGTTGTCCATGAGCACCGGCTCGCAGTCCAGTTCTCCTTTGCCTCTCCTAACAGCGAGTGCAGGTGGTGGTGGTGGGAGTGGAGGAGAGAGTTCTTCATCAGATAACAAGAAGGCCACCCCCCTCGATAGCCAGACCGGTGCCATTGAAACGGTGCCAAGGAAGTCCATTGATACATTTGGACAGAGGACATCCATATACCGTGGTGTAACAAGGCATAGATGGACGGGTAGATATGAGGCTCATCTATGGGACAACAGTTGCAGAAGAGAAGGACAAACTCGAAAGGGAAGGCAAGTTTATTTAGGTGGTTATGACAAAGAAGAAAAGGCAGCTAGGGCTTACGATTTAGCAGCACTGAAGTATTGGGGTACCACCACCACAACAAATTTCCCTATTAGCAACTATGAAAAAGAGATAGAGGAGATGAAGCACATGACAAGGCAGGAGTACGTAGCATCTCTGCGAAGGAAGAGTAGCGGGTTTTCTCGTGGAGCATCCATATATAGAGGAGTGACCAGACACCATCAGCATGGGAGATGGCAGGCAAGGATTGGAAGAGTCGCAGGCAACAAAGATCTTTACTTGGGAACTTTCAGCACCCAAGAGGAAGCAGCAGAGGCCTATGACATTGCTGCCATTAAGTTTCGAGGATTGAATGCGGTGACCAACTTTGATATGAGTAGATATGATGTTAATAGCATTCTAGAGAGCAGTACCTTGCCGATTGGTGGAGCTGCAAAGCGGTTGAAAGATGCTGAGCAGGCTGAAATGACTATAGATGGACAGAGGACAGACGATGAGATGAGCTCACAGCTGACTGATGGAATCAACAACTATGGAGCACACCACCATGGCTGGCCTACTGTTGCATTCCAACAAGCTCAGCCATTTAGCATGCACTACCCTTATGGCCATCAGCAGAGGGCTGTTTGGTGTAAGCAAGAGCAAGACCCTGATGCCACACACAACTTTCAAGATCTTCACCAACTACAATTGGGAAACACTCACAACTTCTTCCAGCCTAATGTTCTGCACAACCTCATGAGCATGGACTCTTCTTCAATGGACCATAGCTCAGGCTCCAATTCAGTCATCTATAGCGGTGGTGGAGCCGCTGATGGCAGCGCTGCAACTGGCGGCAGTGGCAGTGGGAGCTTCCAAGGGGTAGGTTATGGGAACAACATTGGCTTTGTGATGCCCATAAGCACCGTCATCGCTCATGAAGGCGGCCATGGCCAGGGAAATGGTGGCTTTGGAGATAGCGAAGTGAAGGCGATTGGTTACGACAACATGTTTGGATCGACAGATCCTTACCATGCTAGGAGCTTGTACTATCTTTCACAGCAATCATCTGCAGGCATGGTGAAGGGCAGTAGTGCATATGATCAGGGGTCAGGGTGTAACAACTGGGTTCCAACTGCAGTTCCAACCCTAGCTCCAAGGACTAACAGCTTGGCAGTATGCCATGGAACACCTACATTCACAGTATGGAATGATACAtaa.
2300-BBM-Bam-F:5’-ATGGCTTCCATGAACAACTGGT-3’(SEQ ID NO.2)；
2300-BBM-Sal-R:5’-TGTATCATTCCATACTGTGAAGTGT-3’(SEQ ID NO.3)。
2. Preparation of Agrobacterium
The recombinant plasmid which is correctly detected is selected and added into competent cell EHA105 to be evenly mixed and transferred into a electric stun cup. And starting an Eppendorf4308 electric shock conversion instrument to carry out electric shock conversion, and setting conversion parameters: 1.8KV,5ms. Then, the mixture was subjected to shaking culture at 28℃and 180rpm in a shaker for 2 hours, and then Kan + and Rif plates were applied and cultured in an incubator at 28℃for 3 days. Positive monoclonal colonies were picked and cultured for 48h in a shaker at 28℃at 180 rpm.
1ML of the bacterial liquid is sucked and added into 50mL of LB (or YEP) liquid culture medium containing 50mg/L Kan + and 25mg/L rifamycin (Rif), and the bacterial liquid is subjected to shaking culture at 28 ℃ and 180rpm until OD 600 reaches 1.0; the bacterial liquid and 30% of glycerol are mixed according to the following ratio of 1:1 are mixed and placed in a refrigerator at the temperature of minus 80 ℃ for standby.
3. Preparation of grape seed genetic transformation receptor of' Cabernet Sauvignon
Healthy and full 'Cabernet Sauvignon' seeds are selected, ultrasonic treatment is carried out in 2.5g/L gibberellin (GA 3) solution for 2min at 55 ℃ for 13min, then shaking is carried out in a shaking table at 180rpm at 28 ℃ for 24h, then the seeds are treated with 75% alcohol for 30s, sterilized water is used for 3 times, sodium hypochlorite with 1.5% of available chlorine mass fraction is used for 15min, sterilized water is used for 3 times, and in an ultra-clean workbench, the back seed coats of the seeds are cut by a knife, and are broken by force at the beak part, and the seeds are pre-cultured until abdominal shells are broken.
4. Genetic transformation of grape seeds
(1) And preparing an aggressive dyeing liquid. Activating the agrobacterium obtained in the step 2, collecting thalli, re-suspending the thalli to OD 600 =0.6 by using sterile water, simultaneously adding 100mM acetosyringone, and then placing the mixture in a shaking table at 180rpm for dark culture for 2 hours to obtain an invaded dye liquor for later use.
(2) And (5) infection. Clamping seeds of Cabernet Sauvignon which are pre-cultured until the abdomen has obvious shell cracks into an infection liquid in a laboratory with a bacterial environment (i.e. not operated on an ultra-clean workbench), vacuumizing for 6min, shaking the wrist for 14min, sucking the bacterial liquid with filter paper, planting a high-temperature sterilized nutrition matrix, performing dark culture for 25d at 25 ℃ for 16h and 8h, and performing fluorescence detection; seeds not infected with agrobacterium were also set as negative controls.
5. Identification of transgenic grape plants
Under non-tissue culture conditions, by day 20, a total of 54 grape plants germinated, with a germination rate of 75% (fig. 2). By day 40, 14 well-grown and robust plants were selected for transgenic grape plant identification.
(1) The grape plants are observed by using a fluorescence confocal microscope, and positive seedlings excited by fluorescent signals are initially screened. The detection patterns of wild-type grapes (UT) and transgenic grape plants (OE-VvBBM) under a fluorescence confocal microscope are shown in FIG. 4.
(2) Seedlings with fluorescent signals were selected for PCR detection, DNA from leaf blades of grape plants was extracted using DNAsecure Plant Kit (TIANGEN) kit as template, and PCR identification was performed using Semi-BBM-F:5'-GTAAGCAAGAGCA AGACCCTGATG-3' (SEQ ID NO. 4) and 2300-R:5'-CTCGACCAGGATGGGCACC-3' (SEQ ID NO. 5), untransformed 'Cabernet Sauvignon' plants were used as controls, and 6 grapes were given a band of interest, indicating that VvBBM gene had been successfully inserted into the grape genome (FIG. 5).
(3) And (3) selecting 14 transformed grape plants for fluorescent quantitative PCR detection, and finding that the gene expression quantity of the transgenic grape plants VvBB M is obviously higher than that of control plants (figure 6).
(4) By comparison of wild-type and transgenic grape plants, it was found (FIG. 3) that transgenic grape plants grew significantly higher than untransformed plants.
Based on the results, the conversion is carried out by taking Cabernet Sauvignon seeds as acceptor materials in a non-sterile environment, and the conversion rate can reach 42.85%.
EXAMPLE 2 application of genetic transformation to other genes under non-tissue culture conditions
The same transformation method as in example 1 was used, with agrobacterium EHA105 as a bioengineering strain, to successfully transfer the target gene into 'chardonnay' grape plants using a plant expression vector pHZM carrying transcription factor VaMIEL (SEQ ID No. 6) and a screening reporter gene GFP. On day 40 after transformation, grape plants were subjected to protein and genomic DNA extraction and detection by Western blotting (Western blotting) and PCR amplification experiments, with specific protein bands and PCR bands successfully detected (fig. 7). Western blot was again performed at day 60, and bands were also detected, confirming long-term stable expression of the transgene in grape plants.
SEQ ID NO.6：
ATGGAAGGTGGGGTTGATAGCAAATTGCTTGAAATTTGTGGAAGAGAGGTGTCTCTCACGGAGCTTGGATCTGGGAAATATGGGTGCACACATTACAGAAGGAGATGCAAGATTCGGGCTCCTTGTTGTGATGAAATCTTTGATTGTAGGCACTGCCATAATGAAGCCAAGAATTTACTGGAAACTGATCCTCATGATCGCCACGATATTCCAAGGCATGAAGTTAAAAAGGTCATTTGTTCTCTTTGTGACACAGAACAAGATGTCCAACAGAATTGCATTGCCTGTGGCGTTTGCATGGGGGAATACTTTTGCGAGAAATGCAAGTTCTTCGATGATGATGTTTCTAAGAATCAATATCATTGTGAAGAATGTGGAATCTGCAGAACCGGAGGGAAGGAGAATTTCTTTCACTGCAACAAATGTGGATGTTGCTATTCAAATATGATGAAGGATACACACCGATGTGTGGAAAGGGCAATGCACCACAATTGCCCTGTTTGCTTCGAGTTCCTTTTTGATACAACAAAAGATATTACTGTCTTACAATGTGGACACACTATACATTGGGAGTGTGTGAAGGAGATGCAACAGCATTTTTGCTACTCATGCCCTGTTTGCTCAAAATCCATTTGTGATATGTCCAGTTTGTGGGAAAAAATTGACCGAGAGGTTGCTTCAACTCCCATGCCTGAAATGTACCGAAAGAAGATGGTGTGGATCCTCTGCAATGATTGTGGGGCACAATCTGAAGTTCAATTCCATATTGTGGCTCATAAATGCCTGAGCTGCAAGTCCTACAACACTAGACAGATACAAGGAGGTCCAGCTTCTTCATGCTCATCAGGGATTGCTGGGATGGTGAGATGA.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The efficient genetic transformation method of the grape seeds independent of tissue culture is characterized by comprising the following steps of:
Sterilizing grape seeds, performing skin cutting treatment, and pre-culturing until belly shells are cracked;
Placing the grape seeds with split belly shells into a dip dyeing liquid for dip dyeing treatment, then planting the grape seeds into a culture medium, and culturing to obtain transgenic grape plants;
the dip-dyeing liquid contains recombinant engineering strains capable of expressing heterologous genes.
2. The method of high-efficiency genetic transformation of tissue-independent grape seeds of claim 1, wherein the sterilization treatment comprises alcohol sterilization and sodium hypochlorite solution sterilization.
3. The method for efficient genetic transformation of tissue-independent grape seeds of claim 2, wherein the grape seeds are further subjected to gibberellin soaking treatment prior to sterilization.
4. The method of claim 1, wherein the skin-cutting treatment is a scarification of the back seed coat and beak of the grape seed.
5. The method of high-efficiency genetic transformation of tissue-independent grape seeds of claim 1, wherein the dip-dye solution further comprises acetosyringone.
6. The method for efficient genetic transformation of tissue-independent grape seeds of claim 5, wherein the acetosyringone concentration is 100mM.
7. The efficient genetic transformation method of non-tissue culture-dependent grape seeds of claim 5, wherein the dip-dyeing liquid is obtained by adding acetosyringone into a bacterial liquid containing the recombinant engineering strain and culturing in a dark state.
8. The method for efficient genetic transformation of tissue-independent grape seeds of claim 7, wherein the dark culture time is 2 hours.
9. The method for efficient genetic transformation of tissue-independent grape seeds of claim 7, wherein the bacterial liquid has OD 600 = 0.6.
10. The method for efficient genetic transformation of non-tissue culture dependent grape seeds according to claim 1, wherein the dip-dyeing treatment is a vacuum dip-dyeing treatment and a shaking infection, the time of the vacuum dip-dyeing treatment is 6min, and the time of the shaking infection is 14min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410071900.9A CN118006675A (en) | 2024-01-18 | 2024-01-18 | Efficient genetic transformation method for grape seeds independent of tissue culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410071900.9A CN118006675A (en) | 2024-01-18 | 2024-01-18 | Efficient genetic transformation method for grape seeds independent of tissue culture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118006675A true CN118006675A (en) | 2024-05-10 |
Family
ID=90947747
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410071900.9A Pending CN118006675A (en) | 2024-01-18 | 2024-01-18 | Efficient genetic transformation method for grape seeds independent of tissue culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118006675A (en) |
-
2024
- 2024-01-18 CN CN202410071900.9A patent/CN118006675A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113604490B (en) | Kiwi fruit canker susceptibility gene AcBXL1 and application thereof | |
| CN110713529A (en) | Application of VvDUF642 gene in causing abortion of plant seeds | |
| CN104770294B (en) | A kind of protocorm based on the sprouting of iris seed is the breeding method of acceptor | |
| CN107475287B (en) | Eggplant genetic transformation method | |
| CN107630022B (en) | Application of tomato SlMYB75 gene in enhancing rot resistance and prolonging shelf life of tomato fruits | |
| CN101928724A (en) | Mechanical hybrid rice seed production method utilizing transgenic technology of chloroplasts | |
| CN113234751A (en) | Agrobacterium tumefaciens transformation vector based on rhizobium III type effector NopP and application thereof | |
| CN117070557A (en) | Rapid and efficient agrobacterium-mediated iris seed genetic transformation method | |
| CN116103337B (en) | Agrobacterium-mediated passion flower genetic transformation method | |
| CN115896160B (en) | Method for efficiently and rapidly obtaining apple stable transgenic plants by using agrobacterium rhizogenes | |
| CN115044606B (en) | Method for establishing agrobacterium rhizogenes-mediated jujube genetic transformation system | |
| KR100402513B1 (en) | Efficient method for the development of transgenic plants by gene manipulation | |
| CN118006675A (en) | Efficient genetic transformation method for grape seeds independent of tissue culture | |
| CN112889668A (en) | Populus genetic transformation method | |
| CN114958904B (en) | Method for rapidly obtaining radish transgenic material | |
| CN114507679B (en) | Pinus massoniana terpenoid synthesis related enzyme gene PmDXR and application of promoter thereof | |
| KR101198648B1 (en) | Method for transforming Cucumis sativus line through direct shoot induction and Cucumis sativus line transformant produced by the same | |
| CN117187294B (en) | Application of BnaC5.ACBP4 gene in improving flooding resistance of plants | |
| CN115725644B (en) | Genetic transformation method and application of Polygonatum cyrtonema Fabricius | |
| CN116376933B (en) | Rice germination rate regulating gene OsSCYL2 and application thereof | |
| CN116479037A (en) | Natrii sulfas VIGS silencing system and application thereof | |
| CN115807033A (en) | Agrobacterium mediated genetic transformation method using grape seeds as acceptor material | |
| CN115992169A (en) | Efficient lily quilt sheet transient expression method and application | |
| CN105602957A (en) | Cotton fiber specific expression promoter TB7P1 and application thereof | |
| CN118006665A (en) | Genetic transformation method of syzygium samarangense |
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 |