CN113186220A - Light source combination box for promoting kiwi fruit transgenic efficiency and application thereof - Google Patents

Light source combination box for promoting kiwi fruit transgenic efficiency and application thereof Download PDF

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CN113186220A
CN113186220A CN202110459973.1A CN202110459973A CN113186220A CN 113186220 A CN113186220 A CN 113186220A CN 202110459973 A CN202110459973 A CN 202110459973A CN 113186220 A CN113186220 A CN 113186220A
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light source
light
kiwi fruit
combination box
transgenic
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CN113186220B (en
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李大卫
钟彩虹
刘晓莹
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Wuhan Botanical Garden of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
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    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F21—LIGHTING
    • F21K—NON-ELECTRIC LIGHT SOURCES USING LUMINESCENCE; LIGHT SOURCES USING ELECTROCHEMILUMINESCENCE; LIGHT SOURCES USING CHARGES OF COMBUSTIBLE MATERIAL; LIGHT SOURCES USING SEMICONDUCTOR DEVICES AS LIGHT-GENERATING ELEMENTS; LIGHT SOURCES NOT OTHERWISE PROVIDED FOR
    • F21K9/00—Light sources using semiconductor devices as light-generating elements, e.g. using light-emitting diodes [LED] or lasers
    • F21K9/20—Light sources comprising attachment means
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F21—LIGHTING
    • F21V—FUNCTIONAL FEATURES OR DETAILS OF LIGHTING DEVICES OR SYSTEMS THEREOF; STRUCTURAL COMBINATIONS OF LIGHTING DEVICES WITH OTHER ARTICLES, NOT OTHERWISE PROVIDED FOR
    • F21V19/00—Fastening of light sources or lamp holders
    • F21V19/001—Fastening of light sources or lamp holders the light sources being semiconductors devices, e.g. LEDs
    • H—ELECTRICITY
    • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
    • H05B—ELECTRIC HEATING; ELECTRIC LIGHT SOURCES NOT OTHERWISE PROVIDED FOR; CIRCUIT ARRANGEMENTS FOR ELECTRIC LIGHT SOURCES, IN GENERAL
    • H05B45/00—Circuit arrangements for operating light-emitting diodes [LED]
    • H05B45/30—Driver circuits
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F21—LIGHTING
    • F21Y—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES F21K, F21L, F21S and F21V, RELATING TO THE FORM OR THE KIND OF THE LIGHT SOURCES OR OF THE COLOUR OF THE LIGHT EMITTED
    • F21Y2115/00—Light-generating elements of semiconductor light sources
    • F21Y2115/10—Light-emitting diodes [LED]

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Abstract

The invention discloses a light source combination box for promoting kiwi fruit transgenic efficiency and application thereof, and relates to the field of genetic engineering. The light source combination box comprises red and blue LED light sources, and in the same LED light source combination box, total 100 LED lamp pearls are 10X 10 arrangement, and the LED lamp pearls of ruddiness and blue light are evenly arranged in a proportion of 1: 4, the input voltage is 220V, the power is 300W, and the photosynthetic photon flux density of the working environment is 200 +/-5 mu molm‑2s‑1The photoperiod is 12 light/12 h dark, and the temperature is 24 +/-1 ℃. The invention has the following advantages and positive effects: the efficiency of the existing kiwi fruit transgenosis can be obviously improved; secondly, the method is suitable for accelerating the production conditions of tissue culture regeneration and the like of the kiwi fruits; and is suitable for most kiwi fruit molecular laboratories.

Description

Light source combination box for promoting kiwi fruit transgenic efficiency and application thereof
Technical Field
The invention relates to the field of genetic engineering, in particular to a light source combination box for promoting kiwi fruit transgenic efficiency and application thereof.
Background
The kiwi fruit is a fruit with extremely high nutritional value and economic value, and is called fruit king and vitamin C king. The kiwi plant tissue culture and transgenic technology is a key technology for developing kiwi gene function research and crop genetic improvement. However, the kiwi fruit is perennial woody vine, the tissue culture induction differentiation of the kiwi fruit needs a long time, and the research on accelerating the tissue culture and the transgenic efficiency of the kiwi fruit has important significance for the scientific research of the kiwi fruit.
The inventor finds that different illumination influences on the growth of the kiwi fruits through experiments in the earlier stage:
at present, the basic growth requirement of the kiwi fruits can be met by the traditional white light, but the growth is slow, and the period is long; under the condition of pure red light, the growth speed of calluses and plants of the kiwi fruits is obviously improved, but the chlorophyll content is reduced, and the growth state of the plants is poorer; the chlorophyll content of kiwi callus and plants is elevated under pure blue light conditions, but the growth rate is slower. Therefore, the research and development of the optimal illumination combination suitable for the tissue culture of the kiwi fruits and the improvement of the genetic transformation efficiency of the kiwi fruits have important application values.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art, and provides a light source combination box for promoting the kiwi fruit transgenic efficiency and application thereof.
The purpose of the invention is realized as follows:
light source combination box (light source combination box for short) for promoting kiwi fruit transgene efficiency
The light source combination box comprises red and blue LED light sources, and in the same LED light source combination box, total 100 LED lamp pearls are 10X 10 arrangement, and the LED lamp pearls of ruddiness and blue light are evenly arranged in a proportion of 1: 4, the input voltage is 220V, the power is 300W, and the working environment isFor operation the Photosynthetic Photon Flux Density (PPFD) is 200 + -5 μmol m-2s-1The photoperiod is 12 light/12 h dark, and the temperature is 24 +/-1 ℃.
Application of light source combined box
According to the invention, the efficiency of molecular experiments such as kiwi fruit transgenosis is improved by designing a special light source suitable for kiwi fruit genetic transformation.
The following applies:
taking a proper amount of sterile calluses of kiwi fruit (red sun), developing a transgenic experiment in the kiwi fruit by an agrobacterium transformation method, respectively culturing under white light and a designed combined light source after co-culturing, wherein the specific illumination condition is that the Photosynthetic Photon Flux Density (PPFD) is 200 +/-5 mu mol m-2s-1The photoperiod is 12h light/12 h dark, the temperature is 24 +/-1 ℃, and the later verification of a transgenic material shows that the light source combination can improve the transgenic efficiency by more than 50%.
The working mechanism is as follows:
the growth and development of plants are influenced by different light qualities, and in the kiwi fruits, the inventor finds that blue light has remarkable promoting and accumulating effects on the chlorophyll content, the carbon and nitrogen content and the leaf plastid number of the kiwi fruits through earlier experiments; and the red light can induce the vegetative growth of kiwi plants and improve the content of starch and sucrose in leaves. Therefore, the red and blue light sources with the optimal proportion are configured, so that the growth of the kiwi fruit tissue culture material can be rapidly promoted, particularly, in a transgenic experiment, the growth efficiency of a transgenic successful material can be rapidly improved, the quantity of resistance inhibition materials can be reduced, and a better transgenic material can be finally obtained.
The invention has the following advantages and positive effects:
the efficiency of the existing kiwi fruit transgenosis can be obviously improved;
secondly, the method is suitable for accelerating the production conditions of tissue culture regeneration and the like of the kiwi fruits;
and is suitable for most kiwi fruit molecular laboratories.
Drawings
FIG. 1: schematic diagram of light source combination box
The ratio of red light to blue light is 1: 4, the LED lamp beads of the LED lamp box are distributed schematically, red light represents the LED lamp beads emitting red light, and blue light represents the LED lamp beads emitting blue light;
FIG. 2: testing a real scene through an experiment;
FIG. 3: a genetic transformation screening map of the callus of the kiwi fruit under white light treatment, namely a pPOE-3 XFLAG-DN vector schematic diagram;
FIG. 4: a kiwi callus genetic transformation screening diagram under red and blue mixed light treatment, namely a pPOE-3 XFLAG-DN vector schematic diagram;
FIG. 5: one of PCR identification and sequencing analysis of genetically transformed plants,
is PCR identification and sequencing identification of kiwi transgenic plants over-expressing pPOE-3 XFLAG-DN vectors;
FIG. 6: the second of PCR identification and sequencing analysis of genetically transformed plants,
is PCR identification and sequencing identification of kiwi transgenic plants of over-expression pPOE-3 XFLAG-DN vectors.
Detailed Description
The following detailed description is made with reference to the accompanying drawings and examples.
First, the material source of the invention
1. The over-expression vector pPOE-3 XFLAG-DN used by the self-purchased LED lamp box disclosed by the invention comes from a laboratory where the inventor is located (figure 1 and figure 2).
2. All primers were designed by the inventors and synthesized by the Kyoto Biotech Co., Ltd, Beijing (Table 1)
TABLE 1 primers designed in the present invention
Figure BDA0003042050870000031
3. The PCR polymerase used in this experiment was purchased from Biotech, Inc. of Kyoto, Beijing.
Second, example
1. Embodiments of light Source combiner Box
The structure of the light source combination box is as follows: composed of red and blue LED light sources in the same LED light source combined boxThe total number of 100 LED lamp beads is 10 multiplied by 10, the arrangement mode of the LED lamp beads of the red light and the blue light is shown in figure 1, and the proportion of the red light and the blue light is 1: 4; the input voltage of the light source combination box is 220V, the power is 300W, and the operating Photosynthetic Photon Flux Density (PPFD) is 200 +/-5 mu mol m-2s-1。
2. Examples of applications of light source combination boxes
1) Respectively taking a proper amount of sterile calluses of kiwi fruits (red sun), and performing pre-culture for 1 week under control white light and the light source combination box designed by the invention;
2) mu.L of vector (100 ng/. mu.l) pPOE-3 XFLAG-DN was taken, Agrobacterium strain EHA105 competent cells were transformed using standard electroporation method, and PCR amplification and identification were performed with primers POE-F/POE-R, cut into 2-3 mm-sized blocks (about 800-: blue light 1: 4) pre-culturing for 3 days;
3) preparing 2 sterile centrifuge tubes of 2mL, respectively adding LB liquid culture medium of 50ng/mL Kan, lightly selecting a small amount of agrobacterium tumefaciens monoclonally inoculated with the target vector by using toothpicks, placing in a constant temperature oscillator at 28 ℃ and 200r/min for overnight pre-culture (about 12 hours), and obtaining seed liquid;
4) the next morning, 2 sterile 50mL centrifuge tubes were prepared, 35mL LB broth with 50ng/mL Kan added, 35ul 100mM/L Acetosyringone (AS) added, and the overnight pre-cultured Agrobacterium seed solution was mixed AS 1: transferring 100 parts of the culture medium into new culture medium, culturing in a constant temperature oscillator at 28 deg.C and 200r/min for 12-20h, and expanding to OD600 of about 0.6;
5) centrifuging the agrobacterium subjected to amplification culture for 5min at the speed of 10 Xg/min, removing supernatant in a clean workbench, firstly resuspending thalli by using a small amount of liquid MS culture medium containing 100 mu m/mL acetosyringone, then diluting agrobacterium liquid by using the liquid culture medium, sucking 2mL of agrobacterium liquid into a cuvette, and adjusting the bacterium liquid to OD (OD) of 0.4-0.6 in an A600 mode in a spectrophotometer for later use;
6) respectively clamping the pre-cultured two irradiated red-yang callus tissues into an agrobacterium culture solution in a clean workbench, marking correspondingly, and placing the tissues in a constant-temperature oscillator at 28 ℃ and 100r/min for shaking and infecting for 15 min;
7) carefully removing the suspension in a clean workbench, fully washing with sterile water for 4-5 times, taking out the cleaned callus, placing in sterilized filter paper, air drying for 20-30min, transferring into differentiation culture medium containing 100Mm/L acetosyringone, and co-culturing at 24 deg.C in dark for 3 days;
8) after the co-culture is finished, taking out the callus, washing with sterile water for 5-6 times, placing in sterilized filter paper for air drying, transferring into a differentiation culture medium containing 200mg/L timentin for delayed culture, respectively placing under white light and designed light source, and making Photosynthetic Photon Flux Density (PPFD) be 200 + -5 μmol m-2s-1The photoperiod is 12h light/12 h dark, the temperature is 24 +/-1 ℃, and the culture is delayed for 2 days;
9) after the delayed culture is finished, taking out the callus, and inoculating the callus into a differentiation culture medium for resistance screening;
10) repeating the step 8 every 15 days, and carrying out screening culture for 4 times;
11) after screening is finished, inducing and differentiating the screened resistant callus to culture to form a plant, taking a small number of leaves from the screened resistant callus in a clean workbench, extracting DNA by a CTAB method, carrying out PCR (polymerase chain reaction) amplification electrophoresis detection by using a pPOE-3 xFLAG-DN (positive displacement amplification-negative) carrier primer POE-F/POE-R, and sequencing (figure 3) to obtain an individual with successful genetic transformation;
12) by comparing the genetic transformation efficiency of the red kiwi fruits under the white light and the light source combination box designed by the invention (table 2), the genetic transformation efficiency of the kiwi fruits of the illumination combination of the invention is calculated to be 15.63 percent and is obviously higher than the 9.41 percent transformation efficiency of the white light (table 3).
TABLE 2 white light and Red blue Mixed light treatment Actinidia chinensis callus genetic transformation positive callus screening record (unit: granule)
Figure BDA0003042050870000051
TABLE 3 genetic transformation efficiency of Kiwi fruit under white and Red-blue Mixed light conditions
Figure BDA0003042050870000052
Sequence listing
<110> Wuhan plant garden of Chinese academy of sciences
<120> light source combination box for promoting kiwi fruit transgenic efficiency and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atccccgggt accacagtcg aca 23
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aactttattg ccaaatgttt gaacg 25

Claims (2)

1. The utility model provides a promote light source combination box of kiwi fruit transgenic efficiency which characterized in that:
the LED lamp bead is composed of red and blue LED light sources, and in the same LED light source combination box, 100 LED lamp beads are arranged in a10 multiplied by 10 mode, the LED lamp beads of red light and blue light are uniformly arranged, and the proportion is 1: 4, the input voltage is 220V, the power is 300W, and the operating photosynthetic photon flux density of the working environment is 200 +/-5 mu mol m-2s-1The photoperiod is 12 light/12 h dark, and the temperature is 24 +/-1 ℃.
2. Use of a light source module according to claim 1, wherein:
taking a proper amount of sterile callus of the red sun of the kiwi fruit, developing a transgenic experiment in the kiwi fruit by an agrobacterium transformation method, respectively culturing under white light and a designed combined light source after co-culturing, and improving the transgenic efficiency by more than 50% through verification of a transgenic material in the later period.
CN202110459973.1A 2021-04-27 2021-04-27 Light source combination box for promoting kiwi fruit transgenic efficiency and application thereof Active CN113186220B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109630916A (en) * 2019-01-30 2019-04-16 天津斯隆达科技发展有限公司 A kind of Kiwi berry LED light supplement lamp and its application
CN209371079U (en) * 2019-03-01 2019-09-10 天津斯隆达科技发展有限公司 A kind of Kiwi berry light compensating lamp

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109630916A (en) * 2019-01-30 2019-04-16 天津斯隆达科技发展有限公司 A kind of Kiwi berry LED light supplement lamp and its application
CN209371079U (en) * 2019-03-01 2019-09-10 天津斯隆达科技发展有限公司 A kind of Kiwi berry light compensating lamp

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
B.D. MARIANA: "IMPROVING GENE TRANSFER IN APPLE", 《MSC THESIS REPORT OF WAGENINGEN UNIVERSITY》 *
LIU XIAOYINGA ET AL.: "Effect of light on growth and chlorophyll development in kiwifruit ex vitro and in vitro", 《SCIENTIA HORTICULTURAE》 *
T.Y. GELAYE: "IMPROVING GENE TRANSFER IN POTATO", 《MSC THESIS REPORT OF WAGENINGEN UNIVERSITY》 *
袁华玲等: "不同光质对对萼猕猴桃试管苗生长的影响", 《安徽农学通报》 *
解潇冬等: "光质对红阳猕猴桃愈伤组织生长速度和花青素合成的影响", 《山西农业科学》 *

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