CN117448370B

CN117448370B - Construction method of genetic transformation system of North mugwort

Info

Publication number: CN117448370B
Application number: CN202311394863.7A
Authority: CN
Inventors: 崔宇鹏; 张坤朋; 郭可栋; 王静; 郜新强
Original assignee: Anyang Institute of Technology
Current assignee: Anyang Institute of Technology
Priority date: 2023-10-25
Filing date: 2023-10-25
Publication date: 2024-09-17
Anticipated expiration: 2043-10-25
Also published as: CN117448370A

Abstract

The invention provides a construction method of a genetic transformation system of mugwort, and belongs to the technical field of mugwort breeding. The invention provides a construction method of a genetic transformation system of North moxa, which comprises the following steps: (1) Cutting stem segments of sterile north Ai Zupei seedlings for propagation to obtain north Ai Youmiao, and cutting stem segments of the north moxa seedlings as receptor materials; (2) The receptor material is transferred to a co-culture medium for culture after being infected by an infection liquid carrying a target gene; (3) Transferring the receptor material cultured in the step (2) into a bud induction culture medium for culture to obtain a resistant bud; (4) Transferring the resistant buds to a rooting culture medium for culture to obtain complete plants; (5) And transplanting and culturing the plant to obtain a north mugwort genetic transformation system. The invention has the advantages of simple operation, short growth period, good repeatability, unified and simple transformation mode, high transformation efficiency, good genetic stability of the obtained transgenic North mugwort plants, and the like.

Description

Construction method of genetic transformation system of North mugwort

Technical Field

The invention relates to the technical field of mugwort breeding, in particular to a construction method of a genetic transformation system of mugwort.

Background

Mugwort (ARTEMISIA ARGYI) is a perennial herb of the genus Artemisia of the family Compositae and is widely distributed in Asia. Ai Caoquan herbal medicines have effects of warming blood, expelling cold dampness, stopping bleeding, warming channel, and preventing miscarriage. The moxa is obtained by sun-drying and mashing the mugwort leaves, and is not only a raw material for preparing moxa sticks for moxibustion, but also a raw material for preparing inkpad. Based on the characteristics of simple material drawing, low price, obvious efficacy and the like, the wormwood has been widely researched and used at present, but the wormwood is concentrated in the directions of chemical components, pharmacological actions and the like.

The Chinese mugwort herb is not produced by earth but is produced in the field of cloud. Song Shi is preferred for Tangyin to be repeated (note: now Tangyin county vodka.) the modern Wei Tangyin is Bei ai. At present, besides the traditional moxibustion agents which are applied more, various novel compound preparations are gradually accepted by the masses, and not only are the novel compound preparations applied to clinic, but also play a great role in daily chemical, health care, beauty treatment and other industries. Due to the polyploidy and high heterozygosity of Tangyin North moxa, the traditional breeding is limited by difficult directional improvement of beneficial characters, narrow breeding range and the like, and no report of a North moxa genetic transformation system is found at present. The invention establishes a genetic transformation system of the north mugwort, which lays a solid foundation for the genetic engineering breeding and new germplasm resource creation of the north mugwort in the future.

Disclosure of Invention

The invention aims to establish a construction method of a genetic transformation system of the north mugwort, which lays a solid foundation for the genetic engineering breeding and new germplasm resource creation of the north mugwort in the future.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a construction method of a genetic transformation system of North moxa, which comprises the following steps:

(1) Cutting stem segments of sterile north Ai Zupei seedlings for propagation to obtain north Ai Youmiao, and cutting stem segments of the north moxa seedlings as receptor materials;

(2) The receptor material is transferred to a co-culture medium for culture after being infected by an infection liquid carrying a target gene;

(3) Transferring the receptor material cultured in the step (2) into a bud induction culture medium for culture to obtain a resistant bud;

(4) Transferring the resistant buds to a rooting culture medium for culture to obtain complete plants;

(5) And transplanting and culturing the plant to obtain a north mugwort genetic transformation system.

Preferably, in the step (1), the length of the stem segment of the north Ai Youmiao is 1.3-1.7 cm, and the stem segment of the north Ai Youmiao maintains the growth point of the leaf stalk.

Preferably, in the step (2), the dyeing solution is LB medium containing acetosyringone, the concentration of the acetosyringone is 1.95-1.98 mg/ml, and the volume ratio of the acetosyringone to the LB medium is 0.2:20-25; the pH of the dyeing liquid is 5.7-6.0; the infection time is 8-12 min, the temperature is 26-28 ℃, and the rotating speed is 180-200 rpm.

Preferably, the co-culture medium in the step (2) contains IAA 0.1-1 mg/L, GA 30.01.01-0.1 mg/L based on MS culture medium; the culture time of the co-culture medium is 2-3 days.

Preferably, the co-culture medium in step (2) contains IAA 1mg/L and GA30.1mg/L based on MS medium.

Preferably, the bud induction resistance medium in the step (3) contains 0.01 to 0.5mg/L, KT 0.1.1 to 0.5mg/L, NAA 0.01 to 0.1mg/L, kan to 55mg/L of 6-BA and 200 to 400mg/L of cephalosporin on the basis of MS medium, and the pH=5.7 to 6.0; the culture conditions of the bud induction resistance medium in step (3) are: the illumination intensity is 1800-2000 lx, the light period is 16h, the illumination is 8h dark, the temperature is 24+/-2 ℃, and the time is 10-14 days.

Preferably, the bud induction resistance medium in step (3) contains 6-BA 0.5mg/L, KT 0.5mg/L, NAA 0.1mg/L, kan to 55mg/L and cephalosporin 200 to 400mg/L, pH=5.7 to 6.0 based on MS medium.

Preferably, the rooting medium in the step (3) contains NAA 0.1-0.2 mg/L, kan-55 mg/L and cephalosporin 200-400 mg/L based on MS medium; the illumination intensity is 1800-2000 lx, the light period is 16h, the illumination is 8h dark, the temperature is 24+/-2 ℃, and the time is 20-21 days.

Preferably, the rooting medium in the step (3) contains NAA 0.1mg/L, kan-55 mg/L and cephalosporin 200-400 mg/L based on MS medium.

The invention establishes a construction method of a genetic transformation system of Artemisia northwest, which comprises the steps of infecting genetic transformation receptor materials with recombinant agrobacterium tumefaciens containing target DNA to obtain infected explants, and culturing the infected explants through a co-culture medium, a bud induction medium and a rooting medium to obtain transgenic Artemisia northwest plants. The invention has the advantages of simple operation, short growth period, good repeatability, unified and simple transformation mode, high transformation efficiency, good genetic stability of the obtained transgenic North mugwort plants, and the like. Lays a solid foundation for genetic engineering breeding and new germplasm resource creation of the north mugwort in future.

Drawings

FIG. 1 is a diagram of the stems of North moxa after co-cultivation infection on the co-culture medium in example 3;

FIG. 2 is a graph showing the generation of resistant shoots from North moxa stems after induction of infestation by shoot induction medium in example 3;

FIG. 3 is a graph of the resistance to shoot growth in rooting medium in example 3;

FIG. 4 is a graph showing the rooting of resistant shoots in rooting medium in example 3;

FIG. 5 is a plot of transplanting rooted regenerated seedlings into the soil in example 3;

FIG. 6 is a phenotype of the rooted regenerated plantlets of example 3 after 2 months of growth;

FIG. 7 shows the PCR detection result of transgenic plant resistance marker gene Kan gene in example 4;

FIG. 8 is a graph showing the results of GFP detection in the transgenic positive north Ai Zhizhu glandular hairs, leaves and stem segments of example 4.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The tool enzymes, kits, medicines, instruments, vectors and competent cells required in the following examples were as follows:

(1) Enzyme and kit: DNAMARKER III (MD 103) reagents were purchased from Tiangen Biochemical (technology) Beijing Co., ltd; the high-fidelity PCR amplification enzyme KOD-Plus-Neo (KOD-401) was purchased from TOYOBO BioCo., agrobacterium competent GV3101 was purchased from Shanghai.

(2) Other drugs: agarose was purchased from Beijing full gold Biotechnology Co., ltd; peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. are domestic analytical purities; kanamycin (YZ-130556), rifampicin (IR 0110), sucrose (YZ-4454) and the like are purchased from Beijing Soy technologies Co., ltd. Plant expression vector pBI121-GFP was stored for this laboratory.

(3) Preparing a solution: the various reagents mentioned in the invention but not listed are prepared according to the method on the third edition of the molecular cloning experiment guide, and the biochemical reagent is analytically pure or above.

(4) LB medium: tryptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L; LB solid medium: 10g/L of Tryptone (Tryptone), 5g/L of Yeast extract (Yeast extract), 10g/L of sodium chloride (NaCl) and 15g/L of agar powder, and fixing the volume to 1L; LB selection Medium: before LB plate laying, adding antibiotics with corresponding concentration when the culture medium is sterilized under high pressure and cooled to 55 ℃, shaking uniformly, and then plate laying. The basic components of the MS medium include Murashige & Skoog (MS 524): 4.74g/L, MES g/L, 110mg/ml of Vitamin B, 61mg/L of Vitamin B, 100mg/L of inositol and 2% of sucrose, and 7g/L of agar powder is added when the solid culture medium is used, and the pH value is 5.8.

(5) The main instrument is as follows: PCR amplification apparatus (BIO-RAD), high-speed centrifuge (HettichMIKRO R), electrophoresis apparatus (BIO-RAD), gel imaging system (BIO-RAD), vacuum freeze dryer (ALPHAI-5).

Example 1

Preparation of Agrobacterium suspension carrying the Gene of interest for infection of North Artemisia seedlings

Preparing agrobacterium: the plant expression vector pBI121-GFP plasmid is extracted by adopting a full-scale gold company plasmid extraction kit, the plasmid concentration is detected to be 200 ng/. Mu.l, agarose gel electrophoresis detection is carried out, no protein pollution is caused, the test requirement is met, the plant expression vector pBI121-GFP plasmid (HIA with kanamycin resistance gene) is transferred into an agrobacterium strain GV3101, and the plasmid agrobacterium carrying green fluorescent protein GFP gene and kanamycin resistance gene is obtained and named GFP1 and stored at the temperature of minus 80 ℃.

Agrobacterium activation: before infecting the north Ai Youmiao stem segments, the agrobacterium is activated to help the bacterial liquid to infect, and the conversion efficiency is improved, and the specific method is as follows: picking up the stored GFP1 glycerinum from a refrigerator at the temperature of minus 80 ℃, picking up the target agrobacterium in an ultra clean bench by using a 200 mu L sterile gun, streaking on LB solid medium (containing kan (kanamycin) 50mg/L and Rif (rifampicin) 25 mg/L), placing the solid medium in a constant temperature incubator at the temperature of 28 ℃ for culturing for 48 hours, growing colonies to obtain activated agrobacterium, then picking up the target agrobacterium single colony, inoculating the target agrobacterium single colony in 2ml LB liquid medium (containing kan 50mg/L and Rif25 mg/L) and placing the single colony on a shaking table at the temperature of 28 ℃ for shaking for 12 hours at 180 rpm; then transferring into 50ml of the same LB medium, placing on a shaking table at 28 ℃ for shaking at 180rpm for 18 hours, enabling bacterial liquid to be golden yellow, centrifuging at 5000rpm for 5 minutes, discarding supernatant, collecting agrobacterium tumefaciens bacterial blocks, using 25ml of ME to invade a dye solution (LB+200. Mu.l acetosyringone (1.96 mg/ml) and PH 5.7) for resuspension, measuring the agrobacterium tumefaciens suspension by using a spectrophotometer, adjusting the suspension concentration, and obtaining GFP1 suspension when the light absorption value OD _600nm is 0.8 for standby.

Preparation of genetic transformation receptor material for North moxa

Cutting stem segments from sterile north Ai Zupei seedlings stored in the laboratory for propagation, clamping the stem segments by forceps when the stem segments grow to 4 weeks, cutting the stem segments of the north moxa seedlings by using a scalpel to about 1.5cm, and keeping growing points at petioles when cutting the stem segments, wherein the north Ai Youmiao stem segments are used as receptor materials for later use.

Example 2

Optimization of infection time

To optimize the infection time, the north Ai Youmiao stem segments were used as explants, which were placed in sterile triangular flasks with 20ml of ME-invasive solution, the infection time was set to 5min, 10min and 15min, 100 explants were treated per flask, 3 times repeated, the infected explants were placed on a co-culture medium (MS+IAA 1mg/L+GA30.1 mg/L), co-cultured for 2 days, and then transferred to a shoot induction medium (MS+6-BA 0.5 mg/L+KT0.5 mg/L+NAA 0.1mg/L+Kan 50 mg/L+cephalosporin 300 mg/L) to induce resistant shoots, and the rate of induction and conversion efficiency of shoots at the petioles were counted after 10 days. As a result, it was found that explants infected for 5min, 10min and 15min all had a certain transformation effect, among which the budding induction rate at the petiole of the explant infected for 10min was the highest, and the transformation efficiency was the highest (Table 1).

TABLE 1 conversion efficiencies of North moxa stem under different infection conditions

Example 3

Infection and co-cultivation

The prepared 100 North moxa stem explants were soaked in 25ML ME (GFP 1 suspension prepared in example 1) with GFP1 Agrobacterium, shaken for 10min at 28℃and 180rpm in a shaker greenhouse, the invaded solution was discarded, the explants were placed on sterile filter paper to blot the surface bacterial solution, and then co-cultured for 2 days at 24℃based on dark culture, and the specific formulation is shown in Table 2. Statistics were repeated three times with 50 explants per disc, and as a result, it was found that when IAA (0.1-1 mg/L) and GA3 (0.01-0.1 mg/L) were added simultaneously to the basal MS medium, the surface of the explant was significantly reduced, the probability of subsequent budding was increased only if the surface of the explant was green, and no explant browning indicated that the explant was hardly budded. Based on the statistical results of Table 2, MS solid medium +0.1-1 mg/L IAA +0.01-0.1 mg/L GA3, pH 5.7 was selected as co-culture medium, more preferably MS solid medium +1mg/L IAA +0.1mg/L GA3, pH 5.7. At the same time, the results of the dark culture at 24℃for 2 days and 3 days were found to be similar, and there was no statistically significant difference.

TABLE 2 phenotypic statistics of explants co-cultured at different hormone concentrations

Bud induction culture and rooting culture

The stem segments of the seedlings of the mugwort (figure 1) were dark-cultured on the co-culture medium (MS solid medium +1mg/LIAA +0.1mg/L GA3, pH 5.7) at 24℃for 2 days, respectively placed on the bud induction resistant medium (hormone ratio is shown in Table 3) combined with different hormone concentrations, and cultured for 14 days under the conditions of illumination intensity of 2000lx, photoperiod of 16h/8h (light/dark) and 24.+ -. 2 ℃ to induce resistant buds. As a result, it was found that the formulations of these hormone combinations all induced resistant shoots, wherein the shoot induction medium C9, MS solid medium +0.5 mg/L6-BA +0.5mg/L KT +0.1mg/L NAA +50mg/Lkan +300mg/L cephalosporin, had the highest induction rate of resistant shoots at pH 5.7 (induced resistant shoots as shown in FIG. 2).

When the resistant bud of the C9 culture grows to 1cm, the resistant bud is cut from the explant, transferred into rooting culture media with NAA of different concentrations (NAA concentration is shown in Table 4), and grown for 3 weeks under the same illumination condition, and root growth is observed and recorded. As can be seen from Table 4, auxin NAA plays an important role in the induction of mugwort roots, and in the MS culture medium with very low NAA concentration, the rooting rate of seedlings is only 10.2%, the average rooting number is 0.5, and the rooting rate is very short; along with the gradual increase of NAA concentration in the culture medium, the average root number, rooting rate and average root length of the mugwort are changed, the rooting rate is 98.4% when the NAA concentration is 0.1mg/L, the effective average root number is 4.6, the seedling growth condition is better, and the mugwort is the optimal rooting formula; as NAA concentration increases, rooting rate and average root number decrease significantly, and root growth slows down. Therefore, we obtain that the rooting culture medium (MS solid culture medium+0.1-0.2 mg/LNAA+50mg/Lkan +300mg/L cephalosporin) in the range has better condition that the resistant buds of the wormwood grow out of the wormwood root system (figures 3 and 4), the whole transgenic plant of the outdoor germplasm is obtained after the resistant buds root and grow into the plant (figure 5) and the plant grows for 2 months (figure 6).

TABLE 3 Induction of resistant buds from mugwort treated with combinations of different hormone concentrations

TABLE 4 Effect of NAA at different concentrations on rooting of resistant shoots of mugwort

Example 4

Transgenic plant kana resistance marker gene kanPCR detection and GFP fluorescence observation analysis

The N3 group transformed plants of the GFP1 vector transformed in example 3 (rooting medium conditions are MS solid medium+0.1 mg/LNAA+50mg/Lkan +300mg/L cephalosporin) were taken for PCR amplification detection of exogenous Canada resistance marker gene sequences and fluorescent protein luminescence observation analysis of GFP.

Taking transgenic tender leaves, extracting DNA, and using KanF:5'-ATGATTGAACAAGATGGATTG-3' and KanR:5'-TCAGAAGAACTCGTCAAGAAG-3' primers were used for PCR detection. The KOD-Plus-Neo high-fidelity enzyme was used to amplify the target band from the transgenic DNA in the following reaction system:

TABLE 4 target gene amplification System

Table 5PCR reaction procedure was as follows:

1	Predenature	94℃,2min
			2	Denature	98℃,10s
3	Annealing	56℃,30s
			4	Estension	68℃,50s
5	Estension	68℃,10min

note that 30 cycles of amplification were performed from 2 to 4.

After the PCR reaction is finished, 5 mu l of PCR amplification product is sucked for 1% agarose gel electrophoresis, and the PCR amplification product is observed and photographed under a BIO-RAD gel imaging system instrument. As shown in FIG. 7, the transgenic North mugwort plants and the positive control plasmid both amplify a target band (which is identical to the theoretical sequence 795bp of Kan) with a size of about 800bp, while the receptor North Ai Zhizhu (negative control, band 4) does not amplify a band, and the transgenic mugwort plants (bands 2, 3 and 5-10) have obvious amplified bands as the positive plasmid (band 1), indicating that the Kan gene carried by Agrobacterium has been successfully introduced into the North mugwort plants.

Based on kan gene detection, leaves and stem segments of transgenic positive North mugwort plants are further randomly selected, and GFP fluorescent protein markers of glandular hairs, leaves and stem segments of the transgenic plants are observed through a fluorescence microscope and a confocal microscope to emit light, and the result is shown in figure 8, GFP fluorescent signals are observed in the leaves and stem segments of the transgenic North mugwort plants, and the GFP1 vector carrying GFP labels is proved to be successfully genetically transformed and expressed in the North mugwort stem segments

The double detection method of the transformed plants shows that the transgenic North mugwort plants can be obtained by the method of the genetic transformation system, and the double detection method is also an effective guarantee for screening and identifying positive plants.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The construction method of the genetic transformation system of the mugwort is characterized by comprising the following steps:

(3) Transferring the receptor material cultured in the step (2) into a bud induction resistance culture medium for culture to obtain a resistance bud;

(5) Transplanting and culturing the plant to obtain a north mugwort genetic transformation system;

the infection time in the step (2) is 8-12 min;

The co-culture medium in the step (2) contains IAA 0.1-1 mg/L, GA ₃ 0.01.01-0.1 mg/L based on an MS culture medium;

the bud induction resistance culture medium in the step (3) contains 6-BA 0.01-0.5 mg/L, KT 0.1-0.5 mg/L, NAA 0.01.01-0.1 mg/L, kan-55 mg/L and cephalosporin 200-400 mg/L based on an MS culture medium, and the pH=5.7-6.0;

the rooting culture medium in the step (4) contains NAA 0.1-0.2 mg/L, kan-45 mg/L and cephalosporin 200-400 mg/L based on an MS culture medium.

2. The method of claim 1, wherein the length of the stem segment of north Ai Youmiao in step (1) is 1.3 cm to 1.7cm, and the stem segment of north Ai Youmiao retains a growth point at the petiole.

3. The construction method according to claim 2, wherein the dyeing solution in the step (2) is an LB medium containing acetosyringone, the concentration of acetosyringone is 1.95-1.98 mg/ml, and the volume ratio of the acetosyringone to the LB medium is 0.2:20-25; the pH value of the infection liquid is 5.7-6.0; the temperature during the infection is 26-28 ℃, and the rotating speed is 180-200 rpm.

4. The method according to claim 3, wherein the co-culture medium is cultured for 2 to 3 days.

5. The method of claim 4, wherein the co-culture medium in step (2) comprises IAA 1mg/L, GA 3.0.1 mg/L based on MS medium.

6. The method of claim 1, wherein the culture conditions of the shoot induction resistance medium in step (3) are: the illumination intensity is 1800-2000 lx, the photoperiod is 16h, the illumination is 8h dark, the temperature is 24+/-2 ℃, and the time is 10-14 days.

7. The method according to claim 6, wherein the shoot induction-resistant medium in the step (3) contains 6-BA 0.5mg/L, KT 0.5mg/L, NAA 0.1mg/L, kan to 55mg/L and cephalosporin 200 to 400mg/L based on MS medium, and pH=5.7 to 6.0.

8. The construction method according to claim 1, wherein in the step (4), the illumination intensity is 1800-2000 lx, the light period is 16 hours, the illumination is 8 hours of darkness, the temperature is 24+ -2 ℃, and the time is 20-21 days.

9. The method according to claim 8, wherein the rooting medium in the step (4) contains NAA 0.1mg/L, kan-55 mg/L and cephalosporin 200-400 mg/L based on MS medium.