CN110669786B - Method for establishing and optimizing agrobacterium rhizogenes-mediated middle caragana hairy root genetic transformation system - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for establishing and optimizing an agrobacterium rhizogenes-mediated caragana intermedia hairy root genetic transformation system. The hairy root genetic transformation system enables the gene to be over-expressed in the hairy root of the caragana intermedia, has simple operation and high expression level, and can provide technical support for developing gene function verification and screening functional genes; and when the hairy root grows to a certain size, the main root is cut off, and the hairy root replaces the main root to become a part of the middle caragana plant and form a chimera of the transgenic hairy root and the stem and leaf of the original plant.

Description

Method for establishing and optimizing agrobacterium rhizogenes-mediated genetic transformation system of middle caragana hairy roots

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for establishing and optimizing an agrobacterium rhizogenes-mediated intermediate caragana hairy root genetic transformation system.

Background

Compared with traditional crops or transgenic crops planted in outdoor fields, the hairy root tissues are always in an artificially controllable closed environment, and the risk of gene drift is greatly reduced; can grow rapidly and can proliferate at high speed in the culture process; agrobacterium rhizogenes is a gram-negative soil bacterium of the genus Agrobacterium of the family Rhizobiaceae, which is capable of infecting most dicotyledonous plants and a few monocotyledonous plants as well as individual gymnosperms. Compared with agrobacterium tumefaciens, hairy roots generated by agrobacterium rhizogenes have stable genetic property, and a plurality of dicotyledonous plants obtain heritable transgenic plants through a rooting system; in addition, the hairy roots can produce metabolites in the culture medium, thereby providing convenient conditions for extraction and separation. The agrobacterium rhizogenes can cause most plants to root after infecting the plants, so the agrobacterium rhizogenes is applied to various plants, but the agrobacterium rhizogenes only carries out production and application in precious medicinal materials, such as ginseng and the like because the rooting condition is strict.

Middle Caragana (Caragana intermedia Kuang) belongs to leguminous shrubs and is mainly distributed in arid and semiarid desert regions in inner Mongolia, Ningxia and northern Shaanxi in China. The caragana plant has the characteristics of cold resistance, drought resistance, salt and alkali resistance, barren resistance and the like, has strong adaptability to desert regions, not only maintains water and soil, prevents wind and fixes sand, but also has higher feeding value. Therefore, the research on the gene function is urgently needed to discover the stress-resistant mechanism. However, unlike some model plants such as arabidopsis thaliana, tobacco, rice, etc., which already have mature genetic transformation systems, there is an urgent need to establish a gene function verification method based on transgenic hairy roots in middle caragana due to the lack of regeneration systems and transgenic technologies in middle caragana.

At present, the genetic transformation system of the hairy root of leguminous plants is still rarely researched, so that the successful construction examples are not many, the leguminous plants such as soybean, pigeon pea and the like are researched more, and the genetic transformation system of the hairy root of caragana sylvestris is not researched systematically at present. At present, the genetic transformation method of the hairy roots of the plants is mainly combined with the tissue culture technology, the genetic transformation efficiency is relatively high, but the operation process has many careless matters, the operation is relatively complicated, the possibility of pollution is also provided, and the application of the successful practices has certain difficulty; the method for directly infecting agrobacterium rhizogenes on the plant is simple and easy to implement, avoids the problem of bacterial infection, and improves the possibility of application of transgenic hairy roots. In addition, hairy roots generated by the plant which is directly infected by agrobacterium rhizogenes are verified to be transgenic positive roots, and can be used for related researches on gene functions, metabolites and the like.

Disclosure of Invention

The invention aims to establish a method for realizing gene expression in a middle caragana hairy root for functional verification research of genes. The method is realized by the following technical scheme:

(1) electrically transforming an expression vector containing a GUS reporter gene into an agrobacterium K599 competent cell;

(2) the Agrobacterium obtained in (1) and Agrobacterium K599 without any exogenous vector were cultured in LB liquid medium to the desired concentration (expressed as OD value).

(3) Selecting well-grown middle caragana seedlings growing for about 6-9 days, and slowly injecting the agrobacterium liquid prepared in the step (2) into the leaf nodes of the middle caragana and hypocotyl parts above nutrient soil of the leaf nodes.

(4) And observing the growth condition of the hairy roots of the caragana intermedia and detecting the GUS reporter gene expression condition within 21-28 days after transformation.

The step (1) is specifically as follows: the expression vector containing GUS reporter gene used was pCambia1305.2 (laboratory preservation), Agrobacterium K599 (Shanghai Weidi Biotechnology, Inc.). K599 Agrobacterium rhizogenes contains pRi2659 agropine Ri plasmid, has wide host range and simultaneously has streptomycin resistance.

The step (2) is specifically as follows: inoculating agrobacterium K599 carrying pCambia1305.2 vector and no exogenous vector to 4mL LB liquid medium (Tryptone 1g, Yeast extract 0.5g, NaCl 1g, NaOH or HCl to adjust pH value to 7.0, deionized water to 100mL, autoclaving at 121 ℃ for 20 min); among them, LB liquid medium contained 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin. Shaking at 200rpm and culturing at 28 deg.C overnight to OD ₆₀₀ The value is about 1.2, 1mL of the bacterial liquid is taken and transferred into 20mL of fresh LB liquid culture medium again (volume ratio is 1:20), the bacterial liquid is cultured at 28 ℃ and 200rpm with shaking until the OD600 value is 1.2, and then acetosyringone with the final concentration of 100 mu mol/mL is added.

The step (3) is specifically as follows: under the normal growth condition, selecting well-grown middle caragana seedlings growing for 6-9 days, and selecting plants with stout hypocotyls for injection. And (3) sucking the agrobacterium suspension which is added with the acetosyringone and then stands for 24 hours by using a 1mL needle tube, pressing the needle tube with proper force after the needle tube is fully sucked, and pricking holes at the hypocotyl part to inject the bacterial liquid, so that the bacterial liquid is ensured to be injected into the pricked holes, and the pricked holes are vertical to the hypocotyl as much as possible. 1 injection is carried out every 24h, 3 injections are carried out totally, and dark treatment is carried out for 24h after each injection is finished, and dark treatment is carried out for 3d totally.

The step (4) is specifically as follows: after infection, the culture humidity is kept at about 75%, the temperature is kept at about 25 ℃, and the hairy roots of caragana intermedia are selected within 21-28 days to detect the GUS gene expression condition. The leaves were then stained in GUS staining solution.

Compared with the prior art, the invention has the following advantages:

1. according to the invention, the gene is over-expressed in the hairy root of the caragana intermedia by injecting K599 agrobacterium infection, the method is simple to operate, high in expression level and reliable in result, and can provide technical support for developing gene function verification and screening functional genes; and when the hairy root grows to a certain size, the main root is cut off, and the hairy root replaces the main root to become a part of the middle caragana plant and form a chimera of the transgenic hairy root and the stem and leaf of the original plant.

2. The method takes bacterial liquids with different concentrations as initial conditions for exploration, selects acetosyringone with the best effect and the concentration of 100 mu mol/mL from acetosyringone with different concentrations to assist the infection of agrobacterium, increases the infection efficiency of agrobacterium, and further improves the rooting efficiency of hairy roots by increasing the injection times. In conclusion, the optimal genetic transformation efficiency of the hairy roots is finally determined by exploring different conditions.

Drawings

FIG. 1: schematic structure of plant expression vector pCambia 1305.2.

FIG. 2: PCR verification results of the pCambia1305.2 vector, wherein 1-10 are primer I amplification results, and 11-20 are primer II amplification results.

FIG. 3: growing 7-day-old middle caragana seedlings and infecting the hypocotyls of the middle caragana by an injection method, wherein a is the 7-day-old middle caragana seedlings and b is the process of infecting the hypocotyls of the middle caragana by the injection method.

FIG. 4: the difference between the hypocotyl after injection and the hypocotyl before non-injection.

FIG. 5: injecting middle caragana that grows for different time after infection.

FIG. 6: GUS staining condition of caragana intermedia after infection.

Detailed Description

EXAMPLE 1 electrotransformation of pCambia1305.2 expression vector into Agrobacterium K599 competent

(I) Experimental method

(1) The pCambia1305.2 plasmid was removed and thawed and placed on ice. Cleaning the electrode cup (the electrode cup needs to be soaked in 75% ethanol for 10min, then soaked in 100% ethanol for 10min, cleaned by clear water and dried by a fume hood).

(2) The Agrobacterium K599 competent cells were removed from the-80 ℃ freezer, placed on ice and thawed.

(3) Add 1. mu.L of plasmid to the competent cells, gently mix, and aspirate all Agrobacterium with a sheared and sterilized tip into a pre-cooled electrode cup (U1400V, T5.4-5.8 ms).

(4) All the transformation solution was aspirated, added to a 1.5mL EP tube containing 800. mu.L of LB liquid medium, and cultured at 28 ℃ for 2 hours with shaking at 160 rpm.

(5) And sucking 50 mu L of bacterial liquid, spreading the bacterial liquid on a double-resistance plate containing 50 mu g/mL kanamycin and 50 mu g/mL streptomycin, and performing inverted culture at 28 ℃ for 36-48 h.

(6) And selecting a single colony on the plate, and identifying whether the expression vector is successfully transferred into the agrobacterium K599 by colony PCR. Designing PCR primers according to 2 sequence designs of a 35S promoter, GRP-GusPlus coding gene and the like on a pCambia1305.2 vector, and designing 2 pairs of primers in total, wherein the design position of the primer (i) is the 35S promoter, and the length of an amplified target fragment is 884 bp; the design position of the primer II is the GRP-GusPlus coding gene, and the length of the amplified target fragment is 1023 bp.

(2) Results of the experiment

As shown in FIG. 2, 20 Agrobacterium K599 single clones were picked up, and the detection shows that 2 pairs of different primers can amplify a band with a fragment size corresponding to the size of the target fragment, indicating that the pCambia1305.2 vector has been transferred into Agrobacterium GV 3101. Adding the correctly identified agrobacterium into 4mL of LB liquid culture medium, carrying out shaking culture at 28 ℃ and 200rpm for 16-24 h, preserving the seeds, and storing at-80 ℃ for later use.

Example 2 Agrobacterium-mediated optimization of the genetic transformation System for hairy roots of intermediate Caragana

(I) Experimental method

(1) Planting and selecting of caragana intermedia

Seeds of Caragana intermedia are collected from Nemontage Hohaote City and Lin county and from Queen Siziqi. Seeds with full seeds and no insect eyes are selected and sown in a pot of nutrient soil and vermiculite (V: 1:3) and cultured in a greenhouse with the temperature of 25 ℃ and the illumination time of 16 h/the darkness of 8 h. Healthy intermediate caragana seedlings growing for about 7d and growing in vigor were selected for injection (fig. 3).

(2) Preparation of Agrobacterium suspension containing expression vector

The GUS gene carried by pCambia1305.2 vector (figure 1) is used as a reporter gene to optimize a transformation system. The vector was electrically transformed into Agrobacterium K599 competent cells, and Agrobacterium identified as a positive clone was inoculated into 4mL of LB liquid medium (containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin), shaken overnight at 28 ℃ and 200 rpm. The next day, a part of the bacterial liquid is preserved, and the preservation steps are as follows: 1mL of the bacterial liquid is taken in a 1.5mL EP tube, centrifuged for 2min at 4000rpm, the supernatant is discarded, 800 mu L of fresh LB liquid culture medium and 200 mu L of 75% glycerol are added, mixed evenly, frozen by liquid nitrogen and stored in a refrigerator at minus 80 ℃ for a long time. Performing amplification culture on the other part of the bacterial liquid, transferring 0.5-1 mL of the bacterial liquid into 20mL of LB liquid medium (containing 50 ug/mL kanamycin and 50 ug/mL streptomycin), shaking at 28 deg.C and 200rpm, and detecting OD ₆₀₀ 0.6 to 1.8. In addition, acetosyringone with the final concentration of 100 mu mol/mL is added as an inducer and is fully mixed evenly), and the mixture is kept stand for 24 hours in the dark at room temperatureAnd (5) performing injection. Wherein, 100mmol/L acetosyringone preparation: weighing 0.3924g acetosyringone, dissolving in 10mL DMSO, diluting to 20mL with ultrapure water after completely dissolving, subpackaging in 1mL centrifuge tube, and storing at-20 deg.C for use.

(3) Injecting the hypocotyl of caragana intermedia

During injection, the injection is carried out at a position with darker light as much as possible, so that the Vir gene in the agrobacterium can be better induced by acetosyringone, and in addition, the interaction between the bacterial cells of the agrobacterium and plant cells is more facilitated in a dark environment. After the Agrobacterium suspension was filled up using a 1mL disposable needle-less syringe, the Agrobacterium suspension was injected at the hypocotyl site by pressing the syringe (FIG. 3). After infection, the plants were cultured for 3 days in the dark under moisture-preserving conditions. And (4) observing and shearing hairy roots from the 14 th day after infection, carrying out GUS staining, and photographing after 14-16 h of staining.

The GUS staining solution comprises the following formula:

(1)0.2M NaH ₂ PO ₄ ·2H ₂ o: weighing 1.56g, and fixing the volume to 50mL by using sterile water;

(2)0.2M Na ₂ HPO ₄ : weighing 1.42g, and metering to 50mL by using sterile water;

(3)0.1M K ₃ Fe(CN) ₆ : weighing 0.82g, diluting to 25mL with sterile water, and storing at 4 deg.C in dark place;

(4)0.1M K ₄ Fe(CN) ₆ ·3H ₂ o: weighing 1.05g, fixing the volume to 25mL by using sterile water, and storing at 4 ℃ in a dark place;

(5)100mg/ml X-GLUC: weighing 5mg of X-GLUC, dissolving with 50 mu L of dimethylformamide, and storing at 4 ℃ in a dark place; the X-GLUC is prepared at present at 100 mg/ml.

Preparing 100mL of GUS buffer:

after preparation, the product can be stored at 4 ℃ in dark for 2 months, and the storage time is not suitable to be overlong.

GUS staining solution:

to 100mL of GUS buffer, 500. mu.L of 100mg/mL X-GLUC was added and used as it is.

GUS histochemical staining method:

GUS staining is carried out in an incubator at 37 ℃ for 14-16 h. GUS staining was visually observed and photographed.

(II) results of the experiment

The results show that: after 14 days of infection (fig. 5), the injected traces were clearly visible on the hypocotyl of the middle caragana, the injection site swelled or a healed wound was formed. After 21 days of infection (fig. 5), hairy roots of around 0.5cm were produced at the site of middle caragana injection, and after 28 days of infection (fig. 5), hairy roots, which were longer and branched, were produced at the site of middle caragana injection. In order to improve the conversion efficiency, the invention firstly sets different bacterial liquid concentrations (OD) ₆₀₀ The values are 0.3, 0.6 and 1.2) respectively, injecting the hypocotyl of the middle caragana seedling which grows for about 7 days, observing and counting the result, and showing that the OD is ₆₀₀ Values of 0.6, respectively, were able to induce more hairy roots, a percentage of 16.7%. In addition, at different bacterial liquid concentrations (OD) ₆₀₀ The values are respectively 0.6 and 1.2), different concentrations of acetosyringone (0, 100 and 200 mu mol/mL) are respectively added to observe and count the results, and the results show that the OD value of the bacterial liquid concentration is 1.2, and the acetosyringone concentration is 100 mu mol/mL, so that more hairy roots can be induced and generated, and the percentage is 52.8%. As shown in FIG. 6, obvious hairy roots expressing GUS reporter genes were detected 14 to 28 days after infection under various infection conditions. These results demonstrate that, under the current conditions, OD was added to 100. mu. mol/mL acetosyringone ₆₀₀ The injection of a bacterial solution with a value of 1.2 best infects the hypocotyl of the middle caragana. All the above experiments were performed in 3 biological replicates.

The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims (4)

1. A method of effecting gene expression in the hairy root of caragana intermedia, comprising the steps of:

(1) electrically transforming an expression vector containing a GUS reporter gene into an agrobacterium K599 competent cell;

(2) culturing the agrobacterium obtained in the step (1) and agrobacterium K599 which does not contain any exogenous vector in an LB liquid medium; shaking at 200rpm and culturing at 28 deg.C overnight to OD ₆₀₀ The value is 1.2, the bacterial liquid is taken and transferred into a fresh LB liquid culture medium again, the volume ratio of the bacterial liquid to the LB liquid culture medium is 1:20, the bacterial liquid and the LB liquid culture medium are subjected to shaking culture at 28 ℃ and 200rpm until OD is reached ₆₀₀ A value of 1.2; then acetosyringone with the final concentration of 100 mu mol/mL is added;

(3) selecting middle caragana seedlings which grow well for 6-9 days, and slowly injecting the agrobacterium liquid prepared in the step (2) into the hypocotyl part above the middle caragana leaf node and the nutrient soil thereof; the method specifically comprises the following steps: under the normal growth condition, selecting well-grown middle caragana seedlings growing for 6-9 days, and selecting plants with thicker hypocotyls for injection; sucking the agrobacterium suspension which is added with the acetosyringone and then stands for 24h by using a 1mL needle tube, and punching a hole at the hypocotyl part to inject a bacterial liquid; performing 1 injection every 24h for 3 times, performing dark treatment for 24h after each injection, and performing dark treatment for 3 d;

(4) and observing the growth condition of hairy roots of caragana intermedia after transformation, and detecting the expression condition of GUS (glucuronidase) reporter genes.

2. The method for realizing gene expression in the hairy roots of caragana intermedia as claimed in claim 1, wherein the expression vector containing the GUS reporter gene is pCambia 1305.2.

3. The method for realizing gene expression in the hairy roots of caragana intermedia according to claim 1, wherein the LB liquid culture medium of the step (2) is prepared as follows: 1g of Tryptone, 0.5g of Yeast extract, 1g of NaCl, and NaOH or HCl to adjust the pH value to 7.0, diluting deionized water to 100mL, and autoclaving at 121 ℃ for 20 min; among them, LB liquid medium contained 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin.

4. The method for realizing gene expression in hairy roots of caragana intermedia, as claimed in claim 1, wherein after infection, the culture humidity is kept at 75% and the temperature is kept at 25 ℃, and the hairy roots are observed and cut from 14 days after infection to carry out GUS staining, so as to detect the GUS gene expression condition.

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