CN117947092A

CN117947092A - Agrobacterium-mediated-based water yeast Liu Shunshi transformation system and establishment method thereof

Info

Publication number: CN117947092A
Application number: CN202410150446.6A
Authority: CN
Inventors: 及晓宇; 聂显辉; 聂显光; 李磊; 王玉成
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2024-02-02
Filing date: 2024-02-02
Publication date: 2024-04-30

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a water yeast Liu Shunshi transformation system based on agrobacterium mediation and an establishment method thereof. The agrobacterium-mediated-based water yeast Liu Shunshi transformation system is characterized in that 5-azacytidine (Azac), dithiothreitol (DTT) and Acetosyringone (AS) are added into agrobacterium liquid to obtain agrobacterium infection liquid, water yeast Liu Mojun seedlings are used AS explants, and instant transformation seedlings are obtained through instant infection of the agrobacterium infection liquid, so that the agrobacterium-mediated-based water yeast Liu Shunshi transformation system is formed. The transformation system based on the agrobacterium tumefaciens mediated water yeast Liu Shunshi provided by the invention can obtain water yeast Liu Shunshi transformed seedlings with high transformation efficiency in a short time, and can be used for verifying the gene functions of the water yeast willow. The system provides a high-efficiency and rapid technical platform for researching the gene function of the fraxinus mandshurica and provides a technical support for molecular breeding of the fraxinus mandshurica.

Description

Agrobacterium-mediated-based water yeast Liu Shunshi transformation system and establishment method thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a water yeast Liu Shunshi transformation system based on agrobacterium mediation and an establishment method thereof.

Background

Agrobacterium-mediated transient transformation is a highly efficient method for studying gene function, and as a supplement to stable transformation systems, provides a powerful tool for rapidly studying gene function, and provides the possibility for homologous expression and reverse genetic studies in plants without stable genetic transformation systems. Previous studies on transient transformation have mostly focused on herbaceous plants, and in woody plants, the studies are relatively weak. Woody plants have great differences with herbaceous plants in physiological and morphological structures, growth and development and the like, and stress resistance mechanisms of woody plants are also greatly different from those of herbaceous plants.

The fraxinus mandshurica (Fraxinus mandshurica) is a hermaphrodite anemone pollinated tree species of Oleaceae. Due to its excellent wood quality and special grain, it is widely used in furniture and special construction materials. The fraxinus mandshurica not only has higher economic value, but also has important roles in stabilizing the ecological system, protecting the biodiversity and sustainability development of forests. Therefore, an efficient and stable water yeast Liu Shunshi transformation technology is established, excellent resistance genes are screened, and the creation of the novel water yeast Liu Kang germplasm has important significance.

At present, the existing water yeast Liu Shunshi has a too long conversion period, and the instant conversion method of the water yeast Liu Yuan has low conversion efficiency. Therefore, an agrobacterium-mediated-based water yeast Liu Shunshi transformation technology system is established, a high-efficiency and rapid technology platform can be provided for researching the gene function of the water yeast, and a technical support is provided for molecular breeding of the water yeast.

Disclosure of Invention

In order to solve the problem that the transformation period of the water yeast Liu Shunshi is too long and the transformation efficiency of the instant transformation method of the water yeast Liu Yuan is low in the prior art, the invention takes the water yeast willow as a research object, establishes a water yeast Liu Shunshi transformation system based on agrobacterium mediation through an instant transformation technology of agrobacterium mediation, and further identifies the stress resistance function of related genes of the water yeast willow by means of the technology. In order to achieve the above purpose, the invention adopts the following technical scheme:

One of the purposes of the invention is to provide an agrobacterium-mediated-based water yeast Liu Shunshi transformation system, wherein the agrobacterium-mediated-based water yeast Liu Shunshi transformation system is that 5-azacytidine, dithiothreitol and acetosyringone are added into agrobacterium liquid to obtain agrobacterium infection liquid, water yeast Liu Mojun seedlings are used as explants, and instant transformed seedlings are obtained through instant infection of the agrobacterium infection liquid, so that the agrobacterium-mediated-based water yeast Liu Shunshi transformation system is formed.

According to the agrobacterium-mediated-based water yeast Liu Shunshi transformation system provided by the invention, the stress-resistant genes can be rapidly and efficiently screened out after the explants are transformed in a transient way without a subsequent callus induction culture process, and a large amount of redundant tissue culture work is omitted.

Preferably, the concentration of agrobacterium in the agrobacterium solution is od=0.4-1.0.

Preferably, the infestation time is from 2 to 10 hours.

Preferably, the addition amount of 5-azacytidine is 10-50 mug/L based on the volume of the agrobacterium tumefaciens bacteria liquid; the addition amount of dithiothreitol is 100-500 mg/L; the addition amount of acetosyringone is 50-250 mg/L.

The second object of the invention is to provide a method for establishing the transformation system based on the agrobacterium-mediated water yeast Liu Shunshi, which comprises the following steps:

taking fraxinus mandshurica seeds for culture to obtain fraxinus mandshurica tissue culture seedlings;

Preparation of agrobacterium infection solution: inoculating agrobacterium containing target gene into a culture medium, culturing at 28 ℃ and 220rpm for 24-36 hours until the concentration of the agrobacterium is OD=0.6-0.8; centrifuging bacterial liquid with OD=0.6-0.8, collecting bacterial cells, dissolving in sucrose with volume fraction of 2% to adjust the bacterial liquid concentration to OD=0.4-1.0, standing to obtain agrobacterium liquid, and adding 5-azacytidine, dithiothreitol and acetosyringone into the agrobacterium liquid to prepare agrobacterium infection liquid;

transferring the tissue culture seedlings of the fraxinus mandshurica into an agrobacterium infection solution for infection to obtain fraxinus mandshurica Liu Shunshi transformed seedlings, and obtaining the fraxinus mandshurica Liu Shunshi transformation system based on agrobacterium mediation.

Taking out the sterilized fraxinus mandshurica seeds, culturing in a WPM solid culture medium without any hormone for 24-48 hours at the temperature of 25 ℃ in the dark, and culturing the seed embryos by illumination for 2 days to obtain fraxinus mandshurica tissue culture seedlings.

Agrobacterium containing the target gene is cultured on LB medium.

Preferably, the agrobacterium containing the target gene is agrobacterium transformed with an expression vector containing the target gene, wherein the target gene comprises a stress-resistant gene FmMYB.

Preferably, the agrobacterium comprises agrobacterium EHA105.

Preferably, transferring the tissue culture seedlings of the fraxinus mandshurica into an agrobacterium infection solution, and culturing for 1-1.5 hours at the temperature of 25-26 ℃ and the rpm of 85-95 rpm for the first time; adding 1/2 volume of fresh conversion solution into the agrobacteria invasion solution, and performing secondary co-culture for 1.3-1.7 h under the conditions of 25-26 ℃ and 85-95 rpm; and further culturing the roots of the tissue culture seedlings of the fraxinus mandshurica under a dark condition to obtain the fraxinus mandshurica Liu Shunshi transformed seedlings.

Wherein, the roots of the tissue culture seedlings of the fraxinus mandshurica after the secondary co-culture are inserted on a 1/2WPM solid culture medium, and are subjected to dark culture for 24-48 hours at 25-26 ℃ to obtain the fraxinus mandshurica Liu Shunshi transformed seedlings.

Preferably, the 1/2WPM solid medium comprises the following components in percentage by weight: 28-32 g/L of sucrose, 2.16-2.18 g/L of MS powder, 7.4-7.6 g/L of agar and water as solvent.

Preferably, the conversion solution is sucrose with a volume fraction of 2%.

Compared with the prior art, the invention has the following beneficial effects:

The invention provides a transformation system based on agrobacterium-mediated hydrologic Liu Shunshi, which can rapidly and efficiently screen stress-resistant genes after transient transformation of explants without a subsequent callus induction culture process, and saves a large amount of redundant tissue culture work. The transformation system based on the agrobacterium tumefaciens-mediated water yeast Liu Shunshi provided by the invention has the advantages of short transformation period and high transformation efficiency, and provides a certain technical support for realizing large-scale high-quality stress-resistant gene screening and establishing a general technical platform for molecular breeding.

The invention provides a method for establishing a transformation system of the water yeast Liu Shunshi based on agrobacterium mediation, which utilizes a high-efficiency instant transformation water yeast willow technology, can not interfere with the stability of host genome, and is not influenced by position effect in gene expression; it does not require regenerated transformed cells to analyze gene function. In addition, the water yeast Liu Shunshi transformation system based on agrobacterium mediation provided by the invention is a transient gene transformation system, and can remarkably improve the research speed, because the genes can be differentially expressed in a large amount in a short time.

Drawings

FIG. 1 shows the effect of different bacterial concentrations and different infection times on GUS activity for transient transformation in the present invention, wherein A: od=0.4; b is: od=0.6; c: od=0.8; d: od=1.0;

FIG. 2 shows the effect of different bacterial liquid concentrations and different infection times on GUS gene expression level in transient transformation, wherein A is as follows: od=0.4; b: od=0.6; c: od=0.8; d is: od=1.0;

FIG. 3 shows the effect of different drug concentrations on GUS enzyme activity for transient transformation in the present invention, wherein A is: azac; b is: DTT; c is: AS;

FIG. 4 shows the effect of transient transformation at different drug concentrations on GUS gene expression levels in the present invention, wherein A is: azac; b is: DTT; c is: AS;

FIG. 5 shows growth before transient infection and transient infection GUS expression of fraxinus mandshurica in the invention, wherein A is a seed embryo germinated by fraxinus mandshurica; b is the germination of the fraxinus mandshurica strain-free embryo; c is a sterile bud of fraxinus mandshurica; d is a sterile seedling of fraxinus mandshurica; E-H is the expression condition of the water yeast Liu Shunshi infected GUS;

FIG. 6 shows PCR detection results of pROKII infection, non-transient infection and pFGC5941-FmMYB bacteria liquid in the present invention;

FIG. 7 shows the result of Liu Zhizhu qRT-PCR detection of FmMYB gene transient transformation of water yeast in the present invention;

FIG. 8 shows the physiological index analysis of FmMYB gene transient OE, RNAi and CK hydrologic curve Liu Zhizhu under drought stress, wherein A is the content of hydrologic curve Liu Zhizhu O ² -; b is the content of water yeast Liu Zhizhu H ₂O₂; c is the MDA content of the water yeast Liu Zhizhu; d is the activity of the sodium hydrosulfite Liu Zhizhu SOD; e is the activity of water yeast Liu Zhizhu POD; f is the CAT activity of the water yeast Liu Zhizhu.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

The following are the experimental materials used in the examples:

(1) Plant material

The seeds of fraxinus mandshurica are preserved by the Liaoning forest genetic breeding and cultivation key laboratory (Shenyang agricultural university).

(2) Carrier and strain

Agrobacterium EHA105, E.coli DH 5. Alpha. Competent cells, pROKII vector, pFGC5941 vector, pCAMBIA1301 vector were maintained by Liaoning forest genetic breeding and cultivation emphasis laboratory (Shenyang agricultural university).

(3) Main reagent

The E.coli plasmid extraction kit, gel recovery kit and PCR purification kit were all purchased from OMEGA Biotechnology.

In-FusionAdvantage PCR Cloning Kit was purchased from Clontech.

5-Azacytidine (Azac) was purchased from Solarbio company; dithiothreitol (DTT) is available from Solarbio company; acetosyringone (AS) was purchased from Solarbio company.

X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucoside) was purchased from Solarbio company.

(4) Preparation of main solution and culture medium

PEG: PEG powder was dissolved in sterile water at a working concentration of 250mM/L.

50Mg/LKan: kanamycin powder is dissolved in sterile water, the concentration is 50mg/L, and the kanamycin powder is preserved at-20 ℃.

50Mg/L Rif: rifampicin powder was dissolved in DMSO at a concentration of 50mg/L and stored at-20deg.C.

AS: dissolving acetosyringone powder in DMSO, filtering, sterilizing, and storing at-20deg.C.

Solid LB medium: 5g/L yeast extract powder, 10g/L peptone, 10g/L sodium chloride and water as solvent.

Liquid LB medium: 5g/L yeast extract powder, 10g/L peptone, 10g/L sodium chloride and 12g/L agar, and the solvent is water.

1/2WPM liquid Medium: 30g/L sucrose, 2.17g/L MS powder, water as solvent, pH 5.8, and high pressure sterilizing at 121deg.C for 20min, and storing at normal temperature.

1/2WPM solid Medium: 30g/L of sucrose, 2.17g/L of MS powder, 7.5g/L of agar, water as solvent, pH 5.8, and high-pressure sterilization at 121 ℃ for 20min, and preserving at normal temperature.

Example 1

1. Germination of fraxinus mandshurica seeds

Washing the seeds of fraxinus mandshurica with running water for 4 days after removing pericarp, stirring the seeds in an ultra-clean workbench with 75% ethanol for 2min, washing with sterile distilled water for 3 times, sterilizing the seeds in 10% (v/v) sodium hypochlorite solution for 10min, and washing with sterile distilled water for 5 times. Taking out the sterilized seeds, and horizontally placing the seeds in a 1/2WPM solid culture medium plate for culture, wherein the seeds are cultured for about 2 days in dark at 25 ℃. The specific cultivation time is the cut-off cultivation time when the cotyledons of the seeds turn green. Transferring the seed embryo cultured in the darkroom into a tissue culture bottle without any hormone-containing WPM solid culture medium, and placing the seed embryo in the tissue culture room for illumination culture for 2 days to obtain the tissue culture seedling of the fraxinus mandshurica for subsequent instant transformation.

2. Establishment of water yeast Liu Shunshi transformation system based on agrobacterium mediation

2.1 Strain activation

The pCAMBIA1301 empty agrobacterium is streaked in three areas on a solid LB medium containing 50mg/L Kan and Rif, inverted in an incubator for 2 days at 28 ℃, single colonies on the third area are picked up, inoculated in a liquid LB medium added with 50mg/L Kan and Rif, and cultured for 24 hours at 28 ℃ and 220 rpm.

2.2 Pre-culture of bacterial liquid

Culturing agrobacterium with 50mg/L Kan and Rif added liquid LB medium to od=0.8, and taking bacterial liquid with od=0.8, wherein the bacterial liquid and fresh liquid LB medium are 1:49, shaking again to od=0.7 at 28 ℃; and (3) taking bacterial liquid with OD=0.7, and shaking with fresh LB liquid at 28 ℃ until the OD=0.4, 0.6, 0.8 and 1.0 are respectively achieved, so that bacterial liquids with different concentrations are obtained.

2.3 Comparative study of concentration and time of bacterial liquid in Water Yeast Liu Shunshi transformation method

Bacterial solutions with OD=0.4, 0.6, 0.8 and 1.0 are centrifugally collected at 3000rpm, the concentration of the bacterial solution is adjusted to OD=0.8 by dissolving sucrose with volume fraction of 2%, and the bacterial solutions with different concentrations are obtained after standing at room temperature for one hour. Transferring the tissue culture seedlings of the fraxinus mandshurica into the agrobacterium liquid after standing, and performing first co-culture for 1h at 25 ℃ and 90 rpm. Adding 1/2 volume of 2% sucrose into the agrobacterium liquid, and performing secondary co-culture for 1.5h at 25 ℃ and 90 rpm; and then, the roots of the tissue culture seedlings of the fraxinus mandshurica are inserted into a 1/2WPM solid medium, and are cultivated for 24 hours at the temperature of 25 ℃ in darkness, so as to obtain the transformed seedlings of the fraxinus mandshurica Liu Shunshi.

3. Comparative study of different factor combinations of the transformation method of water yeast Liu Shunshi

In order to establish a water yeast Liu Shunshi transformation system and study the optimal conditions of bacterial liquid concentration, infection time and different drug concentrations, the following two groups of experiments are set on the basis of a pCAMBIA1301 agrobacterium strain activation and bacterial liquid pre-culture method:

(1) Different agrobacterium bacteria liquid concentration and infection time are set in bacteria liquid preculture, and the OD value of the bacteria liquid concentration is as follows: 0.4, 0.6, 0.8 and 1.0, the infection times are respectively: 2. 4, 6, 8 and 10h, a grouping experiment was performed.

(2) Based on the initially established optimal agrobacterium infection solution concentration and optimal infection time based on an agrobacterium-mediated water yeast Liu Shunshi transformation system, sequentially adding 5-azacytidine (Azac) (10, 20, 30, 40, 50 mug/L), dithiothreitol (DTT) (100, 200, 300, 400, 500 mg/L) and Acetosyringone (AS) (50, 100, 150, 200, 250 mug/L) into the agrobacterium solution according to the volume of the agrobacterium solution, and carrying out instantaneous infection on water yeast Liu Shunshi transformed seedlings by the invasion solution. After infection, obtaining a water yeast Liu Shunshi transformed plant, namely obtaining the water yeast Liu Shunshi transformation system based on agrobacterium mediation. Wherein, the bacterial solution concentration of the optimal agrobacterium infection solution is od=0.8. The optimal infection time is 0.8h.

The above test uses wild type fraxinus mandshurica seedlings infected with the infection liquid of sterile liquid as a control.

4. GUS histochemical detection based on agrobacterium-mediated water yeast Liu Shunshi transformation system

After carrying out dark culture on the instant transformed water yeast Liu Zhizhu for 2 days, taking out, placing into a centrifuge tube, adding GUS buffer solution until the water yeast is beyond hypocotyl, soaking for 1h in a dark environment at 37 ℃, sucking out the GUS buffer solution by using a pipette, adding GUS detection solution again, dyeing in the dark environment at 37 ℃ until blue color appears, decoloring the material by using 75% (v/v) ethanol, and observing and recording the dyeing condition.

The preparation method of the GUS buffer solution comprises the following steps: 6.2mL of 0.2M NaH ₂PO₄、28mL 0.2MNa₂HPO₄ was mixed to prepare a pH7.0 of 0.2M phosphate buffer; 25mL was mixed with 0.25mL 0.1MK₃[Fe(CN)₆]、0.25mL 0.1M K₄[Fe(CN)₆]、0.5mL 0.1MNa₂EDTA and distilled water to a volume of 50mL.

The preparation method of the GUS detection liquid comprises the following steps: GUS buffer solution and X-Gluc 1:1.

5. Determination and quantitative analysis of GUS enzyme Activity

A series of standard solutions were prepared, 1mM MU mother solution was diluted to a concentration ranging from 0 to 10. Mu.M by using a reaction terminating solution, and a standard curve was drawn based on the fluorescence intensity results obtained by the measurement. The GUS enzyme extraction and determination method specifically comprises the following steps:

(1) Into a 1.5mL centrifuge tube, 0.1g of liquid nitrogen ground water yeast Liu Shunshi was added to transform the plants, and 600. Mu.L of enzyme extract was added.

(2) Centrifuge at 13000rpm at 4℃for 0min and aspirate the supernatant.

(3) Protein content was obtained by measuring the appropriate amount of supernatant by coomassie brilliant blue method.

(4) After preheating at 37 ℃, 50 μl of the supernatant was added to 450 μl of the test solution. Immediately after mixing, 50. Mu.L of the mixture was added to 950. Mu.L of the reaction-terminated solution, and the sample was used as the 0-point of the enzymatic reaction. Wherein the detection solution is 2mmol/LMUG solution; the stop solution was 0.2mol/LNa ₂CO₃.

(5) Then, at 5, 10, 20, 30, 45 and 60 minutes, respectively, 50. Mu.L of the reaction solution was taken out and transferred to 950. Mu.L of the reaction termination solution.

(6) Fluorescence spectrophotometry was set to 365nm excitation wavelength and 455nm emission wavelength, respectively, and fluorescence values at different time points were measured.

Furthermore, we refer to the following 6.1-6.2 to extract total RNA of transiently transformed fraxinus mandshurica, and reverse transcribe into cDNA as a template used in fluorescent real-time quantitative RT-qPCR.

Wherein, the fluorescent real-time quantitative RT-PCR amplification primers are shown in Table 1:

TABLE 1 primers for use in the water yeast Liu Dingliang RT-PCR analysis

The fluorescent real-time quantitative RT-qPCR reaction system is as follows, as shown in Table 2:

TABLE 2 real-time fluorescent quantitative RT-PCR reaction system

Fluorescent real-time quantitative RT-PCR reaction conditions:

55℃to 99℃read every 0.5℃，hold 1s。

6. construction of plant overexpression vector pROKII-FmMYB 9

6.1 Extraction of RNA from fraxinus mandshurica

(1) Adding 600 μL of 2% CTAB extract into a centrifuge tube, putting 0.2g of the fraxinus mandshurica material into a mortar, repeatedly adding liquid nitrogen for full grinding, adding the grinded fraxinus mandshurica into the centrifuge tube, reversely and uniformly mixing, placing the fraxinus mandshurica into a water bath at 65 ℃ for 20min, reversely and uniformly mixing for several times during the period, rapidly transferring the centrifuge tube to ice, and centrifuging at 4 ℃ and 12000rpm for 5min.

(2) The supernatant was transferred to a fresh tube, 300. Mu.L of chloroform and 300. Mu.L of LTris-saturated phenol were added thereto, and after shaking for 2 minutes, the mixture was centrifuged at 12000rpm for 5 minutes at 4 ℃.

(3) The supernatant was taken, 600. Mu.L of chloroform was added thereto, and the mixture was vigorously shaken for 2min, and centrifuged at 12000rpm at 4℃for 5min.

(4) The supernatant was kept, 1mL of absolute ethanol and a few drops of 3M NaAc were added, and the mixture was mixed well, left at-70℃for 15min, and centrifuged at 12000rpm for 10min at 4 ℃.

(5) Each tube was added with 0.5. Mu.L of digestive enzyme, and reacted at 37℃for 30 minutes.

(6) Adding 200 μl of chloroform, mixing, and centrifuging at 12000rpm at 4deg.C for 5min;

(7) Washing the precipitate with 75% absolute ethanol for 2 times, adding about 20 times to the precipitate, and adding water to the precipitate to dissolve the precipitate to obtain the water yeast Liu Zong RNA extract.

(8) Detecting quality of the aquatic yeast Liu Zong RNA by 1% agarose gel electrophoresis, detecting purity and concentration of the aquatic yeast Liu Zong RNA by an ultraviolet spectrophotometer, and preserving at-80 ℃ for standby.

6.2 Reverse transcription of Water Yeast Liu Zong RNA

The cDNA was obtained by reverse transcription using the above-obtained water yeast Liu Zong RNA as a template and the following reaction system and reaction conditions.

(1) The reaction system is shown in table 3:

TABLE 3cDNA reverse transcription System

(2) Reaction conditions: reacting at 37 ℃ for 25min; the reaction was carried out at 80℃for 10s.

The cDNA obtained by reverse transcription is diluted 10-100 times as a template and used at-20 ℃.

6.3 Design of primers

The restriction enzyme SmaI site was introduced according to the fusion characteristics of the plant overexpression vector pROKII and the target gene FmMYB, and pROKII-FmMYB 9 overexpression vector primers are designed as shown in Table 4.

TABLE 4 construction of plant overexpression vector pROKII-FmMYB Gene primer sequences

SmaI single cleavage and purification of 6.4 pROKII vector

Extraction of pROKII plasmid pROKII vector was subjected to single cleavage with restriction enzyme SmaI.

(1) The cleavage reaction system is shown in Table 5:

TABLE 5 cleavage reaction System

(2) Incubation was carried out at 25℃for 4h.

3 Mu L of enzyme digestion products and plasmids before enzyme digestion are respectively separated by 1% agarose gel electrophoresis, the positions of bands can be seen to have certain difference, and the bands of the enzyme digestion products are single and specific, so that the enzyme digestion products are directly recovered by using a PCR purification kit.

6.5 Obtaining of Gene FmMYB of interest

CDNA obtained by reverse transcription of the water yeast Liu Zong RNA is used as a template, and FmMYB-OE-F, fmMYB-OE-R primer is used as PCR.

(1) The reaction system is shown in Table 6:

TABLE 6PCR reaction System

(2) Reaction conditions:

And taking 5 mu L of the product, detecting a PCR result by using 1% agarose electrophoresis, carrying out gel recovery on the specific strip with the correct length by using a gel recovery kit, detecting recovery efficiency by electrophoresis, and measuring the concentration of the specific strip. .

6.6 Fragment ligation

The recovered FmMYB gene fragment and the pROKII fragment recovered by single cleavage were ligated with In-Fusion enzyme to construct the plant vector pROKII-FmMYB 9.

(1) In-Fusion enzyme ligation system as shown In Table 7:

TABLE 7In-Fusion enzyme ligation System

(2) Connection conditions: incubation was carried out at 37℃for 30min and at 50℃for 15min.

6.7 Identification of ligation products transformed E.coli and Positive clones

Mu.L of the liquid after the ligation in 6.6 was transferred into E.coli DH 5. Alpha. Competent cells. The culture was spread on a solid LB medium containing 50mg/LKan for selection. Single colonies on the selection medium were randomly picked for expansion culture, and bacterial liquid PCR was performed using pROKII-F and pROKII-R as primers (Table 8).

(1) Amplification primers, as shown in table 8:

table 8PCR identification of primers for pROKII-FmMYB 9 positive clones

(2) The reaction system is shown in Table 9

TABLE 9PCR reaction System

(3) The reaction procedure:

6.8 sequencing of Positive clones

The positive strain identified by PCR extracts plasmids which were sent to Shanghai Biotechnology company for sequencing.

6.9 Construction of the expression vector pFGC5941-FmMYB for inhibition

Corresponding restriction enzyme sites (NcoI, ascI) and (XbaI, bamHI) were introduced based on the fusion properties of the plant expression vector pFGC5941 and the target gene FmMYB-Cis/Anti, namely: fmMYB 9-Cis/Anti-sense/antisense strand primers

6.10 Cloning of FmMYB 9-Cis/Anti-target fragment

The pROK II-FmMYB plasmid is used as a template to PCR amplify the FmMYB-Cis/Anti target fragment with the cleavage site.

(1) PCR reaction systems, as shown in Table 10

TABLE 10PCR reaction System

(2) Reaction conditions:

FmMYB 9A 9-Cis/Anti-Cis fragment and pFGC5941 vector were digested and PCR amplified using the American OMEGA Biotechnology company PCR purification kit to purify the FmMYB-Cis fragment. FmMYB9-Cis/Anti and pFGC5941 vector were double digested with restriction enzymes (Nco I, asc I) and (Xba I, bam HI), respectively.

(1) The cleavage reaction system is shown in Table 11:

TABLE 11 cleavage reaction System

Incubation was carried out at 37℃for 4h.

(2) Fragment of interest ligation

The digested product was separated by 1% agarose gel electrophoresis, and the FmMYB-Cis/Anti digested fragment and the large pFGC5941 vector fragment were recovered from the gel cut.

The recovered FmMYB-Cis/Anti-gene fragment and the large pFGC5941 vector fragment were ligated by TaKaRa T4 DNA ligase to construct plant vector pFGC5941-FmMYB9-Cis/Anti.

Wherein, T4 DNA ligation system, as in Table 12:

Table 12T4 DNA ligation System

(2) Connection conditions: the reaction was carried out at 16℃overnight.

Positive clone PCR detection:

Randomly picking single bacterial plaque, adding 1mL LB liquid culture medium containing 50mg/Lkan, shaking and culturing at 37 ℃ and 200rpm for 4 hours, performing PCR detection positive cloning by taking escherichia coli bacterial liquid as a template, selecting bacterial liquid with correct strips, propagating, extracting plasmids, and sequencing.

8. FmMYB9 Gene overexpression and inhibition of expression vector transformation of Agrobacterium tumefaciens EHA105

100Ng of recombinant plasmids pROKII-FmMYB and pFGC5941-FmMYB are respectively added into 100 mu L of agrobacteria competent cells, sucked and stirred uniformly and then transferred into a electric shock cup precooled on ice for 10 min; rapidly taking out the electric shock cup after electric shock at 1800V, adding 600 μl of LB liquid medium, rapidly sucking, beating and mixing; sucking the LB liquid culture medium after electric shock into a sterile 1.5mL sterile centrifuge tube, culturing for 1.5h at 28 ℃ with 220rpm in a shaking way, and resuscitating agrobacterium; 200. Mu.L of the transformed bacterial liquid was spread on LB solid medium containing 50mg/L of Rif and 50mg/L of kanamycin, and cultured upside down at 28℃for 2 days.

9. FmMYB9 Gene overexpression and inhibition of expression vector transient transformation and drought tolerance analysis

9.1 Transient transformation of the FmMYB9 Gene and the suppression expression vector

The EHA105 strain containing FmMYB gene over-expression vector pROKII-FmMYB 9, the EHA105 strain inhibiting expression vector pFGC5941-FmMYB and the EHA105 strain of empty vector pBI121 are respectively and transiently infected with the water yeast Liu Youmiao according to the obtained water yeast Liu Shunshi transformation system based on agrobacterium mediation. The water yeast Liu Youmiao infected by the EHA105 bacterial liquid of pROKII-FmMYB 9 and the water yeast Liu Youmiao transiently infected by the EHA105 bacterial liquid of pFGC5941-FmMYB are used as treatment groups; the fraxinus mandshurica seedlings transiently infected with the EHA105 bacterial liquid of pBI121 were used as control groups.

The invention establishes a transformation system based on agrobacterium-mediated water yeast Liu Shunshi, and specific transient transformation conditions are as follows: bacterial solution concentration OD=0.8 of the agrobacterium infection solution, optimal infection time is 8h, addition amount of 5-azacytidine (Azac) is 30 mug/L, DTT and addition amount of 200mg/L, AS is 150mg/, and after infection is finished, the water yeast Liu Shunshi transformed plant is transferred to a 1/2WPM solid culture medium for recovery culture for 48h. Half of three transient infection plants of the obtained over-expression vector, the inhibition expression vector and the empty vector are respectively transferred to a 1/2WPM solid culture medium containing 250mmol/L mannitol, and the rest of the transient infection plants are replaced by a new 1/2WPM solid culture medium to serve as a control group. After culturing in 1/2WPM solid medium for 48h, taking the whole strain of fraxinus mandshurica material of the control group and the treatment group respectively, and partially freezing by liquid nitrogen and then storing in a refrigerator at-80 ℃ for storage, wherein the partial strain is used for subsequent experiments.

9.2 Analysis of stress-resistant physiological index of transient expression of FmMYB9 Gene

The various stress-resistant physiological indexes of the over-expression plants (labeled OE), the inhibition expression plants (labeled RNAi, RNA interference) and the empty vector control plants (labeled CK) are analyzed, and the specific method is as follows:

The content of Malondialdehyde (MDA) was determined by thiobarbituric acid colorimetric method: 0.5g of the water yeast Liu Shunshi transformed plants were ground to homogenate with 5mL of 10% TCA (trichloroacetic acid) and centrifuged at 1000g for 10min at 4 ℃. 2mL of the supernatant was mixed with 2mL of TBA (thiobarbituric acid) and then the mixture was boiled in water for 20 minutes. Absorbance values were measured at 532 nm.

Determination of O ^2- content: 1g of the water yeast Liu Shunshi transformed plants were ground, placed in 10mL of 3% trichloroacetic acid (TCA), and centrifuged at 10000g for 10min at 4 ℃. 1mL of the supernatant was mixed with 1mL of 50mm pH7.0 potassium phosphate buffer containing 1mm hydroxylamine hydrochloride. Absorbance values were measured at 530 nm.

The determination of the H ₂O₂ content is carried out by ultraviolet spectrophotometry: 0.4g of the transformed plants of the water yeast Liu Shunshi were ground to a homogenate with 10mL of 50mM potassium phosphate buffer pH 6.5 and centrifuged at 10000g for 10min at 4 ℃. Then 1mL of the supernatant was mixed with 1mL of 10% (v/v) TiCl ₄. Absorbance values were measured at 412 nm.

Superoxide dismutase (superoxide dismutase, SOD) activity was measured using the NBT method: 1g of transiently transformed plants of fraxinus mandshurica was homogenized in 50mM phosphate buffer pH 7.8 containing 5mM EDTA, 5mM dithiothreitol and 1% (v/v) polyvinylpyrrolidone. The homogenate was centrifuged at 10000g for 5min at 4℃and 0.05mL of the supernatant was added to 2mL of a reaction solution of NBT (nitrotetrazolium chloride) at 0.002g/L, and the reaction was carried out under light for 30 minutes. One unit of SOD activity was defined as the enzyme concentration required to inhibit NBT reduction by 50%, and the change in absorbance at 560nm was measured within 1 minute.

The catalase activity (catalase, CAT) was determined by uv spectrophotometry: 1g of transiently transformed plants of fraxinus mandshurica was homogenized in 50mM phosphate buffer (pH 7.8) containing 5mM EDTA, 5mM dithiothreitol and 1% (v/v) polyvinylpyrrolidone. The homogenate was centrifuged at 10000g for 5min at 4℃and 0.05mL of the supernatant was taken and 3mL of the reaction solution containing 1mL of 50mM phosphate buffer pH 6.8, 2% guaiacol, 2% H ₂O₂ and 100. Mu.L of enzyme solution was added. The change in absorbance within 1min was measured at 470 nm.

Peroxidase (POD) activity assay was determined using the guaiacol method: 1g of transiently transformed plants of fraxinus mandshurica was homogenized in 50mM phosphate buffer pH 7.8 containing 5mM EDTA, 5mM dithiothreitol and 1% (v/v) polyvinylpyrrolidone. The homogenate was centrifuged at 10000g for 5min at 4℃and 0.05mL of the supernatant was taken, and 3mL of the reaction mixture was added as 200mM phosphate buffer pH 7.8, 100mM H ₂O₂ and 50. Mu.L of enzyme solution. The change in absorbance was measured at 240nm over 1 min.

3. Results

3.1 Comparative study of concentration and time of bacterial liquid in Water Yeast Liu Shunshi transformation method

As shown in fig. 1-a, when od=0.4, the GUS enzyme activity increased first and then decreased with increasing agrobacterium infection time, and when the infection time was 8h, the GUS enzyme activity was highest and significantly higher than at other time points. Similar to FIG. 1-A, FIGS. 1-B, 1-C, 1-D all increased over time, with increasing GUS enzyme activity followed by decreasing GUS enzyme activity, but at an Agrobacterium concentration of OD=0.8 for 8 hours, GUS enzyme activity was highest and higher than GUS enzyme activity after infection with other Agrobacterium concentrations.

The results show that the GUS enzyme activity is expressed as a phenomenon of increasing and then inhibiting along with the increase of the concentration of the agrobacterium tumefaciens liquid and the increase of the infection time, and the result of initially establishing a water yeast Liu Shunshi transformation system with the optimal agrobacterium concentration and time combination is shown in figure 1.

To further verify the above results, the relative expression level of GUS gene was also measured in this experiment, and the results are shown in FIG. 2.

2-A, 2-B, 2-C, 2-D, the GUS gene expression level was the highest and higher than that after infection with other Agrobacterium concentrations when the Agrobacterium concentration was OD=0.8 and the time was 8h, as the Agrobacterium concentration was increased and the infection time was increased.

The results show that the GUS gene expression level and the GUS enzyme activity change trend are consistent, and the reliability of the initially established water yeast Liu Shunshi transformation system based on agrobacterium mediation is demonstrated.

3.2 Comparative study of different drugs by the transformation method of Water Yeast Liu Shunshi

Azac, DTT, AS is added in sequence based on the initially established optimal agrobacterium concentration and time conditions, and a transformation system based on agrobacterium-mediated hydrologic yeast Liu Shunshi is obtained, and the result is shown in fig. 3.

As shown in FIG. 3-A, the GUS enzyme activity increased and then decreased with increasing Azac concentration, and the GUS enzyme activity was highest when Azac was 30. Mu.g/L; as shown in FIG. 3-B, the GUS enzyme activity increased and decreased with increasing concentration of DTT, and the GUS enzyme activity was highest when the DTT was 200 mg/L; FIG. 3-C shows that the GUS enzyme activity increases and decreases with increasing AS concentration, and that the GUS enzyme activity is highest when AS is 150. Mu.M. The results show that the GUS enzyme activity is gradually increased after Azac, DTT, AS is added in sequence, and the GUS enzyme activity is improved by more than 10 times compared with the initially established transformation system based on the agrobacterium-mediated hydroyeast Liu Shunshi, so that the research establishes the transformation system based on the agrobacterium-mediated hydroyeast Liu Shunshi.

To further verify the above results, the present invention measured the relative expression level of GUS gene, and the results are shown in FIG. 3.

As shown in FIGS. 3-A, 3-B and 3-C, the GUS gene expression level was observed AS a phenomenon of increasing and suppressing the level of GUS gene expression with increasing Azac, DTT, AS concentration, and was highest when Azac (30. Mu.g/L), DTT (200 mg/L) and AS (150. Mu.M) were added.

The results show that the GUS gene expression level and the GUS enzyme activity change trend are consistent, and the reliability and the repeatability of a water yeast Liu Shunshi transformation system based on agrobacterium mediation are demonstrated.

Using an established transformation system based on agrobacterium-mediated hydrologic Liu Shunshi: bacterial liquid concentration OD=0.8, optimal infection time 8h, adding Azac (30 μg/L), DTT (200 mg/L), AS (150 μM/L). GUS histochemical staining was performed 2d after dark culture, with varying degrees of blue color appearing on the roots, stems, and leaves of 57 out of 60 explants (see FIG. 5), with a conversion of 95% for transient infection.

The results show that the established fraxinus mandshurica infected by the fraxinus mandshurica Liu Shunshi transformation system mediated by agrobacterium has GUS activity, can increase genetic transformation rate, and also shows that bacterial liquid has activity.

3.3 Construction of plant overexpression vector pROKII-FmMYB 9 and inhibition expression vector pFGC5941-FmMYB9

Gene FmMYB was amplified by PCR. The fraxinus mandshurica cDNA is used as a template, a PCR amplified gene FmMYB product is subjected to homologous fusion pROK II, and is transformed into escherichia coli, and after the sequence is correct, the fraxinus mandshurica cDNA is transferred into agrobacterium EHA105 for PCR detection, and is used for a fraxinus mandshurica transformation test (figure 6).

According to the fusion characteristics of a plant inhibition expression vector pFGC5941 and a target gene FmMYB, respectively introducing Noc I and Asc I restriction sites at the 5 'end and the 3' end of FmMYB-Cis; endonuclease Xba I and Bam HI cleavage sites were introduced at the 5 'and 3' ends of FmMYB-Anti, respectively. Ligation, transformation, PCR, sequencing. See fig. 6.

As can be seen from FIG. 6, the plant over-expression vector pROKII-FmMYB 9 reaches the vector and the inhibition expression vector pFGC5941-FmMYB is constructed successfully.

3.4 Transient transformation of the Salix integra FmMYB Gene and drought tolerance analysis

The related plants of the stress-resistant gene FmMYB-9 fraxinus mandshurica are obtained by a water yeast Liu Shunshi transformation system based on agrobacterium mediation, and after drought stress for 48 hours, RT-qPCR analysis is carried out on FmMYB gene expression amounts (figure 7) in 3 transient transformation water yeast Liu Zhizhu, and the results are shown in figure 7.

As can be seen from FIG. 7, fmMYB gene was expressed in the highest level in the transient over-expression (OE) plants and in the repressed expression (RNAi) plants under normal conditions. After drought stress treatment, fmMYB gene has the highest expression level in OE plants, and is higher than that in non-stress treatment OE plants, and the expression level in RNAi plants is lower than that of CK plants.

The results show that the high-efficiency transient over-expression and the inhibition expression FmMYB gene hydroyeast Liu Zhizhu which are successfully obtained can be used for subsequent experiments.

The stress-resistant gene FmMYB is obtained by an agrobacterium-mediated transient transformation system, and the physiological index analysis is carried out on the plant related to the fraxinus mandshurica, and the result is shown in figure 8.

Under normal growth conditions, the O ^2-、H₂O₂ content in the OE plant, the RNAi plant and the CK plant has no obvious difference; after drought stress treatment, the O ^2-、H₂O₂ content of RNAi and CK plants was significantly higher than that of OE plants, with the RNAi plants having the highest O ^2-、H₂O₂ content. The above results indicate that overexpression of FmMYB gene in fraxinus mandshurica can reduce O ^2-、H₂O₂ production under drought stress.

The MDA content measurement result of OE, RNAi, CK plants shows that under normal conditions, the MDA content of OE, RNAi, CK plants has no obvious difference; after drought stress treatment, the MDA content of the OE plants is significantly lower than that of RNAi and CK plants, and the MDA content of RNAi plants is highest.

The result shows that the over-expression FmMYB gene can reduce the accumulation of MDA content of the fraxinus mandshurica under drought stress, thereby increasing the stress-resistant function of the fraxinus mandshurica.

Under normal growth conditions, the activity of SOD, POD, CAT enzymes in an OE plant, an RNAi plant and a CK plant has no obvious difference; under drought stress treatment, the SOD, POD, CAT enzymatic activity of OE plants was significantly higher than that of RNAi and CK plants, with the RNAi plants having the lowest SOD, POD, CAT enzymatic activity.

The result shows that the over-expression FmMYB gene in the fraxinus mandshurica can enhance the SOD, POD, CAT enzyme activity under drought stress, enhance the capability of the fraxinus mandshurica in scavenging active oxygen free radicals generated in the fraxinus mandshurica seedling cells, and further improve the drought tolerance of the fraxinus mandshurica.

The results show that the expression system based on the agrobacterium-mediated hydrologic yeast Liu Shunshi established in the laboratory can effectively and truly reflect the stress-resistance regulation and control of the stress-resistance gene on the hydrologic yeast Liu Zhizhu.

The invention establishes a water yeast Liu Shunshi transformation system based on agrobacterium mediation: the optimal agrobacterium tumefaciens bacteria solution concentration and optimal infection time (OD=0.8, the infection time is 8 h) are obtained by using the growth point of the sterile seedlings of the fraxinus mandshurica as an explant through a transient transformation method. Based on the initially established water yeast Liu Shunshi transformation system with optimal agrobacterium concentration and time combination, azac, DTT, AS with different concentrations is sequentially added to establish a high-efficiency water yeast Liu Shunshi transformation system. The GUS enzyme activity and GUS gene fluorescence quantification show that: azac (30. Mu.g/L), DTT (200 mg/L), AS (150. Mu.M/L) were added to maximize the GUS enzyme activity and GUS gene fluorescence quantification of the aquatic yeast Liu Tina.

The agrobacterium-mediated-based water yeast Liu Shunshi transformation system provided by the invention can obtain the transient transformed seedlings of the fraxinus mandshurica in 3 days, has a short transformation period, and has high transformation efficiency of transient infection, which reaches about 95%. Compared with the existing water yeast Liu Shunshi transformation technology, which has a transformation period of more than 7 days and a transient infection transformation rate of 77.58%, the transformation period of the water yeast Liu Shunshi transformation system based on agrobacterium mediation, which is established by the invention, is short and the transformation efficiency is high.

According to the invention, drought-resistant physiological index analysis is performed on the FmMYB gene transient expression plant based on an agrobacterium-mediated water yeast Liu Shunshi transformation system, and FmMYB is identified to have obvious drought-resistant capacity. The results of this study further demonstrate the reliability of the agrobacterium-mediated-based transformation system of water yeast Liu Shunshi established in the present invention.

It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A transformation system based on agrobacterium-mediated water yeast Liu Shunshi is characterized in that 5-azacytidine, dithiothreitol and acetosyringone are added into agrobacterium liquid to obtain agrobacterium-mediated water yeast infection liquid, water yeast Liu Mojun seedlings are used as explants, and instant transformed seedlings are obtained through instant infection of the agrobacterium-mediated water yeast infection liquid, so that the transformation system based on agrobacterium-mediated water yeast Liu Shunshi is formed.

2. The agrobacterium-mediated-based water yeast Liu Shunshi transformation system of claim 1, wherein the concentration of agrobacterium in the agrobacterium solution is OD = 0.4-1.0.

3. The transformation system based on agrobacterium-mediated hydrological yeast Liu Shunshi according to claim 1, wherein the time of infection is 2-10 h.

4. The transformation system based on agrobacterium-mediated water yeast Liu Shunshi according to claim 1, wherein the addition amount of 5-azacytidine is 10-50 μg/L based on the volume of agrobacterium liquid; the addition amount of dithiothreitol is 100-500 mg/L; the addition amount of acetosyringone is 50-250 mg/L.

5. The method for establishing a transformation system based on agrobacterium-mediated water yeast Liu Shunshi according to claim 1, comprising the steps of:

Preparation of agrobacterium infection solution: preparing agrobacterium liquid containing target genes until the concentration of the bacterial liquid reaches OD=0.4-1.0, and adding 5-azacytidine, dithiothreitol and acetosyringone into the agrobacterium liquid to prepare agrobacterium infection liquid;

6. The method for establishing a transformation system based on agrobacterium-mediated hydrological Liu Shunshi as claimed in claim 5, wherein the first co-cultivation of the tissue culture seedlings of fraxinus mandshurica into the agrobacterium invasion solution is carried out for 1-1.5 h; adding 1/2 volume of fresh conversion solution into the agrobacteria invasion solution for secondary co-culture for 1.3-1.7 h; and further culturing the roots of the tissue culture seedlings of the fraxinus mandshurica under a dark condition to obtain the fraxinus mandshurica Liu Shunshi transformed seedlings.

7. The method for establishing a transformation system based on agrobacterium-mediated hydrologic Liu Shunshi according to claim 6, wherein the roots of the tissue culture seedlings of the fraxinus mandshurica after the secondary co-culture are inserted on a 1/2WPM solid medium, and are subjected to dark culture at 25-26 ℃ for 24-48 hours, so as to obtain the transformed seedlings of the hydrologic flexure Liu Shunshi.

8. The method for establishing a transformation system based on agrobacterium-mediated water yeast Liu Shunshi according to claim 7, wherein the 1/2WPM solid medium comprises the following components in percentage by weight: 28-32 g/L of sucrose, 2.16-2.18 g/L of MS powder and 7.4-7.6 g/L of agar.

9. The method for establishing a transformation system based on agrobacterium-mediated water yeast Liu Shunshi as claimed in claim 6, wherein the transformation solution is 2% sucrose.

10. The method for establishing a transformation system based on agrobacterium-mediated hydrologic Liu Shunshi according to claim 6, wherein the first co-culture conditions of the tissue culture seedlings of fraxinus mandshurica are: conditions of 25-26 ℃ and 85-95 rpm; the conditions for the secondary co-culture were: 25-26 ℃ and 85-95 rpm.