Genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in slash pine

Abstract

The invention discloses a genetic transformation method of a gene SPX4 capable of efficiently utilizing nitrogen and phosphorus in slash pines, and relates to the technical field of forest tree genetic engineering.The method comprises the following steps that 1, embryogenic calluses are obtained; (2) preparing an agrobacterium infection solution; (3) genetic transformation; (4) screening of resistant calluses; and (5) identifying the transgenic embryogenic callus. According to the method, a stable transformation genetic system of slash pines is established for the first time, slash pines embryogenic calluses are re-suspended and then infected with a GV3101 agrobacterium solution containing a plant binary expression vector PBI121-GFP-SPX4 for 20 min, co-culture is conducted for 2 d, calluses with kanamycin resistance are obtained through screening culture, then it is confirmed through PCR detection that the exogenous SPX4 gene is successfully transferred into slash pines genome DNA, and the slash pines with kanamycin resistance can be obtained. And a theoretical foundation is laid for improved variety cultivation of slash pine with efficient nitrogen and phosphorus utilization.

Description

Technical Field

[0001] The invention relates to the technical field of forestry gene engineering, and in particular to a genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii pine. Background Art

[0002] Longleaf pine is an important timber, resin and ecological tree species in the southern region. It has good economic and ecological benefits. Due to its rapid growth, high resin yield and strong adaptability, it has rapidly developed into a high-quality resin-producing tree species that is promoted preferentially in the southern region. However, in the cultivation and growth process of longleaf pine, the problem of low nitrogen and phosphorus utilization efficiency is currently faced. In the red soil area in the south, the nitrogen and phosphorus content in the soil is relatively low and the effectiveness is not high, which is difficult to meet the needs of longleaf pine growth. At the same time, since nutrients are easily lost due to factors such as rainfall and irrigation, the utilization rate of nutrients is further reduced. Traditional fertilization methods are not only costly, but may also have a negative impact on the soil environment and cannot achieve sustainable development. Therefore, screening and cultivating new varieties of longleaf pine with efficient nitrogen and phosphorus utilization has great practical value and economic significance.

[0003] With the continuous development of biotechnology, genetic transformation technology has provided a new way to improve the efficient use of nitrogen and phosphorus in slash pine. By introducing specific genes into slash pine, its ability to absorb, transport and utilize nitrogen and phosphorus nutrients can be enhanced, thereby improving growth rate and quality. Agrobacterium-mediated genetic transformation has become the preferred method in transgenic strategies due to its low cost, simple technology, stable expression of exogenous genes, and low incidence of gene silencing.

[0004] The action mechanism of SPX4 transcription factor mainly includes regulating the expression of transport proteins on the cell membrane of plant roots, enhancing the absorption capacity of nitrogen and phosphorus ions in the soil; promoting the transport and distribution of nitrogen and phosphorus in the plant body, ensuring that nutrients are preferentially supplied to parts with vigorous growth and active metabolism; it may also participate in regulating the metabolic pathways of plants, optimizing the utilization of nitrogen and phosphorus in the body, and improving their utilization rate in biosynthesis and energy metabolism. In recent years, the introduction of SPX4 genes to improve the efficient utilization of nitrogen and phosphorus in plants has been successful in Arabidopsis, rice and other plants, but the research on the transformation of slash pine using SPX4 genes has not been reported. The main reasons are that the embryonic callus regeneration of slash pine is slow, it is sensitive to antibiotics, and the transformation system is imperfect. These factors directly affect the transformation success rate of slash pine. Therefore, the development of a genetic transformation technology for efficient utilization of nitrogen and phosphorus suitable for slash pine has important practical significance and broad application prospects. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and propose a genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliot pine by systematically and deeply studying the genetic transformation conditions of elliot pine.

[0006] The present invention is achieved by the following technical solution, which comprises the following steps:

[0007] The present invention provides a genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii pine, comprising the following steps:

[0008] (1) Obtaining embryonic callus

[0009] Using immature seeds of Pinus elliottii as materials, embryonic callus was first induced on an induction medium, and then the embryonic callus was inoculated into a proliferation medium for proliferation culture.

[0010] (2) Preparation of Agrobacterium infection solution

[0011] Constructing a recombinant plasmid carrying the target gene SPX4 and transforming it into Agrobacterium competent cells, taking the Agrobacterium bacterial liquid containing the recombinant plasmid verified by enzyme digestion and resuspending it to obtain the Agrobacterium infection liquid for the next step of genetic transformation;

[0012] (3) Genetic transformation

[0013] Select the embryonic callus tissue that has been proliferated and cultured in step (1) and perform a light-proof suspension pre-culture, then infect the embryonic callus tissue with the Agrobacterium infection solution prepared in step (2), dry the bacterial solution on the surface of the embryonic callus tissue, transfer it to a co-culture medium, and perform co-cultivation in the dark;

[0014] (4) Screening of resistant callus

[0015] The embryonic callus tissue after co-cultivation in step (3) is sterilized and then placed on a screening medium containing kanamycin for resistance screening;

[0016] (5) Identification of transgenic embryonic callus

[0017] After the embryonic callus with kanamycin resistance screened in step (4) is stably proliferated, DNA is extracted and primers are designed. PCR amplification is performed using the DNA of the resistant callus as a template. The PCR identification result shows that the target band can be amplified. At the same time, RNA is extracted and reversed into cDNA. qRT-PCR expression is performed using the cDNA of the resistant callus as a template. The resistant callus is highly expressed, indicating that the target gene SPX4 is successfully transferred into the embryonic callus of Pinus elliottii.

[0018] As a further optimization scheme of the present invention, in step (1), the formula of the induction medium is MLP, 1.0 mg·L -16-BA, 1.0mg·L -1 KT, 1.0mg·L -1 NAA, 0.048mg·L -1 BR, 5.0mg·L -1 ABA, 450mg·L - 1 L-glutamine, 500mg·L -1 Inositol, 500mg·L -1 Hydrolyzed casein, 30g·L -1 Sucrose and 4.0g·L -1 Gellan gum.

[0019] As a further optimization scheme of the present invention, in step (1), the formula of the proliferation medium is MLP, 0.25 mg·L -1 6-BA, 0.25mg·L -1 KT, 4.00mg·L -1 NAA, 450mg·L -1 L-glutamine, 500mg·L -1 Inositol, 500mg·L -1 Hydrolyzed casein, 30g·L -1 Maltose and 4.0g·L -1 Gellan gum.

[0020] As a further optimization scheme of the present invention, the recombinant plasmid carrying the target gene SPX4 described in step (2) is constructed by the following method:

[0021] (2-1) SPX4 gene cloning

[0022] Primers of SPX4 gene were designed, and cDNA of Pinus elliottii was used as template to synthesize cDNA open reading frame ORF of SPX4 gene by RT-PCR amplification. The target product fragment of RT-PCR was recovered and purified by agarose gel electrophoresis. The target product fragment was connected with Blunt-Zero vector and transformed into competent Escherichia coli DH5α. Resistance screening was performed by LB+50mg / L kanamycin. The culture conditions were 37℃ for 16h. White clones were picked and inoculated into LB medium for liquid culture. The culture conditions were 37℃ for 16h. Blunt-Zero vector plasmid containing SPX4 gene fragment was extracted.

[0023] (2-2) Construction of PBI121-GFP-SPX4 plant binary expression vector

[0024] The PBI121-GFP vector was linearized using Xba I and BamH I restriction endonucleases, and the target product fragment recovered in step (2-1) was connected using seamless cloning technology and transformed into Escherichia coli DH5α competent cells. Positive clones were picked and inoculated into LB medium for liquid culture at 37°C for 16 h. The plasmid was extracted and the PBI121-GFP-SPX4 recombinant plasmid was obtained by single clone sequencing verification.

[0025] As a further optimization scheme of the present invention, in step (2), the Agrobacterium infection solution is prepared by the following method: the Agrobacterium bacterial solution containing the PBI121-GFP-SPX4 recombinant plasmid is inoculated into a YEP liquid medium containing 50 mg / L of kanamycin and rifamycin, and inverted and cultured in the dark at 28°C, a single colony is picked, inoculated into 3 mL YEP liquid medium containing the same antibiotics, and shaken overnight, and then the culture solution is drawn at a ratio of 1:50 and placed in a new YEP liquid medium, and cultured for 6-8 hours until the OD 600 The cells were centrifuged and resuspended in liquid proliferation medium supplemented with 50-100 μM acetosyringone until the OD value of the infection medium was 0.6-0.8. 600 is 0.3 to obtain the Agrobacterium infection solution for later use.

[0026] As a further optimization scheme of the present invention, in step (3), the genetic transformation conditions are specifically as follows: temperature is 24°C ± 2°C, proliferation culture time is 14 days, pre-culture time is 7 days, Agrobacterium infection solution OD is 600 is 0.3, the infection time is 20 min, and the co-cultivation time under dark conditions is 2 days.

[0027] As a further optimization scheme of the present invention, in step (3), the formula of the co-culture medium is MLP, 0.25 mg·L -1 6-BA, 0.25mg·L -1 KT, 4.00mg·L -1 NAA, 450mg·L -1 L-glutamine, 500mg·L-1 inositol, 500mg·L-1 hydrolyzed casein, 30g·L -1 Maltose, 4.0 g·L -1 Gellan gum, 100 μM acetosyringone, 300 mg / L cephalosporin and 300 mg / L timentin.

[0028] As a further optimization scheme of the present invention, in step (4), the formula of the screening medium containing kanamycin is MLP, 0.25 mg·L -1 6-BA, 0.25mg·L -1 KT, 4.00mg·L -1NAA, 450mg·L -1 L-glutamine, 500mg·L -1 Inositol +500mg·L -1 Hydrolyzed casein, 30g·L -1 Maltose, 4.0 g·L -1 Gellan gum, 100 μM acetosyringone, 300 mg / L cephalosporin, 300 mg / L timentin and 60 mg / L kanamycin.

[0029] As a further optimization scheme of the present invention, in step (4), the degerming treatment is specifically as follows: the embryonic callus after co-cultivation is washed twice with sterile water containing 300 mg / L timentin and 300 mg / L cephalosporin antibiotics, and then the embryonic callus is filtered to remove water.

[0030] As a further optimization scheme of the present invention, in step (4), the embryonic callus is screened and cultured in the screening medium for three consecutive times, with a single screening and culture time of 20 days, a temperature of 24°C±2°C, and light-proof treatment.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] (1) The present invention provides a stable genetic transformation method based on the embryonic callus of slash pine, and at the same time conducts a systematic and in-depth study on the genetic transformation conditions of SPX4 gene transformation of slash pine, laying a theoretical foundation for the breeding of high-quality varieties of slash pine with efficient utilization of nitrogen and phosphorus.

[0033] (2) The present invention uses embryonic callus as transformation material, and the transformation efficiency is relatively high, up to 8.4 cells / g, which provides a good foundation for the subsequent acquisition of transgenic plants.

[0034] (3) The genetic transformation method provided by the present invention is simple to operate, has a short cycle, and has high repeatability, and provides a good research platform for functional gene verification, molecular design breeding, and new germplasm creation of slash pine. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the selected callus before transformation;

[0036] Figure 2 This is a schematic diagram of the selected callus tissue observed under a microscope before transformation;

[0037] Figure 3 Schematic diagram of the resistant callus grown on the screening medium;

[0038] Figure 4 This is a schematic diagram of the state of resistant callus after proliferation;

[0039] Figure 5 The figure is the electrophoresis detection diagram of resistant callus DNA (in the figure, Plasmid is the positive plasmid; CK is the untransformed slash pine callus; SPX4 (1-6) is the positive callus);

[0040] Figure 6 Schematic diagram of qRT-PCR detection results of resistant callus (in the figure, CK is untransformed Elliot pine callus, SPX4(1-6) is positive callus). DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Technicians in this field can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0042] 1. Reagents and Materials

[0043] Unless otherwise specified, the reagents and materials used below are commercially available.

[0044] The composition and standard formula of MLP medium are: KNO3 909.9mg / L, Ca(NO3)2·4H2O 236.2mg / L, NH4NO3 200.0mg / L, KH2PO4 136.1mg / L, MgSO4·7H2O 246.5mg / L, Mg(NO3)2·6H2O 256.5mg / L, MgCl2·6H2O 101.7mg / L, H3BO3 15.5mg / L, ZnSO4·7H2O 14.668mg / L, MnSO4·H2O10.5mg / L, Na2MoO4·2H2O 0.125mg / L, KI 4.15mg / L, CuSO4·5H2O 0.1725mg / L, CoCl2·6H2O0.125mg / L, FeSO4·7H2O 13.93mg / L, EDTA-Na2 18.65mg / L, Glycine 2.0mg / L, Nicotinic acid 0.5mg / L, Thiamine HCl 1.0mg / L, Tocopherol 0.5mg / L;

[0045] The formula of YEP liquid medium: 10 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl;

[0046] The formula of YEP solid medium: 10g / L yeast extract, 10g / L peptone, 5g / L NaCl, 15g / L agar;

[0047] The formula of LB medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl;

[0048] Formula of proliferation medium: MLP, 0.25 mg·L -1 6-BA, 0.25mg·L -1 KT, 4.00mg·L -1 NAA, 450mg·L -1 L-glutamine, 500mg·L -1 Inositol, 500mg·L -1 Hydrolyzed casein, 30g·L -1 Maltose, 4.0 g·L -1 Gellan gum;

[0049] Formula of screening medium: MLP, 0.25 mg·L -1 6-BA, 0.25mg·L -1 KT, 4.00mg·L -1 NAA, 450mg·L -1 L-glutamine, 500mg·L -1 Inositol, 500mg·L -1 Hydrolyzed casein, 30g·L -1 Maltose, 4.0 g·L -1 Gellan gum, 100 μM acetosyringone, 300 mg / L cephalosporin, 300 mg / L timentin, 60 mg / L kanamycin;

[0050] Formula of induction medium: MLP, 1.0 mg·L -1 6-BA, 1.0mg·L -1 KT, 1.0mg·L -1 NAA, 0.048mg·L -1 BR, 5.0mg·L -1 ABA, 450mg·L -1 -Glutamine, 500mg·L -1 Inositol, 500mg·L -1 Hydrolyzed casein, 30g·L -1 Sucrose, 4.0 g·L -1 Gellan gum;

[0051] Formula of co-culture medium: MLP, 0.25 mg·L -1 6-BA, 0.25mg·L -1 KT, 4.00mg·L -1 NAA, 450mg·L -1 L-glutamine, 500mg·L-1 Inositol, 500mg·L -1 Hydrolyzed casein, 30g·L -1 Maltose, 4.0 g·L -1 Gellan gum, 100 μM acetosyringone, 300 mg / L cephalosporin, 300 mg / L timentin.

[0052] 2. Methods

[0053] Unless otherwise specified, the following methods are conventional methods known to those skilled in the art.

[0054] Example 1 Agrobacterium genetic transformation

[0055] 1.1. Kanamycin (Kan) concentration screening test

[0056] Embryonic callus of Pinus elliottii of uniform size was selected and inoculated into proliferation medium containing different concentrations of kanamycin, which were 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, and 80 mg / L. The culture was subcultured once every 14 days, and the results were observed and statistically analyzed after 42 days.

[0057] The results are shown in Table 1. At low concentrations, kanamycin had no obvious inhibitory effect on the callus of Pinus elliottii, and the proliferation rate was fast. When the concentration reached 40-60 mg / L, the periphery of the callus began to brown, and the proliferation rate was slow. As the concentration of kanamycin increased, the inhibitory effect was proportional to its concentration. When the concentration reached 80 mg / L, the growth was extremely slow, which might inhibit the growth of resistant callus. Therefore, the screening concentration of kanamycin should be 60 mg / L.

[0058] Table 1 Effect of kanamycin concentration on callus proliferation of Pinus elliottii

[0059]

[0060]

[0061] 1.2. Determination of optimal genetic transformation conditions

[0062] Using the embryonic callus of stably proliferating Pinus elliottii as the material, a single-factor experimental design was used to explore the effects of pre-culture time, infection solution OD value, infection time and co-culture time on transformation efficiency. The number of resistant callus obtained was counted in about 3 months, and the number of resistant callus was used as a standard to measure the efficiency of genetic transformation. The number of resistant callus (individuals / g) = total number of resistant callus / weight of infected callus (g). The pH of the culture medium used in the experiment was 5.8, the culture temperature was 24℃±2℃, and the culture was dark.

[0063] 1.2.1 Effects of different pre-culture times on genetic transformation efficiency of Pinus elliottii

[0064] The callus that proliferated on the proliferation medium for 14 days was clipped and transferred to the liquid proliferation medium. The callus was suspended and pre-cultured for 3, 5, 7, 10, 12, and 14 days, and then the callus was infected with Agrobacterium liquid with an OD value of 0.3 for 30 minutes. The callus was cultured for 2 days and then transferred to the screening medium. Screening was performed once every 20 days, and the transformation efficiency was statistically calculated after three consecutive screenings. The results showed that the transformation efficiency of callus pre-cultured for 7 days was the highest, reaching 7.00 / g. Therefore, the optimal time for pre-culture of slash pine callus before transformation is 7 days (Table 2).

[0065] Table 2 Effect of different pre-culture time on genetic transformation efficiency

[0066]

[0067] 1.2.2 Effect of Agrobacterium tumefaciens concentration on callus transformation of Pinus elliottii

[0068] The callus tissue that had been proliferating on the proliferation medium for 14 days was clipped and transferred to the liquid proliferation medium for pre-culture for 7 days. The concentration of the bacterial solution (OD 600 ) were 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6, respectively, and the embryonic callus of Pinus elliottii was infected for 30 minutes. The culture was carried out for 2 days and then transferred to the screening medium. The screening was performed once every 20 days, and the transformation efficiency was calculated after 3 consecutive screenings. The experimental results showed that the bacterial solution concentration (OD 600 ) was 0.3, the highest transformation efficiency was 7.40 cells / g (Table 3). Therefore, 0.3 was the best infection concentration for transformation of Pinus elliottii callus.

[0069] Table 3 Effects of different bacterial concentrations on genetic transformation efficiency

[0070]

[0071] 1.2.3 Effect of Agrobacterium infection time on callus transformation of Pinus elliottii

[0072] The callus tissue that had proliferated on the proliferation medium for 14 days was clipped and transferred to the liquid proliferation medium for pre-culture for 7 days. 600) was 0.3. The infection time was 10, 20, 30, 40, 50, 60 min, respectively. The culture was continued for 2 days and then transferred to the screening medium. The culture was screened once every 20 days. The transformation efficiency was calculated after 3 consecutive screenings. The experimental results showed that the transformation efficiency was the highest when the infection time was 20 min, which could reach 8.40 cells / g (Table 4). When the callus was inoculated with the bacterial solution for too short a time, the Agrobacterium did not fully contact the wound, and the growth of the Agrobacterium was slow during the co-culture, resulting in low transformation efficiency. When the infection time was too long, the Agrobacterium grew too fast, and the callus rotted and died due to the lack of oxygen caused by the poisoning of the Agrobacterium.

[0073] Table 4 Effects of different infection times on callus transformation of Pinus elliottii

[0074]

[0075] 1.2.4 Effect of co-cultivation time on callus transformation of Pinus elliottii

[0076] The callus tissue that had proliferated on the proliferation medium for 14 days was clipped and transferred to the liquid proliferation medium for pre-culture for 7 days. 600 ) was 0.3 to infect the embryonic callus of Pinus elliottii, and the infection time was 20 minutes, and the co-culture was 1, 2, 3, 4, 5, and 6 days, respectively, and then transferred to the screening medium. Screening was performed once every 20 days, and the transformation efficiency was statistically calculated after 3 consecutive screenings. The experimental results showed that the transformation efficiency was the highest when co-cultured for 2 days (Table 5). Co-cultivation is an important process for Agrobacterium to integrate its T-DNA carrying foreign genes into the cells of plants to achieve genetic transformation. If the co-cultivation time is too short, the efficiency of Agrobacterium infecting plant cells is low; if the co-cultivation time is too long, the callus will die due to excessive growth of Agrobacterium.

[0077] Table 5 Effect of co-cultivation time on callus transformation of Pinus elliottii

[0078]

[0079] In summary, the optimal genetic transformation conditions for Agrobacterium GV3101-SPX4 to transform Pinus elliottii callus were to proliferate the callus for 14 days on the proliferation medium, transfer the newly proliferated part to the liquid proliferation medium, pre-culture for 7 days, and use the concentration (OD 600 ) is 0.3 to infect the embryonic callus of Pinus elliottii, the infection time is 20 min, and the callus is co-cultured in the dark for 2 days. Then the callus is transferred to the screening medium for resistance screening, and the screening is performed every 20 days for 3 consecutive times at the temperature of 24℃±2℃. The callus with kanamycin resistance will grow in clumps.

[0080] Example 2 SPX4 Gene Transformation Genetic Transformation of Pinus elliottii

[0081] 2.1. Obtaining embryonic callus

[0082] Using immature seeds as materials, embryonic callus was induced on induction medium. After 60 days, the induced embryonic callus was inoculated into proliferation medium.

[0083] 2.2 Preparation of Agrobacterium infection solution

[0084] 2.2.1 Cloning and connection of SPX4 gene of Pinus elliottii

[0085] Using elliottii cDNA as a template, the forward primer SPX4-F described in SEQ ID No.1 and the reverse primer SPX4-R shown in SEQ ID No.2 were used to perform RT-PCR amplification to synthesize the cDNA open reading frame ORF of the SPX4 gene, and the RT-PCR target product fragment with a length of 1164 bp was recovered and purified by agarose gel electrophoresis, and then connected with the Blunt-Zero vector to transform the competent Escherichia coli DH5α, and LB+50 mg / L kanamycin was used for resistance screening. The culture conditions were 37°C for 16 hours, and the white clones were picked and inoculated into LB medium for liquid culture at 37°C for 16 hours. The Blunt-Zero vector plasmid containing the SPX4 gene fragment was extracted, and the sequence of the cDNA open reading frame ORF of the SPX4 gene was verified by sequencing as shown in SEQ ID No.3.

[0086] 2.2.2 Construction of PBI121-GFP-SPX4 plant binary expression vector

[0087] The PBI121-GFP vector was linearized with restriction endonucleases Xba I (Takara, 1093S) and BamH I (Takara, 1010S). The target fragment recovered by electrophoresis gel cutting was connected to the linearized vector using seamless cloning technology, and the competent E. coli DH5α was transformed. The positive clones were picked and inoculated in LB medium for liquid culture at 37°C for 16 hours. The plasmid was extracted and the PBI121-GFP-SPX4 recombinant plasmid was obtained by single clone sequencing verification. The Xba I forward primer is shown in SEQ ID No.4, and the BamH I reverse primer is shown in SEQ ID No.5.

[0088] 2.2.3. Agrobacterium transformation and identification of PBI121-GFP-SPX4 plant binary expression vector

[0089] The PBI121-GFP-SPX4 plant binary expression vector was transformed into the competent strain of Agrobacterium GV3101 by liquid nitrogen freezing method. The culture was carried out on a YEP solid medium containing 50 mg / L of Kan and Rif at 28°C for 48 hours. A single colony was picked and inoculated into a YEP liquid medium containing the same Kan and Rif antibiotics for overnight shaking at a speed of 200r / min. The plasmid was extracted using a plasmid extraction kit (Shenggong, B300872-0050) and sequenced for verification. It was verified that the Agrobacterium GV3101-SPX4 bacterial solution containing the PBI121-GFP-SPX4 plasmid can be used for the next callus infection. Glycerol with a final concentration of 20% (volume ratio) was added to the Agrobacterium GV3101-SPX4 bacterial solution and stored at -80°C for a long time. The Agrobacterium GV3101-SPX4 culture liquid stored at -80℃ was thawed at room temperature and then plated on YEP solid medium containing 50mg / L Kan and Rif at 28℃ for 48h. A single colony was picked and inoculated into 3mL LB liquid medium containing the same antibiotics and shaken overnight. The culture liquid was aspirated at a ratio of 1:50 and placed into new YEP liquid medium for 6-8h until OD 600 = 0.6-0.8, centrifuge, and resuspend the cells in liquid proliferation medium supplemented with 50-100 μM acetosyringone to an OD of 0. 600 =0.3, obtain Agrobacterium infection solution and set aside.

[0090] 2.3. Establishment of genetic transformation system

[0091] Select the callus tissue cultured in the proliferation medium for about 14 days in step 2.1 for suspension culture for about 7 days at a temperature of 24℃±2℃ and protected from light. The schematic diagram of the selected callus tissue before transformation is shown in the figure. Figure 1 The schematic diagram of observation under a microscope is shown in Figure 2 shown.

[0092] The callus was then infected with the Agrobacterium infection solution prepared in step 2.2 for 20 minutes. The callus surface solution was dried and transferred to the co-culture medium for co-cultivation at a temperature of 24°C ± 2°C for 2 days in the dark. Afterwards, sterilization was performed, and the co-cultured embryonic callus was washed twice with sterile water containing 300 mg / L timentin and 300 mg / L cephalosporin antibiotics. The sterilized embryonic callus was filtered and placed on a screening medium for resistance screening after removing the water. The screening culture was performed continuously in the screening medium for 3 times, each screening culture lasting 20 days at a temperature of 24°C ± 2°C and protected from light. The schematic diagram of the resistant callus grown on the screening medium is shown in FIG. Figure 3 shown.

[0093] 2.4. Obtaining transgenic embryonic callus

[0094] Six calli with kanamycin resistance obtained in step 2.3 were selected and stably proliferated for 60 days. The schematic diagram of the state of the resistant callus after proliferation is as follows: Figure 3 DNA was extracted using a DNA extraction kit (Tian Gen, DP360), and the forward primer 35S-F shown in SEQ ID No.6 and the reverse primer SPX4-R shown in SEQ ID No.2 were designed and synthesized. PCR amplification was performed using the DNA of the resistant plant as a template. The PCR results showed that the PCR identification results were able to amplify the target band, and all 6 callus tissues were positive ( Figure 5 ), indicating that the target gene SPX4 was successfully transferred into the embryonic callus of Pinus elliottii.

[0095] Subsequently, the expression of the target gene was verified by qRT-PCR in the six positive calli. The internal reference gene used in qRT-PCR was Actin, and the expression of the SPX4 gene in the callus was detected. The sequence of Actin-RT-F is shown in SEQ ID No.7, the sequence of Actin-RT-R is shown in SEQ ID No.8, the sequence of SPX4-RT-F is shown in SEQ ID No.9, and the sequence of SPX4-RT-R is shown in SEQ ID No.10. The results of qRT-PCR were also consistent with the expected results and the results of PCR amplification ( Figure 6 ).

[0096] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii pine, characterized in that: The following steps are involved: (1) Obtaining embryonic callus Using immature seeds of Pinus elliottii as materials, embryonic callus was first induced on an induction medium, and then the embryonic callus was inoculated into a proliferation medium for proliferation culture. (2) Preparation of Agrobacterium infection solution Constructing a recombinant plasmid carrying the target gene SPX4 and transforming it into Agrobacterium competent cells, taking the Agrobacterium bacterial liquid containing the recombinant plasmid verified by enzyme digestion and resuspending it to obtain the Agrobacterium infection liquid for the next step of genetic transformation; (3) Genetic transformation Select the embryonic callus tissue that has been proliferated and cultured in step (1) and perform a light-proof suspension pre-culture, then infect the embryonic callus tissue with the Agrobacterium infection solution prepared in step (2), dry the bacterial solution on the surface of the embryonic callus tissue, transfer it to a co-culture medium, and perform co-cultivation in the dark; (4) Screening of resistant callus The embryonic callus tissue after co-cultivation in step (3) is sterilized and then placed on a screening medium containing kanamycin for resistance screening; (5) Identification of transgenic embryonic callus After the embryonic callus with kanamycin resistance screened in step (4) is stably proliferated, DNA is extracted and primers are designed. PCR amplification is performed using the DNA of the resistant callus as a template. The PCR identification result shows that the target band can be amplified. At the same time, RNA is extracted and reversed into cDNA. qRT-PCR expression is performed using the cDNA of the resistant callus as a template. The resistant callus is highly expressed, indicating that the target gene SPX4 is successfully transferred into the embryonic callus of Pinus elliottii.

2. The genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii according to claim 1, characterized in that: In step (1), the formula of the induction medium is MLP, 1.0 mg·L -1 6-BA, 1.0mg·L -1 KT, 1.0mg·L -1 NAA, 0.048mg·L -1 BR, 5.0mg·L -1 ABA, 450mg·L -1 L-glutamine, 500mg·L -1 Inositol, 500mg·L -1 Hydrolyzed casein, 30g·L -1 Sucrose and 4.0g·L -1 Gellan gum.

3. The genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii according to claim 1, characterized in that: In step (1), the formula of the proliferation medium is MLP, 0.25 mg·L -1 6-BA, 0.25mg·L -1 KT, 4.00mg·L -1 NAA, 450mg·L -1 L-glutamine, 500mg·L -1 Inositol, 500mg·L -1 Hydrolyzed casein, 30g·L -1 Maltose and 4.0g·L -1 Gellan gum.

4. The genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii according to claim 1, characterized in that: The recombinant plasmid carrying the target gene SPX4 described in step (2) is constructed by the following method: (2-1) SPX4 gene cloning Primers of SPX4 gene were designed, and cDNA of Pinus elliottii was used as template to synthesize cDNA open reading frame ORF of SPX4 gene by RT-PCR amplification. The target product fragment of RT-PCR was recovered and purified by agarose gel electrophoresis. The target product fragment was connected with Blunt-Zero vector and transformed into competent Escherichia coli DH5α. Resistance screening was performed by LB+50mg / L kanamycin. The culture conditions were 37℃ for 16h. White clones were picked and inoculated into LB medium for liquid culture. The culture conditions were 37℃ for 16h. Blunt-Zero vector plasmid containing SPX4 gene fragment was extracted. (2-2) Construction of PBI121-GFP-SPX4 plant binary expression vector The PBI121-GFP vector was linearized using Xba I and BamH I restriction endonucleases. The target product fragment recovered in step (2-1) was connected to the linearized vector using seamless cloning technology and then transformed into Escherichia coli DH5α competent cells. Positive clones were picked and inoculated into LB medium for liquid culture at 37°C for 16 h. The plasmid was extracted and the PBI121-GFP-SPX4 recombinant plasmid was obtained by single clone sequencing verification.

5. The genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii according to claim 1, characterized in that: In step (2), the Agrobacterium infection solution is prepared by the following method: inoculating the Agrobacterium culture solution containing the PBI121-GFP-SPX4 recombinant plasmid into a YEP solid culture medium containing 50 mg / L of kanamycin and rifamycin, inverting and culturing in the dark at 28°C, picking a single colony, inoculating it into 3 mL of YEP liquid culture medium containing the same antibiotics, shaking the culture overnight, then aspirating the culture solution at a ratio of 1:50 and placing it into a new YEP liquid culture medium, and culturing for 6-8 hours until the OD 600 The cells were centrifuged and resuspended in liquid proliferation medium supplemented with 50-100 μM acetosyringone until the OD value of the infection medium was 0.6-0.

8. 600 is 0.3 to obtain the Agrobacterium infection solution for later use.

6. The genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii according to claim 1, characterized in that: In step (3), the genetic transformation conditions are as follows: pre-culture time is 7 days, Agrobacterium infection solution OD is 600 is 0.3, the infection time is 20 min, the co-cultivation time is 2 days under dark conditions, and the temperature is 24℃±2℃.

7. The genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii according to claim 1, characterized in that: In step (3), the formula of the co-culture medium is MLP, 0.25 mg·L -1 6-BA, 0.25mg·L -1 KT, 4.00mg·L -1 NAA, 450mg·L -1 L-glutamine, 500mg·L-1 inositol, 500mg·L-1 hydrolyzed casein, 30g·L -1 Maltose, 4.0 g·L -1 Gellan gum, 100 μM acetosyringone, 300 mg / L cephalosporin and 300 mg / L timentin.

8. The genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii according to claim 1, characterized in that: In step (4), the formula of the screening medium containing kanamycin is MLP, 0.25 mg·L -1 6-BA, 0.25mg·L - 1 KT, 4.00mg·L -1 NAA, 450mg·L -1 L-glutamine, 500mg·L -1 Inositol +500mg·L -1 Hydrolyzed casein, 30g·L -1 Maltose, 4.0 g·L -1 Gellan gum, 100 μM acetosyringone, 300 mg / L cephalosporin, 300 mg / L timentin and 60 mg / L kanamycin.

9. The genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii according to claim 1, characterized in that: In step (4), the degerming treatment is specifically as follows: the embryonic callus after co-cultivation is washed twice with sterile water containing 300 mg / L timentin and 300 mg / L cephalosporin antibiotics, and then the embryonic callus is filtered to remove water.

10. The genetic transformation method of nitrogen and phosphorus efficient utilization gene SPX4 in elliottii according to claim 1, characterized in that: In step (4), the embryonic callus is continuously screened and cultured in the screening medium for 3 times, with a single screening culture time of 20 days, a temperature of 24°C±2°C, and protected from light.

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