Method for improving agrobacterium tumefaciens-mediated instantaneous conversion efficiency

Abstract

The invention discloses a method for improving agrobacterium tumefaciens-mediated instantaneous conversion efficiency, and belongs to the technical field of biology. The method comprises the following steps: (1) cloning a NahG gene for coding salicylate hydroxylase and a P19 protein coding gene into a plant expression vector to construct a double-infection factor vector; (2) cloning a target protein coding gene segment to be verified into an expression vector to construct a target protein expression vector; and (3) respectively transforming the double-infection factor vector and the target protein expression vector into agrobacterium cells, mixing in an infection solution, and infecting target plant tissues for transient expression. The invention provides a combination with extremely high agrobacterium-assisted infection factors, a vector with double infection factors is constructed, and efficient gene transformation and gene function research in various plant bodies are realized by utilizing the vector. The invention provides a new feasible scheme for realizing efficient agrobacterium instantaneous transformation in different plant bodies.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for improving the efficiency of plant Agrobacterium-mediated transient transformation and an application thereof in plants. Background Art

[0002] Plant gene function research usually involves one or more transgenic methods to express or silence specific target genes, but the transformation and regeneration experiments of most higher plants are time-consuming and require a relatively high amount of materials. It still takes several months to produce sufficient transgenic plants that can be used for research and analysis (Horsch et al., 1985). In addition, due to gene silencing or differences in the location of genes in chromatin, the level of transgenic expression may vary from transgenic plant to transgenic plant (Vaucheret et al., 1998), so multiple transgenic individuals need to be obtained to reliably analyze the actual functional effects of genes. Transient transformation technology can quickly and efficiently realize plant genetic engineering. By taking advantage of the fragility of plant cell walls, exogenous target genes can be introduced into plant cells in a short period of time to achieve gene transfer and transient expression. As a rapid gene transfer technology, plant transient transformation technology is suitable for most cellular studies such as protein function.

[0003] At present, plant transient transformation technology has been widely used in many plant species, including Arabidopsis, petunia, tobacco, etc. (Krenek et al. 2015). In terms of application technology, the most widely used transient transformation technology is Agrobacterium-mediated genetic transformation. There are usually three methods for Agrobacterium-mediated transient transformation, Agrobacterium infiltration, cell co-culture and Agrobacterium infection, which have a wider range of applications and more convenient technical operations. Agrobacterium transient transformation of plant tissues (mostly leaves) is mainly used for subcellular localization, expression of exogenous genes, interaction between proteins, and interaction between transcription factors and cis-elements (Krenek et al. 2015).

[0004] However, Agrobacterium-mediated transient assays have certain limitations. To this day, researchers' understanding of the feasibility of Agrobacterium infection is still limited to specific species and tissues that are biologically compatible and physically accessible (Wixom et al. 2018). Many crops and horticultural crops still lack an effective Agrobacterium transient transformation technology system, mainly because two organisms are involved in the process of Agrobacterium infection. In general, the efficiency of transient exogenous gene expression assays is affected by variables such as the virulence of Agrobacterium strains and the physiological state of the plant. Some Agrobacterium strains are more toxic to specific plant species than other strains. Conversely, some plant species or genotypes are more sensitive to infection by specific Agrobacterium strains (Wroblewski et al. 2005). Relevant researchers speculate that this variation may be partly due to differences in the ability of bacteria to attach to plant cells or differences in the T-DNA transfer mechanism encoded by Agrobacterium (Lippincott et al. 1977, Nam et al. 1997).

[0005] The effects of Agrobacterium-mediated genetic transformation on different plants vary greatly between different plant species and tissues (Krenek et al. 2015). In addition to the infection ability of the strain itself and the possible differences in the growth state of the plants due to different growth periods, researchers believe that one of the main reasons for the low efficiency of Agrobacterium-mediated genetic transformation in some plants is that Agrobacterium infection has different activation levels of immune responses in different plant species. Plants detect and respond to attacks by exogenous microorganisms through two layers of innate immune systems: the pattern recognition receptors PRRs on the surface of plant cells recognize pathogen-associated molecular patterns PAMPs or damage-associated molecular patterns DAMPs, and promote the expression of immune defense genes based on signal transduction. This type of triggered first-layer immunity is called PTI response; in order to overcome the first-layer immune response of plant PTI, pathogens will secrete effector proteins to inhibit the immune response of plants while infecting plants. In order to overcome the secretion of effector proteins to inhibit plant immune responses and make plants susceptible to diseases, plants have evolved NLRs receptors that recognize potential pathogens to recognize effector proteins, and trigger plant-specific immune recognition by sensing the effector factors of pathogens. The second-layer immunity activated by the above effectors is called ETI response (Yu et al. 2024). In nature, different types of plants or different varieties of the same plant have certain specialized characteristics in disease resistance genes and their mediated immune effects. Therefore, in theory, researchers can also weaken the rejection of plants to Agrobacterium infection by destroying the first-layer immune response of plants or the effector protein function of reference pathogens to enhance the effect of exogenous gene expression.

[0006] For a long time, there has been a lack of systematic research and verification of the actual application effects and combination effects of gene transfer promoting factors derived from different pathogens and used in different plant research fields in different plant species. Therefore, screening and verifying the promoting effects and combination effects of the above factors on Agrobacterium-mediated transient transformation is expected to develop new technologies to improve the effect of Agrobacterium-mediated genetic transformation. Summary of the invention

[0007] The purpose of the present invention is to provide an infection factor combination that can improve the efficiency of Agrobacterium-mediated transient transformation, and to apply the combination to the transformation of exogenous genes in various crops and ornamental plants.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention provides a double infection factor vector, which is used to improve the efficiency of plant Agrobacterium-mediated transient transformation. The double infection factor vector is constructed by cloning the NahG gene encoding salicylate hydroxylase and the P19 protein encoding gene into a plant expression vector.

[0010] The dual infection factor vector provided by the present invention is transformed into plant tissue for expression. The salicylate hydroxylase encoded by the NahG gene can inhibit the accumulation of salicylic acid in the plant body, weaken the salicylic acid signaling pathway on which the plant immune system depends, thereby weakening the plant immune response and promoting the infection of plant tissue by Agrobacterium. The P19 protein is a protein encoded by the Tomatobushy stunt virus (TBSV). As an important means for the virus to counter plant RNAi, it can combine with siRNA in the plant body to prevent the formation of RNA induced silencing complex (RISC), which helps to promote the successful introduction of foreign genes.

[0011] Furthermore, the nucleotide sequence of the NahG gene is shown in SEQ ID NO.1; the nucleotide sequence of the P19 protein encoding gene is shown in SEQ ID NO.2.

[0012] Preferably, the NahG gene fragment and the P19 protein coding gene are connected by a P2A peptide coding sequence, and the nucleotide sequence of the P2A peptide coding sequence is shown in SEQ ID NO. 3. Specifically, the coding sequence of salicylate hydroxylase NahG and P19 protein connected by P2A peptide is shown in SEQ ID NO. 4.

[0013] Preferably, the promoter in the expression module of the dual infection factor vector is a 35S promoter.

[0014] Furthermore, the application includes: cloning the target protein encoding gene fragment to be verified into an expression vector to construct a target protein expression vector, and then using Agrobacterium-mediated transient transformation technology to co-transform the target protein expression vector and the double infection factor vector into plant tissue for expression, so as to improve the expression efficiency of the exogenous target protein.

[0015] In the present invention, the target protein expression vector and the dual infection factor vector are co-transformed into plant tissues for expression through Agrobacterium-mediated transformation. Under the joint action of the dual infection factors salicylate hydroxylase NahG and P19 protein, the Agrobacterium infection efficiency in plant tissues is significantly improved.

[0016] The dual infection factor vector provided by the present invention is used to realize gene transformation operations such as subcellular localization, protein interaction, protein expression level detection and transcriptional regulation in plants with low feasibility of Agrobacterium infection.

[0017] The plants may be, but are not limited to, model plants (such as tobacco), field crops (such as sunflower, cotton, mung bean), and ornamental plants (such as Phalaenopsis, Chinese rose, Rieger begonia, Desert rose, and tulip). The plant tissues may be, but are not limited to, plant leaves and plant petals; the target protein to be verified may be, but is not limited to, transcriptional regulatory factors.

[0018] The present invention also provides a method for improving the efficiency of plant Agrobacterium-mediated transient transformation, comprising the following steps:

[0019] (1) The NahG gene encoding salicylate hydroxylase and the P19 protein encoding gene were cloned into a plant expression vector to construct a dual infection factor vector;

[0020] (2) Cloning the target protein encoding gene fragment to be verified into an expression vector to construct a target protein expression vector;

[0021] (3) The dual infection factor vector and the target protein expression vector are transformed into Agrobacterium cells respectively, mixed in an infection solution, and then infect the target plant tissue for transient expression.

[0022] Preferably, the NahG gene fragment and the P19 protein encoding gene in the dual infection factor vector are connected by a P2A peptide encoding sequence, and the nucleotide sequence is shown in SEQ ID NO.4.

[0023] In the present invention, a suitable plant expression vector is selected according to the characteristics of the target plant to construct a double infection factor vector and a target protein expression vector. Preferably, in step (1), the original vector used to construct the double infection factor vector is a pCAMBIA1300 vector; in step (2), the original vector used to construct the target protein expression vector is a pCAMBIA1300 vector. The expression of the target gene is achieved by using a strong promoter, and the expression vector applicable to the present invention is not limited thereto.

[0024] Preferably, the vector is constructed by homologous recombination technology. Specifically, in step (1), NahG and P19 genes are connected by P2A peptide, and the NahG-2A-P19 gene fragment is obtained by PCR amplification using Overlap PCR technology, and then the double infection factor gene fragment is inserted into the multiple cloning site of the pCAMBIA1300 vector. In step (2), the target protein encoding gene to be verified is inserted into the multiple cloning site of the pCAMBIA1300 vector using homologous recombination technology.

[0025] In the present invention, appropriate plant tissues are selected according to the characteristics of the target plant for Agrobacterium-mediated transient transformation. Preferably, the target plant tissue is a plant leaf or a plant petal.

[0026] Preferably, the plant is tobacco, Phalaenopsis, rose, Rieger begonia, desert rose, sunflower, cotton, mung bean, tulip. The recipient plants applicable to the present invention are not limited to the above 9 plants.

[0027] In the present invention, the appropriate instantaneous transformation concentration of the double infection factor Agrobacterium bacterial solution and the static treatment time after the target plant is infected are selected according to the characteristics of the target plant.

[0028] Preferably, the Agrobacterium strain used is EHA105.

[0029] Preferably, the infection solution comprises: 1 / 4 MS salts, 30 g / L sucrose, 100 mM acetosyringone (As), and 0.005% silicone surfactant (Silwet L-77).

[0030] Preferably, in step (3), the concentration of Agrobacterium containing the dual infection factor carrier in the mixed infection solution is OD 600 The concentration of Agrobacterium containing the target protein expression vector is OD 600 The results show that the instantaneous conversion efficiency is significantly improved under the above concentration conditions.

[0031] Preferably, the method further comprises: using the TransGene Stacking II system for multi-gene assembly to integrate the HispS gene, CPH gene, H3H gene, NPGA gene, and Luz gene into a receptor vector to construct a plant body self-luminescent reporter system vector based on the fungal bioluminescence pathway; then transforming the plant body self-luminescent reporter system vector into Agrobacterium cells, mixing it with Agrobacterium containing a double infection factor vector in an infection solution, and then infecting the target plant tissue for transient expression, and indicating the Agrobacterium infection effect by observing the self-luminescence of the transformed area.

[0032] Preferably, after the injection of Agrobacterium is completed, the cells are cultured at room temperature for 48 hours and then fluorescent imaging is performed using a plant imaging system to calculate photons.

[0033] When constructing a dual infection factor vector, the present invention uses a plant self-luminescence reporter system to indicate the conversion efficiency of exogenous genes to verify the effect of the dual infection factor vector. The proteases encoded by the HispS gene, H3H gene, NPGA gene, CPH gene, and Luz gene in the plant self-luminescence reporter system utilize caffeic acid in the plant body to participate in the plant self-luminescence closed-loop pathway cycle. Caffeic acid is catalyzed by hispidin synthase (HispS) to form hispidin. Hispidin is catalyzed by hispidin-3-hydroxylase (H3H) to undergo a hydroxylation reaction under the condition of consuming NAD(P)H and oxygen to generate 3-hydroxyl hispidin, i.e., fungal luciferin. 3-hydroxyl hispidin is used as a FBP bioluminescent substrate. Under the catalysis of fungal luciferase Luz, fungal luciferin is oxidized to gradually form a high-energy intermediate in a singlet electron excited state, releasing 520nm green fluorescence and CO2, and finally converting fungal luciferin into caffeoylpyruvic acid. Caffey pyruvate hydrolase (CPH) converts caffey pyruvate into caffeic acid, realizing the biological self-luminescence cycle. The protein encoded by the NPGA gene from Aspergillus nidulans can increase the activity of the polyketide synthase family HispS, thereby increasing the synthesis rate of milk tree alkaloids. The present invention uses the above five genes to form a new generation of plant self-luminescence reporter system, which can effectively indicate the conversion effect of exogenous genes through the luminescence intensity of plant tissues as an exogenous reporter system for plants.

[0034] In the feasibility verification stage of Agrobacterium infection, the present invention, based on the above-mentioned self-luminous reporter system FBP, uses Agrobacterium-mediated plant transient transformation technology in different plants and different tissues, and injects the double infection factor vector and the reporter system into plant tissues together. An empty vector without infection factor is used as a blank treatment group. The two detect the feasibility of Agrobacterium infection of target plants and plant tissues based on luminescence intensity detection and real-time fluorescence quantitative PCR technology, verifying that the double infection factor vector has a promoting effect on the efficiency of Agrobacterium transient transformation in different plant species.

[0035] Another object of the present invention is to use the above-mentioned plant body self-luminescence reporter system to quickly screen plant species that can be infected by Agrobacterium and verify the actual effectiveness of different Agrobacterium auxiliary infection factors.

[0036] The present invention has the following beneficial effects:

[0037] The present invention uses the fungal bioluminescence pathway as a new generation of plant reporter system for the first time, and obtains an infection factor combination with extremely high Agrobacterium infection auxiliary effect through a simple and efficient gene transformation verification process. The NahG gene encoding salicylate hydroxylase and the P19 protein encoding gene are cloned into a plant expression vector to construct a double infection factor vector, and the plant body transient transformation operation is performed in multiple species. Combined with real-time fluorescence quantitative technology RT-qPCR and subcellular localization, protein interaction, and Western blot technology, the Agrobacterium infection effect of different plant species is auxiliaryly verified, and efficient gene transformation and gene function research in multiple plant bodies are realized. The present invention provides a new feasible solution for realizing efficient Agrobacterium transient transformation in different plant bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the principle of validating the efficacy of the combination of NahG and P19 dual infectious factors based on the fungal luminescence system.

[0039] Figure 2 Schematic diagram of the NahG single infection factor vector.

[0040] Figure 3 Schematic diagram of the P19 single infectious factor vector.

[0041] Figure 4 Schematic diagram of the NahG-2A-P19 dual infectious factor vector.

[0042] Figure 5 Schematic diagram of the multi-gene vector pYLTAC380MF-G4.

[0043] Figure 6 This is a schematic diagram of the multi-gene vector pYLTAC380MF-G5.

[0044] Figure 7 This is the result of co-transfection of P19 with the luminescent reporter system. The left picture is a plant fluorescence imaging picture, and the right picture is a light quantum statistics chart.

[0045] Figure 8 These are the results of co-transfection of NahG and the luminescent reporter system. The left picture is a plant fluorescence imaging image, and the right picture is a light quantum statistics graph.

[0046] Fig. 9 This is the result of detecting the expression levels of NahG and P19 proteins.

[0047] Fig.10 This is the result of the P19 Agrobacterium injection concentration screening. The left picture is a plant fluorescence imaging picture, and the right picture is a light quantum statistics chart.

[0048] Fig.11 This is the result of co-transfection of the dual infection factor NP19 and the luminescent reporter system. The left picture is a plant fluorescence imaging picture, and the right picture is a light quantum statistics chart.

[0049] Fig.12 This is the result of detecting the transcription levels of H3H and Luz.

[0050] Fig.13 The figure shows the effect of Agrobacterium transient transformation promotion of dual infection factors in multiple plants. (a) is Phalaenopsis, (b) is Desert Rose, (c) is Begonia, (d) is Tulip, (e) is Rose, (f) is Sunflower, (g) is Cotton, and (h) is Mung Bean.

[0051] Fig.14 For cotton GhbHLH121A T -Schematic diagram of eGFP vector.

[0052] Fig.15 For cotton cLuc-GhbHLH121D T Schematic diagram of the vector.

[0053] Fig.16 Schematic diagram of cotton PYE-nLuc vector.

[0054] Fig.17 Schematic diagram of cotton ZmABP9 vector.

[0055] Fig.18 This is the result of observation of subcellular localization in cotton leaves.

[0056] Fig.19 This is a diagram showing the interaction results between the three types of cotton leaf proteins.

[0057] Fig. 20 This is a diagram showing the results of transient transcriptional regulation in leaves of three types of cotton.

[0058] Fig.21 Schematic diagram of the sunflower HaHB10 vector.

[0059] Fig. 22 This is the result of observation of subcellular localization of sunflower petals.

[0060] Fig.23 This is the result of detecting protein expression level in sunflower petals.

[0061] Fig.24 This is the result of transient transcriptional regulation of sunflower petals. DETAILED DESCRIPTION

[0062] The technical scheme of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, the modification or replacement of the inventive method, steps or conditions, all belong to the scope of the present invention.

[0063] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0064] pYL322d1, pYL322d2 and pYLTAC380GW were donated by Professor Liu Yaoguang's laboratory at South China Agricultural University. The construction method can be found in the Chinese patent application number 2017103841977. pCAMBIA1300-cLuc-GhbHLH121D T , pCAMBIA1300-GhPYE-nLuc vector and GhbHLH121A T The coding gene fragment was donated by Professor Zhang Tianzhen's laboratory at Zhejiang University. The D53-mCherry vector was donated by Researcher Chen Tao's laboratory at Hangzhou Normal University. For the specific composition of the vector, please refer to the article published by Cai et al. in Mol Plant in 2021, doi: 10.1016 / j.molp.2020.11.013. The ZmABP9 coding gene fragment was donated by the research group of Zhao Jun, Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0065] Example 1: Creation of plant self-luminescence reporter system FBP, infection factor vector and protein expression vector to be verified

[0066] The present embodiment provides a method for improving the effect of Agrobacterium-mediated transient transformation in plants, including: verifying the promoting effect of P19 infection factor based on the plant self-luminescence reporter system FBP, verifying the promoting effect of NahG infection factor based on the plant self-luminescence reporter system FBP, screening of P19 Agrobacterium injection conditions, construction of a double infection factor vector NP19, and verifying the promoting effect of NP19 infection based on the plant self-luminescence reporter system FBP.

[0067] P19 binds to siRNA in plants to prevent the formation of RNA-induced silencing complexes, reducing the silencing probability of Agrobacterium-mediated exogenous gene introduction. However, the use of P19 in plants may cause damage to the normal growth and development of plant tissues. The accumulation of salicylic acid in plants may enhance the plant's immune response to a certain extent. NahG inhibits the accumulation of salicylic acid in plants and weakens the salicylic acid signaling pathway on which the plant immune system depends, thereby weakening the plant's immune response and promoting Agrobacterium's infection of plant tissues. The combination of P19 and NahG to form NP19 may further improve the efficiency of Agrobacterium's transient transformation on the basis of using the two types of infection factors alone.

[0068] Plant self-luminescent reporter system FBP: Caffeic acid is an intermediate compound in the production of lignin and other key metabolites by most plants. Its content is abundant and diverse in different plants. It is also the starting material for the expression of the FBP pathway. The caffeic acid synthesis pathway in plants is as follows: Figure 1 As shown. In theory, the FBP reporter system and auxiliary infection factors are co-transformed into the recipient plant using transient transformation technology, and the multi-gene vector expresses H3H, HispS, CPH, NPGA, and Luz. The endogenous caffeic acid in the plant is used as a raw material to transform and generate fungal luciferin: HispS converts the endogenous caffeic acid in the plant into galactose, which produces 3-hydroxygalactose under the catalysis of H3H; 3-hydroxygalactose is used as a bioluminescent substrate of FBP, and under the catalysis of fungal luciferase Luz, it releases 520nm green fluorescence and CO2, and finally converts fungal luciferin into caffeoylpyruvate; caffeoylpyruvate hydrolase (CPH) converts caffeoylpyruvate into caffeic acid, realizing the supply cycle of the luminescent substrate caffeic acid in the plant; NPGA is used to increase the activity of the polyketide synthase family HispS, increase the synthesis rate of galactose, and enhance the basic luminescence intensity and stability of the reporter system.

[0069] The following is a detailed description of the construction method of the above expression vector:

[0070] 1. Construction process of infection factor vector

[0071] The NahG and P19 gene fragments were amplified by PCR technology, and the NahG gene fragment was inserted into the multiple cloning site of the pCAMBIA1300 vector backbone by homologous recombination technology to construct the NahG single infection factor expression vector; the P19 gene fragment was inserted into the multiple cloning site of the pCAMBIA1300 vector backbone by homologous recombination technology to construct the P19 single infection factor expression vector; the NahG encoding gene and the P19 encoding gene were connected by P2A peptide, and the NahG-2A-P19 gene fragment was inserted into the multiple cloning site of the pCAMBIA1300 vector backbone by homologous recombination technology to construct the NP19 double infection factor expression vector. When constructing the single infection factor expression vector, the YFP gene fragment was inserted after the P19 or NahG gene fragment, which can form a fusion protein with the upstream gene for antibody recognition in subsequent Western blot experiments to detect whether NahG and P19 are normally expressed in plant leaves.

[0072] The sequence information of P19 can be found in gene accession number: AJ288942.1; the sequence information of NahG can be found in gene accession number: M60055.1; the sequence information of P2A peptide can be found in gene accession number: QKV27547.1; the sequence information of YFP can be found in gene accession number: AAO48599.1.

[0073] The coding sequences of the above genes were codon-optimized and then the corresponding gene fragments were synthesized. Specifically, the NahG coding sequence was synthesized based on the NahG coding sequence in Pseudomonas putida, and the coding sequence was shown in SEQ ID NO.1; the P19 gene coding sequence was shown in SEQ ID NO.2; the P2A peptide gene coding sequence was shown in SEQ ID NO.3; the NahG-2A-P19 gene coding sequence was shown in SEQ ID NO.4, and the YFP gene coding sequence was shown in SEQ ID NO.5.

[0074] The coding sequence fragments of NahG, P19, and NahG-2A-P19 were amplified by PCR reaction, and YFP was connected with the NahG and P19 gene fragments by Overlap PCR (overlapping PCR) technology. All gene fragments were inserted into the pCAMBIA1300 vector backbone through the restriction sites XbaⅠ and SacⅠ. The above vector map is shown in Figure 2 , 3 , as shown in Figure 4.

[0075] 2. Construction process of multigene vectors pYLTAC380GW-G4 and pYLTAC380GW-G5

[0076] In this study, the TransGene Stacking II system was used for multi-gene assembly. The NPGA gene in the 35S-driven Aspergillus nidulans genome and the H3H gene, HispS gene, CPH gene, and Luz gene in the Neonothopanus nambi genome were integrated into the pYLTAC380GW plasmid, and finally the selection marker gene expression cassette element was connected through a one-step BP recombination reaction to form the multi-gene vector pYLTAC380MF-G4 for the blank control treatment of the plant self-luminescence reporter system and the multi-gene vector pYLTAC380MF-G5 for the plant self-luminescence reporter system. Compared with pYLTAC380MF-G4, pYLTAC380MF-G5 added the Luz gene to make its luminescence pathway complete and have the ability to release fluorescence after exogenous introduction into the target plant. YL322d1, pYL322d2, and pYLTAC380GW were donated by Professor Liu Yaoguang's laboratory at South China Agricultural University.

[0077] The sequence information of HispS can be found in gene accession number: QJQ48095.1; the sequence information of CPH can be found in gene accession number: QJQ48093.1; the sequence information of H3H can be found in gene accession number: QJQ48094.1; the sequence information of NPGA can be found in gene accession number: QJQ48097.1; the sequence information of Luz can be found in gene accession number: QJQ48096.1.

[0078] The coding sequence of the above-mentioned gene was codon optimized and the corresponding gene fragment was synthesized. For the specific coding sequence and construction method, please refer to the Chinese patent application number 202111228803.9.

[0079] The specific construction process of the multi-gene vectors pYLTAC380GW-G4 and pYLTAC380GW-G5 is as follows:

[0080] (1) Construction of donor vectors, pYL322d1-HispS, pYL322d2-CPH, pYL322d1-H3H, pYL322d2-NPGA, pYL322d1-Luz.

[0081] (2) The donor vector pYL322d1-HispS and the recipient vector pYLTAC380GW (at a ratio of 1:1 to 2:1) were mixed in the NS3529 competent cell for co-transfection. Heat shock was used with an ice bath for 30 min, heat shock for 90 s, and an ice bath for 2-3 min. The cells were revived in LB without antibiotics at 37°C, 200 rpm, for 2 h. The cells were then spread on LA plates containing kanamycin (Km, 25 mg / L) and chloramphenicol (Chl, 15 mg / L). After culturing at 37°C for 18 h, single clones were grown. All single clones were rinsed into tubes with ddH2O, and the mixed plasmids were extracted.

[0082] (3) Take about 50-100 ng of the mixed plasmid and digest it with 0.5 μL I-Sce I (NEB) in a 10 μL system for 4-5 hours, transform it into Escherichia coli NEB10-β (Bomade Biotechnology Co., Ltd.), spread it on LA plates containing kanamycin (Km, 25 mg / L), and culture it at 37°C for 15 hours. Pick a single clone, culture it in LB (containing 25 mg / L Km and 0.5 mM IPTG), and perform PCR identification on the bacterial liquid. Use Green Taq Mix to further extract the plasmid that can amplify bright bands. Take 200 ng of each and use 0.2 μL NotⅠ to digest it in a 20 μL reaction system for verification. Four bands appear, containing the target gene 6.2k bp, which is the required positive clone pYLTAC380GW-HispS.

[0083] (4) The donor vector pYL322d2-CPH and the acceptor vector pYLTAC380GW-HispS obtained in step (3) were mixed (at a ratio of 1:1 to 2:1) in a NS3529 competent cell for co-transfection, transformed according to method (2), and plated on LA plates containing kanamycin (Km, 25 mg / L) and ampicillin (Amp, 70 mg / L). After culturing at 37°C for 18 h, single clones were grown. All single clones were rinsed into a centrifuge tube with ddH2O, and the mixed plasmid was extracted.

[0084] (5) Take about 40-90 ng of the mixed plasmid and use 0.5 μL PI-Sce I (NEB) and 0.5 μL BSA to digest in a 10 u L system for 4-5 h. Then transform and verify according to the method in (3). Five bands appear, containing the target genes 1.7 k bp CPH and 6.2 k bp HispS, which is the positive clone pYLTAC380GW-HispS-CPH.

[0085] (6) Repeat steps (2) to (5), cross-use d1 and d2 donor vectors containing different target genes and co-transform the receptor vector constructed in the previous round to construct pYLTAC380GW-G4 and pYLTAC380GW-G5;

[0086] Finally, pYLTAC380GW-G4 or pYLTAC380GW-G5 (200 ng) and PYLMFH (100 ng) were mixed with 1 μL of 5×BP enzyme mixture in a 5 μL reaction at 25°C for 5 h. Then 1 μL of proteinase K solution was added to terminate the reaction at 37°C for 10 min. The cells were transferred to NEB10-β competent cells, and positive clones were selected for full plasmid sequencing analysis. The correct pYLTAC380MF-G4 and pYLTAC380MF-G5 vectors were selected for subsequent experiments. The vector map is shown in Figure 5 , 6 shown.

[0087] 3. Construction process of protein expression vector to be verified

[0088] The target protein encoding gene fragment was amplified by PCR technology and inserted into the multiple cloning site of the pCAMBIA1300 vector backbone by homologous recombination technology to construct a protein expression vector.

[0089] Example 2: Verification of the effect of promoting transient transformation of Agrobacterium tumefaciens by P19 and NahG based on the plant self-luminescence reporter system

[0090] In this example, pCAMBIA1300-P19, pCAMBIA1300-NahG, pCAMBIA1300-NahG-2A-P19, and pYLTAC380MF-G5 were transformed into EHA105 Agrobacterium competent cells, and pYLTAC380MF-G5 was used as a plant self-luminous reporter system and transformed into tobacco together with various infectious factor vectors. The actual instantaneous transformation promoting effect of the infectious factors was verified by detecting the luminescence intensity and gene transcription level of tobacco leaves.

[0091] The operation of verifying the effect of promoting the transient transformation of Agrobacterium tumefaciens by P19 and NahG in this example is as follows:

[0092] 1. Construct pCAMBIA1300-NahG according to the method of Example 1 (vector map as shown in Figure 2 As shown), pCAMBIA1300-P19 (vector map as shown Figure 3 As shown), pCAMBIA1300-NahG-2A-P19 (vector map as shown Figure 4 As shown), pYLTAC380GW-G5 (vector map as shown Figure 6 Then pCAMBIA1300-NahG, pCAMBIA1300-P19, pCAMBIA1300-NahG-2A-P19, pYLTAC380GW-G5, and pCAMBIA1300 empty vector were transformed into EHA105 Agrobacterium competent cells respectively.

[0093] 2. Activate Agrobacterium bacterial suspension containing pCAMBIA1300-P19, pCAMBIA1300-NahG, pCAMBIA1300-NahG-2A-P19, pYLTAC380GW-G5, and pCAMBIA1300 on LA+Kana+Rif plates respectively, at 28°C for 36 h, pick Agrobacterium colonies from each plate, resuspend the Agrobacterium cells in resuspension solution (containing 10 mM MgCl2, 100 mM As), and adjust the OD 600 = 0.8-1.0. Agrobacterium cells were collected by centrifugation, resuspended in infection solution (1 / 4MS salts, 30 g / L sucrose, 100 mM acetosyringone, 0.005% Silwet L-77, pH 5.8), and allowed to stand at room temperature for 1 hour.

[0094] 3. Verify the effect of P19 promotion based on the plant self-luminescence reporter system FBP. Specifically, set up the experimental group: pYLTAC380GW-G5+pCAMBIA1300-P19; control group: pYLTAC380GW-G5+pCAMBIA1300. The concentration of pYLTAC380GW-G5 and pCAMBIA1300 in the infection solution was OD 600 The concentration of pCAMBIA1300-P19 cells was OD 600 Adjust to 0.5, and mix the above bacterial infection solutions in equal proportions. Use Nicotiana benthamiana as the receptor material for instantaneous transformation. Use a 1mL sterile syringe without a needle to draw an equal amount of the required mixed bacterial solution. Select the 3rd to 5th complete leaves with good tobacco growth and flat leaf surfaces. Use the syringe needle to make a small hole on the back of the tobacco leaf. Inject the infection solution containing the mixed bacteria into the leaf through the syringe hole. After culturing at room temperature for 48 hours, use the LB985 plant living imaging system to shoot for 30 seconds and calculate the photons.

[0095] 4. The promoting effect of NahG was verified based on the plant self-luminescence reporter system FBP. Specifically, the experimental group was set up as pYLTAC380GW-G5+pCAMBIA1300-NahG; the control group was set up as pYLTAC380GW-G5+pCAMBIA1300. The concentration of pYLTAC380GW-G5, pCAMBIA1300-NahG, and pCAMBIA1300 in the infection solution was OD 600 The ratio of the above bacterial infection solutions was 0.8 to 1.0, and the above bacterial infection solutions were mixed in equal proportions. Nicotiana benthamiana was used as the receptor material for transient transformation. An equal amount of the required mixed bacterial solution was drawn with a 1 mL sterile syringe without the needle. The 3rd to 5th complete leaves with good tobacco growth and flat leaf surfaces were selected. A small hole was made on the back of the tobacco leaf with the needle of the syringe. The infection solution containing the mixed bacteria was injected into the leaf through the small hole of the syringe. After culturing at room temperature for 48 hours, the LB985 plant living imaging system was used to shoot for 30 seconds and calculate the photons.

[0096] 5. Detect the expression level of P19 and NahG proteins in tobacco leaves based on Western blot technology. Specifically, use a mortar and pestle to grind tobacco leaves transiently expressing infection factor proteins in liquid nitrogen. Mix each 0.1g homogenized leaf sample with 450μL 4×Protein SDS sample loading buffer (containing 250mM Tris-HCl, 8% SDS, 40% glycerol, 400mM DTT, 1% bromophenol blue, pH 6.8). Mix the mixture thoroughly and denature it at 95-100℃ for 10min.

[0097] The total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a PVDF membrane polyvinylidene fluoride membrane (polyvinylidene fluoride), and blocked with 5% skim milk powder. Subsequently, the PVDF membrane was incubated with GFP antibody and then incubated with the corresponding endogenous immunoglobulin (IgG) secondary antibody. The protein expression level was detected using ECL Western blotting detection reagent, and plant actin was used as an internal reference.

[0098] 6. Verify the injection concentration of P19 Agrobacterium tumefaciens based on the plant self-luminescence reporter system FBP. Specifically, set up experimental group 1: pYLTAC380GW-G5+pCAMBIA1300-P19 (OD 600 Experimental group 2: pYLTAC380GW-G5+pCAMBIA1300-P19 (OD600 Experimental group 3: pYLTAC380GW-G5+pCAMBIA1300-P19 (OD 600 The concentration of pYLTAC380GW-G5 and pCAMBIA1300 in the infection solution was OD 600 is 0.8-1.0. The above bacterial infection solutions were mixed in equal proportions. Nicotiana benthamiana was used as the receptor material for transient transformation. An equal amount of the required mixed bacterial solution was drawn into a 1 mL sterile syringe without the needle. The 3rd to 5th complete leaves with good tobacco growth and flat leaf surfaces were selected. A small hole was made on the back of the tobacco leaf with the needle of the syringe. The infection solution containing the mixed bacteria was injected into the leaf through the small hole of the syringe. After culturing at room temperature for 48 hours, the LB985 plant living imaging system was used to shoot for 30 seconds and calculate the photons.

[0099] 7. Based on the plant self-luminescence reporter system FBP, the promotion effect of NahG-2A-P19 (NP19) was verified. Specifically, experimental group 1 was set up: pYLTAC380GW-G5+pCAMBIA1300-P19 (OD 600 adjusted to 0.5); Experimental group 2: pYLTAC380GW-G5+pCAMBIA1300-NahG; Experimental group 3: pYLTAC380GW-G5+pCAMBIA1300-NahG-2A-P19 (OD 600 Experimental group 4: pYLTAC380GW-G5+pCAMBIA1300-NahG-2A-P19 (OD 600 Experimental group 5: pYLTAC380GW-G5+pCAMBIA1300-NahG-2A-P19 (OD 600 The concentration of pYLTAC380GW-G5, pCAMBIA1300-NahG, and pCAMBIA1300 in the infection solution was OD 600is 0.8-1.0. The above bacterial infection solutions were mixed in equal proportions. Nicotiana benthamiana was used as the receptor material for transient transformation. An equal amount of the required mixed bacterial solution was drawn into a 1 mL sterile syringe without the needle. The 3rd to 5th complete leaves with good tobacco growth and flat leaf surfaces were selected. A small hole was made on the back of the tobacco leaf with the needle of the syringe. The infection solution containing the mixed bacteria was injected into the leaf through the small hole of the syringe. After culturing at room temperature for 48 hours, the LB985 plant living imaging system was used to shoot for 30 seconds and calculate the photons.

[0100] 8. The transcription-promoting effect of the NahG-2A-P19 (NP19) double infection factor combination was verified based on RT-qPCR technology. The tobacco leaves after Agrobacterium-mediated transient transformation were quickly frozen and ground in liquid nitrogen, and then RNA was extracted using VeZol reagent. The extracted RNA was synthesized into the first-strand cDNA using MonScriptTM RTIII Super Mix with dsDNase. RT-qPCR detection was performed using a LightCycler480 IIPCR instrument, and the transcription level of the target gene was measured by using SYBR Premix and gene-specific primers. NtEF1a was used as a reference gene for tobacco RT-qPCR.

[0101] 9. Results Analysis

[0102] P19 and the luminescent reporter system were injected into tobacco leaves through Agrobacterium-mediated transient transformation. Figure 7 As shown, the light intensity of the experimental group with added infection factor P19 was about twice that of the control group, and the luminescence intensity was significantly higher than that of the control group, confirming that P19 has the functional effect of promoting the transient transformation of Agrobacterium.

[0103] NahG and the luminescent reporter system were injected into tobacco leaves through Agrobacterium-mediated transient transformation. Figure 8 As shown, the light intensity of the experimental group with the infection factor NahG added was about 4 times that of the control group, and the luminescence intensity was significantly higher than that of the control group, confirming that NahG also has the functional effect of promoting the transient transformation of Agrobacterium.

[0104] The results of verifying the expression levels of P19 and NahG proteins in tobacco leaves are as follows Fig. 9 As shown, in the control group without added infection factors, no protein bands were observed, while clear protein bands were observed in the experimental group, indicating that the two types of infection factor proteins can be normally expressed in the leaves.

[0105] The use of P19 in plant tissues will have a certain impact on the normal growth and development of plant tissues. The verification results of the appropriate concentration of Agrobacterium for P19 injection are as follows: Fig.10 As shown in the figure, under the three injection concentration gradients, the luminescence intensity of the experimental group was higher than that of the control group, indicating that under the three injection concentrations, P19 could improve the transient transformation effect of Agrobacterium to a certain extent. 600 The final injection concentration was 0.5 (OD 600 Under the condition of 0.25), the effect of promoting Agrobacterium transient transformation by using P19 alone was the best, and the luminescence intensity of the experimental group was significantly higher than that of the control group, indicating that under this condition, the damage caused by P19 to plant tissues and its own promotion of gene transformation were in a good balance.

[0106] NahG-2A-P19 and the luminescent reporter system were injected into tobacco leaves through Agrobacterium-mediated transient transformation. Fig.11 As shown in the figure, among all the injection combinations, the luminescence intensity of the NahG-2A-P19 double infection factor combination was significantly higher than that of the single infection factor and the control group, indicating that the double infection factor combination has a stronger ability to enhance the effect of Agrobacterium-mediated transient transformation. Among them, pCAMBIA1300-NahG-2A-P19 had an OD of 1.37 before premixing the bacterial solution. 600 0.2 and 0.5 (final injection concentration OD 600 The enhancement effect of the luminescence intensity was similar under the injection conditions of OD 0.1 and 0.25. 600 The final injection concentration is 1.0 (OD 600 The enhancement effect of the luminescence intensity under the injection condition of OD 0.5 was lower than that of the final injection concentration OD 600 There are two injection conditions of 0.1 and 0.25.

[0107] The results of gene transcription level detection of 6 injection combinations are as follows Fig.12 As shown in the figure, the NahG-2A-P19 dual infection factor combination can increase the transcription level of exogenous genes in plant tissues at three injection concentrations. 600 The enhancement effect was strongest under the injection condition of 0.25. The above results show that the NahG-2A-P19 dual infection factor combination can greatly enhance the effect of Agrobacterium transient transformation, so as to realize more gene function research in plants.

[0108] Example 3: Feasibility verification of transient transformation of multiple plants by Agrobacterium based on plant self-luminescence reporter system and dual infection factor combination NP19

[0109] The operation of this example to verify the effectiveness of the dual infection factor combination in transient transformation of multiple plants by Agrobacterium is as follows:

[0110] 1. Construct pCAMBIA1300-NahG-2A-P19 according to the method of Example 1 (vector map as shown in Figure 4 As shown), pYLTAC380GW-G4 (vector map as shown Figure 5 As shown), pYLTAC380GW-G5 (vector map as shown Figure 6 Then pCAMBIA1300-NahG-2A-P19, pYLTAC380GW-G4, pYLTAC380GW-G5, and pCAMBIA1300 empty vector were transformed into EHA105 Agrobacterium competent cells respectively.

[0111] 2. According to the method of Example 2, Agrobacterium containing pCAMBIA1300-NahG-2A-P19, pYLTAC380GW-G4, pYLTAC380GW-G5, and pCAMBIA1300 were streaked on LA+Kana+Rif plates for activation at 28°C for 36 h. Agrobacterium colonies were picked from each plate and resuspended in a resuspension solution (containing 10 mM MgCl2, 100 mM As) to adjust the OD 600 = 0.8-1.0. Agrobacterium cells were collected by centrifugation, resuspended in infection solution (1 / 4MS salts, 30 g / L sucrose, 100 mM acetosyringone, 0.005% Silwet L-77, pH 5.8), and allowed to stand at room temperature for 1 hour.

[0112] Specifically, experimental group 1: pYLTAC380GW-G5+pCAMBIA1300; experimental group 2: pYLTAC380GW-G5+pCAMBIA1300-NahG-2A-P19; control group: pYLTAC380GW-G4+pCAMBIA1300. The bacterial concentrations of pYLTAC380GW-G5 and pCAMBIA1300 in the infection solution were OD 600 The concentration of pCAMBIA1300-NahG-2A-P19 cells was OD 0.8-1.0. 600 Adjust to 0.5, and mix the above bacterial infection solutions in equal proportions.

[0113] 3. Phalaenopsis aphrodite, Adenium obesum, Begonia×hiemalis, Tulipa×gesneriana, Gossypium hirsutum, Vigna radiata, Rosa hybrida, and Helianthus annuus were used as receptor materials for transient transformation. An equal amount of the required mixed bacterial solution was drawn with a 1 mL sterile syringe without a needle. Leaves and petals of the above plants with good growth and smooth surfaces were selected. A small hole was made on the back of the tissue with a syringe. The infection solution containing the mixed bacteria was injected into the target tissue through the small hole. After culturing at room temperature for 48 hours, the LB985 plant in vivo imaging system was used to shoot for 1-5 minutes and calculate the photons. Specifically, Phalaenopsis petals, Adenium obesum leaves, Begonia×hiemalis petals, Tulipa×gesneriana petals, cotton leaves, mung bean leaves, Rosa hybrida petals, and sunflower petals were selected as the target plant tissues for Agrobacterium injection.

[0114] 4. Using VrActin, GhUBQ7, HaEF1a, Ao5Srrna, TgUBQ10, PaUbi, BhACT2, and RhUBC as reference genes for various plants, the NahG-2A-P19 (NP19) double infection factor combination was verified for its transcriptional promotion effect according to the method of Example 2. The target plant tissue after Agrobacterium-mediated transient transformation was rapidly frozen and ground in liquid nitrogen, and then RNA was extracted using VeZol reagent. The first-chain cDNA was synthesized using MonScriptTM RTIII Super Mix with dsDNase. RT-qPCR was performed using a LightCycler480 IIPCR instrument, and the transcription level of the target gene was measured by using SYBR Premix and gene-specific primers.

[0115] 5. Results Analysis

[0116] The results are as follows Fig.13As shown in the figure, NahG, P19 and the luminescent reporter system were injected into different plant tissues through Agrobacterium-mediated transient transformation in Phalaenopsis, Desert Rose, Rieger Begonia, Tulip, Cotton, Mung Bean, Rose and Sunflower. There was no luminescence phenomenon in the control group, and the luminescence intensity of the experimental group with double infection factors was significantly higher than that of the experimental group without infection factors. From the luminescence phenomenon, the simultaneous addition of NahG and P19 can effectively expand the luminescence area of ​​the plant injection tissue and increase the luminescence intensity of the plant injection tissue. In addition, from the results of the transcription level detection of H3H and Luz genes, except for the H3H gene of Phalaenopsis, the transcription levels of the genes of other plant species were significantly increased under the condition of adding NahG and P19. The above results show that the transient transformation effect of the exogenous luminescent reporter system in 8 plant tissues is significantly improved under the promotion of Agrobacterium infection by the double infection factors, which further shows that the combination of NahG and P19 double infection factors can improve the infection efficiency of Agrobacterium transient transformation.

[0117] Example 4: Development of cotton leaf transient transformation technology based on the dual infection factor combination NP19

[0118] The operation of this embodiment to create the Agrobacterium transient transformation technology in cotton leaves is as follows:

[0119] 1. Construct pCAMBIA1300-NahG-2A-P19 according to the method of Example 1 (vector map as shown in Figure 4 As shown), pCAMBIA1300-GhbHLH121A T -eGFP (vector map as Fig.14 As shown), pCAMBIA1300-ZmABP9 (vector map as shown Fig.17 Then the above vector and pCAMBIA1300-cLuc-GhbHLH121D T (Carrier map such as Fig.15 As shown), pCAMBIA1300-GhPYE-nLuc (vector map as shown Fig.16 The pCAMBIA1300 vectors were transformed into EHA105 Agrobacterium competent cells.

[0120] The sequence information of eGFP can be found in gene accession number: PP378488.1; GhbHLH121A T For sequence information, see gene accession number: XM_041103027.1; GhbHLH121D TThe sequence information of eGFP can be found in gene accession number: XM_016835912.2; the sequence information of GhPYE can be found in gene accession number: XM_041080577.1; the sequence information of ZmABP9 can be found in gene accession number: GU237073.1. Specifically, the eGFP gene coding sequence is shown in SEQ ID NO.6; GhbHLH121-A T The gene coding sequence is shown in SEQ ID NO.7; GhbHLH121D T The gene coding sequence is shown in SEQ ID NO.8; the GhPYE gene coding sequence is shown in SEQ ID NO.9; and the ZmABP9 gene coding sequence is shown in SEQ ID NO.10.

[0121] Specifically, pCAMBIA1300-cLuc-GhbHLH121D T (Carrier map such as Fig.15 As shown), pCAMBIA1300-GhPYE-nLuc vector (vector map as shown Fig.16 shown) and GhbHLH121A T The gene fragment was donated by Professor Zhang Tianzhen's research group at Zhejiang University, and the ZmABP9 encoding gene fragment was donated by Professor Zhao Jun's research group at the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. T -eGFP (vector map as Fig.14 ) and pCAMBIA1300-ZmABP9 (vector map as shown Fig.17 The construction method of the invention comprises: obtaining GhbHLH121A by PCR amplification T and ZmABP9 encoding gene fragments, and overlap PCR was used to clone eGFP and GhbHLH121A T The coding gene fragments were connected to form GhbHLH121A T -eGFP gene coding fragment, and the above two gene fragments were respectively inserted into the pCAMBIA1300 vector backbone through the restriction sites XbaⅠ and SacⅠ.

[0122] 2. Place pCAMBIA1300-NahG-2A-P19, pCAMBIA1300-GhbHLH121A T -eGFP, pCAMBIA1300-ZmABP9, pCAMBIA1300-cLuc-GhbHLH121D TAgrobacterium cultures of pCAMBIA1300, pCAMBIA1300-GhPYE-nLuc, and pCAMBIA1300 were streaked on LA+Kana+Rif plates for activation at 28°C for 36 h. Agrobacterium colonies were picked from each plate and resuspended in resuspension solution (containing 10 mM MgCl2, 100 mM As). The OD 600 = 0.8-1.0. Agrobacterium cells were collected by centrifugation, resuspended in infection solution (1 / 4MS salts, 30 g / L sucrose, 100 mMacetosyringone, 0.005% Silwet L-77, pH 5.8), and allowed to stand at room temperature for 1 hour.

[0123] 3. Verify the feasibility of Agrobacterium infection of cotton varieties and plant tissues based on the combination of NP19 double infection factors. Specifically, set up the experimental group: pCAMBIA1300-GhbHLH121A T -eGFP+pCAMBIA1300-NahG-2A-P19. In the infection medium, pCAMBIA1300-GhbHLH121A T -eGFP bacterial concentration OD 600 The concentration of pCAMBIA1300-NahG-2A-P19 cells was OD 0.8-1.0. 600 Adjust to 0.5, and mix the above bacterial infection solutions in equal proportions. Use cotton varieties 3-79, Jinmian 668 and H7124 as receptor materials for transient transformation. Use a 1mL sterile syringe without a needle to draw an equal amount of the required mixed bacterial solution. Select the first complete leaf with good cotton growth and flat leaf surface. Use the syringe needle to make a small hole on the back of the cotton leaf. Inject the infection solution containing the mixed bacteria into the leaf through the syringe hole. After culturing at room temperature for 48 hours, use a Zeiss laser confocal microscope LSM880 to observe the intracellular protein fluorescence signal.

[0124] 4. Feasibility verification of protein interaction in cotton leaves based on NP19 combination. Specifically, set up the experimental group: pCAMBIA1300-cLuc-GhbHLH121D T +pCAMBIA1300-GhPYE-nLuc+pCAMBIA1300-NahG-2A-P19; Control group 1: pCAMBIA1300-NahG-2A-P19+pCAMBIA1300-cLuc-GhbHLH121D T+pCAMBIA1300-nLuc; control group 2: pCAMBIA1300-NahG-2A-P19+pCAMBIA1300-cLuc+pCAMBIA1300-GhPYE-nLuc. In the infection solution, pCAMBIA1300-cLuc-GhbHLH121D T 、pCAMBIA1300-GhPYE-nLuc、pCAMBIA1300-nLuc、pCAMBIA1300-cLuc bacterial concentration OD 600 The concentration of pCAMBIA1300-NahG-2A-P19 cells was OD 0.8-1.0. 600 Adjust to 0.5, and mix the above bacterial infection solutions in equal proportions. Similarly, cotton varieties 3-79, Jinmian 668 and H7124 were used as receptor materials for transient transformation. An equal amount of the required mixed bacterial solution was drawn with a 1mL sterile syringe without a needle. The first complete leaf with good cotton growth and flat leaf surface was selected. A small hole was made on the back of the cotton leaf with a syringe needle, and the infection solution containing the mixed bacteria was injected into the leaf through the syringe hole. After culturing at room temperature for 48 hours, the luciferase complementary fluorescence signal was observed using the Tianneng 5200 fully automatic chemiluminescence image analyzer.

[0125] 5. The feasibility of transient transcriptional regulation in cotton leaves based on NP19 combination was verified. Specifically, the experimental group was set up as pCAMBIA1300-ZmABP9-NOS+pCAMBIA1300-NahG-2A-P19. The control group was set up as pCAMBIA1300+pCAMBIA1300-NahG-2A-P19. The OD value of pCAMBIA1300-ZmABP9-NOS and pCAMBIA1300 in the infection solution was 0.04477 μg / mL. 600 The concentration of pCAMBIA1300-NahG-2A-P19 cells was OD 0.8-1.0. 600 Adjust to 0.5, and mix the above bacterial infection solutions in equal proportions. Similarly, cotton varieties 3-79, Jinmian 668 and H7124 were used as receptor materials for transient transformation. An equal amount of the required mixed bacterial solution was drawn with a 1mL sterile syringe without a needle. The first complete leaf with good cotton growth and flat leaf surface was selected. A small hole was made on the back of the cotton leaf with a syringe needle. The infection solution containing the mixed bacteria was injected into the leaf through the syringe hole. The cotton leaves after Agrobacterium-mediated transient transformation were sampled after culturing at room temperature for 48 hours.

[0126] 6. According to the method of Example 2, the transcriptional level promotion effect of the downstream genes of the cotton leaves injected with ZmABP9 transcription factor was verified based on RT-qPCR technology. According to previous studies, ZmABP9 transcription factor can cause the expression of endogenous GhERF1 and GhZFP1 genes in cotton to be upregulated (Wang et al. 2017). The cotton leaves after Agrobacterium-mediated transient transformation were quickly frozen and ground in liquid nitrogen, and then RNA was extracted using VeZol reagent. The extracted RNA was synthesized into the first-strand cDNA using MonScriptTM RTIII Super Mix with dsDNase. RT-qPCR detection was performed using LightCycler480 IIPCR instrument, and the transcription level of the target gene was measured by using SYBR Premix and gene-specific primers. GhUBQ7 was used as the tobacco RT-qPCR reference gene.

[0127] 7. Results Analysis

[0128] The results of Agrobacterium-mediated subcellular localization observation in the leaves of three varieties of cotton are as follows Fig.18 As shown, after adding the double infection factor combination, a clear intracellular protein fluorescence signal can be observed in the first complete leaf of cotton varieties 3-79, Jinmian 668 and H7124, confirming that the protein is expressed normally in the cotton leaf body after the exogenous gene is introduced.

[0129] The results of the observation of Agrobacterium-mediated luciferase complementary protein interaction in the leaves of three cotton species are as follows Fig.19 As shown, on the first leaf after injection of the three types of cotton, there was no obvious fluorescence signal in the two control groups, while obvious luciferase complementary signals appeared in the experimental group. This shows that by applying a combination of double infection factors, the interaction of the cotton endogenous interaction protein combination can be verified on the cotton leaf body, and an interaction signal with a good visualization effect can be obtained.

[0130] The results of Agrobacterium-mediated transient transcriptional regulation detection in leaves of three cotton species are shown in Figure 2. Fig. 20 As shown, when the double infection factor combination was applied, the exogenous introduction of ZmABP9 significantly led to the upregulation of GhZFP1 gene expression in the three cotton varieties. For the GhERF1 gene, Jinmian 668 and 3-79 showed expression inhibition, while H7124 showed upregulation, indicating that the NP19 double infection factor can be used to quickly study gene function in cotton leaves.

[0131] Example 5: Development of a sunflower petal transient transformation technology based on the dual infection factor combination NP19

[0132] The operation of this embodiment to create the Agrobacterium transient transformation technology in the sunflower petals is as follows:

[0133] 1. Construct pCAMBIA1300-NahG according to the method of Example 1 (vector map as shown in Figure 2 As shown), pCAMBIA1300-NahG-2A-P19 (vector map as shown Figure 4 As shown), pCAMBIA1300-HaHB10-NOS (vector map as shown Fig.21 ). The sequence information of HaHB10 is shown in gene accession number: L48485.1. Specifically, the coding sequence of HaHB10 gene is shown in SEQ ID NO.11. Specifically, HaHB10 coding gene fragments were amplified from sunflower petals by PCR reaction, and the above two gene fragments were inserted into the pCAMBIA1300 vector backbone through restriction sites XbaⅠ and SacⅠ. The D53-mCherry vector was donated by the laboratory of Researcher Chen Tao of Hangzhou Normal University.

[0134] Then pCAMBIA1300-NahG-2A-P19, pCAMBIA1300-HaHB10-NOS, D53-mCherry and pCAMBIA1300 were transformed into EHA105 Agrobacterium competent cells, respectively.

[0135] 2. Activate the Agrobacterium bacterial suspensions of pCAMBIA1300-NahG, pCAMBIA1300-HaHB10-NOS, pCAMBIA1300, D53-mCherry, and pCAMBIA1300-NahG-2A-P19 by streaking on LA+Kana+Rif plates at 28°C for 36 h. Pick Agrobacterium colonies from each plate and resuspend the Agrobacterium bacterial suspensions in resuspension solution (containing 10 mM MgCl2, 100 mM As). Adjust the OD 600 = 0.8-1.0. Agrobacterium cells were collected by centrifugation, resuspended in infection solution (1 / 4MS salts, 30 g / L sucrose, 100 mM acetosyringone, 0.005% Silwet L-77, pH 5.8), and allowed to stand at room temperature for 1 hour.

[0136] 3. The feasibility of Agrobacterium infection of sunflower petals was verified based on the combination of NP19 double infection factors. Specifically, the experimental group was set up: D53-mCherry+pCAMBIA1300-NahG-2A-P19. The concentration of D53-mCherry cells in the infection solution was OD 600 The concentration of pCAMBIA1300-NahG-2A-P19 cells was OD 0.8-1.0. 600Adjust to 0.5, and mix the above bacterial infection solutions in equal proportions. Use sunflower petals as the receptor material for transient transformation, use a 1mL sterile syringe without the needle to draw an equal amount of the required mixed bacterial solution, select sunflower petals with good growth conditions and flat petals, use the syringe needle to make a small hole on the back of the petals, and inject the infection solution containing the mixed bacteria into the petals through the syringe hole. After culturing at room temperature for 48 hours, use a Zeiss laser confocal microscope LSM880 to observe the intracellular protein fluorescence signal.

[0137] 4. The expression level of NahG protein in sunflower petals was detected based on Western blot technology according to the method of Example 2. Specifically, an experimental group was set up: pCAMBIA1300-NahG + pCAMBIA1300-NahG-2A-P19; a control group was set up: pCAMBIA1300 + pCAMBIA1300-NahG-2A-P19. The concentration of pCAMBIA1300-NahG cells in the infection solution was OD 600 0.8~1.0. Transient transformation was performed using sunflower petals as receptor materials. An equal amount of bacterial solution was drawn with a 1mL sterile syringe without a needle. A small hole was made on the back of the petal with a needle of the syringe. The infection solution containing mixed bacteria was injected into the petal through the small hole of the syringe. The petals were cultured at room temperature for 48 hours before sampling. Sunflower petals transiently expressing NahG-YFP protein were ground in liquid nitrogen using a mortar and pestle. Each 0.1g homogenized leaf sample was mixed with 450μL 4×Protein SDS sample loading buffer (containing 250mM Tris-HCl, 8% SDS, 40% glycerol, 400mM DTT, 1% bromophenol blue, pH 6.8). The mixture was thoroughly mixed and denatured at 95-100℃ for 10min. The total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a PVDF membrane polyvinylidene fluoride membrane (polyvinylidene fluoride), and blocked with 5% skim milk powder. Subsequently, the PVDF membrane was incubated with GFP antibody and then incubated with the corresponding endogenous immunoglobulin (IgG) secondary antibody. The protein expression level was detected using ECL Western blotting detection reagent, and plant actin was used as an internal reference.

[0138] 5. According to the method of Example 2, the transcriptional promotion effect of the downstream genes of the sunflower petals injected with the HaHB10 transcription factor was verified based on the RT-qPCR technology. According to previous studies, HaHB10 will lead to upregulation of the expression of the endogenous HaFT and HaSEP3 genes of the sunflower and inhibition of the expression of the HaLOX2 gene (Dezar et al. 2011). The petals after Agrobacterium-mediated transient transformation were quickly frozen and ground in liquid nitrogen, and then RNA was extracted using VeZol reagent. The extracted RNA was used to synthesize the first-chain cDNA using MonScriptTM RTIII Super Mix with dsDNase. RT-qPCR detection was performed using a LightCycler480IIPCR instrument, and the transcription level of the target gene was measured by using SYBR Premix and gene-specific primers. HaEF1a was used as the reference gene for tobacco RT-qPCR.

[0139] 6. Results Analysis

[0140] The results of Agrobacterium-mediated subcellular localization observation in sunflower petals are as follows Fig. 22 As shown, after adding the double infection factor combination, a clear nuclear localization fluorescence signal of mCherry can be observed in the sunflower petals, confirming that the protein is expressed normally in the sunflower petals after the introduction of the exogenous gene.

[0141] The results of the detection of NahG protein expression levels in sunflower petals are as follows Fig.23 As shown, NahG protein bands can be detected in petals injected with NahG encoding gene, indicating that normal expression of heterologous proteins in sunflower petals can be achieved through Agrobacterium-mediated transient transformation technology based on the combination of double infection factors.

[0142] The results of Agrobacterium-mediated transient transcriptional regulation detection in sunflower petals are as follows Fig.24 As shown, when the double infection factor combination was applied, the exogenous introduction of HaHB10 significantly led to the upregulation of HaFT and HaSEP3 gene expression in sunflower petals, while the HaLOX2 gene was downregulated, indicating that the NP19 double infection factor can be used to quickly study gene function in sunflower petals.

Claims

1. Application of a dual infection factor vector in improving the efficiency of plant Agrobacterium-mediated transient transformation, characterized in that: The double infection factor vector is constructed by cloning the NahG gene encoding salicylate hydroxylase and the P19 protein encoding gene into a plant expression vector.

2. The use according to claim 1, characterized in that The nucleotide sequence of the NahG gene is shown in SEQ ID NO.1; the nucleotide sequence of the P19 protein encoding gene is shown in SEQ ID NO.

2.

3. The use according to claim 1 or 2, characterized in that: The NahG gene and the P19 protein encoding gene fragment are connected by a P2A peptide encoding sequence, and the nucleotide sequence of the P2A peptide encoding sequence is shown in SEQ ID NO.

3.

4. The use according to claim 1, characterized in that The promoter in the expression module of the dual infection factor vector is the 35S promoter.

5. The use according to claim 1, characterized in that The application includes: cloning the target protein encoding gene fragment to be verified into an expression vector to construct a target protein expression vector, and then using Agrobacterium-mediated transient transformation technology to transform the target protein expression vector and the double infection factor vector into plant tissue for expression, so as to improve the expression efficiency of the exogenous target protein.

6. A method for improving the efficiency of plant Agrobacterium-mediated transient transformation, characterized in that: The following steps are involved: (1) The NahG gene encoding salicylate hydroxylase and the P19 protein encoding gene were cloned into a plant expression vector to construct a dual infection factor vector; (2) Cloning the target protein encoding gene fragment to be verified into an expression vector to construct a target protein expression vector; (3) The dual infection factor vector and the target protein expression vector are transformed into Agrobacterium cells respectively, mixed in an infection solution, and then infect the target plant tissue for transient expression.

7. The method for improving the efficiency of plant Agrobacterium-mediated transient transformation according to claim 6, characterized in that: The NahG gene fragment and the P19 protein coding gene in the dual infection factor vector are connected by a P2A peptide coding sequence, and the nucleotide sequence is shown in SEQ ID NO.

4.

8. The method for improving the efficiency of plant Agrobacterium-mediated transient transformation according to claim 6, characterized in that: In step (1), the original vector used to construct the double infection factor vector is the pCAMBIA1300 vector; in step (2), the original vector used to construct the target protein expression vector is the pCAMBIA1300 vector; in step (3), the concentration of Agrobacterium containing the double infection factor vector in the infection solution after mixing is OD 600 The concentration of Agrobacterium containing the target protein expression vector is OD 600 It is 0.4~0.

5.

9. The method for improving the efficiency of plant Agrobacterium-mediated transient transformation according to claim 6, characterized in that: In step (3), the target plant tissue is plant leaves or plant petals.

10. The method for improving the efficiency of plant Agrobacterium-mediated transient transformation according to claim 6, characterized in that: The method also includes: using the TransGene Stacking II system for multi-gene assembly to integrate the HispS gene, the CPH gene, the H3H gene, the NPGA gene, and the Luz gene into a receptor vector to construct a plant body self-luminescence reporter system vector based on the fungal bioluminescence pathway; then transforming the plant body self-luminescence reporter system vector into Agrobacterium cells, mixing it with Agrobacterium containing a double infection factor vector in an infection solution, and then infecting the target plant tissue for transient expression, and indicating the Agrobacterium infection effect by observing the self-luminescence of the transformed area.

Images