Novel agrobacterium vector system and use thereof

Abstract

Provided are an agrobacterium vector system and use thereof. The agrobacterium vector system comprises an expression vector and a helper vector. The expression vector comprises elements such as a nucleotide sequence of a replication origin pVS1oriV from a pVS1 replication system. The helper vector comprises elements such as a nucleotide sequence of a replication origin oriV from a pRK2 replication system. The provided agrobacterium vector system can maintain a high copy number in both E. coli and A. tumefaciens, exhibits very high plant transformation efficiency, features a very small backbone, and is easily and flexibly constructed, making it very suitable for library construction. Moreover, due to its small backbone and high construction efficiency, this agrobacterium vector system can be directly transformed into A. tumefaciens without the need for an E. coli transformation first, and the positive rate is high, thereby shortening the entire experimental cycle and reducing the workload.

Description

A novel Agrobacterium vector system and its applications Technical Field

[0001] This invention belongs to the field of plant genetic engineering and relates to a novel Agrobacterium vector system and its uses. Background Technology

[0002] An ideal plant transformation system should possess the following characteristics: high transformation efficiency, high transformation throughput, good uniformity, multiplex transformation capability (i.e., it is not necessary to construct all elements on the same vector), short transformation cycle, convenient operation, and good reproducibility. Currently commonly used plant transformation systems include: protoplast transformation, callus transformation, Agrobacterium infiltration, and gene gun transformation.

[0003] Protoplasts are plant cells with their cell walls removed. They can accept exogenous DNA fragments and achieve transformation using polyethylene glycol (PEG). The transformation efficiency of protoplasts can reach over 40%, and target gene expression can be observed as early as 12 hours post-transformation. However, protoplast preparation is relatively cumbersome, and the number of cells used for each transformation is small, approximately 10. 5 Up to 10 6 However, the stability of the culture is poor, and the culture time is generally limited to within 48 hours. Gene gun transformation refers to the process of bombarding plant explants (leaves, callus, embryos, etc.) with nanoparticles adsorbed with exogenous DNA under external force (high-pressure gas) to achieve transformation. Gene gun transformation can achieve transformation of multiple species without genotype limitations, and the transformation efficiency can reach over 10%. However, gene gun transformation is costly, cumbersome, and has poor reproducibility. Callus transformation refers to the process of infecting plant callus tissue with Agrobacterium tumefaciens to achieve transformation. Plant cells transformed by this method can regenerate into complete plants under suitable conditions; however, the transformation efficiency of this method is generally no higher than 2% (without antibiotic selection), and target gene expression can only be observed 3 to 7 days after transformation. Furthermore, all elements need to be constructed into the same vector.

[0004] In comparison, agroinfiltration has significant advantages. Agrobacterium infiltration involves injecting a solution of Agrobacterium containing the target gene into plant leaves (typically leaves of Nicotiana benthamiana) using a syringe without the needle. This method is simple to operate, has high transformation efficiency (over 95% under suitable conditions), and high transformation throughput (10^10 transformations can be achieved in a single step). 7 Up to 10 8It can transform individual cells, and multiple transformations are possible (i.e., Agrobacterium strains containing different elements can be co-injected). The transformation cycle is short (target gene expression can be observed as early as 24 hours after transformation, and the target gene expression level reaches its peak 3 to 5 days after transformation). However, compared with protoplast transformation and gene gun transformation, Agrobacterium infiltration transformation requires Agrobacterium as a medium, which means that this study needs to construct all the required elements into Agrobacterium vectors.

[0005] *Agrobacterium tumefaciens* is a soil-dwelling Gram-negative bacterium that causes crown gall disease in plants by inducing the formation of tumor-like growths on plant roots. Subsequent research revealed that the pathogenic mechanism of *Agrobacterium* involves the delivery and integration of a pathogenic DNA molecule into the plant genome, thereby leading to crown gall formation. *Agrobacterium* contains a tumor-inducing (Ti) plasmid that exists outside the genomic DNA. The Ti plasmid contains two main regions: a cluster of virulence genes (Vir) and a transferred DNA (T-DNA) region. The T-DNA region is defined by two 25 bp repetitive sequences, designated the left border (LB) and right border (RB). This region can be delivered into plant cells and integrated into the plant genome by *Agrobacterium* using the type IV secretion system (T4SS). The T-DNA region encodes genes for plant hormone synthesis and crown gall synthesis. After transformation into plant cells, the expression of the former leads to hormonal imbalances and tumor formation; while the latter induces the synthesis of crown gall, which can be utilized by Agrobacterium, further leading to Agrobacterium infection. The Vir gene region contains seven gene clusters, named VirA, VirB, VirC, VirD, VirE, VirF, and VirG. They play a crucial role in T-DNA transformation (Tiwari et al., 2022).

[0006] After the pathogenicity of Agrobacterium was initially elucidated, its potential in plant biotechnology was immediately recognized. In currently engineered Agrobacterium strains (EHA105, LBA4404, GV3101, etc.), the T-DNA region on the Ti plasmid is deleted, retaining only the Vir gene cluster; the oncogenes in the T-DNA are also deleted, retaining only the LB and RB sequences, which are then loaded onto a smaller plasmid, thus forming the most commonly used Agrobacterium binary vector system (Gelvin, 2017). Currently, the Agrobacterium transformation system has been used for genetic transformation in various plants such as Arabidopsis, tobacco, tomato, cotton, soybean, rice, maize, and sorghum, and has become one of the most commonly used methods for plant genetic transformation.

[0007] Agrobacterium-mediated transformation has been widely used for plant genetic transformation. Since the invention of binary vectors in 1983, researchers have developed a series of vector systems. Based on the replication system, common Agrobacterium vector systems can be classified into the following categories:

[0008] First, there's the pRK2 series. The pRK2 replication system originates from Klebsiella aerogenes. This system contains two core components: oriV and trfA. The former is the replication initiation site, while the latter is responsible for initiating replication, i.e., the replication initiation protein. pRK2 vectors can maintain a high copy number in Agrobacterium (approximately 10 copies per cell) (Zhang et al., 2020). The pRK2 replication system has a wide host range, replicating in both Agrobacterium and Escherichia coli. However, to increase the copy number in E. coli, the ColE1 replication initiation site of E. coli is often added. A classic pRK2 vector series is the pBin series, but its vector backbone is relatively long (approximately 10 kb), making vector modification inconvenient (C in Figure 1). In recent years, a series of mini-pRK2 vector systems have been developed, mainly including the pCB301 and pEAQ series (E and F in Figure 1). These vectors retain only the oriV and trfA necessary for replication, while removing the redundant parts, and the skeleton size can be reduced to about 3kb (Watson et al., 2016).

[0009] Next is the pVS1 series. The pVS1 replication system originates from *Pseudomonas syringae*. The core components of this system include oriV, StaA, and RepA. oriV is the replication initiation site, StaA is responsible for plasmid segregation stability (stabilizing protein), and RepA is responsible for replication initiation (replication initiation protein). The pVS1 replication system is relatively stable in *Agrobacterium*, and its copy number is also high (approximately 20 copies per cell) (Zhang et al., 2020). The most commonly used vector for the pVS1 system is the pCambia series vector (G in Figure 1). It adds an resistance gene and pUC ori, responsible for maintaining high copy numbers in *E. coli*, to the pVS1 system, with a backbone length of approximately 7 kb. pCambia is also currently the most commonly used vector backbone for *Agrobacterium* transformation. In addition, vectors for the pVS1 replication system also include pLSU, pPZP, and the pGD series (Goodin et al., 2002; Lee et al., 2012).

[0010] The pSa replication system originates from *Escherichia coli* and is derived from the *P15A* replication system of *E. coli*. The core components of this system include pSa ori, the origin of replication, and RepA, which is responsible for initiating replication. Typical vectors for the pSa replication system are the pGreen series vectors (A and B in Figure 1). In these vectors, the coding sequence for RepA is transferred to a pRK2 replication system vector as an auxiliary plasmid, named pSoup. Therefore, the pGreen series vectors, containing only pSa ori, have the smallest vector backbone (2.6 kb) among current *Agrobacterium* vectors. However, the copy number of pSa series vectors in *Agrobacterium* is low (approximately 2 copies per cell), and their stability is also poor (Zhang et al., 2020).

[0011] The pRi replication system originates from the Ri plasmid (D in Figure 1) of *Agrobacterium rhizogenes*. It can stably coexist with the Ti plasmid of *Agrobacterium tumefaciens*. In fact, the pRi replication system is extremely stable. Another characteristic of the pRi replication system is its low copy number, with only about 1 to 2 copies per cell. Based on this, the pRi plasmid can be used to create transgenic plant lines with single-copy insertions (Ye et al., 2011).

[0012] In recent years, the pBBR1 replication system has also been used to construct Agrobacterium vectors. pBBR1 is derived from *Bordetella bronchiseptica*. The core components of this replication system include the replication initiation site pBBR1 oriV and the replication initiation protein pBBR1 Rep. This system has a broad host range, enabling plasmid replication in both *Escherichia coli* and *Agrobacterium*. In 2007, researchers developed the pLX series of vectors based on the pBBR1 replication system. This series of vectors has a very small vector backbone (approximately 2.7 kb) and does not require helper plasmids, thus enabling efficient vector construction and applications in fields such as plant synthetic biology (Pasin et al., 2017) (H in Figure 1). Furthermore, because the pBBR1 replication system is compatible with other current Agrobacterium replication systems, researchers can also implement transformation strategies using multiple vectors within a single *Agrobacterium* strain (Pasin et al., 2017).

[0013] While Agrobacterium can achieve high transformation efficiency in plants such as Arabidopsis, tomato, and tobacco, its transformation efficiency is very low in calcitrant species, especially certain varieties, such as maize, sorghum, and quinoa. To address this issue, researchers have inserted a highly toxic vir gene copy into binary vectors, creating superbinary vector systems to increase the infectivity of Agrobacterium (Anand et al., 2018; Zhang et al., 2020). However, this also results in binary vectors with a backbone length exceeding 36 kb, making construction very difficult. Therefore, a ternary vector system for Agrobacterium was constructed, introducing two mutually replicable plasmids into Agrobacterium: one expressing an additional vir gene copy, and the other carrying T-DNA. This ternary vector system has improved the transformation efficiency of Agrobacterium in calcitrant species (Zhang et al., 2019). Summary of the Invention

[0014] The problem the invention aims to solve

[0015] As mentioned earlier, commonly used Agrobacterium vectors include the pBin, pCB301, pEAQ, pCambia (Hajdukiewicz et al., 1994), pLSU, pGD, pGreenII, pLX, and pRi series (Figure 1). According to the previous description, the pCambia vector of the pVS1 replication system features high copy number (approximately 20 copies per Agrobacterium) and high stability, and is currently the most widely used and suitable Agrobacterium vector backbone. However, the pCambia series backbone is relatively large, approximately 6.3 kb (backbone size refers to the size of the region in the vector excluding T-DNA), which makes its construction inconvenient and unsuitable for constructing high-throughput vector libraries. Therefore, the purpose of this invention is to develop a smaller Agrobacterium vector system based on the pCambia series vectors, more suitable for Agrobacterium introgression transformation.

[0016] Solution for solving the problem

[0017] [1]. An Agrobacterium vector system, wherein the Agrobacterium vector system comprises an expression vector and an auxiliary vector;

[0018] The expression vector comprises:

[0019] a1) The pVS1 oriV nucleotide sequence from the replication origin site of the pVS1 replication system; and

[0020] a2) T-DNA region, wherein the T-DNA region comprises the nucleotide sequence of the right boundary (RB) and the left boundary (LB) of the T-DNA of the Ti plasmid of Agrobacterium tumefaciens;

[0021] The auxiliary carrier comprises:

[0022] b1) The nucleotide sequence of the replication initiation site oriV from the pRK2 replication system;

[0023] b2) The nucleotide sequence encoding trfA, a replication initiation protein from the pRK2 replication system;

[0024] b3) The nucleotide sequence encoding pVS1 StaA, a stable protein derived from the pVS1 replication system; and

[0025] b4) The nucleotide sequence encoding the replication initiation protein pVS1 RepA from the pVS1 replication system.

[0026] [2]. According to the Agrobacterium vector system described in [1], wherein the expression vector does not contain the nucleotide sequence encoding the stable protein pVS1 StaA and the replication initiation protein pVS1 RepA from the pVS1 replication system.

[0027] [3]. The Agrobacterium vector system according to [1] or [2], wherein the auxiliary vector does not contain Agrobacterium virulence gene clusters.

[0028] [4]. The Agrobacterium vector system according to any one of [1] to [3], wherein at least one of the expression vector and the helper vector contains a nucleotide sequence having a functional replication initiation site in Escherichia coli;

[0029] Optionally, the functional replication initiation site in Escherichia coli includes replication initiation site ColE1 or replication initiation site pMB1;

[0030] Preferably, the replication initiation site pMB1 includes the replication initiation site of the pGEM vector or the pUC vector.

[0031] [5]. The Agrobacterium vector system according to any one of [1] to [4], wherein at least one of the expression vector and the helper vector contains a nucleotide sequence encoding a selection marker;

[0032] Optionally, the selection marker is functional in Escherichia coli and / or Agrobacterium.

[0033] [6]. The Agrobacterium vector system according to [5], wherein the nucleotide sequence encoding the selection marker comprises a nucleotide sequence encoding antibiotic resistance;

[0034] Optionally, the antibiotic is selected from ampicillin, chloramphenicol, kanamycin, tetracycline, gentamicin, spectinomycin, bleomycin, buprofen, rifampin, or streptomycin.

[0035] [7]. Agrobacterium vector system according to any one of [1] to [6], wherein the expression vector is obtained by deleting pVS1 StaA and pVS1 RepA from the backbone of the pCambia series vectors.

[0036] [8]. The Agrobacterium vector system according to any one of [1] to [7], wherein the auxiliary vector is obtained by deleting the T-DNA region in the vector of the pRK2 replication system and inserting pVS1 StaA and pVS1 RepA from the pCambia series vectors;

[0037] Preferably, the inserted pVS1 StaA and pVS1 RepA elements are pVS1 StaA and pVS1 RepA elements that have been removed from the backbone of the pCambia series vector from which the expression vector is obtained.

[0038] [9]. The Agrobacterium vector system according to any one of [1] to [8], wherein the auxiliary vector further comprises a T-DNA region, the T-DNA region comprising the nucleotide sequence of the right boundary (RB) and the left boundary (LB) of the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0039]

[0010] . The Agrobacterium vector system according to any one of [1] to [9] further comprises, between RB and LB in the T-DNA region of the expression vector, and / or between RB and LB in the T-DNA region of the helper vector, a nucleotide sequence of the target gene to be expressed, preferably, the target gene being operatively linked to an expression regulatory sequence.

[0040]

[0011] . The Agrobacterium vector system according to any one of [1] to

[0010] , wherein the nucleotide sequence encoding the replication initiation protein pVS1 RepA from the pVS1 replication system in the helper vector is mutated to increase or decrease the copy number of the expression vector.

[0041]

[0012] . The Agrobacterium vector system according to any one of [1] to

[0011] , wherein,

[0042] The pVS1 oriV nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:5, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:5;

[0043] The RB comprises the nucleotide sequence shown in SEQ ID NO:4, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:4; and / or

[0044] The LB contains the nucleotide sequence shown in SEQ ID NO:3, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:3.

[0045]

[0013] . The Agrobacterium vector system according to any one of [1] to

[0012] , wherein the expression vector comprises the following elements:

[0046] T-DNA region, nucleotide sequence encoding antibiotic resistance, replication initiation site of pUC vector, replication initiation site of pVS1 replication system pVS1 oriV;

[0047] Preferably, the sequence of the expression vector is as shown in SEQ ID NO:10, wherein the expression cassette between LB and RB of the expression vector as shown in SEQ ID NO:10 is replaced with the expression cassette of the target gene to be expressed, so as to express the target gene to be expressed.

[0048]

[0014] . The Agrobacterium vector system according to any one of [1] to

[0013] , wherein,

[0049] The oriV comprises the nucleotide sequence shown in SEQ ID NO:7, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:7;

[0050] The amino acid sequence of the replication initiation protein trfA from the pRK2 replication system comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:6.

[0051] The amino acid sequence of the stable protein pVS1 StaA from the pVS1 replication system comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:2; and / or

[0052] The amino acid sequence of the replication initiation protein pVS1 RepA from the pVS1 replication system comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1.

[0053]

[0015] . The Agrobacterium vector system according to any one of [1] to

[0014] , wherein the auxiliary vector comprises the following elements:

[0054] The replication initiation site oriV from the pRK2 replication system, the replication initiation protein trfA from the pRK2 replication system, the nucleotide sequence encoding antibiotic resistance, the replication initiation site of the pUC vector, the stable protein pVS1 StaA from the pVS1 replication system, and the replication initiation protein pVS1 RepA from the pVS1 replication system;

[0055] Preferably, the sequence of the auxiliary vector is shown in SEQ ID NO:9.

[0056]

[0016] . The Agrobacterium vector system according to any one of [1] to

[0015] , wherein the auxiliary vector comprises the following elements:

[0057] The pUC vector contains the replication initiation site, the replication initiation protein pVS1 RepA from the pVS1 replication system, the stable protein pVS1 StaA from the pVS1 replication system, the replication initiation site oriV from the pRK2 replication system, the replication initiation protein trfA from the pRK2 replication system, the T-DNA region, and the nucleotide sequence encoding antibiotic resistance.

[0058] Preferably, the sequence of the helper vector is as shown in SEQ ID NO:12; wherein, the expression cassette between LB and RB of the helper vector as shown in SEQ ID NO:12 is replaced with the expression cassette of the target gene to be expressed, so as to express the target gene to be expressed.

[0059]

[0017] . A genetically engineered cell comprising the Agrobacterium vector system as described in any one of [1] to

[0016] ;

[0060] Optionally, the cells include bacterial cells or plant cells;

[0061] Preferably, the cells comprise Agrobacterium cells;

[0062] Optionally, the plant includes monocotyledonous plants or dicotyledonous plants.

[0063]

[0018] . Any of the following methods (i) to (iii):

[0064] (i) A method for transforming plants, which uses an Agrobacterium vector system as described in any one of [1] to

[0016] , or genetically engineered cells as described in

[0017] ;

[0065] (ii) A method for constructing a library, which uses an expression vector and / or an auxiliary vector in the Agrobacterium vector system as described in any one of [1] to

[0016] ;

[0066] (iii) A method for producing a target protein in plant cells, wherein an Agrobacterium vector system as described in any one of [1] to

[0016] or a genetically engineered cell as described in

[0017] is used to introduce a gene encoding the target protein into a plant cell and to culture the plant cell to allow the production of the target protein.

[0067]

[0019] . According to the method described in

[0018] , the method for transforming plants is Agrobacterium infiltration.

[0068]

[0020] . The method according to

[0018] or

[0019] , wherein the plant comprises a monocotyledonous plant or a dicotyledonous plant.

[0069] The effects of the invention

[0070] The Agrobacterium vector system provided by this invention, such as the pPhi vector used therein, can perform the same function as the pCambia vector and can, to some extent, be a substitute for pCambia. Compared with other Agrobacterium vectors that have been developed, the pPhi vector has the following advantages:

[0071] First, the pUC and pVS1 replication origin sites allow the pPhi vector to maintain high copy numbers in Escherichia coli and Agrobacterium, respectively.

[0072] Secondly, the pPhi vector has a high plant transformation efficiency, which is on par with or higher than that of the currently most efficient pCambia vector.

[0073] Furthermore, because the pPhi vector has a very small skeleton, it is very easy and flexible to construct, making it very suitable for library construction.

[0074] Finally, due to its small size and high construction efficiency, it can skip the transformation of E. coli and directly transform Agrobacterium, with a high positive rate, thus shortening the entire experimental cycle and reducing the workload. Attached Figure Description

[0075] Figure 1 shows a schematic diagram of the current Agrobacterium vector.

[0076] Figure 1A shows a schematic diagram of the Agrobacterium vector pGreenII. Figure 1B shows a schematic diagram of the helper plasmid pSoup. Figure 1C shows a schematic diagram of the Agrobacterium vector pBin19. Figure 1D shows a schematic diagram of the Agrobacterium vector pRi. Figure 1E shows a schematic diagram of the Agrobacterium vector pCB301. Figure 1F shows a schematic diagram of the Agrobacterium vector pEAQ. Figure 1G shows a schematic diagram of the Agrobacterium vector pCambia. Figure 1H shows a schematic diagram of the Agrobacterium vector pLX.

[0077] Figure 2 shows a schematic diagram of the construction of the pPhi vector. In Figure 2, in order to construct the Agrobacterium vector pPhi with a smaller backbone size, the trans-acting factors pVS1 StaA and pVS1 RepA from the most commonly used pCambia backbone were transferred to the helper plasmid pSRK2 derived from pCB301, thereby constructing the compact Agrobacterium vector pPhi.

[0078] Figure 3 shows a comparison of the pPhi vector's backbone size with other current Agrobacterium vectors. In Figure 3, the pPhi vector has the smallest backbone size, only 2.0 kb, compared to other current Agrobacterium vectors.

[0079] Figure 4 shows that the pPhi vector can replicate in Agrobacterium. Figure 4A shows schematic diagrams of the pCambia-RFP and pPhi-RFP vector structures. Figure 4B shows the expression of the reporter genes of the pCambia-RFP and pPhi-RFP vectors in the corresponding Agrobacterium strains. In Figure 4B, pCambia-RFP and pPhi-RFP can replicate in strains EHA105 and EHA105V, respectively, and report red fluorescence. This experiment was performed three independent biological replicates and obtained similar results.

[0080] Figure 5 shows the copy numbers of pPhi and other Agrobacterium vectors in *E. coli* and *Agrobacterium*. Figure 5A shows the copy numbers of the pPhi vector and other current Agrobacterium vectors in *E. coli*. Various Agrobacterium vectors were transformed into *E. coli* strain Fast-T1. Figure 5B shows the copy numbers of the pPhi vector and other current Agrobacterium vectors in *Agrobacterium tumefaciens*. Various Agrobacterium vectors were transformed into *Agrobacterium* strain EHA105 containing the corresponding helper plasmids. Copy numbers in the figures were obtained by qPCR. In Figures 5A and 5B, values ​​are mean ± sem and are based on 6 (Figure 5B) or 9 (Figure 5A) independent biological replicates. Statistical methods used were multiple comparisons Tukey's test based on one-way ANOVA. Lowercase letters in the figures represent statistically distinct groups (P < 0.05).

[0081] Figure 6 shows the working state of the copy number-tunable pPhi vector. Figure 6A shows a schematic diagram of the copy number-tunable pPhi vector. Red asterisks in the figure represent mutations introduced into pVS1 RepA. Figure 6B shows the copy number of the copy number-tunable pPhi vector in Agrobacterium tumefaciens. The pPhi vector was transformed into Agrobacterium EHA105 containing various helper plasmids. Copy numbers in the figures were determined by qPCR. In Figure 6B, values ​​are mean ± sem and based on three independent biological replicates. Statistical analysis was performed using a Tukey test for multiple comparisons based on one-way ANOVA. Lowercase letters in the figures represent statistically significant differences (P < 0.05).

[0082] Figure 7 shows the performance of pPhi and other Agrobacterium vectors in Agrobacterium introgression transformation. A in Figure 7 shows the copy number of T-DNA in *Nicotiana benthamiana* with the pPhi vector compared to other current Agrobacterium vectors. *Agrobacterium tumefaciens* EHA105 containing various Agrobacterium vectors was transformed at 0.1 OD. 600 The concentration of [specific ingredient] was injected into *Nicotiana benthamiana*. The copy number of T-DNA was determined 4 days after injection. Figure 7, B, shows the expression of genes on the pPhi vector and other current *Agrobacterium* vectors in *Nicotiana benthamiana*. The firefly luciferase gene FLuc was inserted into the T-DNA of each *Agrobacterium* vector. Each vector, along with the pCambia vector containing the Renida luciferase RLuc, was co-injected into *Nicotiana benthamiana*. RLuc was used as an internal control. Luciferase activity was determined 4 days after injection. Copy numbers in the figures were obtained by qPCR. In Figures 7, A and B, values ​​are mean ± sem and are based on 3 (Figure 7, A) or 4 (Figure 7, B) independent biological replicates. Statistical methods included Tukey's test for multiple comparisons based on one-way ANOVA. Lowercase letters in the figures represent statistically distinct groups (P < 0.05).

[0083] Figure 8 shows the library construction efficiency of the pPhi vector. In Figure 8, various Agrobacterium backbones were first constructed into entry vectors and then linked to a diversity barcode (DB) using a Golden gate. The transformation efficiency of the library was assessed by colony forming units (CFU). The statistical method used was a Tukey test for multiple comparisons based on one-way ANOVA. Lowercase letters in the figure represent statistically distinct groups (P < 0.05).

[0084] Figure 9 shows pPhi vector-mediated multiple transformations. Figure 9A shows a schematic diagram of pPhi multiple transformations. Two vectors, pPhi-RUBY and pSRK2-HygR, were co-transformed into an Agrobacterium EHA105 strain and then used to transform rice callus. Figure 9B shows the results of pPhi multiple transformations in rice. The left side of the figure shows wild-type callus, and the right side shows callus transformed with pPhi-RUBY and pSRK2-HygR.

[0085] Figure 10 shows a schematic diagram of the experimental workflow based on the pPhi vector. Figure 10A shows the experimental workflow for Agrobacterium introgression transformation based on a traditional Agrobacterium vector. From vector construction to injection into tobacco leaves, at least 6 days are required. Figure 10B shows the experimental workflow for Agrobacterium introgression transformation based on the pPhi vector. After vector construction, the E. coli step can be skipped, and Agrobacterium can be directly electroporated. After correct sequencing, it is injected into tobacco. The entire experimental workflow can be completed in as little as 4 days. Figure 10C shows the experimental workflow for pPhi vectors where high precision is not required. After electroporation of Agrobacterium, the sequencing stage can be skipped, and tobacco can be directly transformed. The entire experimental workflow can be completed in as little as 2 days. Detailed Implementation

[0086] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0087] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0088] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0089] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0090] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0091] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0092] In this instruction manual, when "room temperature" or "room temperature" is used, the temperature can be 15-25℃.

[0093] As used herein, the term "plant" includes the whole plant and any offspring, plant cells, tissues, or parts. The term "plant part" includes any part of a plant, including, for example, but not limited to: seeds (including mature seeds, immature embryos without a seed coat, and immature seeds); plant cuttings; plant cells; plant cell cultures; plant organs (e.g., pollen, embryo, flower, fruit, bud, leaf, root, stem, and related explants). Plant tissues or plant organs can be seeds, callus, or any other group of plant cells organized into structural or functional units.

[0094] Plant "offspring" includes any subsequent generations of a plant.

[0095] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases.

[0096] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0097] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, when describing a specific polypeptide amino acid sequence in this specification and claims, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still included, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0098] Sequence “identity” has a generally accepted meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence similarity can be measured along the full length of the polynucleotide or polypeptide or along a region of the molecule. Although many methods exist for measuring the similarity between two polynucleotides or polypeptides, the term “identity” is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0099] In peptides or proteins, suitable conserved amino acid substitutions are known to those skilled in the art and can generally be performed without altering the biological activity of the resulting molecule. Typically, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0100] As used herein, the term "operably linked" refers to the linking of a regulatory element (e.g., but not limited to, promoter sequences, transcription termination sequences, etc.) to a nucleic acid sequence (e.g., coding sequences or open reading frames) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

[0101] In relation to a sequence, “exogenous” means a sequence that originates from a foreign species, or, if from the same species, a sequence whose composition and / or loci have been significantly altered from its natural form through deliberate human intervention.

[0102] Invention Details

[0103] <Agrobacterium vector system>

[0104] In some aspects of the present invention, an Agrobacterium vector system is provided, wherein the Agrobacterium vector system comprises an expression vector and an auxiliary vector.

[0105] (Vehicle of expression)

[0106] In some embodiments of the present invention, the expression vector comprises:

[0107] a1) The pVS1 oriV nucleotide sequence from the replication origin site of the pVS1 replication system; and

[0108] a2) T-DNA region, wherein the T-DNA region comprises the nucleotide sequence of the right boundary (RB) and the left boundary (LB) of the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0109] In some specific embodiments, the pVS1 oriV comprises the nucleotide sequence shown in SEQ ID NO:5, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:5.

[0110] In some specific embodiments, the RB comprises the nucleotide sequence shown in SEQ ID NO:4, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:4.

[0111] In some specific embodiments, the LB comprises the nucleotide sequence shown in SEQ ID NO:3, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:3.

[0112] In some specific embodiments, the expression vector does not contain the nucleotide sequence encoding the stable protein pVS1 StaA from the pVS1 replication system and the replication initiation protein pVS1 RepA. This reduces the backbone size of the existing pCambia vector. This invention creatively discovers that the expression vector provided by this invention has a high plant transformation efficiency, equal to or higher than the currently highest-efficiency pCambia vector. Furthermore, due to the small backbone of the expression vector, its construction is very easy and flexible, making it very suitable for library construction. Because of its small backbone, its construction efficiency is also very high, allowing it to skip the transformation step of *E. coli* and directly transform *Agrobacterium* with a high positive rate, thus shortening the entire experimental cycle and reducing workload.

[0113] In some specific embodiments, the expression vector is obtained by deleting pVS1 StaA and pVS1 RepA from the backbone of a pVS1 replication system vector (e.g., the pCambia series vectors). The pCambia series vector backbone contains three core elements: pVS1 oriV, StaA, and RepA. This invention transfers the StaA and RepA elements, along with their surrounding regulatory elements, to a single helper plasmid, thereby reducing the pCambia backbone size and forming the expression vector. In this invention, there are no particular limitations on the pCambia series vectors; for example, any commercially available pCambia series vector can be used, such as the pDIRECT series vectors (Cermák et al., 2017), the pGD series vectors (Goodin et al., 2002), the pLSU series vectors (Lee et al., 2012), and the pCambia series vectors (Hajdukiewicz et al., 1994), etc.

[0114] In some specific embodiments, the expression vector comprises the following elements: a T-DNA region (containing RB and LB, and a nucleotide sequence of the target gene to be expressed between RB and LB), a nucleotide sequence encoding antibiotic resistance (e.g., a kanamycin resistance gene), a replication start site of the pUC vector, and a replication start site pVS1 oriV of the pVS1 replication system.

[0115] In the specific implementation schemes described above, the sequence of RB is shown in SEQ ID NO:4, corresponding to nucleotides 164-188 of SEQ ID NO:10; the sequence of LB is shown in SEQ ID NO:3, corresponding to nucleotides 2303-2327 of SEQ ID NO:10; the sequence of the kanamycin resistance gene corresponds to nucleotides 2586-3380 of SEQ ID NO:10; the sequence of the replication initiation site of the pUC vector is shown in SEQ ID NO:8, corresponding to nucleotides 3467-4055 of SEQ ID NO:10; and the sequence of the replication initiation site pVS1 oriV of the pVS1 replication system is shown in SEQ ID NO:5, corresponding to nucleotides 4076-4251 and nucleotides 1-19 of SEQ ID NO:10.

[0116] In some specific embodiments, the sequence of the expression vector is shown in SEQ ID NO:10. Here, SEQ ID NO:10 is an exemplary vector sequence where the nucleotide sequence of the target gene to be expressed between RB and LB is GFP, i.e., the region between RB and LB is the GFP expression cassette. Those skilled in the art can replace the region before RB and LB with the expression cassette of the target gene to be expressed, depending on the target gene to be expressed.

[0117] Therefore, in some embodiments, such as the expression cassette between RB and LB of the expression vector shown in SEQ ID NO:10, the expression cassette of the target gene to be expressed can be replaced with the expression cassette of the target gene to be expressed.

[0118] (Auxiliary carrier)

[0119] In some embodiments of the present invention, the auxiliary carrier comprises:

[0120] b1) The nucleotide sequence of the replication initiation site oriV from the pRK2 replication system;

[0121] b2) The nucleotide sequence encoding trfA, a replication initiation protein from the pRK2 replication system;

[0122] b3) The nucleotide sequence encoding pVS1 StaA, a stable protein derived from the pVS1 replication system; and

[0123] b4) The nucleotide sequence encoding the replication initiation protein pVS1 RepA from the pVS1 replication system.

[0124] In some specific embodiments, the oriV comprises the nucleotide sequence shown in SEQ ID NO:7, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:7.

[0125] In some specific embodiments, the nucleotide sequence encoding the replication initiation protein trfA from the pRK2 replication system comprises the nucleotide sequence shown in positions 1640-2788 of SEQ ID NO:9, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with positions 1640-2788 of SEQ ID NO:9.

[0126] In some specific embodiments, the amino acid sequence of the replication initiation protein trfA from the pRK2 replication system comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:6.

[0127] In some specific embodiments, the nucleotide sequence encoding the stable protein pVS1 StaA from the pVS1 replication system comprises the nucleotide sequence shown in positions 6718-7347 of SEQ ID NO:9, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with positions 6718-7347 of SEQ ID NO:9.

[0128] In some specific embodiments, the amino acid sequence of the stable protein pVS1 StaA from the pVS1 replication system comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:2.

[0129] In some specific embodiments, the nucleotide sequence encoding the replication initiation protein pVS1 RepA from the pVS1 replication system comprises the nucleotide sequence shown in positions 5216-6289 of SEQ ID NO:9, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with positions 5216-6289 of SEQ ID NO:9.

[0130] In some specific embodiments, the amino acid sequence of the replication initiation protein pVS1 RepA from the pVS1 replication system comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1.

[0131] In some specific implementations, the helper vector does not contain the Agrobacterium virulence gene cluster. As mentioned earlier, in the prior art, a highly virulence vir gene copy is inserted into a binary vector to construct a super binary vector system to increase the infectivity of Agrobacterium. However, this also results in a binary vector backbone length exceeding 36kb, making its construction very difficult. The helper vector provided by this invention does not contain the Agrobacterium virulence gene cluster, and when used in combination with the expression vector, it still exhibits an ideally high plant transformation efficiency.

[0132] In some embodiments, the helper vector is obtained by modifying the pCB301 vector of the pRK2 replication system. Using pCB301 as the backbone of the helper plasmid, the pRK2 replication system to which it belongs is very stable in Agrobacterium and can be compatible with the pVS1 replication system in a single bacterium.

[0133] In some specific implementations, the T-DNA region in pCB301 is deleted, and then StaA and RepA elements from a pVS1 replication system (e.g., from the pCambia series vectors) are transferred therein to form the helper vector. Preferably, the inserted pVS1 StaA and pVS1 RepA elements are pVS1 StaA and pVS1 RepA elements deleted from the backbone of the pCambia series vector from which the expression vector is obtained.

[0134] In some specific implementations, the resistance of pCB301 was changed from kanamycin (KanR) to spectinomycin (SpecR), and a replication origin site pUC was added to pCB301 to increase its copy number in E. coli, thus forming the pSRK2 helper vector.

[0135] In some specific implementations, the pSRK2 helper vector includes: a replication initiation site oriV from the pRK2 replication system, a replication initiation protein trfA from the pRK2 replication system, a nucleotide sequence encoding antibiotic resistance (e.g., spectinomycin resistance gene), a replication initiation site of the pUC vector, a stable protein pVS1StaA from the pVS1 replication system, and a replication initiation protein pVS1RepA from the pVS1 replication system.

[0136] In the specific implementation schemes described above, the sequence of the replication initiation site oriV from the pRK2 replication system is shown in SEQ ID NO:7, corresponding to nucleotides 919-1550 of SEQ ID NO:9; the sequence encoding the replication initiation protein trfA from the pRK2 replication system corresponds to nucleotides 1640-2788 of SEQ ID NO:9; the sequence of the spectinomycin resistance gene corresponds to nucleotides 3506-4297 of SEQ ID NO:9; the sequence of the replication initiation site of the pUC vector is shown in SEQ ID NO:8, corresponding to nucleotides 4541-5129 of SEQ ID NO:9; the nucleotide sequence encoding the stable protein pVS1 StaA from the pVS1 replication system corresponds to nucleotides 6718-7347 of SEQ ID NO:9; and the nucleotide sequence encoding the replication initiation protein pVS1 RepA from the pVS1 replication system corresponds to nucleotides 5216-6289 of SEQ ID NO:9.

[0137] In some more specific embodiments, the sequence of the pSRK2 helper vector is shown in SEQ ID NO:9.

[0138] In some more specific embodiments, the nucleotide sequence encoding the replication initiation protein pVS1 RepA from the pVS1 replication system in the helper vector is mutated to increase or decrease the copy number of the expression vector.

[0139] In some embodiments, the helper vector further comprises a T-DNA region containing the nucleotide sequence of the right boundary (RB) and the left boundary (LB) of the T-DNA of the *Agrobacterium tumefaciens* Ti plasmid. As demonstrated in Example 5, in such an embodiment, the *Agrobacterium* vector system provided by the present invention can achieve multiplex transformation of *Agrobacterium*.

[0140] In some exemplary embodiments, the pSRK2 helper vector comprises: a replication initiation site of the pUC vector, a replication initiation protein pVS1 RepA from the pVS1 replication system, a stable protein pVS1 StaA from the pVS1 replication system, a replication initiation site oriV from the pRK2 replication system, a replication initiation protein trfA from the pRK2 replication system, a T-DNA region (containing RB and LB, and the nucleotide sequence of the target gene to be expressed located between RB and LB), and a nucleotide sequence encoding antibiotic resistance (e.g., spectinomycin resistance gene).

[0141] In some more specific embodiments, the sequence of the pSRK2 helper vector is shown in SEQ ID NO:12.

[0142] In some implementations, such as the expression cassette between RB and LB of the helper vector shown in SEQ ID NO:12, the expression cassette of the target gene to be expressed can be replaced with the expression cassette of the target gene to be expressed.

[0143] (A functional replication origin in Escherichia coli)

[0144] In some embodiments of the present invention, at least one of the expression vector and the helper vector contains a nucleotide sequence that has a functional replication initiation site in *Escherichia coli*. The inclusion of a functional replication initiation site in *Escherichia coli* in both the expression vector and the helper vector allows the vector to replicate in both *Agrobacterium* and *Escherichia coli*.

[0145] In some optional embodiments, functional replication initiation sites in Escherichia coli include replication initiation site ColE1 or replication initiation site pMB1.

[0146] In some specific implementations, the replication origin pMB1 encodes two RNAs: RNAI and RNAII, and a protein called Rom or Rop. For example, the replication origin pMB1 can be a high copy number replication origin in a pGEM or pUC vector.

[0147] In some specific embodiments of the present invention, the functional replication origin site in *Escherichia coli* includes a replication origin site pUC. In some specific embodiments, the replication origin site pUC comprises the nucleotide sequence shown in SEQ ID NO:8, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:8.

[0148] (Select marker)

[0149] In some embodiments of the present invention, at least one of the expression vector and the helper vector contains a nucleotide sequence encoding a selection marker.

[0150] In some implementations, the selection marker is functional in Escherichia coli and / or Agrobacterium.

[0151] In some embodiments, the nucleotide sequence encoding the selection marker includes a nucleotide sequence encoding antibiotic resistance;

[0152] In some optional embodiments, the antibiotic is selected from ampicillin, chloramphenicol, kanamycin, tetracycline, gentamicin, spectinomycin, bleomycin, buprofen, rifampin, or streptomycin.

[0153] In some preferred embodiments, both the expression vector and the helper vector contain nucleotide sequences encoding a selection marker. In some more preferred embodiments, the expression vector and the helper vector contain different nucleotide sequences encoding the selection marker, for example, different nucleotide sequences encoding antibiotic resistance.

[0154] (Target gene)

[0155] In some embodiments of the present invention, between RB and LB in the T-DNA region of the expression vector, and / or between RB and LB in the T-DNA region of the helper vector, a nucleotide sequence of the target gene to be expressed is further included, preferably, the target gene being operatively linked to an expression regulatory sequence.

[0156] In some implementations, the "target gene" can be any nucleic acid sequence that needs to be transformed into a plant. For example, the target gene can encode a nucleic acid sequence that encodes traits important to agronomy, insect resistance, disease resistance, herbicide resistance, sterility, and commercial products. Nucleic acid sequences of interest can also include those involved in the metabolism of oil, starch, carbohydrates, or nutrients, as well as those affecting fruit size, sucrose load, etc.

[0157] In this specification, the terms "expression regulatory sequence" and "expression regulatory element" are used interchangeably, referring to a nucleotide sequence located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding sequence and affecting the transcription, RNA processing, or stability or translation of the relevant coding sequence. Plant expression regulatory elements refer to nucleotide sequences capable of controlling the transcription, RNA processing, or stability or translation of a nucleotide sequence of interest in plants. Expression regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. A "promoter" refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the invention, a promoter is a promoter capable of controlling gene transcription in plant cells, regardless of whether it originates from a plant cell. A promoter may be a constitutive promoter, a tissue-specific promoter, a developmental regulatory promoter, or an inducible promoter.

[0158] It is understood that the T-DNA region of the expression vector, between RB and LB, and / or the T-DNA region of the helper vector, further includes restriction endonuclease cleavage sites to facilitate the insertion of the target gene into the vector. In this invention, there are no particular limitations on the type of restriction endonuclease cleavage sites, such as, but not limited to, XbaI, XhoI, BamHI, EcoRI, HindIII, KpnI, etc.

[0159] (Genetically engineered cells)

[0160] In some aspects of the invention, a genetically engineered cell is provided that comprises an expression vector and / or helper vector in the Agrobacterium vector system as described above.

[0161] In some specific implementations, the cells include bacterial cells.

[0162] In some preferred embodiments, the cells comprise Agrobacterium cells, such as Agrobacterium tumefaciens cells.

[0163] In some specific embodiments, the present invention relates to Agrobacterium cells, particularly Agrobacterium tumefaciens cells, said cells comprising expression vectors and / or helper vectors in the Agrobacterium vector system as described above. Agrobacterium cells comprising the Agrobacterium vector system as described above can be used for genetic transformation of plants. For example, the target gene in the expression vector is introduced into plant cells via Agrobacterium introgression. By transforming plant cells with Agrobacterium cells using the Agrobacterium vector system as described above (Agrobacterium introgression), the T-DNA region or its functional portion in the expression vector is integrated into the plant / plant cell genome, thereby stably expressing the target gene in the plant or plant cells.

[0164] In some specific implementations, the cells include plant cells. For example, the expression vector in the Agrobacterium vector system is introduced into plant cells, resulting in transient expression of the target gene.

[0165] (Other components)

[0166] Those skilled in the art will understand that the expression vectors and / or helper vectors in the Agrobacterium vector system described above may also contain other elements of the Agrobacterium vector, such as promoters, enhancers, reporter genes, terminators, signal peptide sequences, etc., as well as non-functional spacer nucleotide sequences between the elements. The combination and design of these elements depend on the purpose and needs of the experiment, and those skilled in the art can select these elements as needed.

[0167] (Methods and Applications)

[0168] In some aspects of the invention, a method for transforming plants is provided, which uses the Agrobacterium vector system as described above, or the genetically engineered cells as described above.

[0169] In some aspects of the invention, the use of the Agrobacterium vector system as described above or the genetically engineered cells as described above in transforming plants or in reagents for preparing transforming plants is provided.

[0170] In some specific implementations, the method is the Agrobacterium infiltration method.

[0171] In some aspects of the invention, a method for producing a target protein in plant cells is provided, which uses the Agrobacterium vector system as described above or the genetically engineered cells as described above, to introduce a gene encoding the target protein into the plant cells and to culture the plant cells to allow the production of the target protein.

[0172] In some aspects of the invention, use is provided in the production of target proteins in plant cells by the Agrobacterium vector system as described above or in the preparation of reagents for producing target proteins in plant cells.

[0173] In some aspects of the present invention, a method for constructing a library is provided, which uses expression vectors and / or auxiliary vectors in the Agrobacterium vector system as described above.

[0174] In some aspects of the invention, the use of expression vectors and / or helper vectors in the Agrobacterium vector system as described above in library construction is provided.

[0175] In the context of this specification, the term "library" is used in its known meaning in the fields of cell biology and molecular biology, referring to a collection of different nucleic acid fragments / molecules. One particular type of library is a library containing random mutants generated through random mutagenesis. Another example is a designed (or synthesized) library containing specially engineered different nucleic acid fragments / molecules.

[0176] The plants described in this invention can be monocotyledonous or dicotyledonous. Suitable plants include, but are not limited to, corn, wheat, rice, barley, sorghum, beans, beets, tomatoes, cassava, cucumbers, Arabidopsis thaliana, and tobacco.

[0177] In some embodiments, the plant cells are isolated plant cells. In some embodiments, the plant cells are protoplast cells.

[0178] In some embodiments, the plant cell is a cell in a plant tissue, plant organ, or plant body; that is, the cell is not separated from the plant tissue, plant organ, or plant body. For example, the plant cell may be a leaf cell.

[0179] Example

[0180] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0181] Example 1: Construction of Agrobacterium vector pPhi

[0182] The pCambia series vectors (the one used in this paper is pDIRECT_22C (Cermak et al., 2017)) have a backbone containing three core elements: pVS1 oriV (represented as pVS1 ori in the figure), StaA, and RepA (Figure 2). pVS1 oriV is the replication initiation site, StaA is responsible for plasmid segregation stability, and RepA is responsible for replication initiation. Additionally, the pCambia backbone contains an ORF3, but it is not essential for replication. Of these elements, only pVS1 oriV is a cis-acting element and must be mounted on the vector; the rest are trans-acting factors. Therefore, in this embodiment, StaA and RepA elements are transferred to an auxiliary plasmid, thus reducing the pCambia backbone size.

[0183] A helper plasmid is a plasmid present in the host bacterial strain in addition to the target plasmid. The helper plasmid does not express the target gene, but only expresses key replication-related genes to help the target plasmid replicate stably in the host strain. In Agrobacterium, vector systems containing helper plasmids (i.e., ternary vector systems) have been developed, including the pGreenII and pGreen3 systems. However, both of these Agrobacterium vector systems have their limitations. The pSa replication system, on which pGreenII depends, has a very low copy number (1 to 2 copies) in Agrobacterium and is unstable; while the pRK2 replication system, on which pGreen3 depends, also has a relatively low copy number in Agrobacterium, and the required helper plasmid pVSI-VIR2 contains an extra copy of the Vir gene, resulting in an excessively large vector size (39.3 kb), making it inconvenient to manipulate.

[0184] Therefore, this embodiment aims to develop an Agrobacterium ternary vector system containing helper plasmids. This embodiment chose pCB301 (Xiang et al., 1999) as the backbone of the helper plasmid because its pRK2 replication system is stable in Agrobacterium and is compatible with the pVS1 replication system within the same bacterium. In this embodiment, the resistance of pCB301 was changed from kanamycin (KanR) to spectinomycin (SpecR), and the T-DNA components (including LB, T-DNA, and RB) in pCB301 were deleted. Then, the StaA and RepA elements from pCambia (including the spacer sequence between StaA and RepA elements and their surrounding regulatory elements in pCambia) were transferred into it, essentially replacing the T-DNA components in pCB301 with the StaA and RepA elements from pCambia. Furthermore, this embodiment added a pUC replication origin site to the vector to increase its copy number in Escherichia coli. In this embodiment, the new vector is named pSRK2, where S represents that the vector is Spec resistant and RK2 represents the replication system to which the vector belongs. After construction, the pSRK2 vector is transformed into Agrobacterium strain EHA105 (purchased from Biomed, BC307-01), and the newly formed strain is named EHA105V (where 'V' indicates that the strain contains helper plasmids that can help the vector of the pVS1 replication system replicate).

[0185] In this embodiment, the pSRK2 carrier includes the following elements:

[0186] The replication initiation site oriV from the pRK2 replication system, the replication initiation protein trfA from the pRK2 replication system, the spectinomycin resistance gene, the replication initiation site of the pUC vector, the stable protein pVS1 StaA from the pVS1 replication system, and the replication initiation protein pVS1 RepA from the pVS1 replication system.

[0187] in:

[0188] The sequence of the replication origin site oriV from the pRK2 replication system is shown in SEQ ID NO:7, corresponding to nucleotides 919-1550 of SEQ ID NO:9;

[0189] The sequence encoding trfA, the replication initiation protein from the pRK2 replication system, corresponds to nucleotides 1640-2788 of SEQ ID NO:9;

[0190] The sequence of the spectinomycin resistance gene corresponds to nucleotides 3506-4297 of SEQ ID NO:9;

[0191] The sequence of the replication initiation site of the pUC vector is shown in SEQ ID NO:8, corresponding to nucleotides 4541-5129 of SEQ ID NO:9;

[0192] The nucleotide sequence encoding the stable protein pVS1 StaA from the pVS1 replication system corresponds to nucleotides 6718-7347 of SEQ ID NO:9;

[0193] The nucleotide sequence encoding the replication initiation protein pVS1 RepA from the pVS1 replication system corresponds to nucleotides 5216-6289 of SEQ ID NO:9.

[0194] In this embodiment, all pVS1-related elements except oriV were deleted from the pCambia backbone (pDIRECT_22C), and the newly generated vector was named pPhi (pφ) (Figure 2). The pPhi vector can only replicate in Agrobacterium strains containing the pSRK2 vector.

[0195] In this embodiment, the pPhi(pφ) vector contains the following elements: a T-DNA region (containing RB and LB, and the nucleotide sequence of the target gene to be expressed located between RB and LB) - a kanamycin resistance gene - a replication start site of the pUC vector - a replication start site of the pVS1 replication system, pVS1 oriV.

[0196] SEQ ID NO:10 is an exemplary vector sequence of the target gene to be expressed between RB and LB, which is the nucleotide sequence of GFP, i.e., the expression cassette between RB and LB is GFP.

[0197] in:

[0198] The sequence of RB is shown in SEQ ID NO:4, which corresponds to nucleotides 164-188 of SEQ ID NO:10;

[0199] The sequence of LB is shown in SEQ ID NO:3, which corresponds to nucleotides 2303-2327 of SEQ ID NO:10;

[0200] The sequence of the kanamycin resistance gene corresponds to nucleotides 2586-3380 of SEQ ID NO:10;

[0201] The sequence of the replication initiation site of the pUC vector is shown in SEQ ID NO:8, corresponding to nucleotides 3467-4055 of SEQ ID NO:10;

[0202] The sequence of the replication origin pVS1 oriV of the pVS1 replication system is shown in SEQ ID NO:5, corresponding to nucleotides 4076-4251 and nucleotides 1-19 of SEQ ID NO:10.

[0203] Analysis revealed that the pPhi vector, while maintaining a high copy number (as demonstrated in subsequent examples), has a backbone size of only 2.0 kb, making it the most compact among all Agrobacterium vectors currently available. This greatly facilitates applications such as library construction using it as a backbone (Figure 3). Furthermore, after removing the StaA and RepA portions, in addition to the shortened backbone length, the overall GC content of the pPhi vector decreased from 59% to 46%. This allows for easy acquisition of the vector backbone via inverse PCR, providing significant flexibility for subsequent vector and library construction.

[0204] Example 2: The pPhi vector can replicate in Agrobacterium.

[0205] Next, this embodiment tests the pPhi vector. First, the mRFP gene was inserted into the pCambia and pPhi vectors constructed in Example 1, respectively, and their expression was driven by the bacterial promoter PLPp, constructing vectors pCambia-RFP and pPhi-RFP (Figure 4A). In this embodiment, both vectors were electrotransformed into Agrobacterium strain EHA105 and Agrobacterium strain EHA105V constructed in Example 1, respectively. After two days of cultivation, both vectors showed similar numbers of Agrobacterium colonies and exhibited strong red fluorescence signals (Figure 4B). This demonstrates that the simplified pPhi vector can replicate and express genes in Agrobacterium.

[0206] Example 3: Performance Comparison of pPhi Vector with Other Current Agrobacterium Vectors

[0207] Then, this embodiment aims to compare the performance of the constructed pPhi with other current Agrobacterium vector backbones and evaluate its various indicators. Therefore, in this embodiment, the green fluorescent protein GFP is fused with the coding sequence of firefly luciferase (FLuc), and the expression cassette of 35Sp-GFP-FLuc (also known as pPhi-35Sp-GFP-p2a-LUC, sequence shown in SEQ ID NO:11) is loaded into the T-DNA regions of pPhi constructed in Example 1, as well as pGreenII (Hellens et al., 2000), pGreen3 (Zhang et al., 2019), pLX (Pasin et al., 2017), pCB301 (Xiang et al., 1999), pEAQ (Sainsbury et al., 2009), pRi (Ye et al., 2011), pCambia (Hajdukiewicz et al., 1994), and pBin19 (Bevan, 1984).

[0208] This embodiment first evaluates the copy number of these Agrobacterium vectors in Escherichia coli. These vectors were transformed into the E. coli Fast-T1 strain (Vazyme, C505-02). Positive clones were selected, and after overnight culture, the bacterial culture was subjected to quantitative real-time PCR. The E. coli genome was used as an internal control for copy number calculation. The results showed that although the pPhi vector did not have the highest copy number in E. coli, it still reached approximately 60 copies per cell, making it a high-copy plasmid (Figure 5, A).

[0209] This embodiment then evaluated the copy number of these Agrobacterium vectors in Agrobacterium tumefaciens. These vectors were transformed into Agrobacterium EHA105 strain. Specifically, pGreenII vector was transformed into Agrobacterium EHA105 containing the pSoup helper plasmid (Biomed, BC313-01); pGreen3 was transformed into Agrobacterium EHA105 containing the pVS1-VIR2 helper plasmid (Zhang et al., 2019); and pPhi vector was transformed into Agrobacterium containing the pSRK2 helper plasmid. Positive Agrobacterium clones were selected, cultured overnight, and then subjected to quantitative real-time PCR. The Agrobacterium Ti plasmid was used as an internal control for copy number calculation. The results showed that the pPhi vector had the highest copy number in Agrobacterium, reaching approximately 15 copies per cell (B in Figure 5). This is partly because pPhi belongs to the pVS1 replication system, which inherently possesses a high copy number, and partly because pPhi's small scaffold also allows for efficient replication in Agrobacterium.

[0210] Building upon this, this embodiment also developed a copy number-tunable version of pPhi. Since pPhi replication depends on the replication-related protein pVS1 RepA (SEQ ID NO:1), this embodiment introduced a series of point mutations into the pVS1 RepA coding sequence on the helper plasmid pSRK2 and tested their effect on the pPhi copy number (Figure 6, A). The results showed that the introduction of the A246V mutation increased the pPhi copy number by approximately 1-fold, reaching approximately 30 copies per cell, while the introduction of point mutations such as D260Q reduced the pPhi copy number to approximately 3 copies per cell (Figure 6, B). Since the expression level of the target gene in plants is related to the copy number of the Agrobacterium vector, the tunable pPhi allows for adjustable target gene expression in plants without modifying the vector; only the type of helper plasmid pSRK2 needs to be adjusted.

[0211] Next, this embodiment evaluated the copy number of T-DNA in *Nicotiana benthamiana* containing these *Agrobacterium* vectors. In this embodiment, *Agrobacterium* strains containing these vectors were analyzed at 0.1 OD0.05. 600 The concentration was injected into the leaves of *Nicotiana benthamiana* (whose seeds were preserved in the inventor's laboratory). Four days after injection, DNA was extracted and used as an internal control for quantitative real-time PCR. The results showed that the copy number of T-DNA in tobacco for each vector was positively correlated with its copy number in *Agrobacterium*. The T-DNA copy number of the pPhi vector reached an average of about 100 copies per tobacco cell (A in Figure 7).

[0212] Finally, this embodiment evaluated the expression levels of the target gene in *Nicotiana benthamiana* using these *Agrobacterium* vectors. Similar to the above, this embodiment used these *Agrobacterium* strains containing these vectors at a concentration of 0.1 OD. 600 The concentration of the drug was injected into the leaves of *Nicotiana benthamiana*. Simultaneously, Renilla luciferase (RLuc), constitutively expressed and carried by the pCambia vector, was also injected into the leaves as an internal control for quantification. Four days after injection, the ratio of the two luciferase activities, FLuc / RLuc, was used to characterize the expression level of the target gene. The results are shown in Figure 7; the expression levels of the target gene by the pPhi vector were not significantly different from those by other *Agrobacterium* vectors (Figure 7, B).

[0213] Example 4: The pPhi vector exhibits the highest library construction efficiency.

[0214] Besides facilitating vector construction and enabling efficient expression of target genes in plants, another important purpose of constructing pPhi is to achieve efficient library construction. To compare the library construction efficiency of pPhi with other Agrobacterium backbones, this study added the toxic gene ccdB to the T-DNA of various vectors, modifying them into entry vectors. Then, the same volume of entry vector was used for Golden Gate ligation with a diversification barcode (DB). After purification, the ligation product was electroporated into *E. coli* DH5α strain. After approximately 1.5 hours of recovery culture, the bacterial culture was plated at different gradients. The vector construction efficiency was deduced based on the number of colony forming units (CFUs). As shown in Figure 8, pPhi achieved the highest construction efficiency among all Agrobacterium backbones, reaching approximately 10. 8 The copy number of pPhi in E. coli is very high, resulting in a high concentration of plasmids for the extracted entry vector. This is also because pPhi has the smallest backbone, naturally leading to the highest vector construction efficiency. This experiment demonstrates that pPhi is well-suited for constructing high-throughput libraries.

[0215] Example 5: pPhi's Multiple Conversion Strategy

[0216] Compared to other vector systems, another advantage of the pPhi system is that it contains two vectors, pPhi and pSRK2. Therefore, pPhi offers significant modification potential; for example, a T-DNA vector can be loaded onto pSRK2 to achieve multiplex transformation. To achieve stable plant transformation, researchers typically place all elements (including the target gene and resistance gene) onto a single Agrobacterium T-DNA vector. While this method ensures genetic linkage between the target gene and the resistance gene, it results in large vectors that are difficult to construct. Furthermore, changing the resistance gene requires rebuilding the vector, which is inconvenient.

[0217] Therefore, in this embodiment, a T-DNA segment (i.e., the T-DNA portion of pPhi, retaining LB and RB) was also inserted at the SalI restriction site of pSRK2, and a plant resistance gene (i.e., pSRK2-Kan, sequence shown in SEQ ID NO:12) was added to it, while the target gene was carried on the pPhi vector. Although the target gene and the resistance gene are not on the same T-DNA segment at this time, since pPhi and pSRK2 coexist in the same Agrobacterium, they can easily enter a plant cell simultaneously. In addition, since the copy number of pSRK2 is lower than that of pPhi (the copy number of the pRK2 replication system is lower than that of pVS1), the copy number of the resistance gene's T-DNA is also lower than that of the target gene, and the corresponding integration frequency will theoretically be lower. Therefore, although the target gene and the resistance gene are not encoded by the same T-DNA segment, the plant explant cells that have integrated the resistance gene have a high probability of also integrating the target gene, thereby achieving multiple transformation by Agrobacterium.

[0218] To verify the feasibility of the multiple transformation strategy, this embodiment inserted the hygromycin resistance gene (HygR) into the T-DNA of the pSRK2 vector (pSRK2-Kan, where the plant kanamycin resistance gene is replaced with the hygromycin resistance gene, abbreviated as pSRK2-HygR), and inserted RUBY as the target gene into the T-DNA of the pPhi vector, i.e., the pPhi-35Sp-Ruby sequence is shown in SEQ ID NO:13, abbreviated as pPhi-RUBY (A in Figure 9). RUBY contains three genes: P450 cytochrome oxidase CYP76AD1, dihydroxyphenylalanine oxidase DODA, and glucosyltransferase (GTF). The expression products of these three genes can catalyze the biosynthesis of betalain in plant cells, thereby making the cells pink.

[0219] In this example, two vectors, pPhi-RUBY and pSRK2-HygR, were co-transformed into Agrobacterium EHA105, and then used to transform rice callus tissue. After approximately 4 weeks, over 60% of the resistant callus tissue exhibited varying degrees of red color (Figure 9, B). This indicates that RUBY and HygR were co-transformed into a single rice cell. This experiment demonstrates the feasibility of pPhi multiple transformation.

[0220] Example 6: pPhi can shorten the cycle of plant experiments.

[0221] Based on the above embodiments, this embodiment pre-tests whether the pPhi vector can shorten the experimental cycle of the current Agrobacterium infiltration method. In the traditional Agrobacterium infiltration method, from the construction of the vector, it is necessary to go through the steps of transforming E. coli, sequencing, culturing E. coli, extracting plasmids, transforming Agrobacterium, and culturing Agrobacterium, which takes at least 6 days to obtain the target Agrobacterium strain and inject it into Nicotiana benthamiana (Figure 10, A). Since the pPhi vector backbone is small and easier to construct, this embodiment explores whether the E. coli transformation step can be skipped using the pPhi vector, that is, the product can be directly transformed into Agrobacterium after construction. To this end, this embodiment attempts to construct several pPhi vectors expressing GFP (35Sp-GFP). After Golden Gate assembly, 1 μL of the reaction product was taken, electrotransformed into Agrobacterium, and plated. After 2 days, many colonies grew. Several colonies were randomly selected and colony PCR was performed using the corresponding primers. The positive rate was found to be higher than 95%. Subsequent sequencing results also showed that the vector construction was correct. The entire process was completed within 4 days (Figure 10, B). These results demonstrate that the construction of the pPhi vector can skip the E. coli step, thereby shortening the experimental cycle and reducing workload. Furthermore, for some experiments with low precision requirements, the sequencing step can be skipped, and the electroporated Agrobacterium can be directly cultured, reducing the entire experimental cycle to 2 days (Figure 10, C).

[0222] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0223] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

[0224] This disclosure relates to partial nucleotide or amino acid sequences.

[0225] pVS1 RepA(SEQ ID NO:1)

[0226] pVS1 StaA(SEQ ID NO:2)

[0227] LB (SEQ ID NO:3)

[0228] RB (SEQ ID NO:4)

[0229] pVS1 oriV (SEQ ID NO:5)

[0230] trfA (SEQ ID NO:6)

[0231] oriV (SEQ ID NO:7)

[0232] pUC origin (SEQ ID NO:8)

[0233] pSRK2 (SEQ ID NO:9)

[0234] in:

[0235] The underlined portion represents the sequence from the oriV replication origin site of the pRK2 replication system;

[0236] The single underlined portion is the sequence encoding trfA, a replication initiation protein from the pRK2 replication system;

[0237] The double-underlined portion represents the sequence of the spectinomycin resistance gene;

[0238] The italicized and bold text indicates the sequence of the replication start site of the pUC vector;

[0239] The uppercase letters represent the nucleotide sequence encoding pVS1 StaA, a stable protein from the pVS1 replication system;

[0240] The gray background represents the nucleotide sequence encoding pVS1 RepA, a replication initiation protein from the pVS1 replication system. pPhi-35Sp-GFP (SEQ ID NO:10)

[0241] in:

[0242] Uppercase letters and single underscores indicate sequences of type RB;

[0243] Uppercase letters and double underscores indicate sequences where the value is LB;

[0244] The lowercase letters and double underscores represent the sequence of the kanamycin resistance gene;

[0245] The italicized and bolded portions are sequences of the replication initiation sites of the pUC vector;

[0246] The gray background represents the sequence of the pVS1 oriV replication origin site of the pVS1 replication system;

[0247] The lowercase letters and single underscores represent the nucleotide sequence encoding GFP;

[0248] pPhi-35Sp-GFP-p2a-LUC (SEQ ID NO:11)

[0249] pSRK2-Kan (SEQ ID NO:12)

[0250] in:

[0251] The underlined portion represents the sequence from the oriV replication origin site of the pRK2 replication system;

[0252] The lowercase letters and single underscores represent sequences encoding trfA, a replication initiation protein from the pRK2 replication system;

[0253] The lowercase letters and double underscores represent the sequence of the spectinomycin resistance gene;

[0254] The italicized and bold text indicates the sequence of the replication start site of the pUC vector;

[0255] The uppercase letters represent the nucleotide sequence encoding pVS1 StaA, a stable protein from the pVS1 replication system;

[0256] The gray background represents the nucleotide sequence encoding the replication initiation protein pVS1 RepA from the pVS1 replication system.

[0257] Uppercase letters and single underscores indicate sequences of type RB;

[0258] Uppercase letters and double underscores indicate sequences of type LB.

[0259] pPhi-35Sp-Ruby(SEQ ID NO:13)

[0260] References

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[0264] Cermák T,Curtin SJ,Gil-Humanes J,Cegan R,Kono TJY,KonecnáE,Belanto JJ,Starker CG,Mathre JW,Greenstein RL,et al.A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants.Plant Cell.2017:29(6):1196-1217.

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Claims

1. An Agrobacterium vector system, wherein, The Agrobacterium vector system includes an expression vector and an auxiliary vector; The expression vector comprises: a1) The pVS1 oriV nucleotide sequence from the replication origin site of the pVS1 replication system; and a2) T-DNA region, wherein the T-DNA region comprises the nucleotide sequence of the right boundary (RB) and the left boundary (LB) of the T-DNA of the Ti plasmid of Agrobacterium tumefaciens; The auxiliary carrier comprises: b1) The nucleotide sequence of the replication initiation site oriV from the pRK2 replication system; b2) The nucleotide sequence encoding trfA, a replication initiation protein from the pRK2 replication system; b3) The nucleotide sequence encoding pVS1 StaA, a stable protein derived from the pVS1 replication system; and b4) The nucleotide sequence encoding the replication initiation protein pVS1 RepA from the pVS1 replication system.

2. The Agrobacterium vector system according to claim 1, wherein, The expression vector does not contain the nucleotide sequence encoding the stable protein pVS1 StaA from the pVS1 replication system or the replication initiation protein pVS1 RepA.

3. The Agrobacterium vector system according to claim 1 or 2, wherein, The auxiliary vector does not contain Agrobacterium virulence gene clusters.

4. The Agrobacterium vector system according to any one of claims 1 to 3, wherein, At least one of the expression vector and the helper vector contains a nucleotide sequence that has a functional replication initiation site in Escherichia coli; Optionally, the functional replication initiation site in Escherichia coli includes replication initiation site ColE1 or replication initiation site pMB1; Preferably, the replication initiation site pMB1 includes the replication initiation site of the pGEM vector or the pUC vector.

5. The Agrobacterium vector system according to any one of claims 1 to 4, wherein, At least one of the expression vector and the helper vector contains a nucleotide sequence encoding a selection marker; Optionally, the selection marker is functional in Escherichia coli and / or Agrobacterium.

6. The Agrobacterium vector system according to claim 5, wherein, The nucleotide sequence encoding the selection marker includes a nucleotide sequence encoding antibiotic resistance; Optionally, the antibiotic is selected from ampicillin, chloramphenicol, kanamycin, tetracycline, gentamicin, spectinomycin, bleomycin, buprofen, rifampin, or streptomycin.

7. The Agrobacterium vector system according to any one of claims 1 to 6, wherein, The expression vector was obtained by deleting pVS1 StaA and pVS1 RepA from the backbone of the pCambia series vectors.

8. The Agrobacterium vector system according to any one of claims 1 to 7, wherein, The helper vector was obtained by deleting the T-DNA region from the vector of the pRK2 replication system and inserting pVS1 StaA and pVS1 RepA from the pCambia series vectors; Preferably, the inserted pVS1 StaA and pVS1 RepA elements are pVS1 StaA and pVS1 RepA elements that have been removed from the backbone of the pCambia series vector from which the expression vector is obtained.

9. The Agrobacterium vector system according to any one of claims 1 to 8, wherein, The helper vector also contains a T-DNA region, which includes the nucleotide sequence of the right boundary (RB) and the left boundary (LB) of the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

10. The Agrobacterium vector system according to any one of claims 1 to 9, wherein, The expression vector contains, between RB and LB in the T-DNA region, and / or between RB and LB in the T-DNA region of the helper vector, a nucleotide sequence of the target gene to be expressed, preferably wherein the target gene is operatively linked to an expression regulatory sequence.

11. The Agrobacterium vector system according to any one of claims 1 to 10, wherein, The nucleotide sequence encoding the replication initiation protein pVS1 RepA from the pVS1 replication system in the helper vector is mutated to increase or decrease the copy number of the expression vector.

12. The Agrobacterium vector system according to any one of claims 1 to 11, wherein, The pVS1 oriV nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:5, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:5; The RB comprises the nucleotide sequence shown in SEQ ID NO:4, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:4; and / or The LB contains the nucleotide sequence shown in SEQ ID NO:3, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:

3.

13. The Agrobacterium vector system according to any one of claims 1 to 12, wherein, The expression vector comprises the following elements: T-DNA region, nucleotide sequence encoding antibiotic resistance, replication initiation site of pUC vector, replication initiation site of pVS1 replication system pVS1 oriV; Preferably, the sequence of the expression vector is as shown in SEQ ID NO:10, wherein the expression cassette between LB and RB of the expression vector as shown in SEQ ID NO:10 is replaced with the expression cassette of the target gene to be expressed, so as to express the target gene to be expressed.

14. The Agrobacterium vector system according to any one of claims 1 to 13, wherein, The oriV comprises the nucleotide sequence shown in SEQ ID NO:7, or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:7; The amino acid sequence of the replication initiation protein trfA from the pRK2 replication system comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:

6. The amino acid sequence of the stable protein pVS1 StaA from the pVS1 replication system comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:2; and / or The amino acid sequence of the replication initiation protein pVS1 RepA from the pVS1 replication system comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:

1.

15. The Agrobacterium vector system according to any one of claims 1 to 14, wherein, The auxiliary carrier includes the following elements: The replication initiation site oriV from the pRK2 replication system, the replication initiation protein trfA from the pRK2 replication system, the nucleotide sequence encoding antibiotic resistance, the replication initiation site of the pUC vector, the stable protein pVS1 StaA from the pVS1 replication system, and the replication initiation protein pVS1 RepA from the pVS1 replication system; Preferably, the sequence of the auxiliary vector is shown in SEQ ID NO:

9.

16. The Agrobacterium vector system according to any one of claims 1 to 15, wherein, The auxiliary carrier includes the following elements: The pUC vector contains the replication initiation site, the replication initiation protein pVS1 RepA from the pVS1 replication system, the stable protein pVS1 StaA from the pVS1 replication system, the replication initiation site oriV from the pRK2 replication system, the replication initiation protein trfA from the pRK2 replication system, the T-DNA region, and the nucleotide sequence encoding antibiotic resistance. Preferably, the sequence of the helper vector is as shown in SEQ ID NO:12; wherein, the expression cassette between LB and RB of the helper vector as shown in SEQ ID NO:12 is replaced with the expression cassette of the target gene to be expressed, so as to express the target gene to be expressed.

17. A genetically engineered cell comprising the Agrobacterium vector system as described in any one of claims 1 to 16; Optionally, the cells include bacterial cells or plant cells; Preferably, the cells comprise Agrobacterium cells; Optionally, the plant includes monocotyledonous plants or dicotyledonous plants.

18. Any of the following methods (i) to (iii): (i) A method for transforming plants, which uses the Agrobacterium vector system as described in any one of claims 1 to 16, or the genetically engineered cells as described in claim 17; (ii) A method for constructing a library, which uses an expression vector and / or an auxiliary vector in the Agrobacterium vector system as described in any one of claims 1 to 16; (iii) A method for producing a target protein in plant cells, wherein the Agrobacterium vector system as described in any one of claims 1 to 16 or the genetically engineered cell as described in claim 17 is used to introduce a gene encoding the target protein into a plant cell and to culture the plant cell to allow the production of the target protein.

19. The method according to claim 18, wherein, The method for transforming plants is the Agrobacterium infiltration method.

20. The method according to claim 18 or 19, wherein, The plants include monocotyledonous plants or dicotyledonous plants.

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