Controllable method for removing exogenous vector skeleton and screening marker

Abstract

The invention discloses a method for controllably removing an exogenous vector skeleton and a selection marker. According to the method, a novel transformation vector sequence containing an isolated expression cassette sequence composed of a non-autonomous transposon and a target gene is designed, and the isolated expression cassette is inserted into a corn kernel purple aleurone layer marker gene sequence to form a target gene expression cassette transposition detection system. Meanwhile, a fluorescent protein coding gene specifically expressed by a grain aleurone layer and a selection marker gene sequence are utilized to form a vector skeleton detection system. According to the method, a plurality of new ultra-pure transformant events can be obtained from one initial transformation event, the safety evaluation cost and time are saved, the number of transformation events is increased, and the method has great value for accelerating and promoting industrialization of transgenic crops.

Description

A method for controllable removal of exogenous vector backbone and screening markers Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for controllable removal of exogenous vector backbones and screening markers. Background Technology

[0002] Corn is my country's largest grain crop and an important feed and industrial raw material, playing a vital role in maintaining food security, promoting livestock development, and meeting industrial raw material needs. In recent years, the demand for corn in my country and globally has continued to increase. Relying solely on conventional breeding methods is insufficient to ensure a stable food supply, necessitating technological innovation to support the development of the corn industry. Transgenic technology is a crucial means of improving crop yield, resistance, and quality, and a significant development direction in agricultural bio-breeding technology. In recent years, my country has gradually advanced the commercialization of transgenic products, facing competition from domestic and international transgenic varieties. Therefore, in addition to continuously optimizing target gene traits, transgenic technology itself still possesses enormous research and development potential.

[0003] Plant transformants typically refer to transgenic plants that can be stably inherited through Agrobacterium-mediated genetic transformation. Most commercially available maize transformants internationally not only carry the target trait gene sequences but also contain selection marker genes for glyphosate or glufosinate resistance, as well as other vector sequences. Subsequent safety assessments require a comprehensive analysis and evaluation of the molecular characteristics, genetic features, and safety of these selection marker genes. The retention of some vector sequences in the transformants also increases the risk of unintended effects, thus increasing the cost of safety assessments. Therefore, creating "ultra-pure transformants" completely free of selection markers and exogenous vector backbones can reduce the number of genes required for safety assessment, lower the cost of transgenic maize safety assessments, accelerate the safety assessment process, and promote the healthy and rapid development of my country's transgenic maize industry. Furthermore, removing selection markers from transgenic maize can prevent safety risks such as herbicide resistance in field weeds and herbicide resistance gene drift.

[0004] Marker-free transgenic technology can eliminate selection markers introduced by traditional genetic transformation, significantly improving the safety of transformants. Therefore, marker-free transformation has always been a key area of ​​research in transgenic technology. To date, gene gun transformation based on mixed exogenous DNA fragments, dual-T vectors, and marker-free transformation systems based on the Cre-LoxP system have been discovered (Zhu et al. 2017; Zhu et al. 2018). However, these technologies all have limitations to varying degrees. Gene gun transformation often involves multiple copies of the inserted fragment, while dual-T vector transformation often involves tight linkage between the target gene and the marker gene, making it difficult to effectively separate the marker gene and remove residual fragments of the T-vector (Leng et al. 2020). Currently, the most widely used system is the marker gene excision system based on the Cre-LoxP system. For example, the excision of selection markers in transgenic rice rich in anthocyanins and astaxanthin was achieved using the Cre-LoxP system. However, the Cre-LoxP technology still has significant limitations. It can only remove the DNA sequence between two LoxP fragments, leaving one LoxP site in the genome. Therefore, it cannot completely remove the exogenous vector backbone. Furthermore, the selection marker removal process in Cre-LoxP technology occurs as the transformant is obtained, making it impossible to monitor and control. Multiple generations of testing are typically required to confirm the absence of residual selection markers, increasing time costs and requiring further efficiency improvements. Simultaneously, Cre-LoxP technology cannot completely remove the exogenous sequences at both ends of the transformation vector, meaning it cannot achieve complete removal of the vector backbone. Moreover, for maize germplasm or vector plasmids that are difficult to genetically transform, obtaining more transgenic events from a single transformation will significantly increase the speed of phenotypic evaluation and marketization of the transformants. Therefore, it is still necessary to develop efficient and controllable new technologies to achieve complete removal of the vector backbone and create more transgenic events, thereby obtaining ultra-pure transformants. This would reduce the safety evaluation costs of genetically modified maize, accelerate the safety evaluation process, and enhance the market competitiveness of genetically modified products. Summary of the Invention

[0005] The purpose of this invention is to provide an efficient, visual, and controllable method for completely removing exogenous vector backbones and selection markers, thereby creating ultrapure transformants containing only the target gene expression cassette.

[0006] A method for controllable removal of exogenous vector backbones and screening markers, comprising the following steps:

[0007] (1) Construct a target gene separation expression cassette, an aleurone layer-specific reporter gene expression cassette, a marker gene expression cassette, and a screening marker gene expression cassette, which consist of a non-autonomous transposon and a target gene. Then, use Agrobacterium-mediated genetic transformation to deliver the vector containing the above expression cassette into the maize recipient line.

[0008] (2) Sow T1 maize seeds that express fluorescent protein and cultivate them to obtain mature maize plants;

[0009] (3) Use maize germplasm with autonomous transposon activity to cross-pollinate the maize in step (2), activate the target gene segregation expression cassette transposition, and judge the kernels that have transposonized by the purple spots of the aleurone layer.

[0010] (4) Collect the purple-spotted maize seeds that have undergone transposition as described in step (3), sow and cultivate them to obtain mature maize plants for self-pollination;

[0011] (5) Collect the seeds obtained by self-pollination in step (4) that do not contain the vector skeleton and only retain the target gene expression cassette, and sow and cultivate them to form maize plants;

[0012] (6) Pollinate the corn plant pollen described in step (5) with the female ear of the initial corn transformant T1 generation described in step (2), and observe the kernel phenotype of the ear of the plant described in step (2). The appearance of purple spots on the kernels indicates that the plant described in step (5) carries autonomous transposase activity. Use this method to screen the plant described in step (5). The self-pollinated offspring of the plant without autonomous transposase activity and only retaining the target gene expression cassette are the ultrapure transformants.

[0013] The selection marker gene expression cassette is a promoter-driven selection marker gene, wherein the promoter is a 35S promoter or a Ubiquitin promoter; and the selection marker gene is a Bar, Hpt, or D-mannose isomerase gene.

[0014] The autonomous transposon sequences are derived from Ac, Piggyback (containing the PB transposon coding sequence and its specifically recognized terminal inverted repeat sequence), Ping / Pong, or Sleepybeauty (containing the SB transposon coding sequence and its specifically recognized terminal inverted repeat sequence); the non-autonomous transposon sequences are derived from Ds or mPing.

[0015] A target gene expression cassette consisting of a promoter, target gene, and terminator is further modified by adding a non-autonomous transposon Ds sequence to its outer side, thus forming a target gene segregation expression cassette.

[0016] The aleurone layer-specific reporter gene is ZmC1, ZmBz1, or ZmR2.

[0017] The fluorescent marker gene is mCherry, DsRed, RFP, or Rubby.

[0018] The trait of lacking autonomous transposase activity described in step (6) is the absence of purple spots on the seeds.

[0019] The maize recipient line mentioned in step (1) is an inbred line such as B73, KN5585, or B104, or a Hi-II hybrid.

[0020] The beneficial effects of this invention: This invention discloses a transposon-mediated, highly efficient, visual, and controllable method for creating ultra-clean maize transformants (TRUST). This method designs a novel transformation vector sequence containing a segregated expression cassette sequence consisting of a non-autonomous transposon (an inverted repeat sequence recognized by the transposon) and a target gene. This segregated expression cassette is inserted into a maize kernel purple aleurone layer marker gene sequence to form a target gene expression cassette transposition detection system. Simultaneously, a vector backbone detection system is constructed using a fluorescent protein-encoding gene and a selection marker gene sequence specifically expressed in the kernel aleurone layer. The above vector is transformed into a maize recipient line, and hybridization with germplasm containing transposase induces the target gene expression cassette to detach from the vector backbone and reintegrate into the maize genome in a controllable manner. The fluorescent protein and purple aleurone layer marker are used to visually track the target gene segregation and reintegration process, and ultra-clean transformants containing only the target gene expression cassette are obtained within three generations of screening. This invention establishes a highly efficient, visual, and controllable system for creating ultrapure transgenic transformants. This technology can generate multiple new ultrapure transformant events from an initial transformation event, saving safety evaluation costs and time while increasing the number of transformation events. This is of great value for accelerating the industrialization of transgenic crops. Attached Figure Description

[0021] Figure 1 is a schematic diagram of the carrier construction for the Corn Ultrapure Transformer Technology (TRUST).

[0022] Figure 2 shows the isolation of the target gene (GOI) mediated by the Maize Transformer Creation Technology (TRUST). In the figure, AB represents the statistical results of the proportion of ears and transgenic positive kernels in the initial transformant (T1 generation) created using TRUST; Bright represents sunlight, and Fluorescence represents fluorescence. C shows the probe locations for digital PCR detection of the Bar, GFP, and DsRed genes; D shows the digital PCR detection of the Bar, GFP, and DsRed genes in the initial transformant (T1 generation) and the target gene (GOI) isolates. Copy number in F1 generation; EF represents the proportion of F1 ears and kernels with target gene segregation (purple-spotted kernels) mediated by TRUST (Transurethral Stimulation Technology for Maize); GH represents the proportion of F2 ears and kernels of ultrapure transformants containing only the target gene (GFP) (F3 generation) mediated by TRUST; I represents the fluorescence distribution of kernels with various phenotypes in F2 ears observed under a stereomicroscope, showing that ultrapure transformant kernels lack the red fluorescent protein (DsRed) carried by the vector backbone and exhibit a green fluorescent phenotype containing the target gene GFP; Whole seed shows an intact kernel, and Longitudinal section shows a longitudinal section of the kernel; En, endosperm; Em, embryo; Al, aleurone layer; Scale bar is 1.0 cm in A, E, and G, and 0.2 cm in I.

[0023] Figure 3 shows the detection of vector insertion sites for two initial transgenic events; where A represents the vector sequence insertion site and primer positions used in initial transgenic event 1; B represents the PCR validation event 1 (…). #1 The original insertion site of the vector sequence, NT in the first lane indicates a non-transgenic plant, T1- #1 The launching line indicates the T1 generation transgenic line (T1 segregator) of Event 1, F1- #1 Launchline indicates lines where the target gene expression cassette separates from the original insertion site; C represents transgenic event 2 ( #2 The vector sequence insertion site and the primer positions used; D represents the original insertion site of the vector sequence in PCR verification event 2; NT in the first lane indicates a non-transgenic plant; T1- #2 The launching line shows the T1 generation transgenic line (T1 segregator) of Event 2, F1- #2 Launch line indicates a line in which the target gene expression cassette separates from the original insertion site.

[0024] Figure 4 shows the ultra-clean maize transformants created by TRUST and the detection of copy number and GOI insertion sites; where A represents the self-pollinated ears of the ultra-clean maize transformants obtained by TRUST. #1-1) and the hybrid ears of the initial transgenic event 1 (T1- #1 x TRUST #1 -1) Phenotype, Bright indicates sunlight, Fluorescence indicates fluorescence, scale bar is 1.0 cm; B is the copy number of Bar, GFP and DsRed genes in the ultrapure transformant detected by digital PCR; C is the copy number of ultrapure transformant seeds detected by third-generation sequencing technology (PacBio Single Molecule Real-Time sequencing). #1 -1) shows the insertion site of the target gene, confirming that the transformant contains only the target gene (GFP) expression cassette; D is the PCR detection of ultrapure transformant grains (TRUST). #1 Insertion site of the target gene in -1). Detailed Implementation

[0025] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0026] Example 1

[0027] A novel transformation vector, pCAMBIA-Ds-GFP (Figure 1), was constructed using standard gene cloning techniques. This vector contains four tandem gene expression cassettes. The first cassette contains a maize zein endosperm-specific promoter sequence driving the expression of the green fluorescent protein (GFP) gene, terminated by a 35S terminator. An inverted repeat sequence of the non-autonomous transposon Ds is attached to the 5' and 3' ends of this cassette, forming a complete target gene segregation expression cassette. The second cassette consists of the promoter and coding region of the maize endogenous anthocyanin synthesis-related gene ZmC1, along with its own terminator. The full sequence of the target gene segregation expression cassette is inserted into the coding region of the ZmC1 gene. When the transposon mediates the separation of the target gene segregation expression cassette from the second cassette, the coding sequence of the ZmC1 gene is restored, producing a functional ZmC1 protein that activates anthocyanin synthesis in the aleurone layer of the grain. Therefore, the presence of transposable targets can be determined by the purple spots on the aleurone layer of the grain. The third expression cassette is driven by the barley aleurone layer-specific promoter (Ltp2) to express the red fluorescent protein gene (DsRed), with a proteinase inhibitor terminator sequence as the terminator for this coding frame. The fourth expression cassette is driven by the tobacco mosaic virus 35S promoter to express the phosphinothricin acetyltransferase (PAT) gene, with expression terminated by the 35S transcription terminator.

[0028] Table 1 Primers used for vector construction

[0029]

[0030] Vector construction method: The barley pLtp2 promoter sequence, DsRed protein-coding sequence, and proteinase inhibitor terminator sequence were amplified separately (primers used are shown in Table 1). The three fragments were ligated by fusion PCR to form the pLtp2-DsRed third expression cassette. The binary vector pCAMBIA3301 containing the resistance expression cassette was digested with HindIII and BstEII restriction endonucleases. The vector backbone fragment was recovered, and the pLtp2-DsRed expression cassette was further ligated to pCAMBIA3301 using recombinant cloning technology. After recombination, the original BstEII restriction endonuclease site was destroyed, forming the pCAMBIA3301-Ltp2-DsRed vector. Based on the sequence of the ZmC1 gene in the W22 genome, a sequence for inserting a non-autonomous transposon (Ds) into the third exon of ZmC1 was designed and synthesized, with a BstEII site reserved in it for subsequent insertion of the target gene expression cassette. Then, the synthesized ZmC1 expression cassette containing the Ds insertion (synthetic sequence shown in SEQ ID NO:1, ZmC1-Ds) was cloned into the EcoRI and HindIII sites of pCAMBIA3301-Ltp2-DsRed, and the EcoRI restriction site was destroyed to form pCAMBIA3301-ZmC1-Ltp2-DsRed. Subsequently, a target gene segregation expression cassette (synthetic sequence shown in SEQ ID NO:2, zein-pro-GFP) consisting of a zein promoter, a GFP coding frame, and a 35S terminator was synthesized, and this complete expression cassette was inserted into the BstEII site of pCAMBIA3301-ZmC1-Ltp2-DsRed via recombination ligation, ultimately forming the novel transformation vector pCAMBIA-Ds-GFP used in this application (primer sequences are shown in Table 1).

[0031] Transgenic maize lines (T0 segregating lines) were obtained by Agrobacterium-mediated transformation of the maize inbred line KN5585. The number of positive and negative kernels on ears from two independent T0 transformation events was statistically analyzed, showing a 1:1 segregation ratio, indicating that the exogenous vector sequence was inserted into the maize genome as a single copy. Further digital PCR (droplet-digital PCR, ddPCR; reaction system and probe sequences are shown in Tables 2 and 3) confirmed that both transgenic events involved single-copy insertion. Second- and third-generation high-throughput sequencing analysis of the insertion site of the transformation vector pCAMBIA-Ds-GFP, combined with PCR verification, clarified that the vector sequence was inserted into maize chromosomes 2 and 10, respectively, in the two transgenic events (Figures 3A-D).

[0032] The experimental procedure for droplet digital PCR (ddPCR) is as follows: First, probes are designed to amplify the Bar, GFP, and DsRed genes in the pCAMBIA-Ds-GFP vector; simultaneously, the maize endogenous gene zSSIIb is used as a single-copy internal reference gene, and its probe sequence is designed. 20 μL digital PCR reaction systems are prepared for each of the four probes. Each reaction system undergoes droplet generation and is then placed in a QX200 Droplet Digital PCR instrument (Bio-Rad) for PCR reaction. The reaction steps are as follows:

[0033] 1: 95℃, 10min;

[0034] 2: 94℃, 30 sec; 60℃, 60 sec; 40 cycles;

[0035] 3: 98℃, 10min.

[0036] After generating the results data, the ddPCR data were analyzed using QuantaSoft analysis software (Bio-Rad).

[0037] Table 2. Reaction system used for ddPCR

[0038]

[0039]

[0040] Table 3. Probe sequences used in ddPCR

[0041]

[0042] By harvesting T1 generation segregators (T1 segregators) through self-pollination of T0 generation segregators, and then hybridizing them with the Activator transposon activator line (Ac) (National Germplasm Bank No.: 0L010780), the target gene segregation expression cassette is activated to detach from the initial integration site of the vector in the genome, restoring the function of the C1 gene in the second expression cassette. Therefore, the occurrence of transposition can be determined by observing the accumulation of anthocyanins in the aleurone layer of maize kernels. 35%-50% of the F1 ears obtained by hybridization had purple-spotted grains (Fig. 2A-F). The purple-spotted grains were named as target gene segregating lines and sown. After the plants matured, they were self-pollinated to obtain F2 ears. The ears of this generation showed four phenotypes of grain segregation (Fig. 2G): (1) Grains containing only green fluorescence, which are potential ultra-pure transformant grains; (2) Red fluorescent grains represent the initial event obtained by genetic transformation (T1 segregating line), whose target gene segregating expression cassette has not transposed, so it has not separated from the second expression cassette; (3) Grains with purple spots that have transposed the target gene expression cassette are chimeras, and the target gene in some of their somatic cells has not yet separated from the second expression cassette; (4) Non-transgenic grains, which did not show GFP and DsRed fluorescence or anthocyanin accumulation.

[0043] Further observation of the complete kernels and longitudinal sections of the above four types using a stereomicroscope revealed the following results (Fig. 2I): (1) The kernels of the potential ultrapure transformants without a vector backbone showed green fluorescence only in the maize endosperm, accounting for 3.6% of the total kernels (Fig. 2H); (2) The kernels of the isolated original transformation event (T1 segregating line) showed red fluorescence in the aleurone layer and green fluorescence in the endosperm, indicating that the segregation expression cassette had not transposed; (3) The kernels that had undergone target gene expression cassette transposition showed purple spots or the entire aleurone layer was purple under sunlight due to the accumulation of anthocyanins, indicating that the target gene segregation expression cassette had transposed and separated from the original vector. Under fluorescence, the aleurone layer showed red fluorescence and the endosperm showed green fluorescence; (4) No anthocyanin accumulation or fluorescent labeling was observed in the non-transgenic kernels under sunlight or fluorescence. Furthermore, digital PCR was used to prove that there was only one copy of the target gene in the obtained ultrapure transformants, and there were no fluorescent labeling genes or Bar selection marker genes in the vector backbone (Fig. 2D).

[0044] To obtain ultrapure transformants lacking Ac transposase activity and to demonstrate the stable inheritance of the target gene expression cassette within these transformants, grains from F2 ears expressing only the target gene (GFP) were sown, and their pollen was simultaneously transferred to female ears of the T1 segregating line through self-pollination. Hybrid ears (T1-) without the purple-spotted grain phenotype were screened. #1 ×TRUST #1 -1), its pollen source TRUST #1The -1 line is considered the ultrapure transformant. Further analysis using PacBio SingleMolecule Real-Time sequencing (PbBio SingleMolecule Real-Time sequencing) was conducted to detect the ultrapure transformant grains (TRUST). #1 The insertion site of the target gene in -1) was identified, confirming that the transformant contained only the target gene (GFP) expression cassette; PCR was also used to detect and verify the purity of the ultrapure transformant seeds (TRUST). #1 Insertion site of the target gene in -1) (Figure 4A-D).

[0045] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for controllable removal of exogenous carrier backbone and screening markers, characterized in that, The following steps were performed: (1) Constructing a target gene segregation expression cassette, an aleurone layer-specific reporter gene expression cassette, a marker gene expression cassette, and a screening marker gene expression cassette, consisting of a non-autonomous transposon and a target gene, and delivering the vector containing the above expression cassette into the maize recipient line using Agrobacterium-mediated genetic transformation; (2) Sowing transgenic T1 generation maize seeds and cultivating mature maize plants; (3) Using maize germplasm with autonomous transposon activity to perform hybrid pollination on the maize in step (2) to activate the transposition of the target gene segregation expression cassette, and identifying transposable kernels by the purple spots in the aleurone layer; (4) Collecting the purple spots in the aleurone layer that have undergone transposition as described in step (3). (5) Seeds with purple spots are sown and cultivated to obtain mature corn plants for self-pollination; (6) Seeds obtained from self-pollination in step (4) without a vector skeleton and only retaining the target gene expression cassette are collected and sown to form corn plants; (7) The pollen of the corn plants in step (5) is used to pollinate the female ears of the initial corn transformant T1 generation in step (2), and the phenotype of the ears and kernels of the plants in step (2) is observed. The appearance of purple spots on the kernels indicates that the plants in step (5) carry autonomous transposase activity; The plants in step (5) are screened using this method. The offspring of plants that do not have autonomous transposase activity and only retain the target gene expression cassette are ultra-pure transformants.

2. The method for controllable removal of exogenous carrier backbone and screening markers according to claim 1, characterized in that, The selection marker gene expression cassette is a promoter-driven selection marker gene, wherein the promoter is a 35S promoter or a Ubiquitin promoter; and the selection marker gene is a Bar, Hpt, or D-mannose isomerase gene.

3. The method for controllable removal of exogenous carrier backbone and screening markers according to claim 1, characterized in that, The autonomous transposon sequences are derived from Ac, Piggyback, Ping / Pong, or Sleepybeauty; the non-autonomous transposon sequences are derived from Ds or mPing.

4. The method for controllable removal of exogenous carrier backbone and screening markers according to claim 1, characterized in that, A target gene expression cassette consisting of a promoter, a target gene, and a terminator is further modified by adding a non-autonomous transposon Ds sequence to its outer side, thus forming a target gene segregation expression cassette.

5. The method for controllable removal of exogenous carrier backbone and screening markers according to claim 1, characterized in that, The aleurone layer-specific reporter gene is ZmC1, ZmBz1, or ZmR2.

6. The method for controllable removal of exogenous carrier backbone and screening markers according to claim 1, characterized in that, The marker gene is mCherry, DsRed, RFP, or Rubby.

7. The method for controllable removal of exogenous carrier backbone and screening markers according to claim 1, characterized in that, The trait of lacking autonomous transposase activity described in step (6) is the absence of purple spots on the seeds.

8. The method for controllable removal of exogenous carrier backbone and screening markers according to claim 1, characterized in that, The maize recipient line mentioned in step (1) is an inbred line of B73, KN5585, or B104, or a Hi II hybrid.

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