A method for constructing and detecting a genetic resource ownership marker of a plant

Abstract

The application belongs to the technical field of plant genetic resource protection, gene editing and molecular tracing, and discloses a plant genetic resource ownership marking construction and detection method based on nano delivery and guided gene editing. The method first selects a repeated sequence region and / or a non-coding safe insertion region in a plant genome as a target insertion region, designs an exogenous ownership identification sequence with a length of 15-20 bp, constructs a guided gene editing system containing a guide RNA and an editing protein, and forms a nano delivery complex by using a plant suitable nano delivery carrier, so as to realize site-specific insertion of the exogenous ownership identification sequence at the target site. Then, the marking is identified through specific amplification, sequencing and nanopore detection, and its stable inheritance is verified. The application has the advantages of high delivery efficiency, little influence on plant growth, stable and heritable marking, convenient detection and the like, and can be used for the right protection, tracing and protection of rare plants, wild close relatives, Chinese medicinal materials and other important plant genetic resources.

Description

Technical Field

[0001] This invention relates to the fields of biological genetic resource protection, plant molecular breeding, gene editing and molecular traceability, and specifically to a method for introducing a guide gene editing system into plant tissue using a nanodelivery carrier, implanting exogenous ownership identifier sequences at predetermined sites in the plant genome, and verifying and detecting the intergenerational stability of the ownership identifier sequences. Background Technology

[0002] Plant genetic resources are a crucial foundation for national biosecurity and germplasm innovation. Existing plant genetic resource conservation technologies mainly focus on morphological identification, DNA barcoding, or conventional molecular marker analysis. However, most of these technologies can only be used for species identification or phylogenetic analysis, and are insufficient to address issues such as "confirmation of resource ownership," "in situ marking in living organisms," "stable intergenerational transmission," and "rapid end-stage detection."

[0003] Especially for easily lost plant resources such as rare wild plants, wild rice, and medicinal herbs, existing technologies have the following shortcomings: First, plant cell walls form a natural barrier to the delivery of exogenous macromolecules, resulting in low efficiency and narrow applicability of traditional transformation systems; second, existing molecular markers are usually "recognition markers" rather than "identification markers," and cannot achieve active coding; third, for materials used in deep processing or ex-situ propagation, there is a lack of stable detection methods that can be used for chain-based monitoring. The document you provided also clearly states that it is necessary to solve the problems of plant cell wall penetration, implanting 15-20 bp exogenous markers in the "hidden regions" of the genome, achieving in-situ encryption of root tip meristems through spray transfection, and constructing a closed-loop system through multi-generation stability and nanopore detection.

[0004] Therefore, there is an urgent need to provide a method for constructing and detecting plant genetic resource ownership markers that can take into account delivery efficiency, site-specific insertion, plant biological neutrality, and rapid detection. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing and detecting plant genetic resource ownership markers based on nanodelivery and guided gene editing, in order to solve the following problems existing in the prior art: Exogenous editing components have difficulty efficiently crossing plant cell walls; Ownership markers are difficult to implant stably and at specific sites in the plant genome; Implanting markers can easily affect the normal growth and development of plants. There is a lack of rapid and accurate testing systems suitable for regulatory scenarios.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for constructing ownership markers for plant genetic resources includes the following steps: Select a target insertion region in the plant genome, wherein the target insertion region is a repetitive sequence region and / or a non-coding safe insertion region that does not affect the expression of important agronomic traits of the plant; Design an exogenous ownership identifier sequence, the exogenous ownership identifier sequence being 15–20 bp in length, as the core information carrier of plant genetic resource ownership markers; A guided gene editing system is constructed, wherein the guided gene editing system includes at least a guide RNA that targets the target insertion region, and an editing protein with cutting and site-specific insertion functions; preferably, the editing protein is a fusion editing protein formed by Cas protein and reverse transcriptase; The guided gene editing system is loaded with a plant-compatible nanodelivery carrier to form a nanodelivery complex; the nanodelivery carrier is preferably a single-walled carbon nanotube, a multi-walled carbon nanotube, or a combination thereof. The nanodelivery complex is delivered to living plant tissues, preferably to root tip meristems, shoot tip meristems, young leaf tissues, or callus tissues; the preferred delivery methods are spray transfection, local dripping, infiltration, or injection. The guided gene editing system is used to act within plant cells to insert the exogenous ownership marker sequence at a specific point in the target insertion region, thereby obtaining plant material with ownership markers. The physiological and ecological effects of the obtained plant materials were evaluated to confirm that the introduction of the ownership markers did not significantly affect plant growth, reproduction, and expression of target traits. The plant material was subjected to intergenerational transmission verification and molecular detection to confirm that the ownership identifier sequence can be stably inherited with cell division and generational transmission.

[0007] Furthermore, the above molecular detection can be performed using specific PCR amplification, sequencing verification, transmethylase-assisted labeling detection, and / or nanopore sequencing detection.

[0008] Compared with the prior art, the present invention has at least the following beneficial effects: High delivery efficiency: The nano-delivery carrier enhances the ability of exogenous editing components to cross plant cell walls and cell membranes, reducing the dependence of traditional transformation methods on tissue culture systems; More stable markers: By guiding gene editing, ownership marker sequences are inserted into a predetermined region of the plant genome, achieving heritable molecular watermarking; Better biological neutrality: By selecting repetitive sequence regions or safe insertion regions, the impact on normal plant growth and development and major traits is reduced; Highly adaptable to various applications: Through PCR, transmethylase-assisted detection, and nanopore detection, both laboratory testing and rapid on-site identification can be achieved. Suitable for regulatory applications: It can be used for the ownership marking, traceability and protection against loss of rare plants, wild closely related species, special Chinese medicinal materials and other important plant genetic resources. Attached Figure Description

[0009] Figure 1 This is a schematic diagram of the overall process of the method for constructing plant genetic resource ownership markers according to the present invention.

[0010] Figure 2 This is a schematic diagram illustrating the nanodelivery complex crossing the plant cell wall and entering the cell nucleus.

[0011] Figure 3 A schematic diagram illustrating the targeted insertion of exogenous ownership identifier sequences into the target insertion region of the plant genome.

[0012] Figure 4 A schematic diagram of the testing and stability verification process for plant materials with ownership markings. Detailed Implementation

[0013] The present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0014] One or both of single-walled carbon nanotubes and multi-walled carbon nanotubes are selected as nanodelivery carriers. The nanodelivery carriers are dispersed in plant compatibility buffer, and then a guided gene editing system is added to form a stable complex. The guided gene editing system includes guide RNA targeting the insertion region and a fusion protein formed by Cas protein and reverse transcriptase.

[0015] The loading performance of the nanodelivery complex was evaluated by particle size analysis, surface potential analysis, and gel retardation experiments; its biocompatibility was evaluated by plant tissue viability assays; and nanodelivery formulations with both high delivery efficiency and low toxicity were selected for subsequent experiments.

[0016] Based on the genomic information of the target plant, repetitive sequence regions or non-coding safe insertion regions are selected as insertion sites. A 15–20 bp exogenous ownership identifier sequence is designed, which is not completely identical to the natural sequence of the target plant genome, and is used to represent specific origin or ownership information.

[0017] After the guided gene editing system is loaded into a nanodelivery vector, it is applied to plant tissues. Once inside the cell nucleus, the editing system recognizes the target insertion region under the guidance of guide RNA, and, with the synergistic action of Cas protein and reverse transcriptase, inserts the exogenous ownership marker sequence into the plant genome at a specific site, obtaining plant material with ownership markers.

[0018] The above-mentioned nanodelivery complex was used to treat plant root tip meristems, shoot tip meristems, or young tissues by spraying, local dripping, or immersion to achieve in-situ densification of living plant tissues.

[0019] After treatment, the experimental group and the control group were compared under the same culture conditions, and plant height, root length, leaf area, chlorophyll content, flowering and fruiting, and major phenotypic traits were measured. If the plant material with the ownership mark showed no significant abnormalities compared with the untreated control, the mark implantation was considered biologically neutral.

[0020] Plant materials bearing ownership markers were propagated to the next generation or multiple generations. Nucleic acids were extracted from parental and progeny plants, and PCR amplification was performed using specific primers located on both sides of the target insertion region. The amplified products were sequenced to confirm the presence of the exogenous ownership marker sequence and the consistency of the insertion site.

[0021] Furthermore, transmethylase-assisted labeling can be used to enrich or identify the target region, and nanopore sequencing platforms can be combined to quickly read and determine the ownership marker. If consistent exogenous ownership marker sequences are detected across consecutive generations, it indicates that the ownership marker is stably inherited.

[0022] This invention can be applied to wild rice, rare Chinese medicinal herbs, germplasm resources of distinctive local plants, and other plant genetic resources that require key protection. By constructing ownership markers for parental materials or original core populations, it is possible to achieve source identification and traceability management of propagation materials, living plants, and their subsequent utilization.

Claims

1. A method for constructing ownership markers for plant genetic resources, characterized in that, Includes the following steps: (1) Select a target insertion region in the plant genome, wherein the target insertion region is a repetitive sequence region and / or a non-coding safe insertion region that does not affect the expression of important plant traits; (2) Design a foreign ownership identifier sequence with a length of 15–20 bp; (3) Construct a guided gene editing system, wherein the guided gene editing system includes a guide RNA that targets the target insertion region and an editing protein with site-specific editing function; (4) The guided gene editing system was loaded onto a plant-compatible nanodelivery carrier to form a nanodelivery complex; (5) The nanodelivery complex is delivered to living plant tissue, and the exogenous ownership identifier sequence is inserted into the target insertion region to obtain plant material with ownership mark; (6) The plant material is subjected to intergenerational stability verification and molecular detection to confirm that the exogenous ownership marker sequence is stable and can be genetically transmitted.

2. The method according to claim 1, characterized in that, The nanodelivery carrier is a single-walled carbon nanotube, a multi-walled carbon nanotube, or a combination of both.

3. The method according to claim 1, characterized in that, The editing protein is a fusion protein formed by Cas protein and reverse transcriptase.

4. The method according to claim 1, characterized in that, The exogenous ownership identifier sequence is not completely identical to the natural sequence of the target plant genome.

5. The method according to claim 1, characterized in that, The living plant tissue is root tip meristem, shoot tip meristem, young leaf tissue, or callus tissue; the delivery method is one or more of spray transfection, local drip application, infiltration, or injection.

6. A method for detecting plant genetic resource ownership markers obtained by the method according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Extract nucleic acids from the plant material to be tested; (2) Specific amplification and / or sequencing of the sequences on both sides of the target interposition region; (3) Determine whether the plant material to be tested has an ownership mark based on whether the exogenous ownership mark sequence is detected.

7. The detection method according to claim 6, characterized in that, The detection also includes transmethylase-assisted labeling of the target region, followed by reading using nanopore sequencing.

8. A kit for implementing the detection method of claim 6 or 7, characterized in that, include: (1) Primers and / or probes designed for both sides of the target insertion region; (2) Transmethylase reagent for labeling target regions; (3) Reagents or consumables used for nanopore sequencing library preparation and detection; (4) Data analysis module for outputting ownership identification results.

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