Method for visually marking transgenic soybean seeds

Abstract

The invention discloses a method for visually marking transgenic soybean seeds, which comprises the following steps: connecting the front 1000bp nucleotide sequence of a seed-specific promoter pGY1 and ATG of a glycinin gene with an RUBY report system, and inserting the seed-specific promoter pGY1 and ATG into a pMDC123 vector to construct a pMDC123-pGY1:: RUBY recombinant vector; according to the method disclosed by the invention, the transgenic soybean seeds are enabled to present a unique grey green phenotype by utilizing an RUBY report system, and the transgenic seeds can be quickly screened out only through visual inspection without the help of complex instruments and equipment, so that the screening process is greatly simplified, the screening efficiency is improved, and even personnel without professional molecular biology knowledge can also easily operate; the selected pGY1 promoter has the characteristic of seed specificity high expression, which means that the RUBY report system is only expressed in seeds and does not express in other tissues or organs of soybean plants.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant breeding, and particularly relates to a method for visual marking of transgenic soybean seeds. BACKGROUND

[0002] The transgenic technology of animals and plants is increasingly mature, but the screening of transgenic plants in the process of plant genetic transformation becomes a very important step. In the process of plant genetic transformation, a screening marker gene is used to improve the transformation efficiency or screening efficiency. Its role is to distinguish between transformed and non-transformed plant materials to screen and identify transgenic plants.

[0003] So far, many screening marker genes have been reported, which are mainly divided into two categories. One is a selection marker gene, such as an antibiotic gene and an herbicide-resistant gene, which confers corresponding resistance to transgenic plants, but this method needs to be optimized in the later stage, and different species are sensitive to different antibiotics or herbicides to different degrees. The other is a reporter gene, such as a fluorescent protein gene. Most of this type of screening gene produces visual color differences in transgenic materials with the help of a reaction substrate or corresponding equipment, but this marker gene may be mis-screened due to subjective judgment, and the use of this method sometimes needs the assistance of expensive instruments or reaction substrate reagents. Therefore, it is very important to find a good screening system.

[0004] Therefore, we propose a method for visual marking of transgenic soybean seeds. SUMMARY

[0005] The purpose of the present application is to provide a method for visual marking of transgenic soybean seeds to solve the problems in the background art.

[0006] To achieve the above purpose, the present application provides the following technical scheme: a method for visual marking of transgenic soybean seeds, comprising the following steps: step one, constructing a recombinant vector: connecting a seed-specific promoter pGY1 of a soybean globulin gene, an ATG 1000 bp nucleotide sequence and a RUBY reporter system, and inserting them into a pMDC123 vector to construct a pMDC123-pGY1::RUBY recombinant vector;

[0007] The RUBY reporter system generates betacyanin with tyrosine as a reaction substrate, so that the transgenic seeds present a red or gray-green phenotype;

[0008] Step two, transgenic operation:

[0009] The recombinant vector in step one is introduced into soybean cells by an Agrobacterium-mediated method, the Agrobacterium strain is GV3101, the bacterial liquid concentration is OD600=0.6-0.8, and the co-culture time is 2-3 days.

[0010] Step three, seed phenotype screening and identification:

[0011] Phenotype observation: Harvest transgenic soybean seeds, screen the seeds presenting gray-green phenotype;

[0012] Molecular verification: Extract the leaf DNA of the seedling after the germination of the gray-green seed, and perform PCR amplification by using the primer pair, wherein the upstream primer is designed for the pGY1 promoter sequence, and the downstream primer is designed for the RUBY reporter system sequence; the positive plant shows specific amplification bands, and the transgenic integration is verified.

[0013] Preferably, the nucleotide sequence in the step one is shown as SEQ ID NO: 1.

[0014] Preferably, the RUBY reporter system catalyzes the generation of betalain from tyrosine by expressing the dopa decarboxylase and cytochrome P450 monooxygenase genes, and the color change of the seed is observed by naked eye without relying on external instruments or reagents.

[0015] Preferably, the specific steps of the agrobacterium-mediated transformation in the step two include:

[0016] Step one: introduce the recombinant vector pMDC123-pGY1::RUBY into the agrobacterium strain, and prepare the competent cell by the liquid nitrogen freeze-thaw method;

[0017] Step two: select the soybean cotyledon node or embryo tip as the explant, co-culture with the agrobacterium bacterial liquid, and then transfer to the screening medium after the sterilization treatment, so as to induce the differentiation to form the transgenic plant.

[0018] Preferably, in the step three, the reaction system of PCR identification is as follows: 2x Taq PCR MasterMix 10 μL, 0.5 μL of each of the upstream and downstream primers, 1 μL of template DNA, and ddH2O is supplemented to 20 μL.

[0019] The reaction program is as follows: 95℃ pre-denaturation for 5min, 95℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 1min, a total of 35 cycles, and finally 72℃ extension for 10min.

[0020] Compared with the prior art, the beneficial effects of the present application are:

[0021] (1) The RUBY reporter system is used to make the transgenic soybean seed present a unique gray-green phenotype, without the aid of complex instruments and equipment, and the transgenic seed can be quickly screened by naked eye observation, which greatly simplifies the screening process and improves the screening efficiency, and even personnel without professional molecular biology knowledge can easily operate;

[0022] (2), the selected pGY1 promoter has the characteristics of high expression of seed specificity, which means that the RUBY report system will only be expressed in the seed, and will not be expressed in other tissues or organs of the soybean plant, on the one hand, unnecessary gene expression in non-seed tissues is avoided, resource waste is reduced, and the potential influence on the normal growth and development of plants is reduced, on the other hand, the accuracy of screening is further improved, only the presence of related phenotype changes in the seed represents the success of transgenic, avoiding the misjudgment of the screening result caused by the interference factors in other parts. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A schematic diagram of the transgenic soybean visual screening marker method is provided for the embodiments of the present application;

[0024] Figure 2 A pMDC123-pGY1: : RUBY recombinant vector diagram is provided for the embodiments of the present application;

[0025] Figure 3 An Arabidopsis thaliana and soybean seed phenotype observation diagram is provided for the embodiments of the present application;

[0026] Figure 4 A seedling PCR identification and phenotype diagram is provided for the embodiments of the present application. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0028] Embodiment one: please refer to Figures 1-4 The present application provides a technical solution: a transgenic soybean seed visual marker method, comprising the following steps: step one, constructing a recombinant vector: connecting the seed-specific promoter pGY1 of the soybean globulin gene, the ATG 1000bp nucleotide sequence and the RUBY report system, and inserting them into the pMDC123 vector to construct the pMDC123-pGY1: : RUBY recombinant vector;

[0029] The pGY1 promoter is connected with the RUBY report system to catalyze betalain with tyrosine as the substrate, so that the transgenic seed presents a red or gray-green phenotype;

[0030] The above expression cassette is inserted into the pMDC123 vector to construct the recombinant vector pMDC123-pGY1: : RUBY;

[0031] Step two, transgenic operation:

[0032] The recombinant vector of step one is introduced into soybean variety recipient cells by using Agrobacterium-mediated method, the Agrobacterium strain is GV3101 or EHA105, the bacterial solution concentration OD600 is 0.6-0.8, and the co-culture condition is 25℃, dark environment, and the culture time is 2-3 days;

[0033] Inducing callus and regenerating plants;

[0034] Step three, seed phenotype screening and identification:

[0035] Harvesting T0 generation plant seeds, screening seeds with gray-green phenotype (non-transgenic seeds are normal yellow) by naked eye observation;

[0036] Phenotype observation: harvesting transgenic soybean seeds, screening seeds with gray-green phenotype;

[0037] Molecular verification: extracting leaf DNA of gray-green seed germinated seedlings, using primer pair, upstream primer for pGY1 promoter sequence design, downstream primer for RUBY report system sequence design for PCR amplification, positive plants show specific amplification bands, verifying transgenic integration.

[0038] The nucleotide sequence in the step one is shown as SEQ ID NO: 1.

[0039] The RUBY report system catalyzes the generation of betalain from tyrosine by expressing dopa decarboxylase and cytochrome P450 monooxygenase genes, without relying on external instruments or reagents, and observing seed color change by naked eye.

[0040] The specific steps of Agrobacterium-mediated transformation in the step two include:

[0041] Step one: introducing the recombinant vector pMDC123-pGY1::RUBY into Agrobacterium strain, and preparing competent cells by liquid nitrogen freeze-thaw method;

[0042] Step two: selecting soybean cotyledon node or embryo tip as explant, co-culturing with Agrobacterium bacterial solution, and then transferring to screening medium after sterilization treatment to induce differentiation and form transgenic plants.

[0043] In the step three, the reaction system of PCR identification is: 2×Taq PCR Master Mix 10 μL, 0.5 μL of each upstream and downstream primer, 1 μL of template DNA, and ddH2O to make up to 20 μL;

[0044] The reaction program is: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, a total of 35 cycles, and finally 72℃ extension for 10 min.

[0045] Example 2

[0046] Construction of recombinant vector

[0047] The pGY1 promoter (1000 bp nucleotide sequence before ATG as shown in SEQ ID NO: 1) was cloned from soybean genome, connected with RUBY reporter system, inserted into pMDC123 vector, and a pMDC123-pGY1: :RUBY recombinant vector was constructed.

[0048] RUBY reporter system mechanism: through expression of dopa decarboxylase (DODA) and cytochrome P450 monooxygenase (CYPAL1), catalyzing tyrosine to betalain, making the transgenic seed present a gray-green phenotype (without instrument assistance).

[0049] Agrobacterium-mediated genetic transformation

[0050] Recipient material: soybean variety.

[0051] Agrobacterium strain GV3101, bacterial solution concentration OD600=0.7, introduced into recombinant vector by liquid nitrogen freeze-thaw method.

[0052] Transformation steps:

[0053] Co-culture of explants and Agrobacterium for 2 days (25°C, dark);

[0054] After degerming treatment, transfer to screening medium to induce callus and regenerate plants.

[0055] Seed phenotype screening and molecular verification

[0056] Phenotype observation: harvest T1 generation seeds, screen gray-green seeds (non-transgenic seeds are yellow), as shown in Figure 3 .

[0057] PCR identification:

[0058] Reaction system: 2x Taq PCR Master Mix 10 μL, primers 0.5 μL each, template DNA 1 μL, ddH2O to 20 μL.

[0059] Program: 95°C pre-denaturation for 5 min, 35 cycles (95°C for 30 s, 58°C for 30 s, 72°C for 1 min), 72°C extension for 10 min.

[0060] Results: 850 bp specific bands were amplified from the DNA of gray-green seedlings, consistent with Figure 4 electrophoresis results, verifying transgene integration.

[0061] Example 3

[0062] Recombinant vector and transformation system

[0063] The same pMDC123-pGY1::RUBY recombinant vector as in Example 1 was used.

[0064] Transformation method: floral infection method (Agrobacterium strain EHA105, OD600=0.6) was used to directly infect flowering plants of Arabidopsis thaliana without tissue culture.

[0065] Seed phenotype screening and verification

[0066] Phenotype observation: T1 generation seeds were harvested, and transgenic positive seeds were red, and non-transgenic seeds were normal brown, as shown in Figure 1. Figure 3

[0067] PCR identification:

[0068] The primer pair and reaction system were the same as in Example 1.

[0069] Results: The 850bp band was amplified from the DNA of the red seed seedlings, proving that the pGY1::RUBY expression cassette was successfully integrated.

[0070] Table 1 is a comparison and commonality of Example 2 and Example 3:

[0071]

[0072] The seed-specific promoter pGY1 of the soybean globulin gene is selected, and the 1000bp nucleotide sequence before the ATG has the characteristics of high expression in seeds. After being connected with the RUBY report system and inserted into the pMDC123 vector to construct a recombinant vector, the promoter is a DNA sequence recognized, combined and started by RNA polymerase. It is like a "switch" that determines the starting position and time of gene expression. The pGY1 promoter can accurately play a role in the development process of soybean seeds and start the transcription process of the RUBY report system connected thereto. The RUBY report system contains dopa decarboxylase and cytochrome P450 monooxygenase genes. After receiving the "start" signal from the promoter, these genes begin to express corresponding enzymes. These enzymes use tyrosine as a reaction substrate and generate betalain through a series of catalytic reactions. This substance can make the seeds present a red phenotype, thereby providing an intuitive visual marker for screening of transgenic seeds.

[0073] ​The agrobacterium strain GV3101 is selected as a carrier, and the constructed recombinant vector is introduced into the agrobacterium. The agrobacterium is a natural gene transfer tool, and the T-DNA (transferable DNA) on the Ti plasmid of the agrobacterium can be transferred and integrated into the plant genome under specific conditions. During the co-culture of the prepared agrobacterium liquid containing the recombinant vector (the concentration of the agrobacterium liquid is controlled at 0.6-0.8, and the activity of the agrobacterium is suitable at this concentration, which is conducive to subsequent transformation operation) and soybean explants (such as cotyledon nodes or embryo tips) for 2-3 days, the agrobacterium will adhere to the surface of the explant cells, and the T-DNA (including pGY1 promoter and RUBY reporter system elements) on the recombinant vector will be transferred to the soybean cells. After degerming treatment, the explants are transferred to the screening medium, and only the soybean cells successfully integrated with the recombinant vector can survive and induce differentiation to form transgenic plants on the screening medium. The cells that are not successfully integrated cannot grow due to lack of resistance.

[0074] After the transgenic soybean seeds are harvested, phenotype observation is performed. Since the RUBY reporter system expresses betalain in the seeds, the transgenic seeds will exhibit a different gray-green phenotype from non-transgenic seeds. The seeds can be preliminarily screened by naked eye. In order to further confirm that the screened seeds are indeed successfully transgenic seeds, molecular verification is required. The DNA of the gray-green seed seedlings is extracted after germination, and the upstream and downstream primers are designed according to the pGY1 promoter sequence and the RUBY reporter system sequence for PCR amplification. PCR (polymerase chain reaction) is a molecular biology technique for amplifying and expanding specific DNA fragments. If the seeds are successfully transgenic, the pGY1-RUBY sequence is integrated into the genome. In the PCR reaction, the primers will bind to the corresponding sequence on the template DNA. After multiple cycles of denaturation, annealing, and extension, specific DNA bands can be amplified. The amplification products are detected by agarose gel electrophoresis and other methods. If the expected size band appears, it indicates that the transgene has been successfully integrated into the soybean genome.

[0075]

[0076] Figure 2 In the method:

[0077] The RB T-DNA repeat is a right border repeat sequence in the transgenic vector designed to improve transformation efficiency and stability. The core function is to ensure the precise transfer of the target gene (pGY1::RUBY) to the plant genome.

[0078] The expression of the GY1 promoter in the visual marker of the transgenic soybean seed means that the GY1 promoter is used to drive the expression of the reporter gene, thereby producing a visual marker in the transgenic soybean seed.

[0079] Expression of CYP76AD1 in visual marker of transgenic soybean seeds means that the gene is used as a reporter gene, and the fluorescent protein produced by its expression is used to visualize the transgenic soybean seed marker;

[0080] DODA is a reporter gene as RUBY gene (also known as doda) in visual marker of transgenic soybean seeds, used for rapid screening of transgenic soybean plants;

[0081] Glucosyltransferase is a core functional component of the RUBY reporter system, which enhances the stability and visibility of the red phenotype by chemically modifying (glycosylating) betalain precursor molecules, ensuring a stable, prominent and easy-to-screen red phenotype;

[0082] lac operator is a gene expression regulatory element, usually used in combination with lac repressor (LacI), for precise control of the opening or closing of reporter genes (such as the RUBY system). Its core role is to dynamically regulate the expression of marker genes through inducers (such as IPTG), thereby improving the specificity or flexibility of screening;

[0083] CAP binding site is a gene expression regulatory element, usually working with CAP (Catabolite Activator Protein) of prokaryotes, to achieve fine regulation of visual marker genes (such as the RUBY system) by enhancing promoter activity or responding to specific metabolic signals. Its core significance lies in improving the sensitivity and specificity of the marker system through metabolic signal coupling or expression enhancement;

[0084] CaMV 35S promoter (enhanced) is a widely used constitutive strong promoter, whose core role is to drive the continuous and efficient expression of marker genes (such as the RUBY system), ensuring the rapid and prominent generation of visual signals (such as red pigments) in seeds. Its design significance lies in breaking through the limitations of tissue specificity or compensating for the low efficiency of weak promoters;

[0085] Basta resistance is a key selection marker system, whose core role is to screen successfully transformed transgenic seeds or plants, while it can also work with visual markers (such as the RUBY system) to achieve a double verification mechanism;

[0086] CaMV poly(A) signal (CaMV poly(A) signal) is a key transcription termination regulatory element, whose core function is to ensure efficient processing, stable existence and completion of translation of mRNA of marker genes (such as RUBY system), so as to improve the intensity and consistency of visual signals (such as pigment accumulation);

[0087] LB T-DNA repeat (LB T-DNA repeat) is a key element in the process of Agrobacterium-mediated transformation, and its core function is to define the cutting and integration boundary of T-DNA, so as to ensure the accurate insertion of target genes (such as visual marker gene RUBY or resistance gene bar / pat) into plant genome;

[0088] KanR (KanR) is a classic antibiotic selection marker, mainly used for screening of transformed cells successfully introducing exogenous genes (such as visual marker gene RUBY). Its core function is to distinguish transformed and non-transformed materials by conferring kanamycin resistance to transgenic individuals;

[0089] ori (origin of replication) is a core element in plasmid vector for regulating DNA replication, and its core function is to ensure efficient replication and amplification of exogenous gene vector (such as plasmid carrying visual marker gene RUBY) in host bacteria (such as E. coli or Agrobacterium);

[0090] BOM (basis of mobility site) usually refers to the origin of transfer (oriT) on plasmid vector, and its core function is to mediate the transfer of plasmid from E. coli to Agrobacterium by bacterial conjugation, so as to ensure that the vector carrying target genes (such as visual marker gene RUBY) efficiently enters Agrobacterium, and then completes plant transformation;

[0091] pVS1 oriV is the origin of replication in plasmid vector, and its core function is to regulate the autonomous replication of plasmid in host bacteria (such as Agrobacterium or E. coli), so as to ensure efficient amplification of the vector carrying target genes (such as visual marker gene RUBY) in bacteria, and provide sufficient DNA material for subsequent plant transformation;

[0092] pVS1 RepA is a key protein in plasmid vector for regulating replication initiation, and its core function is to bind to the origin of replication (oriV) and activate the autonomous replication of plasmid, so as to ensure stable amplification of the vector carrying visual marker genes (such as RUBY) in Agrobacterium or other hosts;

[0093] pVS1 StaA is a key gene in plasmid vector that regulates the stability of plasmid. Its core function is to ensure that the vector carrying visual marker genes (such as RUBY) can exist stably in the host bacteria (such as Agrobacterium) for a long time, avoiding the decrease of transformation efficiency or failure of marker gene expression caused by plasmid loss.

[0094] RB T-DNA repeat: right border T-DNA repeat sequence

[0095] GY1 promoter: GY1 promoter

[0096] M13 fwd: M13 forward primer

[0097] pVS1 StaA: pVS1 site-specific recombinase A

[0098] pVS1 RepA: pVS1 replication protein A

[0099] No. 15 vd: 15th variable region

[0100] bom: bom site

[0101] ori: origin of replication

[0102] Kanr: kanamycin resistance gene

[0103] CaMV poly(A) signal: cauliflower mosaic virus polyadenylation signal

[0104] Basta resistance: glufosinate ammonium resistance

[0105] LB T-DNA repeat: left border T-DNA repeat sequence

[0106] M13 rev: M13 reverse primer

[0107] lac promoter: lactose operon promoter

[0108] nos terminator: nopaline synthase terminator

[0109] 15S 8.18S: 15S and 8.18S fragments that may be related to ribosomal RNA Glucosyltransferase: glucosyltransferase

[0110] lac operator: lactose operon operator

[0111] CAP binding site: catabolite gene activator protein binding site

[0112] CaMV 35S promoter (enhanced): (enhanced) Cauliflower Mosaic Virus 35S promoter

[0113] CYP79AD1 : Cytochrome P450 79AD1 (a cytochrome P450 protein)

[0114] P2A: P2A peptide (a self-cleaving peptide)

[0115] DODA: dopa decarboxylase.

[0116] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are only by way of example and that changes, modifications, substitutions and alterations can be made thereto without departing from the spirit and scope of the application as defined in the following claims, in which:

Claims

1. A method for visual labeling of genetically modified soybean seeds, characterized in that, Includes the following steps: Step 1: Constructing a recombination vector: The seed-specific promoter pGY1 of the soybean globulin gene, the first 1000 bp of the ATG nucleotide sequence, was linked to the RUBY reporter system and inserted into the pMDC123 vector to construct the pMDC123-pGY1::RUBY recombinant vector. The RUBY reporter system uses tyrosine as a reaction substrate to generate betalains, which causes the transgenic seeds to exhibit a red or gray-green phenotype. Step 2, Transgenic Operation: The recombinant vector described in step one was introduced into soybean cells using the Agrobacterium-mediated transformation method. The Agrobacterium strain was GV3101, the bacterial concentration was OD600 = 0.6-0.8, and the co-culture time was 2-3 days. Step 3: Seed phenotypic screening and identification: Phenotypic observation: Harvest transgenic soybean seeds and screen for seeds that exhibit a gray-green phenotype; Molecular verification: DNA was extracted from the leaves of seedlings after the gray-green seeds germinated. PCR amplification was performed using primers designed with the upstream primer targeting the pGY1 promoter sequence and the downstream primer targeting the RUBY reporter system sequence. Positive plants showed specific amplified bands, verifying transgene integration.

2. The method for visual labeling of genetically modified soybean seeds as described in claim 1, characterized in that, The nucleotide sequence in step one is shown in SEQ ID NO:

1.

3. The method for visual labeling of genetically modified soybean seeds as described in claim 1, characterized in that, The RUBY reporter system catalyzes the production of betalains from tyrosine by expressing dopa decarboxylase and cytochrome P450 monooxygenase genes.

4. The method for visual labeling of genetically modified soybean seeds as described in claim 1, characterized in that, The specific steps of Agrobacterium-mediated transformation in step two include: Step 1: The recombinant vector pMDC123-pGY1::RUBY was introduced into Agrobacterium strain, and competent cells were prepared by liquid nitrogen freeze-thaw method; Step 2: Select soybean cotyledon nodes or embryo tips as explants, co-culture them with Agrobacterium tumefaciens solution, and then transfer them to a selection medium after sterilization treatment to induce differentiation into transgenic plants.

5. The method for visual labeling of genetically modified soybean seeds as described in claim 1, characterized in that, In step three, the PCR identification reaction system is as follows: 10 μL of 2×TaqPCRMasterMix, 0.5 μL each of upstream and downstream primers, 1 μL of template DNA, and ddH2O to a final volume of 20 μL. The reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles, and finally 72℃ extension for 10 min.

Images