Genetic transformation visual marking method based on yellow leaf phenotype

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a genetic transformation visual marking method based on yellow leaf phenotypes, the method is based on the functions of forward regulation and control of leaf yellowing of a Cla97C04G068470 gene and negative regulation and control of leaf yellowing of a Cla97C04G068530 gene, visual screening of transgenic plants is realized by constructing two types of recombinant vectors, and the genetic transformation visual marking method based on the yellow leaf phenotypes has the advantages that the genetic transformation visual marking method is high in genetic transformation efficiency and high in yield. The recombinant vector is introduced into an agrobacterium GV3101 strain, a prepared bacterial solution infects leaves of a target plant, and after culture, a leaf infected area of a transgenic positive plant shows an obvious yellow phenotype, and can be quickly identified by naked eyes without depending on a complex instrument. The method is simple and convenient to operate, high in screening efficiency and low in cost, has wide applicability in various plants, and provides a reliable solution for efficient identification of transgenic plants.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a genetic transformation visualization marker method based on the yellow leaf phenotype. Background Technology

[0002] While plant transgenic technology has become increasingly sophisticated, the screening of transgenic plants has become a crucial step in the process of plant genetic transformation. Screening marker genes are used in plant genetic transformation to improve transformation or screening efficiency. Their role is to distinguish between transformed and non-transformed plant materials, thereby screening and identifying transgenic plants.

[0003] To date, existing screening marker genes are mainly divided into two categories. One category consists of selection marker genes, such as antibiotic resistance genes and herbicide resistance genes, which confer corresponding resistance to transgenic plants. However, this method requires subsequent optimization, as different species have different sensitivities to different antibiotics or herbicides. The other category consists of reporter genes, such as fluorescent protein genes. These screening genes mostly rely on reaction substrates or corresponding equipment to predict visual color differences in transgenic materials. However, these marker genes can sometimes be misselected due to subjective judgment, and the use of this method sometimes requires expensive instruments or reaction substrate reagents, and lacks intuitive visual evaluation methods. Therefore, finding a good screening system is very important. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a genetic transformation visualization marker method based on the yellow leaf phenotype. Utilizing key genes controlling yellow leaf vein formation as screening markers, the method enables naked-eye screening and identification of transgenic plants through intuitive changes in leaf color. This provides a convenient and intuitive solution for transgenic plant identification, significantly improving the efficiency and practicality of the screening process while reducing screening costs.

[0005] To achieve the above objectives, this invention provides a genetic transformation visualization marker method based on the yellow leaf phenotype, comprising the following steps: 1) Construction of overexpression vector recombinant plasmid: using enzyme digestion reagents to... Cla97C04G068470 The CDS sequence of the gene was cloned into the pCAMBIA2300 vector, the vector was transformed into E. coli, and positive E. coli were screened by PCR and sequencing. Then, the recombinant plasmid of the overexpression vector pCAMBIA2300-470 was extracted. 2) Constructing a silencing vector recombinant plasmid: Cla97C04G068530 Gene or Cla97C04G068530 After the homologous gene was digested with enzymes, it was inserted into the TRV vector. The vector was then transformed into E. coli, and positive E. coli were screened by PCR and sequencing. Subsequently, the recombinant plasmid of the silencing vector TRV-530 was extracted. 3) Introduction: The recombinant plasmid from step 1) or step 2) is introduced into the Agrobacterium strain; 4) Preparation of bacterial suspension: The strain described in step 3) is cultured, centrifuged, and resuspended to obtain bacterial suspension; 5) Infection and screening: Infect the target plant leaves with the bacterial solution described in step 4), and culture the infected target plants. After culture, observe the leaf phenotype with the naked eye. Plants with yellow leaf infection areas are transgenic positive plants.

[0006] Furthermore, in step 1), the enzyme digestion reagents are BamHI and SaII enzymes; the Escherichia coli is Escherichia coli DH5α.

[0007] Furthermore, in step 2), the enzyme digestion reagent is BamHI enzyme; the Escherichia coli is Escherichia coli DH5α.

[0008] Furthermore, in step 1) Cla97C04G068470 The CDS sequence of the gene is shown in SEQ ID NO.1; in step 2) Cla97C04G068530 The CDS sequence of the gene is shown in SEQ ID NO.2.

[0009] Furthermore, in step 3), the Agrobacterium strain is GV3101.

[0010] Further, in step 4), the culture was carried out using LB liquid medium containing kanamycin; the centrifugation was performed at 5000 rpm for 10 min; the resuspending osmosis buffer consisted of 10 mM MES, 10 mmol / L MgCl2, 200 μM acetylsylcholine, and a pH of 5-6; the bacterial culture OD... 600 It ranges from 0.6 to 1.

[0011] Furthermore, in step 5), the bacterial solution is injected into the target plant leaves using a needle-free syringe. After infection, the target plant is first cultured in the dark for 1-2 days, and then restored to normal growth conditions for continued culture.

[0012] Compared with the prior art, the present invention has the following advantages and technical effects: 1) This invention features visual screening, is easy to operate, does not rely on expensive instruments or reaction substrates, and can quickly identify transgenic plants simply by observing changes in leaf color with the naked eye; 2) This invention has high screening efficiency and short cycle. The phenotype of watermelon transgenic plants can be observed within one week, and that of tomatoes and tobacco can be observed within two weeks. The yellow phenotype of tomatoes lasts for more than a month, which significantly shortens the screening cycle. 3) This invention is low in cost and environmentally friendly, avoiding the environmental risks and species compatibility issues caused by selection markers such as antibiotics and herbicides. At the same time, it does not require additional investment in instruments, equipment and reagents. 4) This invention has wide applicability and has been successfully verified in watermelon, tomato, and tobacco, indicating that this method has broad application potential in different plant species; 5) The markers of this invention have high reliability. By using two strategies—positive regulation of gene overexpression and negative regulation of gene silencing—to achieve yellow phenotype markers, the uncertainty caused by a single strategy is reduced, and the probability of false screening is lowered.

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 The image shows a phenotypic observation of watermelon leaves with the pCAMBIA2300 vector in the example. In the image, A represents pCAMBIA2300-unloaded and B represents pCAMBIA2300-470. Figure 2 The image shows a phenotypic observation of watermelon leaves with TRV vectors in the embodiment. In the image, A represents TRV-unloaded and B represents TRV-530. Figure 3 The image shows a phenotypic observation of tomato leaves with the pCAMBIA2300 vector in the example. In the image, A represents pCAMBIA2300-unloaded and B represents pCAMBIA2300-470. Figure 4 This is a phenotypic observation diagram of tomato leaves with TRV vector in the example. In the diagram, A represents TRV-unloaded and B represents TRVSolyc-530. Figure 5 The image shows a phenotypic observation of tobacco leaves with the pCAMBIA2300 vector in the example. In the image, A represents pCAMBIA2300-unloaded and B represents pCAMBIA2300-470. Figure 6 Phenotypic observation of tobacco leaves with TRV vector in the embodiment, where A represents TRV-unloaded and B represents TRVCHLH-530. Detailed Implementation

[0015] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0016] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0017] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0018] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0019] LB liquid medium containing kanamycin (Kan): 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, and deionized water to a final volume of 1 L. A 50 mg / mL Kan solution is typically prepared (dissolved in sterile water). The commonly used final concentration is 50 μg / mL when added to LB (i.e., to prepare 1 L of LB liquid medium, sterilize and cool to approximately 50°C or below, add 1 mL of 50 mg / mL kanamycin stock solution, mix well, and use).

[0020] Example 1 1.1 Construction of overexpression vectors.

[0021] Using BamHI and SaII enzyme digestion to Cla97C04G068470 The CDS was cloned into the pCAMBIA2300 vector, and PCR amplification was performed using primers with homologous arms (primers 470-F and 470-R are shown in Table 1). The constructed vector was named pCAMBIA2300-470. The vector was transferred into DH5α Escherichia coli, and positive clones were screened by PCR and sequencing. The bacterial culture was stored at -80℃.

[0022] Recombinant plasmids of the above positive strains were extracted, and pCAMBIA2300-470 and pCAMBIA2300-empty vectors were introduced into Agrobacterium GV3101 strain (Shanghai Weidi Biotechnology Co., Ltd.).

[0023] The method for extracting recombinant plasmids from positive bacterial strains is as follows: 1) Column equilibration: Add 200 μl of YS Buffer to the adsorption column that has been loaded into the collection tube, centrifuge at 13000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the tube (Note: the adsorption column must be processed and used on the same day).

[0024] 2) Take 1-5 ml of overnight cultured E. coli solution and add it to a centrifuge tube (provided by yourself). Centrifuge at 12000 rpm for 30 seconds to collect the bacterial pellet (the operation can be repeated to collect the bacterial pellet). Discard the supernatant as much as possible.

[0025] 3) Add 200 μl of colorless PA Buffer (confirm that RNase A has been added) to the tube, mix thoroughly with a pipette or vortex mixer, and suspend the bacterial pellet.

[0026] 4) Add 250 μl of blue PB Buffer to the centrifuge tube, gently invert and mix 8-10 times to fully lyse the bacteria and form a clear, viscous blue solution, indicating complete lysis. Let stand at room temperature for no more than 5 minutes.

[0027] 5) Add 350 μl of yellow PM2 Buffer to the centrifuge tube and immediately invert it 10 times to mix. The solution color will change completely from blue to yellow and yellowish-white flocculent or lumpy precipitate will appear, indicating that it is mixed evenly and neutralized completely. Let it stand at room temperature for 2 minutes.

[0028] 6) Centrifuge at 12000 rpm for 5 min, then transfer the solution to the adsorption column already loaded into the collection tube. Centrifuge at 12000 rpm for 30 s, discard the waste liquid in the tube, and return the adsorption column to the collection tube.

[0029] 7) Add 600 μl of PW Buffer to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 min, discard the waste liquid in the tube, and put the adsorption column back into the collection tube. Repeat this step once.

[0030] 8) Discard the waste liquid in the tube and put the adsorption column back into the collection tube. Repeat this step once.

[0031] 9) Place the adsorption column back into the collection tube, centrifuge at 12000 rpm for 2 min, and discard the waste liquid.

[0032] 10) Place the adsorption column in a new centrifuge tube, add 30~100μl of YEBBuffer to the middle of the adsorption membrane, incubate at room temperature for 3 min, centrifuge at 12000rpm for 2 min, collect the plasmid DNA solution, and store at -20℃ for a long time.

[0033] Cla97C04G068470The CDS sequence (SEQ ID NO.1):

[0034] 1.2 Construction of the silent carrier.

[0035] 1) Construction of the watermelon silencing vector: Avoid Cla97C04G068530 Based on the conserved sequence of the gene, specific primers (primers 530-F and 530-R, as shown in Table 1) were designed to amplify the target fragment using watermelon leaf cDNA as a template. The fragment was digested with BamHI and then inserted into the TRV vector. This vector was transformed into DH5α *E. coli*, and positive strains were screened by PCR and sequencing. The pTRV2-530 recombinant plasmid was extracted and constructed. The TRV2-530, TRV2 empty vector, and TRV1 helper plasmid were transformed into *Agrobacterium* GV3101 cells, respectively, to obtain positive single clones.

[0036] The virus-induced gene silencing (VIGS) assay used vectors TRV1 and TRV2. TRV1 was a helper plasmid. TRV2 had multiple cloning sites for inserting foreign genes. TRV1 played a helper role in the TRV system, enhancing the replication and spread of TRV2.

[0037] Cla97C04G068530

[0038] 2) Construction of the tomato silencing vector: Find tomatoes on the Ensembl Plants online website (https: / / plants.ensembl.org / index.html). Cla97C04G068530 The homologous gene Solyc04g015750.3.1 was used. Based on the conserved sequence of the Solyc04g015750.3.1 gene, specific primers (primers Solyc530-F and Solyc530-R are shown in Table 1) were designed. The target fragment was amplified using tomato leaf cDNA as a template. After digestion with BamHI, the fragment was inserted into the TRV vector. The vector was then transformed into DH5α Escherichia coli. Positive strains were screened by PCR and sequencing. The TRV2-sloyc530 recombinant plasmid was extracted and constructed.

[0039] 3) Construction of tobacco silencing carriers: Find tobacco on the Ensembl Plants online website (https: / / plants.ensembl.org / index.html). Cla97C04G068530 homologous genes CHLH (XM_019394869.1), according to CHLH Specific primers (primers CHLH530-F and CHLH530-R are shown in Table 1) were designed based on the conserved sequence of the gene. The target fragment was amplified using tobacco leaf cDNA as a template. After digestion with BamHI, the fragment was inserted into the TRV vector. The vector was then transformed into DH5α Escherichia coli. Positive strains were screened by PCR and sequencing. The TRV2-CHLH530 recombinant plasmid was extracted and constructed.

[0040] Table 1 Primer Sequences

[0041] 1.3 Genetic transformation.

[0042] 1) Watermelon genetic transformation: Agrobacterium GV3101 strains pCAMBIA2300-470, pCAMBIA2300-empty vector, TRV2-530, TRV2-empty vector, and TRV1-empty vector were amplified in LB broth containing kanamycin. Agrobacterium GV3101 strains were collected by centrifugation (5000 rpm, 10 min) and resuspended in osmotic buffer (10 mM MES, pH 5.6, 10 mM MgCl2, 100 μM acetylsylcholine) to achieve a final OD concentration. 600When the concentration of *Agrobacterium* GV3101 was 0.6–0.8, the TRV1 helper plasmid was mixed 1:1 with TRV2-530 and TRV2 empty vectors, respectively. Then, suspensions of *Agrobacterium* GV3101 under different treatments were injected into watermelon leaves at the four-leaf-one-heart stage using a needle-free injector. After injection, the leaves were incubated in the dark at 23°C for one week.

[0043] 2) Genetic transformation of tomatoes: Agrobacterium GV3101 strains pCAMBIA2300-470, pCAMBIA2300-empty vector, TRV2-530, TRV2-empty vector, and TRV1-empty vector were amplified in LB broth containing kanamycin. Agrobacterium GV3101 strains were collected by centrifugation (5000 rpm, 10 min) and resuspended in osmotic buffer (10 mM MES, pH 5.7, 10 mmol / L MgCl2, 200 μM acetylsylcholine) to achieve the final OD. 600 The values ​​reached 0.8–1.0. The TRV1 helper plasmid was mixed 1:1 with TRV2-530 and TRV2 empty vectors, respectively. Subsequently, suspensions of Agrobacterium GV3101 under different treatments were injected into the cotyledons and true leaves of tomatoes using a needle-free injector. After injection, the plants were incubated in the dark at 23°C for 1–2 days until they recovered to normal growth conditions.

[0044] 3) Genetic transformation in tobacco: Agrobacterium GV3101 strains pCAMBIA2300-470, pCAMBIA2300-empty vector, TRV2-530, TRV2-empty vector, and TRV1-empty vector were amplified in LB broth containing kanamycin. Agrobacterium GV3101 strains were collected by centrifugation (5000 rpm, 10 min) and resuspended in osmotic buffer (10 mM MES, pH 5.7, 10 mmol / L MgCl2, 200 μM acetylsylcholine) to achieve the final OD. 600 The values ​​reached 0.8–1.0. The TRV1 helper plasmid was mixed 1:1 with TRV2-530 and TRV2 empty vectors, respectively. Subsequently, suspensions of Agrobacterium GV3101 under different treatments were injected into the leaves of tobacco plants using a needle-free injector. After injection, the plants were incubated in the dark at 23°C for 1–2 days until they returned to normal growth conditions.

[0045] Example 2 Phenotypic observation and identification of transgenic plants.

[0046] 1) Phenotypic observation and identification of transgenic watermelon plants: By observing the color of watermelon leaves infected with the virus vector within one week, it can be clearly seen that the injected area of ​​the leaves turns yellow on the 4th day, as shown in the following results. Figures 1-2 As shown, on day 6, compared with TRV-infected leaves without a host, overexpression of [a specific gene] was significantly higher. Cla97C04G068470 After gene injection, the injection site turns yellow and becomes silent. Cla97C04G068530 After gene administration, the injected areas of the leaves turned yellow compared to the uninoculated areas.

[0047] 2) Phenotypic observation and identification of transgenic tomato plants: Records were recorded and observed within two weeks after viral vector infection. Experimental results confirmed that the yellowing phenotype was successfully induced in both the cotyledons and true leaves. In tomato plants, the yellowing phenotype in both cotyledons and true leaves appeared within two weeks and persisted for more than a month, with the phenotype recorded on day 26 (e.g., ...). Figures 3-4 (As shown).

[0048] 3) Phenotypic observation and identification of transgenic tobacco plants: The results were recorded and observed within two weeks after viral vector infection, confirming that the yellowing phenotype was successfully induced in all infiltrated areas of the leaves. In tobacco plants, the leaf yellowing phenotype appeared within two weeks after infiltration, and its phenotype was recorded on day 13 (e.g., ...). Figures 5-6 (As shown).

[0049] This invention selects two candidate genes located in the yellow veins of watermelon leaves and inserts them into TRV and pCAMBIA2300 vectors respectively to construct recombinant vectors. After being transformed into watermelon, tomato and tobacco leaves, the corresponding transcription process is initiated. Through a series of reactions, the chlorophyll content is reduced, and the leaves exhibit a yellow phenotype, thus providing an intuitive visual marker for the screening of transgenic plants.

[0050] The constructed recombinant vector was introduced into Agrobacterium. Agrobacterium, as a natural gene transfer tool, allows its Ti plasmid's T-DNA (transferable DNA) to be transferred and integrated into the plant genome under specific conditions. The Agrobacterium culture containing the recombinant vector (OD) was then... 600 Inject the transgenic plant (at a suitable activity concentration for transformation) into the leaves and culture for 1-2 days. Plants that have successfully undergone transient transformation can be observed for phenotypic changes within one week or one month. Because the expression of the yellow leaf marker system in the leaves reduces chlorophyll synthesis, transgenic plants exhibit a yellow phenotype that differs from non-transgenic plants, allowing for preliminary screening of target plants by visual inspection.

[0051] In summary, the yellow leaf marking system developed in this invention employs two strategies. Cla97C04G068530 and Cla97C04G068470 They jointly regulate the leaf yellowing phenotype through opposite molecular mechanisms. On the one hand, silence... Cla97C04G068530The gene causes a noticeable yellowing phenomenon in transgenic plants, providing a new strategy for transgenic identification. Unlike widely used bleaching markers such as PDS (phytene desaturase), our system offers additional visual selection. On the other hand, overexpression... Cla97C04G068470 It also induces a distinct yellow phenotype, providing another method for transgenic identification. Compared to commonly used methods, such as fluorescent proteins (e.g.) GFP ) or betaine synthesis markers (such as RUBY This invention does not require expensive optical equipment and relies on only one exogenous gene ( Cla97C04G068470 Unlike RUBY Three genes are required. It provides a reliable, economical, and user-friendly method for watermelon transformation and genome editing. To test the broad applicability of this system, we first validated it in tomato and tobacco, where successful infiltration induced significant leaf yellowing. These findings suggest that this strategy has potentially wide applicability across different species.

[0052] Compared with traditional technologies, this method not only avoids the environmental and time-related problems associated with antibiotics or herbicides, but also eliminates the need for specific instruments. It allows for direct screening and identification of transgenic plants through leaves and with the naked eye, greatly accelerating the screening speed, reducing screening costs, and providing significant convenience for transgenic research.

[0053] In summary, the yellow leaf marker system developed in this study combines several advantages: it is cost-effective, visually perceptible, and easy to implement. It holds promise as a replacement or supplement to existing marker genes in various transformation systems, thus providing a more reliable, economical, and user-friendly solution for the efficient identification of transgenic plants.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for visualizing genetic transformation markers based on the yellow leaf phenotype, characterized in that, Includes the following steps: 1) Construction of overexpression vector recombinant plasmid: using enzyme digestion reagents to... Cla97C04G068470 The CDS sequence of the gene was cloned into the pCAMBIA2300 vector, the vector was transformed into E. coli, and positive E. coli were screened by PCR and sequencing. Then, the recombinant plasmid of the overexpression vector pCAMBIA2300-470 was extracted. 2) Constructing a silencing vector recombinant plasmid: Cla97C04G068530 Gene or Cla97C04G068530 After the homologous gene was digested with enzymes, it was inserted into the TRV vector. The vector was then transformed into E. coli, and positive E. coli were screened by PCR and sequencing. Subsequently, the recombinant plasmid of the silencing vector TRV-530 was extracted. 3) Introduction: The recombinant plasmid from step 1) or step 2) is introduced into the Agrobacterium strain; 4) Preparation of bacterial suspension: The strain described in step 3) is cultured, centrifuged, and resuspended to obtain bacterial suspension; 5) Infection and screening: Infect the target plant leaves with the bacterial solution described in step 4), and culture the infected target plants. After culture, observe the leaf phenotype with the naked eye. Plants with yellow leaf infection areas are transgenic positive plants.

2. The method according to claim 1, characterized in that, The enzyme digestion reagents mentioned in step 1) are BamHI and SaII enzymes; the Escherichia coli is Escherichia coli DH5α.

3. The method according to claim 1, characterized in that, The enzyme digestion reagent mentioned in step 2) is BamHI enzyme; the Escherichia coli is Escherichia coli DH5α.

4. The method according to claim 1, characterized in that, The steps described in step 1) Cla97C04G068470 The CDS sequence of the gene is shown in SEQ ID NO.1; as described in step 2). Cla97C04G068530 The CDS sequence of the gene is shown in SEQ ID NO.

2.

5. The method according to claim 1, characterized in that, The Agrobacterium strain mentioned in step 3) is strain GV3101.

6. The method according to claim 1, characterized in that, The culture in step 4) uses LB liquid medium containing kanamycin; the centrifugation is performed at 5000 rpm for 10 min; the resuspending osmosis buffer is 10 mM MES, 10 mmol / L MgCl2, 200 μM acetylsylgenone, and pH is 5-6; the OD of the bacterial culture is... 600 It ranges from 0.6 to 1.

7. The method according to claim 1, characterized in that, In step 5), the bacterial solution is injected into the leaves of the target plant using a needle-free syringe. After infection, the target plant is first cultured in the dark at 20-25℃ for 1-7 days, and then restored to normal growth conditions for continued culture.

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