Use of broccoli genes BoSULTR1;1 and BoSULTR1;2 in improving plant stress resistance

Abstract

The application of broccoli genes BoSULTR1;1 and BoSULTR1;2 in improving plant stress resistance belongs to the technical field of plant genetic engineering. In order to solve the technical problem of how to improve the stress resistance of broccoli plants, the application provides the broccoli genes BoSULTR1;1 and BoSULTR1;2, and the application uses an expression vector or a recombinant bacterium overexpressing BoSULTR1;1 and BoSULTR1;2 genes to prepare a transgenic broccoli plant. The stress resistance related detection results prove that the BoSULTR1;1 and BoSULTR1;2 genes have the ability to improve the resistance of the transgenic broccoli plant to cotton bollworm, pathogenic bacteria and drought. The transgenic material obtained by the application has important theoretical significance and practical value for accelerating the breeding process of stress-resistant plants and improving the breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of a gene in improving plant stress resistance. Background Technology

[0002] Broccoli (Brassica oleracea L. var. botrytis L.) is a biennial herbaceous plant belonging to the Brassicaceae family and the Brassica genus. Broccoli has a small surface area and distinct, loose flower buds. The tender stems of the flower buds are harvested for consumption. Broccoli is rich in nutrients, containing protein, sugar, fat, vitamins, and carotene. Its nutritional content ranks first among similar vegetables, earning it the reputation of "vegetable crown".

[0003] Currently, due to increased consumer demand and agricultural restructuring, the planting area of ​​broccoli is increasing year by year. With the development of the broccoli industry and the increase in planting years, various diseases are becoming increasingly apparent. Currently, growers mainly rely on spraying various pesticides to control broccoli pests and diseases, resulting in large amounts of pesticide residues on the broccoli, which is detrimental to the health of consumers. Furthermore, broccoli's poor drought resistance is a significant problem. Broccoli is a semi-hardy vegetable with poor drought resistance; it cannot form flower heads when temperatures exceed 30℃, and growing in dry soil leads to stunted plants and premature formation of small flower heads.

[0004] Therefore, pests, diseases, and drought have become serious problems in broccoli cultivation. Researchers in this field have been dedicated to finding ways to improve the resistance of broccoli plants to pests, diseases, and drought. Thus, exploring the stress resistance of broccoli is of great significance for broccoli production and breeding. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of broccoli genes BoSULTR1;1 and BoSULTR1;2 in improving plant stress resistance.

[0006] One objective of this invention is to provide an application of broccoli genes in improving plant stress resistance, wherein the broccoli genes are BoSULTR1;1 and BoSULTR1;2 genes.

[0007] In a preferred embodiment of the present invention, the nucleotide sequence of the BoSULTR1;1 gene is shown in SEQ ID NO.1.

[0008] In a preferred embodiment of the present invention, the nucleotide sequence of the BoSULTR1;2 gene is shown in SEQ ID NO.2.

[0009] In a preferred embodiment of the present invention, the stress resistance of the application is insect resistance, pathogen resistance and drought resistance, and the application is to obtain transgenic plants using the broccoli genes BoSULTR1;1 and BoSULTR1;2.

[0010] In a preferred embodiment of the present invention, the method for preparing the transgenic plant includes the following steps:

[0011] S1. The BoSULTR1;1 and BoSULTR1;2 genes were cloned using primers. The nucleotide sequence of the BoSULTR1;1 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the BoSULTR1;2 gene is shown in SEQ ID No. 2.

[0012] S2. The BoSULTR1;1 gene clone sequence and BoSULTR1;2 gene clone sequence obtained in S1 are respectively ligated to the vector to obtain the expression vector;

[0013] S3. The expression vectors obtained in S2 were transformed into Agrobacterium rhizogenes to obtain recombinant bacteria;

[0014] S4. Infect plants with the recombinant bacteria obtained in S3 to obtain transgenic plants.

[0015] In a preferred embodiment of the present invention, the upstream primer nucleotide sequence of the primer for the BoSULTR1;1 gene described in S1 is shown in SEQ ID No. 3, and the downstream primer nucleotide sequence is shown in SEQ ID No. 4.

[0016] The upstream primer nucleotide sequence of the primers for the BoSULTR1;2 gene is shown in SEQ ID No. 5, and the downstream primer nucleotide sequence is shown in SEQ ID No. 6.

[0017] In a preferred embodiment of the present invention, the carrier described in S2 is pCAM35S.

[0018] In a preferred embodiment of the present invention, the recombinant bacterial infection method described in S4 is Agrobacterium-mediated cotyledon node culture, and the plant is broccoli.

[0019] A second objective of this invention is to provide an expression vector containing the BoSULTR1;1 gene with the nucleotide sequence shown in SEQ ID NO.1, or the BoSULTR1;2 gene with the nucleotide sequence shown in SEQ ID NO.2.

[0020] A third objective of this invention is to provide a recombinant bacterium containing the aforementioned expression vector.

[0021] The fourth objective of this invention is to provide the application of the above-mentioned expression vector or recombinant bacteria in improving plant stress resistance.

[0022] The beneficial effects of this invention are:

[0023] This invention provides the application of the broccoli BoSULTR1;1 and BoSULTR1;2 genes in improving plant stress resistance. By constructing overexpression vectors and recombinant bacteria, transgenic broccoli plants are prepared to improve the insect resistance, pathogen resistance and drought resistance of broccoli plants.

[0024] This invention tested the stress resistance of transgenic broccoli overexpressing the BoSULTR1;1 and BoSULTR1;2 genes. The results of the bollworm resistance test showed that the transgenic broccoli plants had a higher resistance to bollworms compared to the control group, reducing bollworm damage. The results of the pathogen resistance test showed that the transgenic broccoli plants had a higher resistance to pathogens compared to the control group, reducing pathogen infection. The results of the drought resistance test showed that the transgenic broccoli plants had higher drought resistance compared to the control group and could grow normally under drought conditions.

[0025] In summary, the BoSULTR1;1 and BoSULTR1;2 genes provided by this invention can effectively enhance the insect resistance, disease resistance, and drought resistance of transgenic plants, which has important theoretical significance and practical value for accelerating the breeding process of stress-resistant plants and improving breeding efficiency. Attached Figure Description

[0026] Figure 1 This is a PCR identification diagram of the coding regions of the BoSULTR1;1 and BoSULTR1;2 genes in Example 1.

[0027] Figure 2 This is a PCR identification diagram of the recombinant bacteria containing overexpression of BoSULTR1;1 and BoSULTR1;2 genes in Example 2.

[0028] Figure 3 This is a green fluorescence detection image of the hairy roots of transgenic broccoli in Example 4;

[0029] Figure 4 The graphs shown in Example 5 are for testing the resistance of transgenic broccoli to cotton bollworm. A is a plant observation graph, and B is a cotton bollworm weight statistics graph.

[0030] Figure 5 The graphs shown in Example 5 are for testing the pathogen resistance of transgenic broccoli. A is a plant observation graph, and B is a statistical graph of pathogen numbers.

[0031] Figure 6 This is a graph showing the drought resistance performance of the transgenic broccoli in Example 5. Detailed Implementation

[0032] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0034] Example 1: Cloning of broccoli genes BoSULTR1;1 and BoSULTR1;2

[0035] In this embodiment, cDNA from broccoli cotyledons was used as a template for amplification to obtain PCR amplification products of the BoSULTR1;1 and BoSULTR1;2 genes, respectively. The obtained PCR amplification products were then verified by PCR, and the results are as follows: Figure 1 As shown, the coding regions of the BoSULTR1;1 and BoSULTR1;2 genes were successfully cloned.

[0036] The upstream primer nucleotide sequence of the BoSULTR1;1 gene is shown in SEQ ID No. 3, and the downstream primer nucleotide sequence is shown in SEQ ID No. 4; the upstream primer nucleotide sequence of the BoSULTR1;2 gene is shown in SEQ ID No. 5, and the downstream primer nucleotide sequence is shown in SEQ ID No. 6.

[0037] The amplification system consisted of: 2 μL dNTP Mix, 2 μL 10×Ex Taq Buffer, 0.3 μL Ex Taq Enzyme, 1 μL cDNA template, 1 μL upstream primer, 1 μL downstream primer, and RNase-free H2O to a final volume of 20 μL.

[0038] The amplification program was as follows: 94℃ pre-denaturation for 10 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles, and 72℃ complete extension for 10 min.

[0039] Example 2: Construction of expression vector

[0040] This embodiment uses the SPARK easy Gel DNA Extraction Kit to recover and purify the electrophoretic bands of the PCR amplification products of the BoSULTR1;1 and BoSULTR1;2 genes obtained in Example 1, including the following steps:

[0041] S1: Under UV light irradiation, use a blade to cut off the gel blocks containing the PCR amplification product bands of the BoSULTR1;1 and BoSULTR1;2 genes obtained in Example 1, and place them into 1.5mL EP tubes respectively. Then add 600μL of sol and heat in a metal bath at 65°C until completely melted. Invert the tube every 2 minutes to accelerate melting and obtain gel solutions containing the amplification product bands of the BoSULTR1;1 and BoSULTR1;2 genes respectively.

[0042] S2: Add the gel solution obtained in S1 to the adsorption column EC, place at room temperature for 1 min, centrifuge at 13800×g for 1 min, and discard the effluent; then add 600 μL of washing buffer WB, centrifuge at 13800×g for 1 min, discard the effluent, and repeat the washing once.

[0043] S3: Place the EC adsorption column after rinsing in S2 back into the collection tube and centrifuge at 13800×g for 2 min; then place the EC adsorption column into a new collection tube, add 20 μL RNase Free H2O to the middle of the adsorption membrane, let it stand at room temperature for 2 min, centrifuge at 13800×g for 1 min, and the elution product is the purified target fragment.

[0044] S4: Expression vectors were constructed using homologous recombination. The recombination system consisted of: 2 μL Exnase II, 4 μL 5×CEII Buffer, 1 μL of insert fragment product, 3 μL of linearized vector pCAM35S, and 20 μL of ddH2O. The reaction was carried out at 37 °C for 30 min to obtain pCAM35S-BoSULTR1;1-eGFP and pCAM35S-BoSULTR1;2-eGFP expression vectors, respectively.

[0045] Example 3: Construction of recombinant bacteria

[0046] In this embodiment, 2 μL of the pCAM35S-BoSULTR1;1-eGFP and pCAM35S-BoSULTR1;2-eGFP expression vectors obtained in Example 2 were respectively added to 50 μL of thawed Agrobacterium rhizogenes competent cells ATCC15834. After mixing by pipetting, the mixture was placed in ice. The ice-bathed mixture was then added to a pre-cooled electroporation cuvette (electroporator program: C=25μF, PC=200Ω, V=2400V). The cuvette was capped, dried, and inserted into the electroporator groove. Electroporation was started. After the reaction was completed, the mixture was quickly transferred to an EP tube, and 1 mL of TY liquid medium (each liter of TY liquid medium consists of: 5 g peptone, 3 g yeast extract, and 10 mL 1M calcium chloride aqueous solution) was added. The mixture was incubated at 28°C for 3 h, and then spread onto TY solid medium containing Kana and Rif (each liter of TY solid medium consists of: 5 g peptone, 3 g yeast extract, 15 g agar, and 10 mL...). After incubation in 1M calcium chloride aqueous solution for 48 hours, colony PCR identification was performed, and the results are as follows: Figure 2 As shown, recombinant bacteria containing the expression vectors pCAM35S-BoSULTR1;1-eGFP and pCAM35S-BoSULTR1;2-eGFP were successfully obtained.

[0047] Example 4: Preparation of hairy roots from transgenic broccoli

[0048] In this embodiment, robust broccoli seedlings that had grown to two weeks old and exhibited uniform growth were selected. The recombinant bacteria obtained in Example 3 were used for infection. The infection process involved making a slanted cut 2 cm below the cotyledon node, scraping off Agrobacterium rhizogenes ATCC15834 cells from the cut surface, and then quickly inserting the seedling into a flowerpot containing only vermiculite to absorb OD (oxidative stress). 600 A 0.8% solution of Agrobacterium rhizogenes ATCC15834 was injected into the roots, and a transparent plastic cup was immediately placed over it to maintain moisture and ensure sufficient water. After one week of growth, numerous hairy roots appeared on the cut surface. The roots, except for the oblique cut, were trimmed and the roots were replanted in the mixed soil. Green fluorescence detection was performed on the hairy roots of the transgenic broccoli. The appearance of green fluorescence indicated successful transformation, yielding transgenic broccoli hairy root chimeras that positively overexpressed the BoSULTR1;1 and BoSULTR1;2 genes, respectively. Figure 3 As shown.

[0049] Example 5: Application of the BoSULTR1;1 and BoSULTR1;2 genes in broccoli to enhance plant stress resistance

[0050] 1. Application of BoSULTR1;1 and BoSULTR1;2 genes in improving plant insect resistance. In this example, transgenic broccoli with empty vectors was used as the control group, and transgenic broccoli hairy root chimeras overexpressing BoSULTR1;1 and BoSULTR1;2 genes obtained in Example 4 were used as the experimental group to test the resistance to cotton bollworm. The specific steps included: placing broccoli plants in the control group and experimental group in the same space with cotton bollworms, keeping the experimental environment the same for each group, taking pictures of the leaves of the broccoli plants after 1 day, and counting the weight of the cotton bollworms.

[0051] The results are as follows Figure 4 As shown, the leaf damage of broccoli plants overexpressing BoSULTR1;1 and BoSULTR1;2 genes was significantly reduced compared to the control group. Furthermore, the weight statistics of the transgenic broccoli plants after being eaten by bollworms indicated that the weight of the bollworms that ate the broccoli plants overexpressing BoSULTR1;1 and BoSULTR1;2 genes was significantly lower than that of the bollworms that ate the control group broccoli plants. In conclusion, overexpression of BoSULTR1;1 and BoSULTR1;2 genes significantly improved the resistance of transgenic broccoli plants to bollworms.

[0052] 2. Application of the BoSULTR1;1 and BoSULTR1;2 genes in broccoli to enhance plant disease resistance

[0053] In this embodiment, transgenic broccoli with an empty vector was used as the control group, and the transgenic broccoli hairy root chimera overexpressing the BoSULTR1;1 and BoSULTR1;2 genes obtained in Example 4 was used as the experimental group to test its resistance to pathogens. The specific steps included:

[0054] (1) Take the Pst DC3000 bacterial culture stored in the -80℃ refrigerator, streak it on KB medium containing 50mg / L Kana+Rif, and place it in a constant temperature incubator at 28℃ for 48h.

[0055] (2) Pick another single colony of Pst DC3000 cultured in (1) and place it in an Erlenmeyer flask containing 30 mL of KB liquid medium (each liter of KB liquid medium consists of: 20 g peptone, 1.97 g K2HPO4·3H2O, 1.5 g MgSO4·7H2O, 10 mL glycerol, pH = 7.4; and 50 mg / L kanamycin + 50 mg / L rifampin). Incubate overnight in a constant temperature shaker at 28 °C and 220 rpm. On the second day, measure the OD value of the colonies after the above culture using a spectrophotometer.

[0056] (3) OD in (2) 600Bacterial suspensions with a pH of 0.8-1.2 were transferred to 50 mL EP tubes and centrifuged at 4°C and 4000 × g for 10 min. The supernatant was discarded, and the cells were resuspended in sterile 10 mM MgCl2 solution and diluted to OD200. 600 =0.2, to obtain a resuspension;

[0057] (4) Take 100 mL of the resuspension obtained in (3) and add 4 μL of surfactant Silwet L-77 to the above resuspension. Shake and mix well to obtain a mixture. Treat the leaves of the broccoli plants in the experimental group and the control group with pathogen PstDC3000. The pathogen PstDC3000 treatment is as follows: use the above mixture to spray the front and back of the leaves of the broccoli plants in the experimental group and the control group evenly. Spray continuously for 3 days and observe the yellowing status of the leaves.

[0058] (5) After treating the broccoli leaves with Pst DC3000 pathogen from (4) for 3 days, weigh and record the fresh weight; then, in a clean bench, immerse the weighed leaves in 70% ethanol for 15 seconds, gently shake to kill the surface bacteria, then remove the leaves and rinse them in sterile water, and wipe them dry with sterile filter paper; then put the leaves into a 2mL EP tube, use a tissue homogenizer to homogenize them, and add 900μL of sterile water to dilute them; then take 100μL of the diluted solution and put it into a new 2mL EP tube, and add 900μL of sterile water to dilute and mix; repeat the above steps once, take 50μL of the diluted solution after 3 dilutions, and spread it on KB solid medium (each liter of KB solid medium consists of: 20g peptone, 1.97g K2HPO4·3H2O, MgSO4·7H2O) 1.5g of glycerol, 10ml of agar powder, 15g of agar powder, pH=7.4; and a mixture containing 50mg / L kanamycin + 50mg / L rifampin were placed on a plate and incubated upside down in a 28℃ incubator for 48h. The number of colonies on the plates was recorded, and the bacterial quantification was as follows:

[0059]

[0060] In this embodiment, trypan blue staining was used to stain the leaves of plants infected with Pst DC3000. To visually observe the cell death in the leaves, the following steps were taken: Leaves of broccoli plants from the control group and experimental group that had been infected for 3 days were taken, immersed in 0.4% trypan blue staining solution, wrapped in aluminum foil to protect them from light, and stained at room temperature for 30 minutes; the trypan blue staining solution was removed, and then 98% ethanol was added to decolorize the leaves overnight at room temperature until they were completely decolorized. The staining was observed and photographed to record the staining status.

[0061] The results are as follows Figure 5As shown, compared with the control group, the transgenic broccoli plants overexpressing BoSULTR1;1 and BoSULTR1;2 genes showed significantly reduced leaf damage and lower pathogen counts. This indicates that overexpression of BoSULTR1;1 and BoSULTR1;2 genes significantly improved the resistance of transgenic broccoli plants to pathogen Pst DC3000.

[0062] 3. Application of the BoSULTR1;1 and BoSULTR1;2 genes in broccoli to improve plant drought resistance

[0063] In this embodiment, transgenic broccoli with an empty vector was used as the control group, and the transgenic broccoli hairy root chimera overexpressing the BoSULTR1;1 and BoSULTR1;2 genes obtained in Example 4 was used as the experimental group to test drought resistance. The specific steps included: taking pictures and collecting data on the broccoli plants of the control group and the experimental group before drought treatment; treating and culturing the broccoli plants of the control group and the experimental group together in a 10% polyethylene glycol 6000 aqueous solution for 1 day; and then taking pictures to observe the growth status of the broccoli plants after the above treatment.

[0064] The results are as follows Figure 6 As shown, compared with the control group, the transgenic broccoli plants overexpressing BoSULTR1;1 and BoSULTR1;2 genes showed better growth and less wilting. This indicates that overexpression of BoSULTR1;1 and BoSULTR1;2 genes significantly improved the drought resistance of transgenic broccoli plants.

[0065] The above results indicate that overexpression of the BoSULTR1;1 and BoSULTR1;2 genes can effectively enhance the transgenic plants' resistance to insects, diseases, and drought.

[0066] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of a broccoli gene in improving plant stress resistance, characterized in that, Plant stress resistance is improved by overexpressing the broccoli gene, which is... BoSULTR1;1 Genes and BoSULTR1;2 Gene; The BoSULTR1;1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The BoSULTR1;2 The nucleotide sequence of the gene is shown in SEQ ID NO.2; The aforementioned resistance refers to drought resistance, resistance to bollworms, or resistance to pathogens. Pst ; The plant in question is broccoli.

2. The application according to claim 1, characterized in that, The application utilizes the above BoSULTR1;1 Genes and BoSULTR1;2 Genes were obtained from transgenic plants.

3. The application according to claim 2, characterized in that, The method for preparing the transgenic plant includes the following steps: S1. Using primers to pair the... BoSULTR1;1 Genes and BoSULTR1;2 Gene cloning; S2. Obtained from S1 BoSULTR1;1 Gene cloning sequence and BoSULTR1;2 Gene clone sequences are ligated into vectors to obtain expression vectors; S3. The expression vectors obtained in S2 were transformed into Agrobacterium rhizogenes to obtain recombinant bacteria; S4. Infect plants with the recombinant bacteria obtained in S3 to obtain transgenic plants.

4. The application according to claim 3, characterized in that, S1 as described BoSULTR1;1 The upstream primer nucleotide sequence of the gene primer is shown in SEQ ID No. 3, and the downstream primer nucleotide sequence is shown in SEQ ID No. 4; The BoSULTR1;2 The upstream primer nucleotide sequence of the gene primer is shown in SEQ ID No. 5, and the downstream primer nucleotide sequence is shown in SEQ ID No.

6.

5. The application according to claim 3, characterized in that, The vector described in S2 is pCAM35S; the method of infection of the recombinant bacteria described in S4 is Agrobacterium rhizogenes-mediated method.

6. The application of an expression vector in improving plant stress resistance, characterized in that, The expression vector enhances plant stress resistance by overexpressing a broccoli gene, which is... BoSULTR1;1 Genes and BoSULTR1;2 Gene; The BoSULTR1;1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The BoSULTR1;2 The nucleotide sequence of the gene is shown in SEQ ID NO.2; The aforementioned resistance refers to drought resistance, resistance to bollworms, or resistance to pathogens. Pst ; The plant in question is broccoli.

Images