Application of muskmelon ubiquitin receptor protein CmDSK2b in improvement of plant stress resistance

Abstract

The invention relates to application of muskmelon ubiquitin receptor protein CmDSK2b in improvement of plant stress resistance, and belongs to the technical field of genetic engineering. In order to solve the technical blank of ubiquitin receptor protein in research on stress resistance of horticultural crops, the invention provides muskmelon ubiquitin receptor protein CmDSK2b, the nucleotide sequence of the coding gene of the muskmelon ubiquitin receptor protein CmDSK2b is shown as SEO ID NO.1, and an expression cassette, a recombinant vector and a recombinant bacterium containing the coding gene of the muskmelon ubiquitin receptor protein CmDSK2b are provided. The muskmelon ubiquitin receptor protein CmDSK2b provided by the invention has a function of degrading target protein in a plant body, provides a fundamental research theory for accurately degrading specific protein in the plant by utilizing a genetic engineering means and utilizing application of molecular biology and genetic engineering means in plant stress resistance breeding, and has a wide application prospect. The method has important significance on enriching biological functions of ubiquitin receptor protein.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to application of melon ubiquitin receptor protein CmDSK2b in improving plant stress resistance. Background Art

[0002] The plant ubiquitination mechanism is a complex protein post-translational modification process, and ubiquitination is common in plant biotic stress. Studies have shown that ubiquitination modification can degrade pathogen proteins, thereby inhibiting the infection of pathogens to plants. Similarly, ubiquitination modification can also regulate the stability and degradation of the plant's own proteins, thereby affecting the plant's ability to resist stress. Studies have also found that plants can modify low-temperature response negative regulatory factors through ubiquitination and promote their degradation, thereby enhancing the plant's ability to resist low temperatures. It can be seen that ubiquitination regulates plant immune responses, hormone signal transduction, protein stability and degradation, protein interactions, etc. through a variety of mechanisms in plant biotic stress, thereby helping plants cope with various biotic stresses.

[0003] DSK2 (Dominant Suppressor of KAR2) is a type of ubiquitin receptor protein that exists in species such as humans, Arabidopsis, rice, Toxoplasma gondii and yeast; DSK2 has UBA (C-terminal ubiquitin-associateddomain) and UBL (N-terminal ubiquitin-like domain) domains, and its UBA and UBL domains can bind to ubiquitinated substrates and proteasome subunits respectively, transporting polyubiquitinated substrates to the proteasome for degradation.

[0004] Researchers found that in Arabidopsis, DSK2a and DSK2b are involved in the degradation of peroxisome membrane-associated proteins, affecting the function of peroxisomes; in rice, OsDSK2b negatively regulates leaf blight and osmotic stress resistance, while the UBL-UBA protein OsDSK2a can mediate rice seedling growth and response to salt stress; given the important role of ubiquitin receptor proteins in plant resistance and the related mechanism research has been mostly carried out in the model crop Arabidopsis, while there are few reports on the research on the stress resistance of horticultural crops. Summary of the invention

[0005] The invention solves the technical gap in the study of ubiquitin receptor proteins in horticultural crop stress resistance and provides the application of melon ubiquitin receptor protein CmDSK2b in improving plant stress resistance.

[0006] One of the purposes of the present invention is to provide an application of melon ubiquitin receptor protein CmDSK2b in improving plant stress resistance. The nucleotide sequence of the gene encoding the melon ubiquitin receptor protein CmDSK2b is shown in SEO ID NO.1.

[0007] In a preferred embodiment of the present invention, the application degrades the target protein in the plant by the melon ubiquitin receptor protein CmDSK2b to improve the stress resistance of the plant, and the stress resistance includes powdery mildew resistance and ventricle number development of watermelon.

[0008] In a preferred embodiment of the present invention, the plant is melon or watermelon.

[0009] The second object of the present invention is to provide an expression cassette, which contains a gene encoding the melon ubiquitin receptor protein CmDSK2b whose nucleotide sequence is shown as SEO ID NO.1.

[0010] The third object of the present invention is to provide a recombinant vector, which contains the above-mentioned expression cassette or contains the gene encoding the melon ubiquitin receptor protein CmDSK2b whose nucleotide sequence is shown as SEO ID NO.1.

[0011] The fourth object of the present invention is to provide a recombinant bacterium, which contains the above-mentioned recombinant vector, expression cassette or contains the melon ubiquitin receptor protein CmDSK2b encoding gene with the nucleotide sequence shown in SEO ID NO.1.

[0012] A fifth object of the present invention is to provide the use of the above-mentioned expression cassette, recombinant vector or recombinant bacteria in improving plant stress resistance.

[0013] A sixth object of the present invention is to provide a method for improving plant stress resistance, the method comprising the following steps:

[0014] S1: Using melon cDNA as a template, PCR amplification was performed using primer pairs to obtain the cloned sequence of the CmDSK2b gene;

[0015] S2: using the CmDSK2b gene clone sequence obtained in S1 to prepare an expression cassette and a recombinant vector;

[0016] S3: The expression cassette or recombinant vector obtained in S2 is transferred into Agrobacterium to obtain recombinant bacteria;

[0017] S4: Infect plants with the recombinant Agrobacterium obtained in S3 to obtain transgenic plants.

[0018] In a preferred embodiment of the present invention, the nucleotide sequence of the primer pair in S1 is shown as SEO ID NO.2-3.

[0019] In a preferred embodiment of the present invention, the Agrobacterium in S3 is GV3101, and the plant in S4 is melon or watermelon.

[0020] Beneficial effects of the present invention: The present invention provides an application of melon ubiquitin receptor protein CmDSK2b in improving plant stress resistance, wherein the application improves plant stress resistance by degrading a target protein in a plant through the melon ubiquitin receptor protein CmDSK2b, and the nucleotide sequence of the encoding gene of the melon ubiquitin receptor protein CmDSK2b is shown in SEO ID NO.1. The present invention also provides an expression cassette, a recombinant vector and a recombinant bacterium containing the encoding gene of the melon ubiquitin receptor protein CmDSK2b.

[0021] The melon ubiquitin receptor protein CmDSK2b provided by the present invention has the function of degrading target proteins in plants. Effect experiments have proved that the melon ubiquitin receptor protein CmDSK2b reduces the accumulation of CmMLO5 protein and Cmpm2.1 protein (melon powdery mildew resistance-related proteins / genes) through degradation, and as the concentration of the recombinant bacterial solution containing the melon ubiquitin receptor protein CmDSK2b increases, its degradation effect on CmMLO5 protein and Cmpm2.1 protein becomes more and more significant. Therefore, the present invention analyzes the molecular regulation mechanism of CmDSK2b-CmMLO5 coordinated regulation of melon powdery mildew resistance, provides an important theoretical basis for the precise degradation of specific proteins in plants using genetic engineering methods, and is of great significance for enriching the biological functions of ubiquitin receptor proteins.

[0022] The melon ubiquitin receptor protein CmDSK2b provided by the present invention has a degrading effect on the ClRPK2 protein in watermelon, and the melon ubiquitin receptor protein CmDSK2b degrades the expression of the ClRPK2 protein, a gene related to the development of the number of ventricles in watermelon. It can be seen that the melon ubiquitin receptor protein CmDSK2b provided by the present invention provides a basic research theory for the application of molecular biology and genetic engineering methods in plant stress resistance breeding, and is expected to promote the further development of plant biotechnology in the field of protein regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram showing the cloning results of the melon ubiquitin receptor protein CmDSK2b in Example 1;

[0024] Figure 2 This is a graph showing the result of the melon ubiquitin receptor protein CmDSK2b degrading the expression of CmMLO5 protein in Example 2;

[0025] Figure 3 This is a diagram showing the result of the degradation of Cmpm2.1 protein expression by the melon ubiquitin receptor protein CmDSK2b in Example 3;

[0026] Figure 4 This is a graph showing the results of the melon ubiquitin receptor protein CmDSK2b degrading ClRPK2 protein expression in Example 4. DETAILED DESCRIPTION

[0027] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0028] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and those skilled in the art can obtain them through commercial channels.

[0029] Example 1: Preparation of melon ubiquitin receptor protein CmDSK2b with protein degradation function

[0030] In this implementation, the melon ubiquitin receptor protein CmDSK2b sequence obtained from the Cucurbitaceae Genome Website (http: / / cucurbitgenomics.org / organism / 18) was used as a template, and primer sequences were designed using Primer 6 to obtain primer pairs for cloning the gene encoding the melon ubiquitin receptor protein CmDSK2b, as shown in Table 1; In this implementation, PCR amplification was performed using melon cDNA as a template to obtain the CmDSK2b gene clone sequence. The PCR amplification system and program used in the above PCR amplification are shown in Tables 2-3.

[0031] Table 1

[0032]

[0033] Table 2

[0034]

[0035] Table 3

[0036]

[0037] In this example, 1% agarose gel electrophoresis was used to detect the PCR amplification products obtained above. The electrophoresis detection results are as follows: Figure 1 As shown, and according to the gel recovery kit ( Quick Gel Extraction Kit, Code No. EG101-01) instruction manual, recover and purify the target fragment gel, and connect the obtained target fragment to the TA cloning vector. The specific steps are as follows:

[0038] S1: Prepare a ligation reaction system to connect the obtained target fragment with the TA vector, mix well, and connect at 25°C for 5 minutes to obtain a ligation product;

[0039] S2: The ligation product obtained in S1 was transformed into E. coli. The transformation method was carried out according to the instructions of the competent cell E. coli DH5α (Code No. 9057, Takara);

[0040] S3: Randomly pick a single colony with complete and full morphology from S2 and perform colony PCR reaction. The PCR product is detected by 1% agarose gel electrophoresis. The bacterial solution identified as a positive clone is sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing to obtain the target gene sequence.

[0041] In this example, the gene encoding the melon ubiquitin receptor protein CmDSK2b sequenced by Shanghai Sangon Biotechnology Co., Ltd. is located on chromosome 6 of melon, has a base length of 1710 bp, and the nucleotide sequence is shown in SEO ID NO.1.

[0042] Example 2: Application of melon ubiquitin receptor protein CmDSK2b in degrading melon powdery mildew resistance-related CmMLO5 protein

[0043] In this example, the coding gene sequence of the melon powdery mildew resistance-related CmMLO5 protein was obtained from the Cucurbitaceae genome website (http: / / cucurbitgenomics.org / organism / 18). The homologous arm primers of the melon ubiquitin receptor protein CmDSK2b and the melon powdery mildew resistance-related CmMLO5 protein were designed by homologous recombination method and PCR amplification was performed. The primer information is shown in Table 4. In this example, the pCAMBIA1300-sGFP plasmid and the pGreen62SK plasmid were double-digested with restriction endonucleases, and the recombination reaction was carried out at 37° C. for 30 minutes. The homologous recombination system is shown in Table 5. The homologous recombination-completed system was transformed into competent cells E. coli DH5α (Code No. 9057, Takara), and the transformation results were sequenced to obtain the pGreen62SK-CmDSK2b fusion expression vector and the pCAMBIA1300-sGFP-CmMLO5 fusion expression vector.

[0044] In this example, the pGreen62SK-CmDSK2b fusion expression vector and the pCAMBIA1300-sGFP-CmMLO5 fusion expression vector obtained above were transformed into Agrobacterium competent GV3101 (Code No. AC1001, WEIDI), and the cell suspension was prepared and injected into 4-week-old tobacco leaves, cultured in the dark at 23°C for 2 days, and observed and imaged using a laser scanning confocal microscope.

[0045] Table 4

[0046]

[0047] Table 5

[0048]

[0049]

[0050] The results are as follows Figure 2 As shown in the figure, when the pCAMBIA1300-sGFP-CmMLO5 fusion expression vector was co-expressed with the pGreen62SK empty vector solution, a strong green fluorescence signal was presented on the cell membrane of tobacco leaves, indicating that the CmMLO5 protein was stably expressed on the cell membrane. However, when pCAMBIA1300-sGFP-CmMLO5 was co-expressed with the pGreen62SK-CmDSK2b vector solution, the fluorescence signal was significantly weakened, indicating that the CmDSK2b protein reduced the expression and accumulation of the CmMLO5 protein by degradation. Moreover, with the increase in the concentration of the pGreen62SK-CmDSK2b expression vector solution, the fluorescence signal of the CmMLO5 protein was further weakened, indicating that the degradation effect of the CmDSK2b protein on the CmMLO5 protein became more and more significant.

[0051] It can be seen that the melon ubiquitin receptor protein CmDSK2b provided by the present invention can achieve the purpose of effectively degrading the melon powdery mildew resistance-related CmMLO5 protein in vivo.

[0052] Example 3: Application of melon ubiquitin receptor protein CmDSK2b in degrading melon powdery mildew resistance-related Cmpm2.1 protein

[0053] In this example, the coding gene sequence of the melon powdery mildew resistance-related Cmpm2.1 protein was obtained from the Cucurbitaceae genome website (http: / / cucurbitgenomics.org / organism / 18). The homologous recombination method was used to design the homologous arm primers of the melon powdery mildew resistance-related Cmpm2.1 protein and perform PCR amplification. The primer information is shown in Table 6. In this example, the pCAMBIA1300-sGFP plasmid was double-digested with restriction endonucleases, and the recombination reaction was performed at 37° C. for 30 minutes. The homologous recombination system is shown in Table 5. The homologous recombination-completed system was transformed into competent cells E. coli DH5α (Code No. 9057, Takara), and the transformation results were sequenced to obtain the pCAMBIA1300-sGFP-Cmpm2.1 fusion expression vector.

[0054] In this example, the pGreen62SK-CmDSK2b fusion expression vector obtained in Example 1 and the pCAMBIA1300-sGFP-Cmpm2.1 fusion expression vector obtained above were transformed into Agrobacterium competent GV3101 (Code No. AC1001, WEIDI), and a cell suspension was prepared and injected into 4-week-old tobacco leaves, cultured in the dark at 23°C for 2 days, and observed and imaged using a laser scanning confocal microscope.

[0055] Table 6

[0056]

[0057]

[0058] The results are as follows Figure 3 As shown, when the pCAMBIA1300-sGFP-Cmpm2.1 fusion expression vector was co-expressed with the pGreen62SK empty vector bacterial solution, a strong green fluorescence signal was presented on the cell membrane of tobacco leaves, indicating that the Cmpm2.1 protein was stably expressed on the cell membrane. When pCAMBIA1300-sGFP-Cmpm2.1 was co-expressed with the pGreen62SK-CmDSK2b vector bacterial solution, the fluorescence signal was significantly weakened, which showed that the CmDSK2b protein reduced the expression and accumulation of the Cmpm2.1 protein by degradation. It can be seen that the melon ubiquitin receptor protein CmDSK2b provided by the present invention can achieve the purpose of effectively degrading the melon powdery mildew resistance-related Cmpm2.1 protein in vivo.

[0059] Example 4: Application of melon ubiquitin receptor protein CmDSK2b in degrading watermelon ventricular number development-related ClRPK2 protein

[0060] In this example, the coding gene sequence of the ClRPK2 protein related to the development of the number of ventricles in watermelon was obtained from the Cucurbitaceae genome website (http: / / cucurbitgenomics.org / organism / 18). The homologous arm primers of the ClRPK2 protein related to the development of the number of ventricles in watermelon were designed by homologous recombination method and PCR amplification was performed. The primer information is shown in Table 7. In this example, the pCAMBIA1300-sGFP plasmid was double-digested with restriction endonucleases, and a recombination reaction was performed at 37° C. for 30 minutes. The homologous recombination system is shown in Table 5. The system completed with the homologous recombination was transformed into competent cells E. coli DH5α (Code No. 9057, Takara), and the transformation results were sequenced to obtain the pCAMBIA1300-sGFP-ClRPK2 fusion expression vector.

[0061] In this example, the pGreen62SK-CmDSK2b fusion expression vector obtained in Example 1 and the pCAMBIA1300-sGFP-ClRPK2 fusion expression vector obtained above were transformed into Agrobacterium competent GV3101 (Code No. AC1001, WEIDI), and a cell suspension was prepared and injected into 4-week-old tobacco leaves, cultured in the dark at 23°C for 2 days, and observed and imaged using a laser scanning confocal microscope.

[0062] Table 7

[0063]

[0064] The results are as follows Figure 4 As shown, when the pCAMBIA1300-sGFP-ClRPK2 fusion expression vector was co-expressed with the pGreen62SK empty vector bacterial solution, a strong green fluorescence signal was presented on the cell membrane of tobacco leaves, indicating the stable expression of the ClRPK2 protein on the cell membrane. When pCAMBIA1300-sGFP-ClRPK2 was co-expressed with the pGreen62SK-CmDSK2b vector bacterial solution, the fluorescence signal was significantly weakened, which showed that the CmDSK2b protein reduced the expression and accumulation of the ClRPK2 protein by degradation. It can be seen that the melon ubiquitin receptor protein CmDSK2b provided by the present invention can achieve the purpose of effectively degrading the ClRPK2 protein related to the development of the ventricle number of watermelon in vivo.

[0065] In summary, the melon ubiquitin receptor protein CmDSK2b provided by the present invention has the function of degrading proteins, and achieves the purpose of improving plant stress resistance by degrading the expression of target proteins in plants.

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

Claims

1. Application of melon ubiquitin receptor protein CmDSK2b in improving plant stress resistance, characterized in that: The nucleotide sequence of the gene encoding the melon ubiquitin receptor protein CmDSK2b is shown in SEO ID NO.

1.

2. The use according to claim 1, characterized in that: The application improves plant stress resistance by degrading target proteins in plants through the melon ubiquitin receptor protein CmDSK2b, and the stress resistance includes powdery mildew resistance and watermelon ventricle number development.

3. The use according to claim 2, characterized in that: The plant is melon or watermelon.

4. An expression cassette, characterized in that The expression cassette contains a gene encoding the melon ubiquitin receptor protein CmDSK2b whose nucleotide sequence is shown as SEO ID NO.

1.

5. A recombinant vector, characterized in that: The recombinant vector contains the expression cassette of claim 4 or contains the gene encoding the melon ubiquitin receptor protein CmDSK2b with the nucleotide sequence shown in SEO ID NO.

1.

6. A recombinant bacterium, characterized in that: The recombinant bacteria contains the recombinant vector according to claim 5, the expression cassette according to claim 4, or contains the gene encoding the melon ubiquitin receptor protein CmDSK2b with a nucleotide sequence shown in SEO ID NO.

1.

7. Use of the expression cassette according to claim 4, the recombinant vector according to claim 5 or the recombinant bacterium according to claim 6 in improving plant stress resistance.

8. A method for improving plant stress resistance, characterized in that: The method comprises the following steps: S1: Using melon cDNA as a template, PCR amplification was performed using primer pairs to obtain the cloned sequence of the CmDSK2b gene; S2: using the CmDSK2b gene clone sequence obtained in S1 to prepare an expression cassette and a recombinant vector; S3: The expression cassette or recombinant vector obtained in S2 is transferred into Agrobacterium to obtain recombinant bacteria; S4: Infect plants with the recombinant Agrobacterium obtained in S3 to obtain transgenic plants.

9. The method according to claim 8, characterized in that The nucleotide sequence of the primer pair described in S1 is shown in SEO ID NO.2-3.

10. The method according to claim 8, characterized in that The Agrobacterium described in S3 is GV3101, and the plant described in S4 is melon or watermelon.

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