Application of protein CsHAC1 in enhancing plant antioxidant activity

Abstract

This invention discloses the application of protein CsHAC1 in enhancing the antioxidant activity of plants. The amino acid sequence of protein CsHAC1 is shown in SEQ ID NO: 2. Experiments have shown that overexpression of the CsHAC1 gene in cucumber variety 9930 can enhance the antioxidant activity of cucumber. Therefore, it is evident that protein CsHAC1 can improve the antioxidant activity of plants. This invention has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of protein CsHAC1 in enhancing the antioxidant activity of plants. Background Art

[0002] Vegetables are an important part of our daily diet. They not only provide vitamins, minerals, and fiber, but also a variety of antioxidants (such as vitamin C, vitamin E, carotenoids, and phenolic compounds). These antioxidants have a certain preventive and therapeutic effect on some chronic diseases caused by oxidative damage from free radicals, and can also delay aging caused by the accumulation of oxidative damage exceeding the body's own antioxidant defense system's repair capacity. The accumulation of antioxidants in crops is regulated by specific functional genes. For example, studies on wild and cultivated camellia species have found that cultivated camellias have higher levels of certain flavonoid antioxidants, and the genes involved in the corresponding metabolic pathways have also changed in both wild and cultivated species, indicating that the antioxidant content of camellias may have been selected during domestication. As another example, in potatoes, overexpression of the anthocyanin-associated R2R3-MYB transcription factor LeAN2 can increase anthocyanin accumulation and enhance resistance to low temperature and oxidative stress. NingSun et al. (2022) demonstrated through mouse experiments that lycopene (LYC) promotes Nrf2 nuclear translocation, increases the expression of antioxidant genes, and reduces the level of reactive oxygen species (ROS).

[0003] Cucumber (Cucumis sativus L.) is one of the world's top ten vegetables and also one of the most widely cultivated vegetables in my country. Compared with other common vegetables, cucumber has the weakest antioxidant activity, and there are significant differences between different varieties. In-depth research into the antioxidant mechanism of cucumber, and thus improving its antioxidant activity, is of great significance. Summary of the Invention

[0004] The purpose of this invention is to improve the antioxidant activity of plants.

[0005] This invention first protects the application of protein CsHAC1 in enhancing the antioxidant activity of plants;

[0006] The amino acid sequence of the protein CsHAC1 is shown in SEQ ID NO: 2.

[0007] This invention also protects the use of protein CsHAC1 in the cultivation of transgenic plants with enhanced antioxidant activity;

[0008] The amino acid sequence of the protein CsHAC1 is shown in SEQ ID NO: 2.

[0009] This invention also protects the application of nucleic acid molecules encoding the protein CsHAC1, which may be S1) or S2):

[0010] S1) Enhances the antioxidant activity of plants;

[0011] S2) Cultivate transgenic plants with enhanced antioxidant activity.

[0012] In the above applications, the nucleic acid molecule encoding the protein CsHAC1 can be b1) or b2):

[0013] b1) The coding region is the DNA molecule shown in SEQ ID NO: 1;

[0014] b2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 1.

[0015] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0016] Of these, SEQ ID NO: 1 consists of 942 nucleotides, and the nucleotides of SEQ ID NO: 1 encode the amino acid sequence shown in SEQ ID NO: 2.

[0017] In any of the above applications, the plant may be a cucumber.

[0018] The present invention also protects a method for cultivating transgenic plants, which may include the following steps: increasing the expression level and / or activity of protein CsHAC1 in a starting plant to obtain a transgenic plant; the transgenic plant exhibits increased antioxidant activity compared to the starting plant;

[0019] The amino acid sequence of the protein CsHAC1 is shown in SEQ ID NO: 2.

[0020] In the above method, the expression level and / or activity of protein CsHAC1 in the starting plant can be increased by methods well known in the art, such as transgenic, multiple copy, promoter alteration, and regulatory factors, to achieve the effect of increasing the expression level and / or activity of any of the above-mentioned proteins CsHAC1 in the starting plant.

[0021] In the above method, the increase in the expression level and / or activity of protein CsHAC1 in the starting plant can be achieved by introducing a nucleic acid molecule encoding the protein CsHAC1 into the starting plant.

[0022] In the above method, the nucleic acid molecule encoding the protein CsHAC1 can be b1) or b2):

[0023] b1) The coding region is the DNA molecule shown in SEQ ID NO: 1;

[0024] b2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 1.

[0025] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0026] Of these, SEQ ID NO: 1 consists of 942 nucleotides, and the nucleotides of SEQ ID NO: 1 encode the amino acid sequence shown in SEQ ID NO: 2.

[0027] In the above method, the introduction of the nucleic acid molecule encoding the protein CsHAC1 into the starting plant can be achieved by introducing a recombinant vector into the starting plant; the recombinant vector can be a recombinant plasmid obtained by inserting the nucleic acid molecule encoding the protein CsHAC1 into an expression vector. Specifically, the expression vector can be the vector Wmv077.

[0028] The recombinant vector may specifically be the recombinant plasmid Wmv077-CsHAC1. The recombinant plasmid Wmv077-CsHAC1 is obtained by inserting the DNA molecule shown in positions 1 to 939 from the 5' end of SEQ ID NO: 1 into the BamHI restriction endonuclease recognition site of the vector Wmv077. The transgenic plant may specifically be CsHAC1-OE1 and CsHAC1-OE2 mentioned in the examples, with the starting plant being cucumber, specifically cucumber variety 9930.

[0029] This invention also protects a plant breeding method, which may include the following steps: increasing the content and / or activity of protein CsHAC1 in plants, thereby increasing the antioxidant activity of plants;

[0030] The amino acid sequence of the protein CsHAC1 is shown in SEQ ID NO: 2.

[0031] In any of the methods described above, the plant may be a cucumber.

[0032] The cucumber mentioned above can be cucumber variety 9930.

[0033] Experiments have shown that overexpression of the CsHAC1 gene in cucumber variety 9930 can enhance the antioxidant activity of cucumber. This demonstrates that the protein CsHAC1 can improve the antioxidant activity of plants. This invention has significant application value. Attached Figure Description

[0034] Figure 1 This is a partial structural diagram of the recombinant plasmid Wmv077-CsHAC1.

[0035] Figure 2 Molecular identification of CsHAC1-OE1 and CsHAC1-OE2.

[0036] Figure 3 The results show the antioxidant activity of CsHAC1-OE1 and CsHAC1-OE2. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0040] The vector Wmv077 is a product of Weimi Biotechnology Co., Ltd., with the product code WMV1311.

[0041] The Total Antioxidant Capacity (T-AOC) assay kit is a product of Solarbio, catalog number BC1315.

[0042] The cucumber variety 9930 is described in the following literature: Miao Tiantian, Li Qiang, Yu Hongjun, Liu Peng, Hao Jia, Jiang Weijie. Effects of exogenous inositol on low temperature resistance of cucumber seedlings. Chinese Vegetables. 2021, Vol.1, Issue(2): 72-79.

[0043] Example 1: Cloning of the gene encoding protein CsHAC1 (i.e., the CsHAC1 gene)

[0044] 1. Total RNA was extracted from the leaves of cucumber variety 9930, and then the first-strand cDNA was reverse transcribed using reverse transcriptase to obtain the cDNA of cucumber variety 9930.

[0045] 2. Using the cDNA of cucumber variety 9930 obtained in step 1 as a template, primer F1 was used:

[0046] 5'-ATGGGGAGAAGGCCGTG-3' and primer R1:

[0047] PCR amplification was performed using a primer pair consisting of 5'-TCATGACAAAAGCCAAGAAATCAC-3', and approximately 942 bp of PCR amplification product was recovered.

[0048] 3. The PCR amplification products recovered in step 2 were sequenced. The sequencing results showed that the nucleotide sequence of the PCR amplification products was as shown in SEQ ID NO: 1.

[0049] The gene shown in SEQ ID NO:1 is named the CsHAC1 gene. The CsHAC1 gene encodes the protein CsHAC1, and the amino acid sequence of the protein CsHAC1 is shown in SEQ ID NO:2.

[0050] Example 2: Obtaining CsHAC1 gene-transgenic cucumber and detecting its antioxidant capacity.

[0051] I. Construction of recombinant plasmid Wmv077-CsHAC1

[0052] 1. Using the PCR amplification product recovered in step 2 of Example 1 as a template, primer F2: 5'- CACGGGGGACgt cgacGGATCC ATGGGGAGAAGGCCGTG-3' (underlined part is the vector's adapter sequence) and primer R2:

[0053] 5'- GTCAGGAACATCGTATGGGTAcat The primer pair consisting of TGACAAAAGCCAAGAAATCAC-3' (the underlined part is the adapter sequence of the vector) was used for PCR amplification, and a DNA fragment of about 985 bp was recovered.

[0054] 2. The vector Wmv077 was digested with the restriction endonuclease BamHI, and the 11kb vector backbone was recovered.

[0055] 3. The DNA fragment recovered in step 1 and the vector backbone recovered in step 2 were recombinantly ligated using recombinase (Full Gold, CU101-01) to obtain the recombinant plasmid Wmv077-CsHAC1.

[0056] The recombinant plasmid Wmv077-CsHAC1 was sequenced. The sequencing results showed that the recombinant plasmid Wmv077-CsHAC1 was obtained by inserting the DNA molecule shown in positions 1 to 939 from the 5' end of SEQ ID NO: 1 into the BamHI restriction endonuclease recognition site of the vector Wmv077.

[0057] The recombinant plasmid Wmv077-CsHAC1 expresses the protein CsHAC1 shown in SEQ ID NO: 2.

[0058] A partial structural diagram of the recombinant plasmid Wmv077-CsHAC1 is shown below. Figure 1 .

[0059] II. Obtaining Recombinant Agrobacterium

[0060] 1. The recombinant plasmid Wmv077-CsHAC1 was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium, which was named GV3101 / Wmv077-CsHAC1.

[0061] 2. The vector Wmv077 was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium, named GV3101 / Wmv077.

[0062] III. Obtaining and Molecular Identification of T1 Generation CsHAC1 Transgenic Cucumbers

[0063] 1. Obtaining T0 generation CsHAC1 gene-transformed cucumber seedlings

[0064] (1) Select plump and intact cucumber variety 9930 seeds. After peeling, disinfect them in 70% (v / v) ethanol aqueous solution for 30s, then sterilize them in 2.0% sodium hypochlorite solution for 15min. Finally, rinse them several times with sterile water and sow them on MS solid medium. After the cotyledons have just broken open (about 2-3 days), cut off the growing point and hypocotyl. Cut off the upper half of both cotyledons and keep the lower half of the cotyledons as cotyledon explants.

[0065] MS solid medium: Dissolve 4.43g MS powder and 30g sucrose in an appropriate amount of deionized water, then add 2.5g plant gel, and bring the volume to 1L with deionized water. Adjust the pH to 5.7-5.8.

[0066] (2) A single colony of GV3101 / Wmv077-CsHAC1 was inoculated into YEB liquid medium containing 50 mg / L kanamycin and 70 mg / L rifampin, and incubated overnight at 28°C with shaking at 200 rpm / min to obtain bacterial suspension 1. 2 mL of bacterial suspension 1 was added to 50 mL of YEB liquid medium containing 50 mg / L kanamycin and 70 mg / L rifampin, and incubated at 28°C with shaking to obtain OD. 600nm Bacterial suspension 2 was prepared with a concentration of 0.6-0.8. The bacterial suspension 2 was centrifuged at 5000 rpm for 5 min to collect the bacterial cells. The cells were then washed once with 1 / 2 MS liquid medium, followed by dilution with 1 / 2 MS liquid medium to obtain the OD value. 600nm The concentration of the inoculum is approximately 0.2.

[0067] 1 / 2MS liquid culture medium: Dissolve 2.2g MS powder and 30g sucrose in an appropriate amount of deionized water, then bring the volume to 1L with deionized water and adjust the pH to 5.7-5.8.

[0068] (3) Infect the cotyledon explants with the infection solution obtained in step (2) for 15 min. After infection, use sterile filter paper to absorb the excess infection solution, and inoculate the cotyledon explants dorsally onto MS differentiation medium and incubate in the dark at 28℃ for 2 days.

[0069] MS differentiation medium: Dissolve 4.43g MS powder, 30g sucrose, 2.5g plant gel, 0.5mg 6-BA and 1mg ABA in an appropriate amount of deionized water, then bring the volume up to 1L with deionized water and adjust the pH to 5.7-5.8.

[0070] (4) After completing step (3), the cotyledon explants were transferred into MS differentiation medium containing 25 mg / L kanamycin and 500 mg / L carbenicillin and cultured at 28°C with alternating light and dark conditions (16 h light culture / 8 h dark culture, light intensity of 2000 lx) for 15-20 days to obtain resistant shoots of about 1-1.5 cm.

[0071] (5) After completing step (4), cut off the resistant bud and transfer it into MS rooting medium containing 100 mg / L kanamycin to induce rooting and obtain resistant cucumber seedlings with 5-6 leaves (at this time, the cucumber root system is well developed).

[0072] MS rooting medium: Dissolve 4.43g MS powder, 30g sucrose and 2.5g plant gel in an appropriate amount of deionized water, then bring the volume up to 1L with deionized water and adjust the pH to 5.7-5.8.

[0073] (6) After completing step (5), the resistant cucumber seedlings are transferred into flower pots filled with sterile soil and covered with plastic wrap to retain moisture. They are then cultured in an artificial climate chamber for one week, and then transferred to a greenhouse to adapt for 3-5 days. Finally, they are transferred to a greenhouse for routine management to obtain T0 generation CsHAC1 gene-transformed cucumber seedlings.

[0074] 2. Obtaining T1 generation CsHAC1 gene-transformed cucumbers

[0075] (1) T0 generation cucumber seedlings transgenic with CsHAC1 gene were self-crossed to obtain T1 generation cucumber seeds transgenic with CsHAC1 gene.

[0076] (2) T1 generation CsHAC1 gene-transformed cucumber seeds were sown on MS solid medium containing hygromycin. The cucumber seedlings that grew normally were T1 generation CsHAC1 gene-transformed cucumbers. The two T1 generation CsHAC1 gene-transformed cucumber lines were named CsHAC1-OE1 and CsHAC1-OE2, respectively.

[0077] Following the steps above, replace GV3101 / Wmv077-CsHAC1 with GV3101 / Wmv077, keeping all other steps unchanged, to obtain T1 generation empty cucumber.

[0078] 3. Molecular identification

[0079] Genomic DNA was extracted from cucumber seedlings to be tested (T1 generation seedlings of CsHAC1-OE1, T1 generation seedlings of CsHAC1-OE2, T1 generation seedlings converted to empty-gene cucumber, or seedlings of cucumber variety 9930, all seedlings having the same number of days of growth) and used as templates. PCR amplification was performed using primer pairs consisting of 5'-ATGGGGAGAAGGCCGTG-3' and 5'-AGCGTAATCTGGAACGTCATAT-3' to obtain PCR amplification products. Then, the following judgment was made: if a PCR amplification product contains a DNA fragment of approximately 1029 bp, the cucumber seedling corresponding to that PCR amplification product is a positive seedling.

[0080] The agarose gel electrophoresis results of PCR amplification products of cucumber variety 9930 seedlings, T1 generation seedlings of CsHAC1-OE1, and T1 generation seedlings of CsHAC1-OE2 are shown in the figure. Figure 2 (Mark stands for DNA Marker)

[0081] The results showed that both the T1 generation seedlings of CsHAC1-OE1 and CsHAC1-OE2 were positive seedlings. The T1 generation seedlings converted to empty cucumbers or those of cucumber variety 9930 were not positive seedlings.

[0082] IV. Antioxidant capacity detection of T1 generation CsHAC1 gene-transformed cucumbers

[0083] The experiment was repeated three times and the average value was taken. The steps for each repetition were as follows:

[0084] 1. Take the seeds of the cucumber to be tested (CsHAC1-OE1, CsHAC1-OE2 or cucumber variety 9930) (for CsHAC1-OE1 and CsHAC1-OE2, take the T1 generation seeds), first disinfect them in 70% (v / v) ethanol aqueous solution for 30 seconds, then place them in 2.0% sodium hypochlorite solution for 15 minutes to sterilize, and finally rinse them several times with sterile water before sowing them in nutrient soil and cultivating them normally until the fruits of the cucumber on day 0, day 10, day 15 and day 30 are obtained.

[0085] 2. The antioxidant capacity of cucumber fruits (day 0, day 10, day 15, or day 30) was determined using the ferric ion reducing antioxidant power (FRAP) method. The FRAP method is a method that measures the antioxidant capacity of samples by utilizing the blue-violet complex formed by the reaction of ferrous ions with TPTZ (tripyridyltriazine) under low pH conditions. The specific steps are as follows:

[0086] (1) Plotting the standard curve

[0087] (1-1) Take a standard solution with a concentration of 40 μmol / mL (a component in the Total Antioxidant Capacity (T-AOC) assay kit), dilute it with distilled water to obtain solutions with concentrations of 0.15 μmol / mL (Solution 1), 0.1 μmol / mL (Solution 2), 0.05 μmol / mL (Solution 3), 0.025 μmol / mL (Solution 4), 0.0125 μmol / mL (Solution 5), 0.00625 μmol / mL (Solution 6), 0.003125 μmol / mL (Solution 7), and 0.00156 μmol / mL (Solution 8). The Fe content in solutions 1-8 is... 2+ The concentrations were 0.075, 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.00156, and 0.00078 μmol / mL, respectively.

[0088] (1-2) Thoroughly mix 100 μL of solution (solution 1, solution 2, solution 3, solution 4, solution 5, solution 6, solution 7, or solution 8) with 100 μL of reagent 2 (a component from the Total Antioxidant Capacity (T-AOC) Assay Kit), then react at room temperature for 10 min. Pipette 200 μL into a microcapacitor and measure the absorbance at 593 nm using a spectrophotometer (using A...). 标准 express).

[0089] (1-3) Thoroughly mix 100 μL of distilled water and 100 μL of reagent II, then react at room temperature for 10 min. Pipette 200 μL into a microvolume glass cuvette and measure the absorbance at 593 nm using a spectrophotometer (using A). 空白对照 express).

[0090] (1-4) After completing steps (1-2) and (1-3), ΔA is obtained. 标准 ΔA 标准 =A 标准 -A 空白对照 .

[0091] (1-5) Fe in solutions 1-8 2+ The concentration is plotted on the x-axis, and the corresponding ΔA 标准 Use the vertical axis to plot the standard curve.

[0092] The results show that the standard curve is: y = 13.078x + 0.0009(R²). 2 =0.9999), where x is Fe 2+ Concentration (μmol / mL), y is the absorbance value.

[0093] (2) Sample detection

[0094] (2-1) Take 0.1g of cucumber fruit to be tested, add 0.5mL of pre-cooled extraction solution (a component in the Total Antioxidant Capacity (T-AOC) Detection Kit), homogenize in an ice bath, then centrifuge at 4℃ and 10000rpm for 10min and collect the supernatant.

[0095] (2-2) Thoroughly mix 6 μL of supernatant, 18 μL of distilled water, and 180 μL of a mixture (the mixture is prepared by mixing reagents 1, 2, and 3 in a 7:1:1 ratio; prepare fresh and use immediately; preheat at 37°C for 10 min before use; reagents 1, 2, and 3 are all components of the Total Antioxidant Capacity (T-AOC) assay kit). React at room temperature for 10 min. Pipette 200 μL into a microvolume glass cuvette and measure the absorbance at 593 nm using a spectrophotometer. This absorbance is the sample absorbance at 593 nm (using A...). 样品 express).

[0096] (2-3) Thoroughly mix 24 μL of distilled water and 180 μL of the mixture, then react at room temperature for 10 min. Pipette 200 μL into a microvolume glass cuvette and measure the absorbance at 593 nm using a spectrophotometer; this is the absorbance of the blank control at 593 nm (using A). 空白对照 express).

[0097] (2-4) After completing steps (2-2) and (2-3), ΔA is obtained. 样品 ΔA 样品 =A 样品 -A 空白对照 .

[0098] (2-5) The ΔA obtained in step (2-4) 样品 Substitute the standard curve drawn in step (1) (i.e., y is ΔA) 样品 ), to obtain Fe in the sample 2+The concentration of Fe in the sample (μmol / mL) was determined; further, the antioxidant capacity of the sample (μmol / g) was calculated based on the sample mass, i.e., the antioxidant capacity of the sample (μmol / g) = Fe in the sample. 2+ Concentration (μmol / mL) × Total volume of sample after reaction (mL) / Sample mass (g).

[0099] Test results are shown Figure 3 (9930 represents cucumber variety 9930, OE1 represents CsHAC1-OE1, and OE2 represents CsHAC1-OE2; ** indicates p<0.01, **** indicates p<0.0001). The results showed that compared with cucumber variety 9930, the antioxidant capacity of T1 generation CsHAC1 gene-transgenic cucumbers (CsHAC1-OE1 and CsHAC1-OE2) was significantly enhanced; moreover, the antioxidant capacity continued to increase with the growth of cucumber fruits.

[0100] The above results indicate that by introducing the CsHAC1 gene into cucumbers to increase the expression level of the protein CsHAC1, the antioxidant capacity of cucumbers can be enhanced.

[0101] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of CsHAC1 overexpression in enhancing the antioxidant activity of cucumber; The amino acid sequence of the protein CsHAC1 is shown in SEQ ID NO:

2.

2. Application of CsHAC1 overexpression in the cultivation of transgenic cucumbers with enhanced antioxidant activity; The amino acid sequence of the protein CsHAC1 is shown in SEQ ID NO:

2.

3. The application of overexpressing a nucleic acid molecule encoding the protein CsHAC1 of claim 1, as S1) or S2). S1) Enhances the antioxidant activity of cucumber; S2) Develop transgenic cucumbers with enhanced antioxidant activity.

4. The application according to claim 3, characterized in that: The nucleic acid molecule encoding the protein CsHAC1 of claim 1 is a DNA molecule whose coding region is shown in SEQ ID NO:

1.

5. The application according to claim 3, characterized in that: The nucleic acid molecule encoding the protein CsHAC1 of claim 1 is a DNA molecule with the nucleotide sequence shown in SEQ ID NO:

1.

6. A method for cultivating transgenic cucumbers, comprising the following steps: increasing the expression level of protein CsHAC1 in a starting cucumber to obtain a transgenic cucumber; compared with the starting cucumber, the transgenic cucumber exhibits increased antioxidant activity; The amino acid sequence of the protein CsHAC1 is shown in SEQ ID NO:

2.

7. The method according to claim 6, characterized in that: The expression level of protein CsHAC1 in the starting cucumber is increased by introducing a nucleic acid molecule encoding protein CsHAC1 into the starting cucumber.

8. A cucumber breeding method, comprising the following steps: increasing the content of protein CsHAC1 in cucumber, thereby increasing the antioxidant activity of cucumber; The amino acid sequence of the protein CsHAC1 is shown in SEQ ID NO: 2.

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