Use of RcWOX8a in mediating anthocyanin accumulation in roses under abiotic stress

Abstract

The application provides application of a gene RcWOX8a in mediation of anthocyanin accumulation of Chinese rose under abiotic stress, and the gene RcWOX8a provided by the application plays an important role in mediation of anthocyanin accumulation of Chinese rose under abiotic stress. In a Chinese rose transient expression strain, the tolerance of Chinese rose to abiotic stress is obviously increased, thereby providing an important basis for Chinese rose breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of RcWOX8a in mediating anthocyanin accumulation in roses under abiotic stress. Background Technology

[0002] The rose (Rosa chinensis Jacq.), belonging to the genus Rosa in the family Rosaceae, is a perennial woody plant. Known for its strong fragrance and vibrant colors, it is often called the "Queen of Flowers" and is widely used as a cut flower, potted plant, and in landscaping. Roses are highly adaptable to different environments, have a long flowering period, and come in a variety of shapes and colors, making them widely cultivated throughout my country.

[0003] Anthocyanins are water-soluble pigments found in many plants, algae, and bacteria, and are important plant secondary metabolites. Anthocyanins play a vital biological role in attracting pollinators and dispersing seeds. Furthermore, they have physiological functions such as scavenging free radicals and reactive oxygen species to protect chloroplasts from photo-oxidative and photoinhibitory damage, and to resist stress conditions or pathogen infection. However, under abiotic stress conditions, the anthocyanin content in roses decreases significantly, affecting the color of rose petals and reducing their ornamental and practical value. Summary of the Invention

[0004] This invention provides the application of RcWOX8a in mediating anthocyanin accumulation in roses under abiotic stress, which can be used to increase the amount of anthocyanins accumulated in roses under abiotic stress.

[0005] The first aspect of this invention provides the application of gene RcWOX8a in increasing anthocyanin accumulation in roses under abiotic stress, the nucleotide sequence of gene RcWOX8a being shown in SEQ ID NO:1.

[0006] As described above, the gene RcWOX8a is overexpressed in roses.

[0007] The application described above, specifically overexpressing the gene RcWOX8a in roses, includes the following steps:

[0008] The open reading frame of gene RcWOX8a is operatively linked to an expression vector to form a recombinant vector containing the gene RcWOX8a fragment.

[0009] The recombinant vector was transformed into Agrobacterium to obtain positive transformants;

[0010] The positive transformants were used to infect roses and cultured to obtain roses that overexpressed the gene RcWOX8a.

[0011] In the applications described above, the abiotic stress includes drought.

[0012] As described above, the rose is the 'White Snow Mountain' rose.

[0013] A second aspect of the present invention provides a method for increasing anthocyanin accumulation in roses under abiotic stress, wherein the gene RcWOX8a is overexpressed in the rose, and the nucleotide sequence of the gene RcWOX8a is shown in SEQ ID NO:1.

[0014] The method described above, specifically for overexpressing the gene RcWOX8a in the rose, includes the following steps:

[0015] The open reading frame of gene RcWOX8a is operatively linked to an expression vector to form a recombinant vector containing the gene RcWOX8a fragment.

[0016] The recombinant vector was transformed into Agrobacterium to obtain positive transformants;

[0017] The positive transformants were used to infect roses and cultured to obtain roses that overexpressed the gene RcWOX8a.

[0018] In the method described above, the abiotic stress includes drought.

[0019] As described above, the rose is the 'White Snow Mountain' rose.

[0020] Using the method described above, after being placed at a temperature of 18°C ​​for 4 days, the anthocyanin content in roses overexpressing the RcWOX8a gene was above 80 μg / g.

[0021] The transcription factor RcWOX8a, cloned from ancient rose pollen, can promote anthocyanin accumulation in roses by mediating abiotic stress, providing an important foundation for rose breeding. Attached Figure Description

[0022] Figure 1 Images of different textures of 'Monthly Powder';

[0023] Figure 2 The content of RcWOX8a in different tissues of 'Yueyue Powder';

[0024] Figure 3 The phenotypes of the control and overexpression petal discs were obtained by vacuum inoculating 'White Snow Mountain' petal discs with pCAMBIA2300-RcWOX8a after 4 days.

[0025] Figure 4 The anthocyanin content in the control and overexpression petal discs after 4 days was determined by vacuum inoculation of 'White Snow Mountain' petal discs with pCAMBIA2300-RcWOX8a.

[0026] Figure 5The 'White Snow Mountain' petal discs were vacuum-infected with pCAMBIA2300-RcWOX8a, and after 3 days, they were air-dried in an incubator at 18℃ under continuous light for 9 hours to obtain the phenotypes of the control and overexpression petal discs.

[0027] Figure 6 'White Snow Mountain' petal discs were vacuum-infected with pCAMBIA2300-RcWOX8a. After 3 days, they were air-dried in an incubator at 18℃ under continuous light for 9 hours. Then, samples of control and overexpression petal discs were taken for DAB and NBT determination.

[0028] Figure 7 'White Snow Mountain' petal discs were vacuum-infected with pCAMBIA2300-RcWOX8a. After 3 days, they were air-dried in an incubator at 18℃ under continuous light for 9 hours. The relative conductivity of the control and overexpression petal discs was then measured.

[0029] Figure 8 The content of abscisic acid and reactive oxygen species-related genes in the 'White Snow Mountain' petal discs was determined by vacuum inoculation with pCAMBIA2300-RcWOX8a for 3 days, followed by air drying in an incubator at 18℃ under continuous light for 9 hours. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0031] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise specified, all reagents used were commercially available or publicly available.

[0032] In this invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids.

[0033] Example 1: Cloning of the RcWOX8a gene in roses

[0034] Total RNA was extracted from the petals of the ancient rose 'Yueyuefen' using a commercially available RNAplant extraction kit. The total RNA was then reverse transcribed into cDNA using a commercially available reverse transcription kit. Primers were designed based on the transcriptome sequencing results, and their sequences are shown in SEQ ID NO:3 and SEQ ID NO:4. An 825 bp band was amplified from the 'Yueyuefen' cDNA using RT-PCR. The PCR product was recovered, and the RcWOX8a gene was obtained. Its nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by this nucleotide sequence is shown in SEQ ID NO:2. The protein consists of 274 amino acid pairs and has a molecular weight of 31.1 kilodaltons (kDa).

[0035] Example 2: Validation of RcWOX8a expression profiles in different rose tissues

[0036] Step 1, extract as follows Figure 1 The RNA from different tissues of the rose 'Monthly Pink' shown (where ad represents leaves at different developmental stages; e represents petals; f represents pistils; g represents stamens; h represents fruits; i represents young stems; j represents mature stems; k represents young thorns; and l represents mature thorns) was extracted using RNAplant (commercially available). The total RNA was reverse transcribed into cDNA using a reverse transcription kit (commercially available).

[0037] Step 2: Design primers based on transcriptome sequencing data. Primer sequences are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0038] Step 3: Using cDNA obtained from reverse transcription of 'Yueyuefen' at different developmental stages as templates, the expression profiles of RcWOX8a in different tissues were verified. The verification results are as follows: Figure 2 As shown, the expression of RcWOX8a varies in different tissues, with higher expression in young leaves (a).

[0039] Example 3: Overexpression of the RcWOX8a gene in the 'White Snow Mountain' rose variety

[0040] Step 1: The open reading frame 825 bp of the RcWOX8a gene is operatively ligated into the pCAMBIA2300 vector to form the pCAMBIA2300-RcWOX8a vector containing the gene fragment.

[0041] Step 2: Transform the vector from Step 1 into Agrobacterium GV3101.

[0042] Step 3: Take Agrobacterium GV3101 from Step 2 and incubate it in 5 ml of LB medium containing 100 μM acetylsuccinone and 50 μg / ml kanamycin at 28°C and 200 rpm for 16 h.

[0043] Step 4: Take the Agrobacterium from Step 3 and subculture it in 50 ml of LB medium containing 100 μM acetylsuccinone and 50 μg / ml kanamycin. Incubate it at 28℃ and 200 rpm for 13-16 h until the OD600 reaches 0.8-1.0.

[0044] Step 5: Take the Agrobacterium tumefaciens bacterial suspension from Step 4, centrifuge at 3700 rpm for 9 min at 4℃, and discard the supernatant; in a clean bench, resuspend the bacterial cells with 5 mL of MgCl2, centrifuge at 3700 rpm for 9 min at 4℃, discard the supernatant, and repeat once;

[0045] Step 6: Resuspend the bacterial cells in an appropriate amount of MgCl2. Inject Agrobacterium containing pCAMBIA2300-Ev or pCAMBIA2300-RcWOX8a plasmid with an OD600 of 1.2. Add 1.5 times the volume of AS and 20 times the volume of MES to the bacterial culture to resuspend the cell pellet. Let stand at room temperature for 2 hours.

[0046] Step 7: Use a hole punch to take the second and third layers of petals from the 'White Snow Mountain' rose, punch holes in them, and obtain small round petal pieces.

[0047] Step 8: Use a vacuum instrument to inoculate the Agrobacterium transformation solution from Step 6 into small round discs of rose petals and culture for 4-7 days;

[0048] Step 9: Air dry in an incubator at 18℃ under continuous light, and observe the color of the rose petals. The results after 4 days are as follows... Figure 3 As shown, compared with the control group EV, the experimental group RcWOX8a has significantly darker petal color, indicating that the drought resistance of the rose is significantly enhanced.

[0049] Example 4: Verification of anthocyanin content in small round petals of the 'White Snow Mountain' rose overexpression.

[0050] Step 1, as follows Figure 3 The control and overexpression petal discs shown were weighed and placed in 1.5 mL centrifuge tubes. 1 mL of hydrochloric acid-methanol (1:99) was added, and the mixtures were stored in the dark at 4°C overnight. The absorbance of the extracts was measured at 530 nm and 657 nm, and the anthocyanin content was calculated using the formula: (A530 - 0.25 × A657) / fresh weight.

[0051] The results of anthocyanin content determination in control and overexpression petal discs are as follows: Figure 4 As shown, anthocyanins were expressed at high levels in the overexpressed petal discs, reaching over 80 μg / g.

[0052] Example 5: DNB and NBT staining verification of the 'White Snow Mountain' rose overexpression petal disc control group and the RcWOX8a gene overexpression experimental group.

[0053] Step 1: Prepare PBS solution: In a container, prepare 80 mL of pure water, add 1.5483 g of disodium hydrogen phosphate heptahydrate, then add 0.5066 g of sodium dihydrogen phosphate monohydrate. Adjust the pH to 7, and add pure water to bring the volume to 800 mL. Prepare two separate containers. Divide the prepared PBS solution into 400 mL portions, add 0.4 g of DAB and 0.4 g of NBT to each, mix well, and store at 4°C protected from light.

[0054] Step 2: Randomly select 10 small round petals and place them into 50ml centrifuge tubes. Add NBT / DAB to each tube, vacuum for 15 minutes, and incubate overnight in the dark. The next day, pour the solution into a waste container, add anhydrous ethanol, and boil until the leaves turn white, changing the anhydrous ethanol continuously. After decolorization, prepare 50% glycerol, pour it into the tubes, and incubate until the leaves unfold. Let it stand for 1-2 hours, then remove it and wipe off the glycerol from the leaf surface with a paper towel before photographing.

[0055] Step 3, as follows Figure 6 As shown, the color of the RcWOX8a overexpression experimental group was significantly lighter than that of the control group, indicating that the RcWOX8a overexpression experimental group accumulated less reactive oxygen species under drought stress, proving that RcWOX8a can improve the drought resistance of roses.

[0056] Example 6: Relative conductivity determination of the 'White Snow Mountain' rose overexpression petal disc control group and the RcWOX8a gene overexpression experimental group.

[0057] Step 1: After culturing the petals of the control group and the RcWOX8a overexpression group in Example 3 for 3 days under normal conditions, the petals were subjected to drought treatment (18℃, air-dried with the lid off for 9 hours). The results are as follows: Figure 5 As shown in the figure, it is clear that the wrinkling was more severe in the control group after drought. (The text then abruptly shifts to a seemingly unrelated topic: selecting...) Figure 5 The relative conductivity of the control and overexpressed petal discs was measured.

[0058] Step 2: Place 6 small round petals into each tube (repeat 3 times for each), and add sterile water (do not overfill). Vacuum the tube and incubate overnight in the dark. The next day, measure the conductivity using a conductivity meter. After boiling for 20 minutes, cool for 5-10 minutes, and then measure the conductivity again. Finally, take the relative value.

[0059] The measurement results are as follows Figure 7 As shown, the relative conductivity of the RcWOX8a overexpression experimental group was significantly lower than that of the control group, proving that RcWOX8a overexpression can improve the drought tolerance of roses.

[0060] Example 7: Verification of the expression of the RcWOX8a gene and genes related to reactive oxygen species and abscisic acid synthesis in overexpressed petal discs of the 'White Snow Mountain' rose.

[0061] Step 1, select as follows Figure 5 The control and overexpression petal discs shown were used to extract total RNA using RNAplant (commercially available) extraction kit. The total RNA was then reverse transcribed into cDNA using a commercially available reverse transcription kit.

[0062] Step 2: Design primers based on transcriptome sequencing data. The primer sequences are shown in SEQ ID NO:5-20. Among them, SEQ ID NO:5-6 is a primer for RcWOX8a, SEQ ID NO:7-8 is a primer for RcNCED1, SEQ ID NO:9-10 is a primer for RcABF2, SEQ ID NO:11-12 is a primer for RcABI5, SEQ ID NO:13-14 is a primer for RcLEA14, SEQ ID NO:15-16 is a primer for RcDREB1, SEQ ID NO:17-18 is a primer for RcRD22, and SEQ ID NO:19-20 is a primer for RcSPCH.

[0063] Step 3: Using cDNA obtained by reverse transcription of the control and overexpression petal discs as templates, the gene overexpression efficiency of RcWOX8a and genes related to reactive oxygen species and abscisic acid synthesis was verified.

[0064] The verification results are as follows Figure 8 As shown, the expression of RcWOX8a and genes related to reactive oxygen species and abscisic acid synthesis were significantly increased in the petals of the 'Bai Xue Shan' mutant line, proving that RcWOX8a overexpression efficiency was high, and further indicating that RcWOX8a is involved in the formation of reactive oxygen species and abscisic acid-related genes in the 'Bai Xue Shan' rose.

[0065] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Overexpression of genes in the 'White Snow Mountain' rose. RcWOX8a Its application in enhancing anthocyanin accumulation in the 'White Snow Mountain' rose variety under drought stress is characterized by... Gene RcWOX8a The nucleotide sequence is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, Gene overexpression in the 'White Snow Mountain' rose RcWOX8a Specifically, it includes the following steps: Genes RcWOX8a The open reading frame is operatively linked to the expression vector to form a structure containing the gene. RcWOX8a Recombination vectors for fragments; The recombinant vector was transformed into Agrobacterium to obtain positive transformants; The positive transformants were infected with the 'White Snow Mountain' rose variety and cultured to obtain overexpressed genes. RcWOX8a The 'White Snow Mountain' rose.

3. A method for increasing anthocyanin accumulation in the 'White Snow Mountain' rose variety under drought stress, characterized in that, Genes overexpressed in the 'White Snow Mountain' rose RcWOX8a The gene RcWOX8a The nucleotide sequence is shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that, Genes overexpressed in the 'White Snow Mountain' rose RcWOX8a Specifically, it includes the following steps: Genes RcWOX8a The open reading frame is operatively linked to the expression vector to form a structure containing the gene. RcWOX8a Recombination vectors for fragments; The recombinant vector was transformed into Agrobacterium to obtain positive transformants; The positive transformants were infected with the 'White Snow Mountain' rose variety and cultured to obtain overexpressed genes. RcWOX8a Roses.

5. The method according to claim 3, characterized in that, After being placed at 18°C ​​for 4 days, the overexpressed gene... RcWOX8a The anthocyanin content in the rose is above 80 μg / g.

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