Ofphl5 gene related to osmanthus carotenoid synthesis and application thereof

Abstract

The application discloses a gene related to osmanthus fragrans carotenoid synthesis OfPHL5 and application thereof OfPHL5 The gene is a MYB transcription factor gene, and an overexpression vector containing the gene OfPHL5 is transformed into osmanthus fragrans petals to perform transient expression. OfPHL5 The results show that the gene expression amount of the transiently transformed osmanthus fragrans petals is significantly increased, and the total carotenoid content in the petals is increased, which indicates that the gene plays an important role in promoting osmanthus fragrans carotenoid synthesis. OfPHL5 The gene can be used for improving the color quality of osmanthus fragrans and excellent variety breeding in osmanthus fragrans genetic engineering.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to an OfPHL5 gene related to osmanthus carotenoid synthesis and an application thereof. Background Art

[0002] Carotenoids are secondary metabolites widely found in plants and have a significant impact on their growth and development. Differences in carotenoid types and content in plant organs, such as petals and fruits, result in distinct colors, creating a vibrant world. Carotenoids can also be broken down to produce important aromatic compounds, such as ionones. For plants, bright colors and fragrant aromas effectively attract insects and other animals for pollination, improving reproductive success. Within the photosynthetic system, carotenoids play a crucial role in plant photosynthesis, helping to capture light energy and transfer it to chlorophyll. They also effectively scavenge free radicals, preventing damage caused by photooxidation. Furthermore, the downstream products of carotenoids, the hormones abscisic acid (ABA) and strigolactone, serve as important endogenous signaling substances for plants to resist stress and regulate growth and development. Carotenoids are also a class of highly valuable natural active substances with antioxidant properties and are a vital component of the human diet.

[0003] Sweet osmanthus (Osmanthus fragrans Lour.) is an important ornamental plant and spice plant. The carotenoids in its petals can be decomposed to produce aroma components such as α-ionone and β-ionone, thereby giving sweet osmanthus its strong violet fragrance, woody fragrance and fruity aroma. The MYB transcription factor is one of the largest transcription factor families, playing a key role in regulating the synthesis of primary and secondary metabolites and widely participating in the regulation of aspects such as cell differentiation, secondary metabolism, environmental stress and pest and disease invasion. However, there are no reports on genes related to sweet osmanthus carotenoid synthesis in the MYB family. Therefore, it is of great significance to study and explore the related genes that promote sweet osmanthus carotenoid synthesis in the sweet osmanthus genome. The present invention utilizes bioinformatics technology to screen out genes that promote sweet osmanthus carotenoid synthesis in sweet osmanthus and performs functional verification in sweet osmanthus petals. Summary of the Invention

[0004] In response to the above-mentioned problems existing in the prior art, the present invention provides an OfPHL5 gene related to the synthesis of osmanthus carotenoids. Overexpression of the gene can promote the synthesis of osmanthus carotenoids and can be used in osmanthus genetic engineering to improve the color quality of osmanthus petals and breed excellent varieties.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] The OfPHL5 gene related to the synthesis of osmanthus carotenoids, the CDS sequence of the OfPHL5 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0007] Biological materials containing the OfPHL5 gene include expression cassettes, expression vectors, and recombinant bacteria.

[0008] Applications of the OfPHL5 gene include:

[0009] (1) Application in Osmanthus fragrans breeding;

[0010] (2) Application in promoting the synthesis of carotenoids in Osmanthus fragrans.

[0011] A method for promoting the synthesis of osmanthus carotenoids comprises the following steps:

[0012] (1) Constructing an overexpression vector containing the OfPHL5 gene shown in SEQ ID NO. 1;

[0013] (2) transforming the expression vector into Agrobacterium to obtain recombinant expression Agrobacterium;

[0014] (3) transforming Osmanthus fragrans with the recombinant Agrobacterium to overexpress the OfPHL5 gene in Osmanthus fragrans;

[0015] Preferably, in step (3), recombinant bacteria are used to transform osmanthus flower petals;

[0016] Preferably, the Agrobacterium is Agrobacterium GV3101.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a gene OfPHL5 related to promoting the synthesis of carotenoids in osmanthus, which is a MYB transcription factor gene. An overexpression vector containing the gene OfPHL5 is transformed into osmanthus petals for transient expression. The results show that the expression level of the OfPHL5 gene in the transiently transformed osmanthus petals is significantly increased, the total carotenoid content in the petals is significantly increased compared with the control group, and the ionone content is significantly decreased, indicating that the OfPHL5 gene plays an important role in promoting the synthesis of total carotenoids in osmanthus, and can be used for breeding work such as improving the color quality and genetic quality of osmanthus in osmanthus genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the agarose gel electrophoresis diagram of the amplified product of the target gene OfPHL5.

[0020] Figure 2 To instantaneously convert the total carotenoids and ionone contents in Osmanthus fragrans petals.

[0021] Figure 3 is the expression level of the OfPHL5 gene in transiently transformed Osmanthus fragrans petals. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is described below with reference to the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0023] Example 1 Screening and cloning of target genes

[0024] (1) Obtaining the target gene: Based on the transcriptome data analysis of five osmanthus varieties conducted by Zheng Riru's research group at Huazhong Agricultural University, a gene sequence was screened and compared with the sequence of the model plant Arabidopsis thaliana. It was determined that the gene belongs to the MYB gene family. The gene has a high similarity with the PHL5 gene of the closely related species Olea europaea, and was named OfPHL5.

[0025] (2) Primer design: Specific primers OfPHL5-CDS-F and OfPHL5-CDS-R (see Table 1) for amplifying the full-length CDS of the OfPHL5 gene were designed using Primer 5.0 software. The amplification primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0026] Table 1 Primer sequences

[0027]

[0028] (3) Target gene amplification: Using Osmanthus fragrans cDNA as a template, amplify the target gene CDS with reference to the instructions of Phanta high-fidelity enzyme. The PCR reaction system and procedure are shown in Tables 2 and 3.

[0029] Table 2 PCR reaction system

[0030]

[0031] Table 3 PCR reaction program

[0032]

[0033]

[0034] (4) Gel electrophoresis and gene sequencing: 0.3 g agarose powder was added to 30 ml TAE, and after melting in a microwave oven, 1.5 μL of 10000× nucleic acid dye was added. The sample was then added to the gel plate and spotted after solidification. After electrophoresis at 120 V and 150 mA for 25 min, the bands were observed on a Gel-Logie 200 gel scanner. The target band was 1152 bp (e.g. Figure 1The target band was recovered and ligated to a T-vector, and transformed into E. coli for sequencing. The CDS sequence of the OfPHL5 gene is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2. The plasmid was extracted from the correctly sequenced bacterial solution and named OfPHL5-CDS-Plasmid, which was used as a template for subsequent vector construction.

[0035] Example 2 Construction of an overexpression vector for the Osmanthus fragrans OfPHL5 gene

[0036] (1) Target gene cloning

[0037] Using the plasmid OfPHL5-CDS-Plasmid as a template, homology primers containing Kpn1 / Sal1 restriction sites were designed using CE Design V1.04 (Table 1). PCR amplification was performed according to the instructions for the Phanta high-fidelity enzyme. The PCR reaction system and procedures are shown in Tables 4 and 3. After the PCR reaction, all PCR products were run on a gel to detect the presence of clear target bands and then excised and recovered.

[0038] Table 4 PCR reaction system

[0039]

[0040] (2) Super1300 vector linearization

[0041] The Super1300 vector was double-digested with Kpn1 and Sal1 to obtain a linearized vector. The digestion reaction system is shown in Table 5. Incubate at 37°C for 15 minutes and heat-inactivate at 65°C for 20 minutes. All digestion products were run on a gel and recovered.

[0042] Table 5 Super1300 vector double enzyme digestion reaction system

[0043]

[0044] (3) Homologous recombination

[0045] The gel-recovered products of the Super1300 linearized vector and OfPHL5 homology arm clones were reacted at 37°C for 30 minutes with the homologous recombinase Exnase II at 37°C to ligate the OfPHL5-CDS to the Super1300 vector, forming a circular plasmid. The recombination reaction system is shown in Table 6. The recombinant product was transformed into E. coli and plated on LB solid medium (supplemented with 50 μg / ml kanamycin). After transformation, the cells were cultured at 37°C until a single colony emerged. PCR was performed using Super-YXJC-F and Super-YXJC-R. Positive colonies were selected, expanded, and sequenced. After sequencing, the plasmid was extracted and named Super1300-OfPHL5. An equal volume of the positive bacterial suspension was stored in 50% glycerol.

[0046] Table 6 Homologous recombination reaction system

[0047]

[0048] Example 3 Transformation of Agrobacterium GV3101

[0049] (1) Remove the competent Agrobacterium GV3101 cells stored in a -80°C freezer and thaw on ice. Add 1 μL of plasmid Super1300-OfPHL5 to every 100 μL of competent cells, pipette to mix thoroughly, and then place on ice for 20 minutes, freeze in liquid nitrogen for 5 minutes, bathe in 37°C water for 5 minutes, and then place on ice for 5 minutes.

[0050] (2) Add 500 μL of LB liquid medium without resistance and culture on a shaker at 28°C and 200 rpm for 1 h.

[0051] (3) After the culture is completed, the bacterial solution is centrifuged at 6000 rpm for 1 min, part of the supernatant is discarded, and 100 μL is evenly spread on LB solid medium (containing 50 μg / ml kanamycin), sealed with a sealing film, and inverted in a 28°C incubator for 40-48 h.

[0052] (4) Bacterial inspection and backup: Use Super-YXJC-F and Super-YXJC-R for PCR identification. If the target bands in the bacterial inspection are correct and the brightness is consistent, the corresponding colonies in the backup plate are picked into LB liquid culture medium (containing 50 μg / ml kanamycin) and shaken. Then, the bacterial liquid and 50% glycerol are preserved in the same volume ratio, quickly frozen in liquid nitrogen, and stored in a -80℃ ultra-low temperature refrigerator.

[0053] Example 4 Infection of Osmanthus Fragrance Petals and Determination of Total Carotenoid and Ionone Contents in Osmanthus Fragrance Petals

[0054] (1) Primary shaking: Agrobacterium-positive transformation liquid was inoculated into LB liquid medium (containing 50 μg / ml kanamycin) at a volume ratio of 1:50 and cultured overnight at 28°C in a shaking incubator.

[0055] (2) Second shake culture: Take the overnight bacterial solution and inoculate it into 100 mL LB liquid medium (containing 50 μg / mL kanamycin) at a volume ratio of 1:50, add 200 μL of 10 mmol / L AS (to make the final concentration 20 μmol / L) and 2 mL of 1 mol / L MES (to make the final concentration 20 mmol / L), and culture at 28 °C in a shaker until the OD 600 =0.6-0.8 (Agrobacterium activity is highest at this time).

[0056] (3) Prepare the infection solution: add 1 mL of 10 mmol / L AS (to a final concentration of 20 μmol / L), 5 mL of 1 mol / L magnesium chloride solution (to a final concentration of 10 mmol / L), and 5 mL of 1 mol / L MES (to a final concentration of 10 mmol / L) in sequence, then add distilled water to make up to 500 mL. Adjust the pH to 5.6.

[0057] (4) Collect the cells and resuspend: OD 600 = 0.6-0.8 Agrobacterium positive transformation bacteria liquid was placed in a 50mL sterilized centrifuge tube, centrifuged at 4°C, 4000r / min for 10min, and the supernatant was discarded. The bacteria were gently suspended in the infection solution until the turbid liquid was uniform, and the OD was adjusted. 600 =0.5-0.6, and let it stand in the dark at room temperature for 2h.

[0058] (5) Infection: Take fresh osmanthus petals, remove the pedicels, and leave only the petals; place the osmanthus petals in a 50mL centrifuge tube containing positive Agrobacterium infection solution. This process should be rapid, perform vacuum infiltration at 0.06Mpa for about 10 minutes, and slowly release the air; remove the osmanthus petals and wash the remaining bacterial solution on the petals with sterile water.

[0059] (6) Cultivation: Place the washed petals in a 50 mL centrifuge tube containing 5% sucrose solution, cover the top with sterile gauze so that the petals are completely immersed in it, and place it in a dark room at room temperature for 60 hours.

[0060] (7) Extraction and determination of total carotenoids in osmanthus petals: 0.2 g of petals were ground into powder under liquid nitrogen conditions, added with 2 mL of 80% acetone, and extracted in the dark for 24 h. After centrifugation at 4°C and 8000 r / min for 15 min, the supernatant was collected and measured using a UV spectrophotometer. Using 80% acetone as a blank control, the absorbance values ​​at wavelengths of 663 nm, 645 nm, and 470 nm were read respectively. Each test was repeated 3 times, and the pigment content in each sample was calculated using the following formula:

[0061] C a (chlorophyll a concentration) = 12.21 × A 663 -2.81×A 645

[0062] C b (chlorophyll b concentration) = 20.13 × A 645 -5.03×A 663

[0063] C x,c (total carotenoid concentration) = (1000 × A 470 -3.27×C a -104×C b ) / 229Unit: mg / L.

[0064] The results showed that the total carotenoid content in the petals of osmanthus fragrans transformed by the super-expression vector Super1300-OfPHL5 was significantly higher than that in the control group CK ( Figure 2 ).

[0065] (7) GC-MS detection of ionone content in transiently converted osmanthus petals: 0.2 g of petals and 2 μL of 10,000-fold methyl nonanoate were sealed in a 20 mL extraction bottle and equilibrated for 10 min. Then, a 2 cm extraction head (50 / 30 μm, DVB / Carboxen / PDMS, Supelco, USA) was inserted and extracted in a 55°C water bath for 30 min. Finally, the extraction head was inserted into the gas chromatography injection port and desorbed at 230°C for 5 min, followed by GC-MS.

[0066] Detection conditions: A DB-5MS column (30 m × 0.25 mm × 0.25 μm, Themo Scientific, Bellefonte, PA, USA) was used, and the carrier gas was high-purity helium (99.999%) with a split ratio of 20:1 and a flow rate of 1 mL / min. The ion source and inlet temperatures were 280°C and 230°C, respectively, and the transfer line temperature was 250°C.

[0067] The GC temperature program was as follows: 40 °C for 3 min, then heated to 120 °C at a rate of 3 °C / min and held for 3 min, then heated to 220 °C at a rate of 8 °C / min and held for 2 min.

[0068] The MS conditions were as follows: EI (electron impact) ion source, electron impact energy 70 eV, positive ion scan mode, mass scan range m / z 40-450 amu.

[0069] The content of ionone in the Osmanthus fragrans petals transiently transformed by the super vector Super1300-OfPHL5 was obviously lower than that of the control group CK, as detected by GC-MS. Therefore, it was inferred that the OfPHL5 gene inhibited the degradation of carotenoids to produce ionone, thereby increasing the content of carotenoids. Figure 2 )。

[0070] Example 5 qRT-PCR verification of transiently transformed Osmanthus fragrans petals

[0071] The total RNA of the transiently transformed Osmanthus fragrans petals was extracted by using a Trizol super-pure RNA extraction kit (Beijing Kangwei Century Biotechnology Co., Ltd.). The extracted RNA was reverse transcribed into cDNA by using a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Quanxi Gold, Beijing) reverse transcription kit. The obtained cDNA was diluted 5 times with water, and 1 μL thereof was taken as a template. The Osmanthus fragrans OfActin gene was used as an internal reference. A 2×SYBR Green qPCR Mix kit from Beijing Aidley Biotechnology Co., Ltd. was used to prepare a qRT-PCR reaction system. The reaction system was as shown in Table 7. The PCR reaction program adopted a three-step method: 95℃ for 2 min; 95℃ for 15 s, 60℃ for 15 s, and 72℃ for 20 s, with a cycle number of 40. After the completion of the fluorescence quantitative PCR program, the results were analyzed by using Roche LC96 software, and the relative expression amount of the OfPHL5 gene was calculated by using the internal reference gene OfActin as a reference and by using an Excel tool. ΔΔCt The results showed that the expression amount of OfPHL5 in the Osmanthus fragrans petals transiently transformed by the super expression vector Super1300-OfPHL5 was obviously higher than that of the control group CK. Figure 3

[0072] Table 7 qRT-PCR reaction system

[0073] ​

Claims

1. Related to the biosynthesis of carotenoids in osmanthus petals OfPHL5 A gene characterized by described OfPHL5 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1 OfPHL5 The protein encoded by the gene is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Containing the composition according to claim 1 OfPHL5 Genetic biomaterial, characterized in that The biological materials are expression cassettes, expression vectors and recombinant bacteria.

4. The method according to claim 1 OfPHL5 Use of the gene or the encoded protein according to claim 2 or the biological material according to claim 3 in promoting the synthesis of carotenoids in osmanthus petals.

5. The method according to claim 1 OfPHL5 Use of the gene or the coded protein according to claim 2 or the biological material according to claim 3 in osmanthus breeding.

6. A method for promoting the synthesis of carotenoids in osmanthus flower petals, characterized in that: Overexpression of the method of claim 1 in Osmanthus fragrans OfPHL5 Gene.

7. The method according to claim 6, characterized in that The following steps are involved: (1) Construct the protein containing the protein shown in SEQ ID NO. OfPHL5 Gene overexpression vector; (2) transforming the overexpression vector into Agrobacterium to obtain recombinant Agrobacterium; (3) Transforming Osmanthus fragrans with the recombinant Agrobacterium to overexpress OfPHL5 Gene.

8. The method according to claim 7, characterized in that In the step (3), the recombinant bacteria are used to transform the osmanthus petals.

9. The method according to claim 7, characterized in that The Agrobacterium is Agrobacterium GV3101.

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