OfPHL5 gene related to synthesis of osmanthus carotenoid and application of OfPHL5 gene

Abstract

The invention discloses an OfPHL5 gene related to synthesis of osmanthus carotenoid and application of the OfPHL5 gene, the OfPHL5 gene is an MYB transcription factor gene, an overexpression vector containing the gene OfPHL5 is transformed into osmanthus petals for transient expression, and the result shows that the expression quantity of the OfPHL5 gene in the osmanthus petals subjected to transient transformation is remarkably increased, so that the OfPHL5 gene can be applied to the synthesis of the osmanthus carotenoid. The OfPHL5 gene plays an important role in promoting the synthesis of the carotenoids in the osmanthus fragrans, and can be used for improving the color and quality of the osmanthus fragrans, breeding excellent varieties and the like in the genetic engineering of the osmanthus fragrans.

Description

Technical Field

[0001] The 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 an important impact on plant growth and development. The differences in carotenoid types and contents in plant organs such as petals and fruits lead to different colors, thus forming a colorful world. At the same time, carotenoids can be cleaved to produce important aroma substances such as ionones. For plants, bright colors and fragrant smells can effectively attract insects or other animals for pollination and improve reproductive success. In the photosynthetic system, carotenoids play an important role in plant photosynthesis. They can help capture light energy and transfer it to chlorophyll; at the same time, they can also effectively remove free radicals and avoid damage to plants caused by photooxidation. In addition, the downstream products of carotenoids, hormones abscisic acid (ABA) and strigolactone, are important endogenous signal substances for plants to resist adversity and regulate growth and development. Carotenoids are also a class of high-value natural active substances with antioxidant capacity and are an important part of human dietary nutrition.

[0003] Sweet osmanthus (Osmanthus fragrans Lour.) is an important ornamental seedling and spice plant. The carotenoids in its petals can be cleaved to produce aroma components such as α-ionone and β-ionone, thereby giving sweet osmanthus a strong violet fragrance, woody fragrance and fruity fragrance. MYB transcription factor is one of the largest transcription factor families, plays a key role in regulating the synthesis of primary and secondary metabolites, and is widely involved in the regulation of aspects such as cell differentiation, secondary metabolism, environmental stress and pests and diseases. However, the genes related to the synthesis of sweet osmanthus carotenoids in the MYB family have not been reported. Therefore, it is of great significance to study and explore the related genes that promote the synthesis of sweet osmanthus carotenoids in the sweet osmanthus genome. The present invention utilizes the technology of bioinformatics to screen out the genes that promote the synthesis of sweet osmanthus carotenoids in sweet osmanthus, and performs functional verification in sweet osmanthus petals. Summary of the invention

[0004] In view of the above 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 the breeding of excellent varieties.

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

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

[0007] Biological materials containing OfPHL5 gene include expression cassette, expression vector 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 as 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 sweet osmanthus 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 an OfPHL5 gene 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 transform the total carotenoids and ionone contents in osmanthus petals.

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

[0022] The technical solution of the present invention is described below in conjunction with 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 belonged to the MYB gene family. The gene had 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 Primer5.0 software, and 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, the target gene CDS was amplified 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 detection and gene sequencing: 0.3 g agarose powder was added to 30 ml TAE, and 1.5 μL 10000× nucleic acid dye was added after melting in a microwave oven. The sample was then spotted after solidification. After electrophoresis at 120 V and 150 mA for 25 min, the bands were observed on a Gel-Logie200 gel scanning imager. The target band was 1152 bp (e.g. Figure 1The target band was recovered and connected to a T-vector, and transformed into E. coli for sequencing. The sequence of the CDS 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 bacterial solution with the correct sequencing and named OfPHL5-CDS-Plasmid, which was used as a template for constructing the vector later.

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

[0036] (1) Target gene cloning

[0037] Using plasmid OfPHL5-CDS-Plasmid as a template, CE Design V1.04 was used to design homology arm primers containing Kpn1 / Sal1 restriction sites (Table 1), and PCR amplification was performed with reference to the instructions of Phanta high-fidelity enzyme. The PCR reaction system and procedures are shown in Tables 4 and 3. After the PCR reaction was completed, all PCR products were run on a gel to detect whether there were clear target bands, and the gel was cut 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 min and heat-inactivate at 65°C for 20 min. All digestion products were run on a gel and then cut and recovered.

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

[0043]

[0044] (3) Homologous recombination

[0045] The gel recovery products of the Super1300 linearized vector and the OfPHL5 homology arm clones were reacted at 37°C for 30 minutes under the catalysis of the homologous recombinase ExnaseII, and OfPHL5-CDS was connected to the Super1300 vector to form a circular plasmid. The recombination reaction system is shown in Table 6. The recombinant product was transformed into Escherichia coli and coated with LB solid medium (containing 50μg / ml kanamycin). After transformation, it was cultured at 37°C until a single clone grew. PCR identification was performed using Super-YXJC-F and Super-YXJC-R, and positive single clones were selected for expansion and sequencing. After the sequencing was correct, the plasmid was extracted and the plasmid name was Super1300-OfPHL5. The positive bacterial solution was stored in 50% glycerol according to the same volume.

[0046] Table 6 Homologous recombination reaction system

[0047]

[0048] Example 3 Transformation of Agrobacterium GV3101

[0049] (1) Take out the competent cells of Agrobacterium tumefaciens GV3101 stored in a -80℃ ultra-low temperature freezer and thaw them on ice. Add 1μL of plasmid Super1300-OfPHL5 to every 100μL competent cells, pipette and mix well, then place in an ice bath for 20 minutes, quick freeze in liquid nitrogen for 5 minutes, place in a 37℃ water bath for 5 minutes, and place in an ice bath 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 was completed, the bacterial solution was centrifuged at 6000 rpm for 1 min, part of the supernatant was discarded, and 100 μL was 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 have consistent brightness, 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 equal volume ratios, quickly frozen in liquid nitrogen, and stored in a -80°C 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: The positive transformation culture of Agrobacterium 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) Secondary shaking: 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 shaking incubator until the OD reaches 0. 600 =0.6-0.8 (Agrobacterium activity is highest at this time).

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

[0057] (4) Collect the cells and resuspend them: OD 600 = 0.6-0.8 of Agrobacterium positive transformation bacteria liquid into a 50mL sterilized centrifuge tube, centrifuge at 4°C, 4000r / min for 10min, and discard the supernatant. Gently suspend the bacteria with infection solution until the turbid liquid is uniform, and adjust the OD 600 =0.5-0.6, and let stand in dark place 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 50 mL centrifuge tube containing positive Agrobacterium infection solution. This process should be rapid, perform vacuum penetration at 0.06 MPa for about 10 minutes, and slowly release the air; take out 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 the solution, and place in a dark room at room temperature for 60 h.

[0060] (7) Extraction and determination of total carotenoids in osmanthus petals: 0.2 g of petals were ground into powder under liquid nitrogen conditions, 2 mL of 80% acetone was added, and the mixture was extracted in a dark place for 24 h. After centrifugation at 4°C and 8000 r / min for 15 min, the supernatant was taken 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 group of experiments 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 osmanthus petals transiently transformed with 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 instantaneous conversion osmanthus petals: 0.2 g of petals and 2 μL of 10,000-fold nonanoic acid methyl ester 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 head was inserted into the gas chromatography injection port and desorbed at 230°C for 5 min, followed by GC-MS.

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

[0067] The temperature program of GC was as follows: 40 °C for 3 min, then increased to 120 °C at a rate of 3 °C / min and retained for 3 min, then increased to 220 °C at a rate of 8 °C / min and retained 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] GC-MS detection showed that the ionone content in the petals of Osmanthus fragrans transformed by the super vector Super1300-OfPHL5 was significantly lower than that in the control group CK. Therefore, it is inferred that the OfPHL5 gene produces ionone by inhibiting the degradation of carotenoids, thereby increasing the carotenoid content. Figure 2 ).

[0070] Example 5 qRT-PCR verification of transient transformation of osmanthus petals

[0071] The total RNA of transiently transformed osmanthus petals was extracted using Trizol ultrapure RNA extraction kit (Beijing Kangwei Century Biotechnology Co., Ltd.). The extracted RNA was reverse transcribed into cDNA using TransScript One-Step gDNARemoval and cDNASynthesisSuperMix (Quanshijin, Beijing) reverse transcription kit. The cDNA was diluted 5 times with water and 1 μL was taken as a template. The osmanthus OfActin gene was used as an internal reference. The qRT-PCR reaction system was prepared using the 2×SYBR Green qPCR Mix kit of Beijing Aidelai Biotechnology Co., Ltd. The reaction system is shown in Table 7. The PCR reaction program adopts a three-step method: 95℃ reaction for 2min; 95℃ reaction for 15s, 60℃ reaction for 15s, 72℃ reaction for 20s, and the number of cycles is 40. After the fluorescence quantitative PCR program is completed, the results are analyzed using Roche LC96 software, and 2- ΔΔCt The relative expression of OfPHL5 gene was calculated by using the internal reference gene OfActin as a reference. The results showed that the expression of OfPHL5 in the petals of Osmanthus fragrans transiently transformed with the overexpression vector Super1300-OfPHL5 was significantly increased compared with that in 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 Said OfPHL5 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1 OfPHL5 The encoded protein of 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 biological material, characterized in that The biological materials include 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 petals, characterized in that: Super-expression of the method of claim 1 in sweet osmanthus OfPHL5 Gene.

7. The method according to claim 6, characterized in that The following steps are involved: (1) Constructing a protein containing the protein shown in SEQ ID NO. OfPHL5 Gene overexpression vector; (2) transforming the expression vector into Agrobacterium to obtain recombinant expression 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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