Camellia japonica gene cpPAL and application thereof in regulating flavonoid content in plant leaves

Abstract

The application discloses a camellia nitidissima gene CpPAL and application thereof in regulating flavonoid content in plant leaves, wherein the nucleotide sequence of the camellia nitidissima gene CpPAL is shown as SEQ ID NO. 1. The application preliminarily predicts the function of the gene based on previous research and bioinformatics analysis software, obtains the full length of the gene sequence through cloning, and constructs an overexpression vector and performs transient transformation on tobacco on this basis. The results show that the flavonoid content in the leaves of the CpPAL transgenic strain is obviously higher than that of the empty carrier. As the gene CpPAL for regulating the flavonoid content in the leaves of camellia nitidissima, the application can be applied to some plants with poor solarization resistance in genetic engineering, and has practical application value.

Description

Technical Field

[0001] This invention relates to a Camellia chrysantha gene and its application, specifically to a Camellia chrysantha gene CpPAL and its application in regulating the flavonoid content in plant leaves. Background Technology

[0002] Golden camellia is a general term for plants in the section *Camellia* of the genus *Camellia* in the family Theaceae. These are evergreen shrubs or small trees, and are rare varieties within the *Camellia* genus with golden flowers, earning them the titles of "giant panda of the plant kingdom" and "queen of the camellia family." Golden camellia prefers shade and cannot tolerate direct sunlight, therefore it can only grow in highly shaded areas. Due to this biological characteristic, golden camellia is mainly distributed in Guangxi, Yunnan, and Guizhou provinces of my country; its introduction to other regions has resulted in poor growth. Related research indicates that plants with high flavonoid content in their leaves can enhance their sun tolerance, and changes in flavonoid content in leaves are regulated by certain genes. To date, no genes regulating flavonoid content in plant leaves have been reported; therefore, identifying functional genes that regulate flavonoid content in plant leaves is of great significance. Summary of the Invention

[0003] Purpose of the invention: The present invention aims to provide a Camellia chrysantha gene CpPAL that can be used to regulate the flavonoid content in plant leaves; another purpose of the present invention is to provide an application of the gene in regulating the flavonoid content in plant leaves.

[0004] Technical solution: The gene CpPAL for flavonoid content in the golden flower tea leaves of the present invention has the nucleotide sequence shown in SEQ ID NO.1.

[0005] The recombinant expression vector of the present invention contains the above-mentioned Camellia chrysantha gene CpPAL.

[0006] The host bacteria described in this invention contain the above-mentioned Camellia chrysantha gene CpPAL or the above-mentioned recombinant expression vector.

[0007] The Camellia chrysantha gene CpPAL, or the recombinant expression vector, or the host bacterium, can be used to regulate the flavonoid content in plant leaves.

[0008] A method for regulating flavonoid content in plant leaves using the Camellia chrysantha gene CpPAL includes the following steps:

[0009] (1) Construct an overexpression vector for the CpPAL gene of Camellia chrysantha;

[0010] (2) Transformation of Agrobacterium GV3101;

[0011] (3) Infecting large-flowered tobacco and screening to obtain resistant transgenic plants;

[0012] (4) Determination of flavonoid content in the leaves of CpPAL transgenic plants.

[0013] Furthermore, the vector used to construct the CpPAL gene overexpression vector of Camellia chrysantha was pSuper1300-CpPAL.

[0014] Furthermore, the primers used to construct the CpPAL gene overexpression vector for Camellia chrysantha were:

[0015] F-caaatcgactctagaaagcttATGCGGCAGGAAATAAGGG;

[0016] R-accggatccactagtatttaaatTTAGTTATACTCAGGTGGTGGTGGG.

[0017] Furthermore, the PCR amplification reaction system was as follows: 1 μL Forward Primer, 1 μL Forward Primer, 1 μL cDNA, 10 μL Prime STAR, and 7 μL ddH2O; the reaction conditions were: denaturation at 98℃ for 10 s; annealing at 58℃ for 15 s; extension at 72℃ for 1 min, for 35 cycles; total extension at 72℃ for 10 min; and termination of the reaction at 16℃.

[0018] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The *Camellia chrysantha* gene CpPAL can be used to increase the flavonoid content in plant leaves, showing promising application prospects in regulating flavonoid content in plants. Based on previous research and bioinformatics analysis software, this invention preliminarily predicted the function of this gene. The full-length gene sequence was obtained through cloning, and an overexpression vector was constructed and transiently transformed into tobacco. The results showed that the flavonoid content in the leaves of the CpPAL transgenic lines was significantly increased compared to the empty vector. As a gene regulating flavonoid content in *Camellia chrysantha* tea leaves, CpPAL can be applied to some plants with poor sun tolerance in genetic engineering, possessing practical application value. Attached Figure Description

[0019] Figure 1 Diagram showing CpPAL gene amplification;

[0020] Figure 2 This is a positive detection image of E. coli transformed with the CpPAL overexpression vector;

[0021] Figure 3 Image showing double enzyme digestion verification of the CpPAL overexpression vector;

[0022] Figure 4 This is a positive detection image of Agrobacterium tumefaciens transformed with the CpPAL overexpression vector;

[0023] Figure 5 The graph shows the determination of flavonoid content in tobacco leaves after transient infection with the CpPAL gene. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments.

[0025] Example

[0026] The material used in this embodiment is the leaves of 5-year-old Camellia chrysantha. In May 2023, the Camellia chrysantha leaves were placed into sterilized centrifuge tubes, immediately flash-frozen in liquid nitrogen, and then stored in a -80°C freezer.

[0027] In this embodiment, total RNA was extracted from plants using the TIANGEN Plant RNA Extraction Kit (DP432). TaKaRa PrimeScript was used. TM The RT Master Mix (Perfect Real Time) reverse transcription kit reverse transcribes the extracted RNA into cDNA. The resulting cDNA is then diluted 10 times with water and stored at -20°C.

[0028] 1. Constructing an overexpression vector for the CpPAL gene in Camellia chrysantha.

[0029] (1) Obtaining the target gene

[0030] Based on the determined transcriptome data of Camellia chrysantha, one gene sequence was screened and named CpPAL.

[0031] (2) Design primers

[0032] The full-length nucleotide sequence of the gene was analyzed using BioXM software to determine restriction enzyme sites. Xba I and Sma I enzymes were selected as the two restriction endonucleases. Primers were designed using CE design software. The required information was filled in, including the sequence near the restriction sites on the vector, the full length of the target gene, and the two restriction sites (5' and 3' ends) in sequence. The designed primers were then sent to Jereh Biotech for synthesis.

[0033] F-caaatcgactctagaaagcttATGCGGCAGGAAATAAGGG

[0034] R-accggatccactagtatttaaatTTAGTTATACTCAGGTGGTGGTGGG

[0035] (3) Vector double enzyme digestion

[0036] The pSuper1300 vector was activated and cultured after being removed from the -80℃ ultra-low temperature freezer. The pBI21 vector plasmid was extracted according to the kit, followed by double enzyme digestion. The system is as follows:

[0037] Restriction endonuclease 1 1 μL

[0038] Restriction endonuclease 2 2μL

[0039] Buffer 2μL

[0040] vector plasmid XμL

[0041] ddH2O 6μL

[0042] 20μL

[0043] Where X(μL) = 1000 ng / vector plasmid concentration (ng / μL). Gently shake the centrifuge tube to mix, centrifuge briefly for 6 seconds, and incubate in a water bath at 37°C for 1 hour. Perform agarose gel electrophoresis on the obtained double-digested vector, and then use a kit for gel extraction and recovery.

[0044] (4) Target gene amplification (e.g.) Figure 1 (As shown)

[0045] Using cDNA diluted 10-fold as a template, PCR amplification was performed in the following system:

[0046] Forward Primer 1μL

[0047] Forward Primer 1μL

[0048] 1 μL of cDNA

[0049] Prime STAR 10μL

[0050] ddH2O 7μL

[0051] 20μL

[0052] Three 20 μL systems were prepared for each gene. The reaction conditions were: denaturation at 98 °C for 10 s; annealing at 58 °C for 15 s; extension at 72 °C for 1 min, 35 cycles; total extension at 72 °C for 10 min; and termination of the reaction at 16 °C. The obtained amplification products were subjected to agarose gel electrophoresis and then recovered by gel cutting using a kit.

[0053] (5) Connection conversion

[0054] The connection system is as follows:

[0055] 200 ng of target gene recovery product

[0056] 100 ng of plasmid double enzyme digestion product recovered

[0057] 2 μL of ligase

[0058] Buffer 4μL

[0059] XμL

[0060] Add ddH2O to 20μL

[0061] 20μL

[0062] Gently shake the centrifuge tube to mix it, centrifuge briefly for 6 seconds, incubate in a 37°C water bath for 30 minutes, and then on ice for 2 minutes.

[0063] Transformation: In a clean bench, use a pipette to take 5 μL of the ligation product and add it to 50 μL of Trelief™ 5α competent cells. Gently mix, incubate on ice for 5 min, incubate in water at 42°C for 60 s, then incubate on ice for 2 min. Add 250 μL of LB liquid (without Kana) and incubate at 37°C and 200 rpm in a shaker for 30 min.

[0064] Spreading: Take 200 μL of the incubated bacterial solution, spread it evenly on LB solid medium (containing 50 mg / L Kana) with a sterile glass rod, and let it dry. After sealing with film, invert the plate and incubate in a 37°C constant temperature incubator for 12-14 h.

[0065] (6) Detection and sequencing of positive single colonies (e.g.) Figure 2 (As shown)

[0066] After bacteria have grown on the culture medium, single colony detection is performed in a clean bench. Eight plump single colonies are selected for each gene and sequentially backed up on LB solid medium containing Kana resistance. The corresponding single colonies are then transferred to the following system for bacterial testing using a sterile toothpick:

[0067] 35sF 1μL

[0068] Gene R 1μL

[0069] Green Mix 10μL

[0070] ddH2O 8μL

[0071] 20μL

[0072] The PCR reaction conditions were as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; 72℃ total extension for 10 min; and 16℃ to terminate the reaction. The obtained amplification products were subjected to agarose gel electrophoresis, and three correct positive colonies were selected for testing.

[0073] (7) Double enzyme digestion verification (e.g.) Figure 3 (As shown)

[0074] The plasmid with the correct sequence obtained from sequencing was verified by double enzyme digestion, as shown in the following system:

[0075] Restriction endonuclease 1 1 μL

[0076] Restriction endonuclease 2 2μL

[0077] Buffer 2μL

[0078] vector plasmid XμL

[0079] ddH2O 6μL

[0080] 20μL

[0081] Where X(μL) = 1000 ng / vector plasmid concentration (ng / μL). Gently shake the centrifuge tube to mix, centrifuge briefly for 6 seconds, and incubate in a water bath at 37℃ for 1 hour. Perform agarose gel electrophoresis on the obtained double-digested vector to detect the double digestion status.

[0082] 2. Transformation of Agrobacterium GV3101

[0083] (1) Take out the GV3101 competent cells stored in the -80℃ ultra-low temperature freezer and thaw them on ice. Add 1μL of plasmid to every 33μL of competent cells, mix well by pipetting, and then sequentially in an ice bath for 20min, quick freeze with liquid ammonia for 5min, in a 37℃ water bath for 5min, and in an ice bath for 5min.

[0084] (2) Add 500 μL of antibiotic-free LB liquid medium and incubate at 28°C and 200 rpm on a shaker for 1 h;

[0085] (3) After the culture is completed, centrifuge the bacterial solution at 6000r for 1min, discard part of the supernatant, and leave 100μL to spread evenly on LB solid medium (containing 50mg / LKana), seal with sealing film, and invert in an incubator at 28℃ for 40-48h.

[0086] (4) Bacterial detection and backup: If the target band in the bacterial detection is correct and the brightness is consistent, pick the corresponding colonies from the backup plate into LB liquid medium (containing 50 mg / L Kamagra) and shake to incubate. Then, preserve the bacterial culture with 50% glycerol at a volume ratio of 3:7, flash-freeze in liquid nitrogen, and store in an ultra-low temperature freezer at -80℃. The positive detection image of Agrobacterium tumefaciens transformed with the CpPAL overexpression vector is shown below. Figure 4 As shown.

[0087] 3. Infecting and instantly transforming large-flowered tobacco.

[0088] (1) Plants to be infected: The tobacco seedlings used for infection are about 40 days old and healthy;

[0089] (2) Shaking culture: The bacterial cultures successfully transformed into Agrobacterium tumefaciens GV3101 fusion expression vector, pSuper1300::GFP empty expression vector, and helper vector P19 were taken out of the -80℃ freezer and thawed in an ice box. 200 μl of each culture was then added to 20 ml of liquid LB medium containing kanamycin and cultured with shaking at 28℃ and 200 rpm in a constant temperature shaking incubator in the dark. The culture was continued until the bacterial cultures containing the fusion expression vector 35S::CpPAL-GFP and the pSuper1300::GFP empty expression vector reached OD... 600 At a value of 0.7, the OD of bacterial culture containing the helper carrier P19 was... 600 When the value reaches 0.5, the cultivation is complete;

[0090] (3) Preparation of buffer solution: Dissolve 0.0196g acetylsuccinone (AS) in 5ml of dimethyl sulfoxide, then add 95ml of sterile pure water to prepare a stock solution. Take 30ml of the stock solution and 170ml of sterile pure water and put them into a new conical flask. Add 0.407g of magnesium chloride and 0.427g of MES to the conical flask, mix well, and prepare a buffer solution. The buffer solution should be prepared and used immediately. If it is not used temporarily, it should be placed in a refrigerator at 4℃.

[0091] (4) Mixing: Take 5 ml of each of the three bacterial solutions and centrifuge at 6000 r / min at 4℃ for 10 min. Collect the bacteria and discard the liquid. Then add 5 ml of buffer solution to the bacteria for resuspending. Mix the 5 ml of bacterial solution containing the fusion expression vector or empty expression vector with the 5 ml of bacterial solution containing the auxiliary vector P19 and place it in a 28℃ electric thermostatic incubator for 2-3 h.

[0092] (5) Injection: Inject the mixture into the back of the tobacco leaf using a 1ml medical syringe, make corresponding marks, water it, and then put it back into the incubator for 2 days.

[0093] 4. Determination of flavonoid content in leaves of CpPAL transgenic plants

[0094] Leaves from which the gene was injected were removed, the midrib and edges were cleaned, dried at 60℃, and ground into powder. Subsequent experimental procedures were performed according to the instructions of the plant flavonoid detection kit. The results showed that the flavonoid content of CpPAL transgenic tobacco was significantly higher than that of the empty vector. (See [link to kit]). Figure 5 .

Claims

1. A golden camellia gene CpPAL Its characteristics are, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. Contains the Camellia chrysantha gene as described in claim 1 CpPAL Recombinant expression vectors.

3. Contains the Camellia chrysantha gene as described in claim 1 CpPAL Or a host bacterium containing the recombinant expression vector of claim 2.

4. A Camellia chrysantha gene as described in claim 1 CpPAL The application of the recombinant expression vector of claim 2 or the host bacteria of claim 3 in increasing the flavonoid content of tobacco leaves.

5. The application according to claim 4, characterized in that, The application method includes the following steps: (1) Constructing golden camellia CpPAL Gene overexpression vectors; (2) Transformation of Agrobacterium GV3101; (3) Infecting large-flowered tobacco and screening to obtain resistant transgenic plants; (4) CpPAL Determination of flavonoid content in the leaves of transgenic plants.

6. The application according to claim 5, characterized in that, Building Golden Camellia CpPAL The vector used for gene overexpression was pSuper1300- CpPAL .

7. The application according to claim 5, characterized in that, Building Golden Camellia CpPAL The primers used for gene overexpression vectors are: F-caaatcgactctagaaagcttATGCGGCAGGAAATAAGGG; R-accggatccactagtatttaaatTTAGTTATACTCAGGTGGTGGTGGG.

8. The application according to claim 5, characterized in that, Building Golden Camellia CpPAL The PCR amplification conditions for the target gene in the gene overexpression vector were as follows: denaturation at 98℃ for 10s; annealing at 58℃ for 15s; extension at 72℃ for 1min, 35 cycles; total extension at 72℃ for 10min; and termination of the reaction at 16℃.

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