Camellia polymerus gene cpPAO and application thereof in regulating chlorophyll content in plant leaves

Abstract

The application discloses a camellia nitidissima gene CpPAO and application thereof in regulating chlorophyll content in plant leaves, wherein the nucleotide sequence of the camellia nitidissima gene CpPAO 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 genetic transformation on Nicotiana grandiflora. The result shows that the chlorophyll content in the leaves of the CpPAO transgenic strain is significantly reduced compared with the empty load. As an important gene for reducing the chlorophyll content in the leaves of plants, the CpPAO can be applied to some plants with greenish leaves 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 CpPAO and its application in regulating chlorophyll content in plant leaves. Background Technology

[0002] PAO is a key gene regulating chlorophyll degradation. A crucial enzyme in chlorophyll degradation is pheophytin a oxidase, which catalyzes the breakdown of pheophytin a into chlorophyll products, leading to a reduction in green color and the formation of colorless substances. The pheophytin a monooxygenase encoded by PAO is located on the thylakoid membrane of chloroplasts. Structurally, it contains a Rieske-type domain, an N-terminal chloroplast transport peptide sequence, and a single-nuclear iron-binding domain, and its structure is highly conserved in plants.

[0003] Camellia chrysantha, belonging to the genus Camellia in the family Theaceae, is mainly distributed in Guangxi, Yunnan, and Guizhou provinces of my country and has high ornamental value. As an evergreen plant, its leaves have a high chlorophyll content. Chlorophyll is a key component for absorbing light energy in photosynthesis. Through different regulatory mechanisms, chlorophyll metabolism pathways are directly or indirectly affected, causing changes in chlorophyll content within the plant and ultimately leading to leaf color mutation phenotypes. Currently, no genes regulating chlorophyll content in plant leaves have been reported; therefore, identifying functional genes that regulate chlorophyll content in plant leaves is of great significance. Summary of the Invention

[0004] Purpose of the invention: The present invention aims to provide a gene CpPAO that can be used to regulate the chlorophyll content in plant leaves; another purpose of the present invention is to provide an application of the gene in regulating the chlorophyll content in plant leaves.

[0005] Technical solution: The Camellia chrysantha gene CpPAO described in this invention has the nucleotide sequence shown in SEQ ID NO.1.

[0006] The recombinant expression vector of the present invention contains the above-mentioned Camellia chrysantha gene CpPAO.

[0007] The host bacteria described in this invention contain the above-mentioned Camellia chrysantha gene CpPAO or the above-mentioned recombinant expression vector.

[0008] The Camellia chrysantha gene CpPAO, or the recombinant expression vector, or the host bacterium, can be used to regulate the chlorophyll content in plant leaves.

[0009] A method for reducing chlorophyll content in leaves using the CpPAO gene of Camellia chrysantha includes the following steps:

[0010] (1) Construct an overexpression vector for the CpPAO gene of Camellia chrysantha;

[0011] (2) Transformation of Agrobacterium GV3101;

[0012] (3) Infecting large-flowered tobacco and screening to obtain resistant transgenic plants;

[0013] (4) Determination of chlorophyll content in leaves of CpPAO transgenic plants.

[0014] Furthermore, the vector used to construct the Camellia chrysantha CpPAO gene overexpression vector was pBI121-CpPAO.

[0015] Furthermore, the primers used to construct the Camellia chrysantha CpPAO gene overexpression vector were:

[0016] F-gagaacacgggggactctagaATGGCTGTTTCACTAGCATCAGG;

[0017] R-ataagggactgaccacccgggATCAACATCAGCATGCACATAATCA.

[0018] Furthermore, the PCR amplification reaction system was as follows: Forward Primer 1 μL, Forward Primer 1 μL, cDNA 1 μL, Prime STAR 10 μL, dd 7μL.

[0019] Furthermore, the measured chlorophyll content includes the content of chlorophyll a and chlorophyll b.

[0020] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: the gene CpPAO can be used to reduce the chlorophyll content in plant leaves, showing promising application prospects in regulating chlorophyll 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 genetically transformed into Nicotiana macrophylla. The results showed that the chlorophyll content in the leaves of the CpPAO transgenic lines was significantly reduced compared to the empty vector. As an important gene for reducing chlorophyll content in plant leaves, CpPAO can be applied to some plants with predominantly green leaves in genetic engineering, demonstrating practical application value. Attached Figure Description

[0021] Figure 1 Diagram showing CpPAO gene amplification;

[0022] Figure 2 This is a positive detection image of Agrobacterium tumefaciens transformed with the CpPAO overexpression vector;

[0023] Figure 3PCR identification diagram of CpPAO transgenic tobacco;

[0024] Figure 4 The graphs show the phenotypes, positive identification, and relative expression levels of the CpPAO transgenic large-flowered tobacco. Among them, (A) shows the phenotypes of the empty vector and the transgenic large-flowered tobacco; (B) shows the semi-quantitative electrophoresis diagrams of the CpPAO gene in the empty vector and the transgenic large-flowered tobacco; and (C) shows the relative expression levels of the CpPAO gene in the empty vector and the transgenic large-flowered tobacco.

[0025] Figure 5 This is a graph showing the chlorophyll content determination in CpPAO transgenic tobacco leaves. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments.

[0027] Example

[0028] The material used in this embodiment is the tender leaves of 5-year-old Camellia chrysantha. In April 2022, the tender leaves of Camellia chrysantha were placed into sterilized centrifuge tubes, immediately flash-frozen in liquid nitrogen, and then stored in a -80°C freezer.

[0029] In this embodiment, total RNA was extracted from plants using the TIANGEN Plant RNA Extraction Kit (DP432). The extracted RNA was reverse transcribed into cDNA using the TaKaRaPrimeScript™ RT Master Mix (Perfect Real Time) reverse transcription kit. The resulting cDNA was then diluted 10-fold with water and stored at -20°C.

[0030] 1. Constructing an overexpression vector for the CpPAO gene of Camellia chrysantha.

[0031] (1) Obtaining the target gene

[0032] Based on the determined transcriptome data of Camellia chrysantha, one gene sequence was screened and named CpPAO.

[0033] (2) Design primers

[0034] 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.

[0035] F-gagaacacgggggactctagaATGGCTGTTTCACTAGCATCAGG

[0036] R-ataagggactgaccacccgggATCAACATCAGCATGCACATAATCA

[0037] (3) Vector double enzyme digestion

[0038] The pBI21 vector was removed from the -80℃ ultra-low temperature freezer beforehand for activation and culture. The pBI21 vector plasmid was extracted according to the kit, followed by double enzyme digestion experiments. The system is as follows:

[0039] Restriction endonuclease 1 1 μL

[0040] Restriction endonuclease 2 2μL

[0041] Buffer 2μL

[0042] vector plasmid XμL

[0043] dd 6μL

[0044] 20μL

[0045] 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.

[0046] (4) Target gene amplification (e.g.) Figure 1 (As shown)

[0047] Using cDNA diluted 10-fold as a template, PCR amplification was performed in the following system:

[0048] Forward Primer 1μL

[0049] Forward Primer 1μL

[0050] 1 μL cDNA

[0051] Prime STAR 10μL

[0052] dd 7μL

[0053] 20μL

[0054] 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.

[0055] (5) Connection conversion

[0056] The connection system is as follows:

[0057] 200 ng of target gene recovery product

[0058] 100 ng of plasmid double enzyme digestion product recovered

[0059] 2 μL of ligase

[0060] Buffer 4μL

[0061] XμL

[0062] dd Add to 20μL

[0063] 20μL

[0064] 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.

[0065] 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 liquid LB (without Kana) and incubate at 37°C and 200 rpm in a shaker for 30 min.

[0066] 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.

[0067] (6) Detection and sequencing of positive single colonies

[0068] 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:

[0069] 35sF 1μL

[0070] Gene R 1μL

[0071] Green Mix 10μL

[0072] dd 8μL

[0073] 20μL

[0074] 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.

[0075] (7) Double enzyme digestion verification

[0076] The plasmid with the correct sequence obtained from sequencing was verified by double enzyme digestion, as shown in the following system:

[0077] Restriction endonuclease 1 1 μL

[0078] Restriction endonuclease 2 2μL

[0079] Buffer 2μL

[0080] vector plasmid XμL

[0081] dd 6μL

[0082] 20μL

[0083] 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.

[0084] 2. Transformation of Agrobacterium GV3101

[0085] (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.

[0086] (2) Add 500 μL of antibiotic-free LB liquid medium and incubate at 28°C and 200 rpm on a shaker for 1 h;

[0087] (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.

[0088] (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 Kana) 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 CpPAO overexpression vector is shown below. Figure 2 As shown.

[0089] 3. Transgenic plants were obtained by infecting and screening large-flowered tobacco.

[0090] (1) Explant disinfection: After harvesting the tender leaves of Nicotiana macrophylla, the surface of the leaves was cleaned with detergent to remove the dust layer. After rinsing with running water for 30 minutes, the leaves were transferred to a clean bench for disinfection. First, 75% ethanol was poured into a beaker and shaken to ensure that the ethanol was in full contact with the surface of the tender leaves for 30 seconds. The leaves were then rinsed three times with sterile water. Next, the leaves were soaked in 5% NaClO for 10 minutes and rinsed four times with sterile water. The moisture on the surface of the leaves was then absorbed with sterile filter paper. After disinfection, the leaf edges and veins were removed with a sterile scalpel. The remaining leaves were then cut into small pieces of 0.5 × 0.5 cm for inoculation.

[0091] (2) Shaking culture: Take out the empty pBI121 vector and the vector containing the target gene and thaw it on ice. Use a pipette to add the bacterial culture to 20 mL of LB liquid medium (containing 50 mg / L Kana) and incubate in the dark on a shaker at 28℃ and 200 rpm until the bacterial culture OD600 is between 0.4 and 0.45.

[0092] (3) Infection: Use sterile tweezers to transfer the cut tobacco leaves into the infection solution and infect for 10 minutes, shaking the conical flask once every 2 minutes;

[0093] (4) Co-culture: After the leaves have been infected, they are taken out and laid flat on sterile filter paper. After the bacterial solution has dried slightly, the leaves are laid flat on the symbiotic culture medium and incubated in the dark at 25°C for 3 days.

[0094] (5) Screening culture: After co-culturing for 3 days, the leaves are transferred to the screening medium for culture. The medium is replaced every 15 days until resistant callus and resistant buds grow.

[0095] (6) Rooting culture: When the length of the sprouted adventitious buds reaches more than 5cm, cut them off from the tissue and remove the callus tissue connected to the stem tissue. Then transfer them to the rooting culture medium to induce rooting.

[0096] (7) Hardening off and transplanting: When the taproot of the transgenic seedlings has grown to about 5 cm and 7-8 leaves have emerged, remove the tobacco seedlings from the culture medium. Without damaging the roots, rinse away any remaining agar gel between the root tissues and place them in tissue culture bottles containing deionized water for acclimatization culture for 2 days. Change the water regularly during acclimatization to prevent contamination and damage to the tobacco seedlings. After acclimatization, label the tobacco seedlings with numbers and transplant them into sterilized substrate soil for further cultivation.

[0097] (8) Screening of transgenic plants: When the transgenic tobacco seedlings were 30 days old, RNA was extracted from the leaves using a kit and reverse transcribed into cDNA. The cDNA was then diluted 10-fold and semi-quantitative PCR was performed. After confirming the cDNA quality was up to standard, qRT-PCR was performed to detect gene expression levels. Transgenic seedlings with high expression levels and consistent growth were selected for subsequent functional verification. The PCR identification diagram of CpPAO transgenic tobacco is shown below. Figure 3 As shown in the figure, the phenotype, positive identification, and relative expression level analysis of CpPAO transgenic large-flowered tobacco are as follows. Figure 4 As shown.

[0098] 4. Determination of chlorophyll content in leaves of CpPAO transgenic plants

[0099] Transgenic lines with uniform growth were selected. Fresh leaves were cut into strips approximately 1 mm long, and 0.05 g of each strip was weighed and placed in an Erlenmeyer flask. 10 ml of a 1:1 mixture of acetone and ethanol was added, and the mixture was extracted in the dark at room temperature for 24 h until the material turned completely white. The material was then transferred to a cuvette. 10 ml of a 1:1 mixture of acetone and ethanol without the sample was used as a control. The optical density was measured at wavelengths of 663 nm, 645 nm, and 470 nm using a spectrophotometer, and the chlorophyll content was calculated.

[0100] Formula: Chlorophyll a content (mg·g⁻¹) = (12.21A663-2.81A645)×VT×N / (1000×W),

[0101] Chlorophyll b content (mg·g⁻¹) = (30.31A645-5.03A663)×VT×N / (1000×W),

[0102] Total chlorophyll content (mg·g⁻¹) = (7.18A663 + 27.5A645) × VT × N / (1000 × W)

[0103] Where A663, A645, and A470 represent the optical density values ​​of the chlorophyll extract at wavelengths of 663nm, 645nm, and 470nm, respectively; VT—extract volume (ml); W—fresh weight of the sample (g); and N—dilution factor.

[0104] Chlorophyll content analysis of CpPAO transgenic large-flowered tobacco leaves revealed a significant decrease in chlorophyll content compared to the uncrossed vector. Figure 5 As shown.

Claims

1. A golden camellia gene CpPAO, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A recombinant expression vector containing the Camellia chrysantha gene CpPAO as described in claim 1.

3. A host bacterium containing the Camellia chrysantha gene CpPAO as described in claim 1 or containing the recombinant expression vector as described in claim 2.

4. The application of the Camellia chrysantha gene CpPAO as described in claim 1, the recombinant expression vector as described in claim 2, or the host bacterium as described in claim 3 in reducing the chlorophyll content in leaves of Nicotiana macrophylla.

5. The application according to claim 4, characterized in that, The application method includes the following steps: (1) Construct an overexpression vector for the CpPAO gene of Camellia chrysantha; (2) Transformation of Agrobacterium GV3101; (3) Infecting large-flowered tobacco and screening to obtain resistant transgenic plants; (4) Determination of chlorophyll content in leaves of CpPAO transgenic plants.

6. The application according to claim 5, characterized in that, The vector used to construct the CpPAO gene overexpression vector of Camellia chrysantha was pBI121.

7. The application according to claim 5, characterized in that, The primers used to construct the CpPAO gene overexpression vector of Camellia chrysantha were: F-gagaacacgggggactctagaATGGCTGTTTCACTAGCATCAGG; R-ataagggactgaccacccgggATCAACATCAGCATGCACATAATCA.

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