A camphor tree color-related gene CcCHS1 and its encoded protein and application

Abstract

The invention discloses a camphor color-related gene CcCHS1 and its encoded protein and application, and relates to the technical field of plant genetic engineering. The camphor color-related gene CcCHS1 disclosed by the invention has a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2. The invention constructs an expression vector of the camphor color-related gene CcCHS1; transforms the constructed camphor color-related gene CcCHS1 expression vector into Populus shanxin; cultivates, screens and obtains transgenic Populus shanxin plants with red leaves. The leaves of the transgenic poplar overexpressing the CcCHS1 gene show orange-red color. The results show that the gene provided by the invention is involved in the regulation of plant leaf color, and as a gene resource, it enriches the theory of plant color molecular regulation, and has important application value for improving the color of plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a camphor tree color-related gene CcCHS1 and its encoded protein and application. Background Art

[0002] Color is one of the most important ornamental traits of garden plants. With the continuous development of urban landscaping construction, a single green plant can hardly meet people's demand for the color of garden plants. At present, the research on colored plants is mostly focused on herbaceous ornamental plants and shrubs such as coleus, pansy, Photinia fraseri, Ligustrum lucidum, etc., and the development and utilization of colored tree resources is still lacking. The new camphor tree variety "Gantong No. 1" is a new generation of excellent garden colored tree resources. It has a long viewing period, rapid landscape formation, easy to form large color block landscape, and significant ecological benefits. It makes up for the monotony of single green camphor trees in current garden greening and plays an important role in promoting the landscape construction of new cities.

[0003] Anthocyanins are a class of flavonoid compounds widely present in plants. They are important pigments for the coloration of leaves, flowers, fruits and other organs. Different types of anthocyanins in plants can make plants appear red, purple, blue-purple and blue. Studies have found that the transcriptional activation of the biosynthetic pathway cascade from phenylpropanoid compounds-flavonoids-anthocyanins is an important reason why the stems and leaves of "Gan Tong No. 1" are rich in anthocyanins, providing a theoretical basis for the innovation of colored plant germplasm (Zhong et al., 2022). Chalcone synthase gene (CHS) is a key rate-limiting enzyme gene in the biosynthetic pathway of flavonoid substances. Chalcone synthase can be specifically expressed in plant color-forming organs, and changes in its expression in plants may affect the color of plant organs. In recent years, CHS genes related to flavonoid metabolism have been cloned from the flowers of various plants. After transformation of petunia, its flower color has changed, and there are variations that do not exist in nature. However, there are few reports on the study of CHS gene changing plant leaf color. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a camphor tree color-related gene CcCHS1. Another technical problem to be solved by the present invention is to provide a coding protein of the camphor tree color-related gene CcCHS1. Another technical problem to be solved by the present invention is to provide an application of the camphor tree color-related gene CcCHS1 for regulating plant leaf color and obtaining new plant germplasm with variable leaf color.

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

[0006] A camphor tree color-related gene CcCHS1, whose nucleotide sequence is shown in SEQ ID NO.1.

[0007] The amino acid sequence of the protein encoded by the camphor tree color-related gene CcCHS1 is shown in SEQ ID NO.2.

[0008] A vector, recombinant bacteria or host cell containing the camphor tree coloration-related gene CcCHS1.

[0009] Application of Cinnamomum camphora color-related gene CcCHS1 in regulating the leaf color of Populus davidiana.

[0010] The method for regulating the color of the Populus davidianus leaves is to turn the color of the Populus davidianus leaves red.

[0011] The application of CcCHS1, a color-related gene of camphor tree, in regulating the leaf color of Populus davidianus, including:

[0012] 1) Constructing the expression vector of CcCHS1, a color-related gene of camphor trees;

[0013] 2) Transform the constructed expression vector of the camphor tree color-related gene CcCHS1 into Populus davidianus;

[0014] 3) Cultivate, screen and obtain transgenic Populus shanxinensis plants with red leaves.

[0015] The expression vector is a plant expression vector.

[0016] The plant expression vector is pBI121-CcCHS1.

[0017] Application of camphor color-related gene CcCHS1 in regulating anthocyanin content in Populus davidianus leaves.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) The camphor tree color-related gene CcCHS1 disclosed for the first time in the present invention has a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2. The present invention constructs an expression vector of the camphor tree color-related gene CcCHS1; transforms the constructed expression vector of the camphor tree color-related gene CcCHS1 into Populus shanxinensis; cultivates, screens and obtains transgenic Populus shanxinensis plants with red leaves. The leaves of the transgenic poplars overexpressing the CcCHS1 gene show an orange-red color.

[0020] 2) The present invention discloses for the first time the camphor tree color-related gene CcCHS1. The transgenic Populus davidianus that overexpresses the CcCHS1 gene has a higher content of anthocyanins in its leaves, thereby making the leaves of the transgenic seedlings red.

[0021] 3) The camphor tree color-related gene CcCHS1 disclosed for the first time in the present invention is involved in the regulation of the red color of plant leaves. As a gene resource, it can not only enrich the theory of plant color molecular regulation, but also has important application value in forest genetic engineering for improving plant color. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the subcellular localization map of CcCHS1;

[0023] Figure 2 This is a schematic diagram of the pBI121-CcCHS1 overexpression vector;

[0024] Figure 3 Figure 2 is the phenotype of transgenic poplars overexpressing the CcCHS1 gene ( Figure 3 a is the expression level detection of transgenic poplar overexpressing CcCHS1 gene; Figure 3 b is the leaf color observation of transgenic poplar trees overexpressing CcCHS1 gene and non-transgenic poplar trees, NT1, NT2 and NT3 are non-transgenic poplar leaves, T1, T2 and T9 are different transgenic lines of CcCHS1; Figure 3 c is the asexual propagation tissue culture seedling of T1 transgenic line; scale bar: 1 cm);

[0025] Figure 4 Figure 2 shows the anthocyanin content in leaves of CcCHS1 transgenic poplar. Figure 4 a is anthocyanin extract; Figure 4 b is anthocyanin content). DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0027] The camphor trees used in this application were planted in the nursery of the Institute of Biological Resources of Jiangxi Academy of Sciences.

[0028] Example 1

[0029] 1. RNA extraction and cDNA acquisition

[0030] Take appropriate amount of leaf tissue samples from camphor tree and use Huayueyang Plant RNA Extraction Kit to extract RNA from the samples. cDNA was synthesized using 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigester plus) kit (Shanghai Yishen Biotechnology Co., Ltd.).

[0031] 2. Cloning the CDS sequence of CcCHS1 gene from Cinnamomum camphora

[0032] According to the CDS sequence of the CcCHS1 gene in the camphor genome, 5′ and 3′ primers were designed using Oligo7 for PCR amplification. The primer sequences are as follows:

[0033] Forward primer F: 5′-ATGGTGGACGTGGAAGCC-3′;

[0034] Reverse primer R: 5′-TCTAAGCAGATACACTATGGAGC-3′.

[0035] PCR reaction system: TaKaRa LA Taq (5U / μL), 0.5μL; 10×LA PCR Buffer II (Mg2+Plus), 5μL; dNTP Mixture (2.5mM each), 8μL; cDNA template, 50ng; Primer F (10μM), 2μL; Primer R (10μM), 2μL; make up to 50μL with sterile deionized water.

[0036] PCR amplification program: 94°C for 1 min; 98°C for 10 s, 57°C for 30 s, 72°C for 1 min 20 s, 35 cycles; 72°C for 10 min.

[0037] The PCR amplification products were separated by 1.2% agarose gel electrophoresis, photographed by a gel imaging system, and the target fragments were excised and recovered using an agarose gel recovery kit (Shanghai Yisheng Biotechnology Co., Ltd.).

[0038] The recovered PCR product was ligated with the pMDTM19-T (Takara) cloning vector and transformed.

[0039] Cloning and ligation reaction system: target fragment recovery product 1-4 μL (60-80 ng / μL); pMDTM19-TVector 1 μL; Solution I 5 μL; ddH 2 Add 10 μL of HO, mix gently, and react at 16°C for 30 min.

[0040] Finally, a single colony was picked for bacterial liquid detection, and the positive strains were sent to Shanghai Bioengineering for sequencing. The nucleotide sequence of the camphor tree color-related gene CcCHS1 obtained by sequencing is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2.

[0041] Example 2

[0042] 1. PCR amplification of CcCHS1 gene

[0043] Using the cDNA obtained by reverse transcription as a template, the CcCHS1 gene was amplified by PCR using a blunt-ended high-fidelity DNA polymerase Primer STAR MaxDNA polymerase (TaKaRa) and forward and reverse primers. The primer sequences are as follows:

[0044] Forward primer F: 5′-agtggtctctgtccagtcctATGGTGGACGTGGAAGCC-3′;

[0045] Reverse primer R: 5′-ggtctcagcagaccacaagTCTAAGCAGATACACTATGGAGC-3′.

[0046] The PCR reaction system was as follows: 50 ng of cDNA template, 25 μL of PrimerSTAR Max Premix (2×), 1 μL of each of forward and reverse primers (10 μmol / L), and sterile ultrapure water was added to make up to 50 μL.

[0047] The PCR amplification program was: 98°C for 10 s; 55°C for 5 s; 72°C for 10 s; 35 cycles; 72°C for 3 min.

[0048] The PCR amplification products were separated by 1.2% agarose gel electrophoresis, photographed by a gel imaging system, and the target fragment of the CcCHS1 gene was excised and recovered using an agarose gel recovery kit (Shanghai Yisheng Biotechnology Co., Ltd.).

[0049] 2. NC cloning reaction

[0050] The complete CDS sequence of the CcCHS1 gene was connected to the subcellular localization vector pNC-Cam1304-SubC (NC biotech) by Nimble Cloning (NC cloning), and the obtained recombinant expression vector was named pNC-CcCHS1-eGFP. The primer sequences used are as follows:

[0051] Forward primer: 5'-agtggtctctgtccagtcctATGGTGGACGTGGAAGCC-3',

[0052] Reverse primer: 5′-ggtctcagcagaccacaagTCTAAGCAGATACACTATGGAGC-3′.

[0053] The NC cloning reaction system is as follows: 80 ng of the recovered product of the target fragment of the CcCHS1 gene, 120 ng of the pNC-Cam1304-SubC vector plasmid, ddH 2O to 5 μL, add 5 μL Nimble Mix, pipette 10-20 times, mix thoroughly. After reacting at 50℃ for 30-60min, transform 2-5 μL of the reaction product into conventional competent cells (Top10). After overnight culture at 37℃ on the transformation plate, select clones for PCR identification, and sequence the positive clones.

[0054] 3. Instantaneous transformation of tobacco

[0055] Transform the constructed pNC-CcCHS1-eGFP into LBA4404 Agrobacterium competent cells, prepare a bacterial solution with an OD600 of 0.6-0.8, and resuspend the bacterial solution with a suspension to prepare an OD 600 The infection solution was 0.6 (10 mM MES, 10 mM MgCl 2 , 20 mg / L As, pH 5.2), and let stand at 28°C for 2-3 hours. Pick tobacco in good condition and inject the infection solution into the tobacco leaves using a needleless syringe.

[0056] The results are as follows Figure 1 As shown, 36 hours after transient transformation, CcCHS1 was abundantly expressed in tobacco leaves, and its subcellular localization pattern was observed using confocal microscopy, which showed that CcCHS1 was localized in the cytoplasm.

[0057] Example 3

[0058] 1. BP reaction

[0059] 1) PCR specific primers (forward primer F: 5'-ATGGTGGACGTGGAAGCC-3', reverse primer R: 5'-TCTAAGCAGATACACTATGGAGC-3') were used to amplify the cDNA obtained by reverse transcription using the high-fidelity enzyme LATaq (TAKARA). The CDS amplified band of the CcCHS1 gene was excised and recovered and the concentration was determined.

[0060] 2) Using Gateway technology, using pCR TM / 8 GW / TOPO TM The CDS sequence of the CcCHS1 gene was constructed into the entry vector pCR using the TA Cloning kit (Invitrogen, K252002). TM / 8 GW / TOPO TM middle.

[0061] Prepare the reaction solution on ice: Fresh PCR product (purified) 40ng, Salt solution 0.5μL, pCR TM / 8 GW / TOPO TM Vector 0.5μL, nucleic acid-free deionized water to 3μL. Gently blow and mix, transfer to ice after PCR reaction at 22℃ for 30min; add 3μL reaction product to 100μL TOP10 competent cells, gently mix, and let stand on ice for 25min; heat shock in a water bath at 42℃ for 1min, quickly put on ice and let stand for 3min; add the above reaction solution to a centrifuge tube containing 800μL SOC culture medium, 37℃ 150rpm, and recover for 1h; centrifuge at room temperature at 4000rpm for 3min, and aspirate about 700μL of the upper layer SOC, mix the remaining bacterial solution and apply it to the LB screening culture plate containing spectinomycin (Spc) resistance; invert and culture in a 37℃ constant temperature incubator for about 12h, pick a single clone strain for PCR detection, the primers used are the CcCHS1 gene specific upstream primer (F: 5'-ATGGTGGACGTGGAAGCC-3') and the T7 downstream primer on the vector (T7-R: 5'-TAATACGACTCACTATAGGG-3'), and send the positive bacterial solution to Shanghai Bioengineering Co., Ltd. for further sequencing verification. The plasmid of the entry vector with correct sequencing was extracted using a plasmid mini-extraction purification kit (Tiangen) and stored at 4℃ for future use.

[0062] 2.LR reaction

[0063] Prepare the reaction solution on ice: entry plasmid XμL (50 ng), PBI121 plasmid YμL (70 ng), LR Clonase II enzyme mix 0.5μL, 1×TE (pH=8) 2-X-YμL, total reaction volume 2.5μL. Incubate at 25℃ for 1h in a PCR instrument, add 0.25μL Proteinase K solution, and react at 37℃ for 10min to terminate the reaction; then transform with Top10 competent cells, apply to LB solid culture medium plates containing 50mg / mL Km resistance, and invert and culture at 37℃ for 12h; pick bacteria, shake bacteria, and detect. The primers used are 35sf (35sf: 5'-CGCACAATCCCACTATCCTT-3') and CcCHS1 gene downstream (R: 5'-TCTAAGCAGATACACTATGGAGC-3') to perform PCR detection on the bacterial solution. After sequencing verification, the target vector after LR reaction is recovered and purified to obtain the target gene overexpression vector pBI121::CcCHS1.

[0064] The results are as follows Figure 2 As shown, the gene is located behind the promoter CaMV35S. Driven by the promoter CaMV35S, pBI121 can be efficiently expressed in the recipient plant.

[0065] 3. Liquid nitrogen freeze-thaw transformation of Agrobacterium

[0066] After thawing GV3101 competent cells on ice, add 100ng of the successfully constructed target gene overexpression vector pBI121::CcCHS1, mix gently, and let stand on ice for 30min; freeze in liquid nitrogen for 1min; heat shock in a water bath at 37℃ for 3min, then quickly put on ice for 2min; add 800μL LB medium, shake at 28℃, 100rpm for 3min; centrifuge at room temperature at 4000rpm for 3min, and aspirate about 600μL LB medium; blow and mix the remaining bacteria, and evenly spread them on the LB plate added with Km antibiotic (50 mg / L) and rifampicin (100 mg / L); incubate inverted at 28°C in a constant temperature incubator for 30-48 h; pick a single strain, shake and culture at 28°C, 250 rpm for 12 h, use the upstream primer (35sf) on the vector and the downstream primer specific to the CcCHS1 gene (R: 5'-TCTAAGCAGATACACTATGGAGC-3') to perform PCR detection on the bacterial solution, and store the positive clone bacterial solution at 4°C for later use.

[0067] 4. Agrobacterium-mediated transformation of Populus shanxinensis by leaf disc method

[0068] Preparation of bacterial solution: Inoculate the positive strain into 2 mL LB + 50 mg / L Km + 100 mg / L Rif liquid medium, and culture at 28°C in a shaker at 250 rpm for about 12 hours. Then take 0.8 mL and inoculate it into 50 mL LB + 50 mg / L Km + 100 mg / L Rif liquid medium until the OD 600 The value was about 0.5, and the cells were centrifuged at 5000 rpm for 5 min at room temperature. The liquid culture medium was discarded, the bacteria were collected, and the bacteria were resuspended in an equal volume of sugar-free MS liquid culture medium and 20 μM / L acetosyringone (AS) was added.

[0069] Infection: Take leaves of 4-6 weeks old robust tissue culture seedlings of Populus dasyphylla, cut wounds along the leaf edges with scissors, transfer the cut leaf discs into the prepared Agrobacterium bacterial solution for 20 minutes, remove the leaf discs with sterile tweezers, dry the residual bacterial solution with sterile filter paper, transfer to differentiation medium MS1 (MS+0.4mg / L 6-BA+0.1mg / LNAA+0.01mg / L TDZ) plates, co-culture in the dark for 2-3 days, wash the bacteria and transfer the leaf discs to MS1 differentiation culture plates for light culture for 1 week. Wash the bacteria again and transfer the leaf discs to MS1 differentiation screening medium plates containing 200mg / L Timentin and 50mg / L Km for culture. After 3 to 4 weeks, when kanamycin-resistant adventitious buds grow on the edge of the leaf disk, they are transferred to the bud elongation screening medium (MS+0.4mg / L 6-BA+0.1mg / LNAA+50mg / L Km+200mg / L Timentin) plate for further screening and cultivation. After 3 to 4 weeks, kanamycin-resistant buds are cut and transferred to the rooting screening medium (MS+20mg / L Km+300mg / L Timentin) plate for rooting screening of resistant plants.

[0070] The results are as follows Figure 3 As shown, the leaves of the transgenic seedlings overexpressing the CcCHS1 gene inoculated in the basic culture medium used for camphor tissue culture showed orange-red color.

[0071] 5. Anthocyanin content in transgenic poplar leaves

[0072] Take 0.100g of transgenic Populus shanxin (T1, T2, T9) and non-transgenic Populus shanxin (NT1, NT2, NT3) leaves respectively and put them into 15mL round-bottom centrifuge tubes, add 10mL 0.1mol / L hydrochloric acid ethanol solution (8.3mL concentrated hydrochloric acid diluted with 95% ethanol to 1L), and extract in a 60℃ water bath for 1h. Use 0.1mol / L hydrochloric acid ethanol solution as reference solution, and measure the optical density of the extract at 530nm, 620nm, and 560nm wavelengths using a spectrophotometer.

[0073] The optical density value calculation formula of anthocyanin is as follows:

[0074] OD λ =(OD 530 -OD 620 )-0.1(OD 650 -OD 620 )

[0075] Among them, OD λ Anthocyanin content.

[0076]

[0077] Among them, OD λis the optical density of anthocyanin at a wavelength of 530 nm; ε is the molar extinction coefficient of anthocyanin 4.62×10 4 ; V is the total volume of the extract (mL); m is the sample mass (g). 10 6 : The calculation result is converted into multiples of nmol.

[0078] The results are as follows Figure 4 As shown, the content of anthocyanins in the leaves of transgenic Populus shanxinensis overexpressing the CcCHS1 gene is higher, making the leaves of the transgenic seedlings red.

[0079] The above results indicate that the CcCHS1 gene is a key enzyme gene for the coloration of camphor tree leaves and has important application value in forest genetic engineering research.

[0080] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A gene related to camphor tree coloration CcCHS1 , whose nucleotide sequence is shown in SEQ ID NO.

1.

2. The camphor tree color-related gene according to claim 1 CcCHS1 The encoded protein has an amino acid sequence as shown in SEQ ID NO.

2.

3. The camphor tree color-related gene according to claim 1 CcCHS1 Application in making the color of Populus alba leaves red.

4. The camphor tree color-related gene according to claim 3 CcCHS1 The application of the method for making the leaves of Populus dasyphylla red is characterized in that: include: 1) Construction of camphor tree color-related genes CcCHS1 expression vector; 2) The constructed camphor tree color-related gene CcCHS1 The expression vector was transformed into Populus shanxinensis; 3) Cultivate, screen and obtain transgenic Populus shanxinensis plants with red leaves.

5. The camphor tree color-related gene according to claim 4 CcCHS1 The application of the method for making the leaves of Populus dasyphylla red is characterized in that: The expression vector is a plant expression vector.

6. The camphor tree color-related gene according to claim 5 CcCHS1 The application of the method for making the leaves of Populus dasyphylla red is characterized in that: The plant expression vector is pBI121- CcCHS1 .

7. The camphor tree color-related gene according to claim 1 CcCHS1 Application in promoting the increase of anthocyanin content in Populus alba leaves.

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