Poplar PdbHLH57 gene as well as expression protein and application thereof

Abstract

The invention discloses a poplar PdbHLH57 gene as well as an expression protein and application thereof, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the poplar PdbHLH57 gene disclosed by the invention is as shown in SEQ ID NO.1, and the amino acid sequence of the expression protein of the poplar PdbHLH57 gene is as shown in SEQ ID NO.2. The method comprises the following steps: constructing an expression vector of a poplar PdbHLH57 gene, and transforming the expression vector into a poplar leaf; and cultivating and screening to obtain the transgenic poplar with red leaves. Results show that compared with wild poplar, the PdbHLH57 gene for promoting poplar anthocyanin synthesis has the advantages that transgenic poplar leaves become red, and the anthocyanin content in transgenic plant leaves is remarkably increased; the expression level of anthocyanin biosynthesis related genes is remarkably increased, and further, it is shown that the PdbHLH57 gene plays an important role in poplar anthocyanin biosynthesis.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a poplar PdbHLH57 gene and its expression protein and application. Background Art

[0002] The wadding-free colorful poplar not only has the advantages of fast growth, strong adaptability, and easy reproduction, but also has bright and beautiful leaves. The wadding-free colorful poplar plays an important role in urban beautification and road greening, and has an economic value that cannot be underestimated. Therefore, it is of great significance to explore the molecular regulatory mechanism of anthocyanin biosynthesis in colorful poplars, and it also provides a theoretical basis for the rapid cultivation of colorful poplars.

[0003] The bHLH family is named for its highly conserved basic helix-loop-helix domain and has been widely studied in plants and animals. bHLH transcription factors are composed of two conserved motifs, the DNA binding region and the helix-loop-helix region (HLH region). The DNA binding region is involved in DNA recognition and binding, while the HLH region is composed of hydrophobic residues and can form homodimers or heterodimers with other bHLH proteins. In plants, more than 500 known bHLHs are divided into 26 subgroups, and many bHLH transcription factors have been identified as key regulators of anthocyanin biosynthesis. Studies have shown that the bHLH transcription factor DcTT8 in Dendrobium officinale can regulate the expression of DcF3'H and DcUFGT, thereby participating in the biosynthesis of anthocyanins. In Arabidopsis, bHLH proteins TT81901, GL3191, and EGL3 form the MBW complex by binding to MYB proteins. Overexpression of AtbHLH4 in Arabidopsis thaliana leads to down-regulation of many key regulatory genes and enzymatic genes in the anthocyanin biosynthesis pathway, and leaves are darker in color than wild type. Overexpression of NtAnla and NtAnlb genes in tobacco significantly increased the anthocyanin content in tobacco. Since there are many types and functions of bHLH genes in different plants, it is of great significance to explore bHLH genes related to anthocyanin biosynthesis in poplar leaves to produce colorful poplars. 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 poplar PdbHLH57 gene. Another technical problem to be solved by the present invention is to provide an expression protein of the poplar PdbHLH57 gene. Another technical problem to be solved by the present invention is to provide an application of the poplar PdbHLH57 gene for regulating the synthesis of anthocyanin glycosides in plants.

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

[0006] A poplar PdbHLH57 gene, whose nucleotide sequence is shown in SEQ ID NO. 1.

[0007] The amino acid sequence of the expressed protein of the poplar PdbHLH57 gene is shown in SEQ ID NO. 2.

[0008] Vectors and recombinant bacteria containing the poplar PdbHLH57 gene.

[0009] Application of the poplar PdbHLH57 gene in regulating the color of plant leaves, wherein the regulating the color of plant leaves is to make the color of plant leaves turn red, comprising:

[0010] 1) Construction of the expression vector of the poplar PdbHLH57 gene;

[0011] 2) Transform the constructed expression vector of the poplar PdbHLH57 gene into poplar leaves;

[0012] 3) Cultivate, screen and obtain transgenic poplars with red leaves.

[0013] Application of poplar PdbHLH57 gene in regulating anthocyanin content in plants.

[0014] The regulating the anthocyanin content in the plant is to promote the increase of the anthocyanin content in the plant.

[0015] Application of poplar PdbHLH57 gene in regulating the expression of genes related to anthocyanin biosynthesis.

[0016] The regulating the expression of anthocyanin biosynthesis-related genes is to promote the increase of the expression of anthocyanin biosynthesis-related genes.

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

[0018] The poplar PdbHLH57 gene disclosed in the present invention has a nucleotide sequence as shown in SEQ ID NO. 1, and an amino acid sequence of its expressed protein as shown in SEQ ID NO. 2. The present invention constructs an expression vector of the poplar PdbHLH57 gene; transforms the constructed expression vector of the poplar PdbHLH57 gene into poplar leaves; and cultivates, screens, and obtains transgenic poplars with red leaves. The results show that the PdbHLH57 gene promotes the synthesis of anthocyanins in poplars. Compared with wild-type poplars, the leaves of transgenic poplars turn red, and the anthocyanin content in the leaves of transgenic plants is significantly increased; the expression level of anthocyanin biosynthesis-related genes is significantly increased, further indicating that the PdbHLH57 gene plays an important role in the biosynthesis of anthocyanin glycosides in poplars. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1GUS staining identification diagram (A) and PCR identification result diagram (B) of transgenic poplars overexpressing PdbHLH57 (WT is wild-type poplar; OE-1 and OE-3 are transgenic positive lines);

[0020] Figure 2 Comparison of the phenotype and anthocyanin content between transgenic poplars and wild-type poplars (A is a comparison of the growth status of transgenic poplars overexpressing PdbHLH57 and wild-type poplars and a leaf image under a microscope; B is a comparison of the leaf phenotypes of transgenic poplars overexpressing PdbHLH57 and wild-type poplars; C is anthocyanin content);

[0021] Figure 3 This is the expression level diagram of genes related to anthocyanin biosynthesis. DETAILED DESCRIPTION

[0022] 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. Molecular biology experimental methods not specifically described can be carried out by referring to the methods listed in the book "Molecular Cloning Laboratory Guide" (3rd Edition) by J. Sambrook or conventional methods in the art, or according to the kit and product instructions.

[0023] The material used in this application is a new variety of colorful poplar "Zhongshan Caiyun", which is planted in the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province.

[0024] Example 1

[0025] 1. Total RNA extraction and cDNA acquisition

[0026] RNAprep pure Plant Kit (TIANGENBiotech) was used to extract total RNA from leaves of Zhongshan Caiyun plants. Using the extracted total RNA as a template, the FastKing one-step genomic cDNA first-strand synthesis premix was used for reverse transcription to obtain cDNA.

[0027] 2. Target gene amplification

[0028] Using cDNA as template, PCR amplification of target gene was performed. The primer sequences are as follows:

[0029] PdbHLH57-F: 5'-ATGGCTACTAAGCTCCACAA-3',

[0030] PdbHLH57-R: 5'-TCAACATTTCCCAGCAACTC-3'.

[0031] The PCR reaction system was as follows: cDNA template 1 μL, upstream and downstream primers 0.8 μL each (10 μM), Buffer 10 μL, dNTPs 0.4 μL, Novozyme high-fidelity enzyme 0.4 μL, ddH 2 O 6.6 μL.

[0032] The PCR reaction conditions were as follows: 95°C for 3 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 35 cycles; and final extension at 72°C for 10 min.

[0033] The obtained amplified product is subjected to agarose electrophoresis, and then the gel is cut and recovered using a gel extraction kit. After the concentration of the recovered product is determined, it is stored at -20°C, or directly subjected to enzyme digestion, ligation and other reactions.

[0034] The nucleotide sequence of the PdbHLH57 gene obtained by final sequencing is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2.

[0035] Example 2

[0036] 1. Construction of overexpression vector of PdbHLH57 gene

[0037] The pCAMBIA2300 empty plasmid and the PCR product with restriction primers were double-digested with Xba I and Sal I endonucleases.

[0038] The double enzyme digestion reaction system is: Xba I 2μL, Sal I 2μL, DNA ≤1μg, 1×T+BSA 6μL, ddH 2 O up to 40μL.

[0039] The double enzyme digestion reaction conditions were: 37°C for 4 h and 30°C for 4 h.

[0040] The target gene fragment was recombined and connected with the linearized vector fragment to obtain the plant expression vector pCAMBIA2300-PdbHLH57.

[0041] The ligation reaction system is: Xba I 2μL, Sal I 2μL, DNA ≤1μg, 1×T+BSA 6μL, ddH 2 The reaction conditions are: mix and centrifuge, place the sample in a metal bath, and connect overnight at 16°C.

[0042] 2. Transformation of Agrobacterium EHA105

[0043] Take the -80℃ stored Agrobacterium competent cells EHA105 and place them in the palm of your hand for a moment to partially melt. When they are in an ice-water mixing state, insert them into ice; take 100μL competent cells and add 0.01-1.0μg plasmid, tap the bottom of the tube with your fingers to mix, and place them on ice, in liquid nitrogen, in a 37℃ water bath, and in an ice bath for 5 minutes each; add sterile LB liquid culture medium and shake and culture at 28℃ for 2-3 hours; centrifuge at 6000rpm for 1min to collect the bacteria, take 50μL supernatant and gently blow the resuspended bacteria block to apply it on an LB plate containing 50mg / L kanamycin, and invert it on a 28℃ shaker for 2-3 days. Pick a single white colony, inoculate it in LB liquid culture medium containing 50mg / L kanamycin, shake and culture at 250rpm, 28℃ for 4 hours, use primers to perform PCR to detect positive clones, and after identification, shake and wait for transformation of poplar leaves.

[0044] 3. Dyeing poplar leaves

[0045] The correctly identified Agrobacterium transformant pCAMBIA2300-PdbHLH57 was inoculated into 30 mL LB liquid medium (containing 50 mg / mL kanamycin) and cultured in a shaking incubator at 28°C until OD 600 is 0.5-0.8; collect the bacteria by centrifugation, resuspend the Agrobacterium in 30mL transformation medium MSO, and transform the poplar leaves by immersion method; take fresh leaves, cut into small pieces, and soak them in Agrobacterium culture medium for about ten minutes; take out the leaves, place them on MS solid culture medium without antibiotics and culture them in the dark at 28℃ for 2-3 days, and after 2-3 days, move them to MS differentiation medium containing kanamycin and wait for differentiation. Change the culture medium once a week, and wait for the leaves to differentiate and grow resistant buds before testing.

[0046] 4. Identification of transgenic positive plants

[0047] 1) Staining identification

[0048] Leaves of wild-type and transgenic plants were placed in GUS dye solution and stained at 37°C for more than 12 hours, followed by decolorization with anhydrous ethanol.

[0049] The results are as follows Figure 1 As shown in A, the leaves of the wild-type poplar plants were not stained blue, while the leaves of the transgenic lines OE-1 and OE-3 appeared blue; the results showed that the reporter gene was successfully integrated into the plant genome, and the plant was a transgenic plant.

[0050] 2) PCR identification

[0051] The blue-dyed plants were verified by PCR using a PCR kit produced by Novazon Biotech (Beijing) Co., Ltd. The primer sequences are as follows:

[0052] Q-PdbHLH57-F: 5'-TTACGACTCAATGACAAGAAG-3',

[0053] Q-PdbHLH57-R: 5'-GCAGTTAGGGGAAGTTATG-3'.

[0054] Amplification conditions were as follows: 95°C for 3 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 35 cycles; final extension at 72°C for 10 min.

[0055] The reaction system was as follows: cDNA template 1 μL, upstream and downstream primers 0.8 μL each (10 μM), Buffer 10 μL, dNTPs 0.4 μL, Novozyme high-fidelity enzyme 0.4 μL, ddH 2 O 6.6 μL.

[0056] The results are as follows Figure 1 As shown in B, the target band could not be amplified using wild-type poplar leaves and water as templates, but the target band could be amplified using transgenic strains OE-1 and OE-3 leaves as templates, indicating that the gene has been transferred into the transgenic strains OE-1 and OE-3.

[0057] Example 3

[0058] 1. Phenotype of transgenic plants

[0059] Transgenic PdbHLH57 poplar and wild-type poplar tissue culture seedlings were selected 30 days after rooting for phenotypic observation.

[0060] The results are as follows Figure 2 As shown in the figure, compared with wild-type poplars, transgenic PdbHLH57 poplars are shorter in height. Under a microscope, the accumulation of anthocyanins in the leaves of transgenic PdbHLH57 poplars can be clearly seen, and the leaves turn red ( Figure 2 The leaves of wild-type poplars did not turn red and the accumulation of anthocyanins was significantly lower than that of transgenic PdbHLH57 poplars ( Figure 2 A in Figure 2 C in the figure). Compared with wild-type poplar, the leaves of the transgenic poplar plants with PdbHLH57 gene were narrow and long ( Figure 2 B in the figure).

[0061] 2. Determination of anthocyanin content in transgenic plants

[0062] 1) Extract anthocyanins

[0063] Take 0.100g of poplar leaves and put them in numbered test tubes, add 10ml of hydrochloric acid ethanol solution, soak in a 60℃ water bath for 30min, pour the solution into a 25ml test tube, add 5ml of extract solution and soak for 15min, pour the solution into a 25ml test tube, add 5ml of extract solution and soak for 15min, pour the solution into a 25ml test tube, soak for a total of 1h, and finally adjust the volume to 25ml.

[0064] 2) Determination of anthocyanin content

[0065] Using 0.1 mol / L hydrochloric acid ethanol solution as reference solution, the optical density of the extract at wavelengths of 530 nm, 620 nm, and 650 nm was measured using a spectrophotometer.

[0066] Optical density of anthocyanins λ =(OD 530 -OD 620 )-0.1*(OD 650 -OD 620 );

[0067] Anthocyanin content (nmol / g) = (OD λ / ε)*(V / m)*1000000;

[0068] Anthocyanin content (μg / g) = anthocyanin content (nmol / g) × M × 10 -3 .

[0069] OD λ is 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); 1000000 is the multiple of the calculated result converted to nmol. M is the molecular weight of anthocyanin, 287.24; 10 -3 To convert ng to μg.

[0070] The results are as follows Figure 2 As shown in Figure C, compared with the control group, the anthocyanin content in the leaves of the transgenic plants was significantly increased, indicating that the PdbHLH57 gene promoted the synthesis and accumulation of anthocyanins in poplar.

[0071] 3. Detection of expression levels of structural genes in anthocyanin biosynthesis pathway

[0072] Transgenic poplars with PdbHLH57 gene and wild-type poplar tissue culture seedlings 30 days after rooting were selected, total RNA was extracted, and cDNA was obtained after reverse transcription. qPCR primers were designed according to the structural genes of the anthocyanin biosynthesis pathway of poplars. The expression levels of anthocyanin biosynthesis-related genes were determined by real-time fluorescence quantitative PCR reaction to verify the effect of PdbHLH57 gene on the expression of structural genes of the anthocyanin biosynthesis pathway of poplars. The primer sequences are as follows:

[0073] PtrCHS1-F: 5'-AAAACGTCCTGAGTGATTATGGC-3',

[0074] PtrCHS1-R: 5'-ATTGCTGGTCGAACGATCTTAT-3';

[0075] PtrCHI1-F: 5'-CACGAACTTACCTCACCCACC-3',

[0076] PtrCHI1-R: 5'-CGTTAATGGCAATATCATCGTCA-3';

[0077] PtrF3H-F: 5'-ATGGAAAGCAGTGACCGAGGA-3',

[0078] PtrF3H-R: 5'-CTTCAACAGGTTGAACGGTGATC-3';

[0079] PtrF3'HF:5'-ATGGTGACCCCGAAGGCAGAT-3',

[0080] PtrF3'HR:5'-GCAATAGCCCATTCCACTGT-3';

[0081] PtrF3'5'HF:5'-CAAGGAATTGAACGTGGGATG-3',

[0082] PtrF3'5'HR: 5'-GCAGGTATGGAAGTTTTGGTATGT-3';

[0083] PtrDFR2-F: 5'-GGATTTTATCAGCGTCATACCACC-3',

[0084] PtrDFR2-R: 5'-AGACAACACTCGCCAAATCCTC-3';

[0085] PtrANS1-F: 5'-ACTACTACCCCAAGTGCCCTCA-3',

[0086] PtrANS1-R: 5'-ACAATCTCTGCCAATGGCTTG-3';

[0087] PtrANR1-F: 5'-ACAGGGTTTGTGGCATCTTTG-3',

[0088] PtrANR1-R: 5'-GTCATGAGGCTAGGGATGACAGT-3';

[0089] PtrLAR1-F: 5'-TACTGACAGGGAAAACACGGAG-3',

[0090] PtrLAR1-R: 5'-GTCGTAGTAAGGCCAAGAAGCA-3';

[0091] PdbHLH57-F: 5'-TCAGTTGGTGAAGCCAGTCC-3',

[0092] PdbHLH57-R: 5'-CCCTTGGCCAACGTCGAATA-3';

[0093] Actin-F: 5'-CTGGCATACAGGGAAAGGACA-3',

[0094] Actin-R: 5'-GTCTGCGACAATGGAACTGGA-3';

[0095] qPCR amplification was performed using the Applied Biosystems 7500 Fast Real-Time PCR System.

[0096] Amplification conditions were: 95°C for 30 s; 95°C for 5 s, 60°C for 34 s, for 40 cycles.

[0097] The reaction system was as follows: 2 μL cDNA template, 0.4 μL upstream and downstream primers (10 μM each), 10 μL TB Green Premix Ex Taq (Tli RNaseH Plus) (2×), 10 μL ddH 2 O7.2μL.

[0098] After the reaction, the melting curve and amplification curve were checked. After confirmation, the expression level of anthocyanin biosynthesis-related genes was calculated using the 2^(-ΔΔCt) method.

[0099] The results are as follows Figure 3As shown, compared with wild-type poplar, overexpression of the PdbHLH57 gene significantly increased the expression levels of anthocyanin biosynthesis-related genes, further indicating that the PdbHLH57 gene plays an important role in the biosynthesis of anthocyanins in poplar.

[0100] 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 poplar PdbHLH57 gene, whose nucleotide sequence is shown in SEQ ID NO.

1.

2. The expressed protein of the poplar PdbHLH57 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. A vector or recombinant bacterium containing the poplar PdbHLH57 gene according to claim 1.

4. Use of the poplar PdbHLH57 gene according to claim 1 in regulating plant leaf color.

5. The use according to claim 4, characterized in that: The regulating the plant leaf color is to make the plant leaf color turn red.

6. The use according to claim 4, characterized in that: include: 1) Construction of the expression vector of the poplar PdbHLH57 gene; 2) Transform the constructed poplar PdbHLH57 gene expression vector into poplar leaves; 3) Cultivate, screen and obtain transgenic poplars with red leaves.

7. Use of the poplar PdbHLH57 gene according to claim 1 in regulating the anthocyanin content in plants.

8. The use according to claim 7, characterized in that: The regulating the anthocyanin content in the plant is to promote the increase of the anthocyanin content in the plant.

9. Use of the poplar PdbHLH57 gene according to claim 1 in regulating the expression of genes related to anthocyanin biosynthesis.

10. The use according to claim 9, characterized in that: The regulating the expression of anthocyanin biosynthesis-related genes is to promote the increase of the expression of anthocyanin biosynthesis-related genes.

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