Poplar PagDELLA08 gene as well as mutant, expression protein and application thereof

Abstract

The invention discloses a poplar PagDELLA08 gene as well as a mutant, expression protein and application thereof, and relates to the technical field of plant genetic engineering. The nucleotide sequences of the coding regions of the function deletion type gene and the function acquisition type gene of the poplar PagDELLA08 gene disclosed by the invention are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 5. The growth rate of the function deletion type mutant poplar strain constructed by the invention is obviously increased, and the fast-growing property of the poplar is improved. Leaves of the constructed functional acquisition type mutant poplar strain still present green after salt stress, the ROS content in leaf discs is low, and the salt tolerance of the leaves is enhanced; and after salt stress, the functional acquisition type mutant poplar strain has more roots and better growth vigor, which indicates that the functional acquisition type mutant has stronger salt tolerance.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically, relates to the Populus alba PagDELLA08 gene, its mutants, expressed proteins and applications. Background Art

[0002] Plantations are an important part of forest area and play an important role in restoring and reconstructing forest ecosystems, providing forest products, increasing forest carbon sinks, and improving the ecological environment. How to increase wood production and improve the supply-demand gap has always been one of the key tasks of forestry breeding researchers in China. Due to unreasonable land use, over-irrigation and climate change, the salinization phenomenon has gradually worsened, resulting in a significant reduction in forest area. With the increase of saline-alkali land, the growth of many plants is restricted in high-salt environments, seriously affecting plant growth and yield. Although there are currently various anti-saline-alkali measures, such as irrigation technology, application of organic fertilizers and selection of salt-tolerant tree species, these methods have some limitations such as economic burden, poor adaptability and insignificant short-term benefits.

[0003] As the main dominant tree species in plantations, Populus alba has the advantages of strong adaptability, fast growth rate and wide application. It is not only an important fast-growing wood resource in China, but also a model plant in woody molecular breeding research. In recent years, the academic community has conducted in-depth research on the salt tolerance, fast growth and other abilities of Populus alba, accumulating a lot of data and achievements. These studies provide a theoretical basis for improving Populus alba varieties and enhancing their application value in the future. Through modern means such as genomics and biotechnology, it is expected to cultivate new Populus alba varieties with stronger salt tolerance and faster growth in the future, thus promoting the ecological restoration and sustainable development of forestry.

[0004] DELLA proteins are key factors coordinating the action of multiple hormone signals. Located in the nucleus, they belong to the plant-specific GRAS family of proteins. DELLA proteins have highly conserved domains and are similar in structure to most proteins in the GRAS family, all having a conserved RGAs domain. The main function of the RGAs domain is to participate in protein-protein interactions and transcriptional regulation. The RGAs domain contains two leucine heptarepeats (LHRI and LHRII) and three conserved motifs (VHIID, PFYRE, and SAW). Compared with other proteins in the GRAS family, the unique feature of DELLA proteins is that they have two new domains (DELLA domain and TVHYNP domain) at their N-terminus. Mutations in the DELLA and TVHYNP domains interfere with the ability of DELLA proteins to bind to the GA receptor GID1. DELLA proteins are not degraded by the 26S proteasome, continuously inhibiting GA signal transduction and resulting in a semi-dominant GA-insensitive dwarf phenotype. The most classic example is the Arabidopsis gai mutant, in which 17 amino acids are deleted from the DELLA domain. Later, studies in rice found that LHRI in the rice DELLA protein (SLR1) only participates in the inhibitory activity of SLR1, while other domains of GRAS are involved in both its inhibitory function and the interaction between SLR1 and GID1 / GID2. Mutations at certain sites in the GRAS domain also result in a GA-insensitive phenotype.

[0005] Currently, there are research reports that the PagHyPRP1 gene in poplar is a negative regulator of salt and drought stress responses, and the poplar IAA17 / HSFA5a module mediates auxin signal transduction, regulating the biosynthesis of lateral root flavonols and the level of ROS, enhancing the salt tolerance of Populus tomentosa. There are few studies and reports on the DELLA gene. Summary of the Invention

[0006] In view of the above problems existing in the prior art, the technical problems to be solved by the present invention are to provide the poplar PagDELLA08 gene. Another technical problem to be solved by the present invention is to provide the mutant gene of poplar PagDELLA08. Another technical problem to be solved by the present invention is to provide the application of the poplar PagDELLA08 gene. Another technical problem to be solved by the present invention is to provide the application of the mutant gene of poplar PagDELLA08 for regulating plant growth and stress resistance.

[0007] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] The poplar PagDELLA08 gene, the nucleotide sequence of its coding region is shown in SEQ ID NO. 1.

[0009] The expressed protein of the PagDELLA08 gene of Populus has an amino acid sequence as shown in SEQ ID NO. 2.

[0010] The mutant gene of PagDELLA08 of Populus includes a loss-of-function mutant gene of PagDELLA08 of Populus and / or a gain-of-function mutant gene of PagDELLA08 of Populus; the coding region nucleotide sequence of the loss-of-function mutant gene of PagDELLA08 of Populus is as shown in SEQ ID NO. 3; the coding region nucleotide sequence of the gain-of-function mutant gene of PagDELLA08 of Populus is as shown in SEQ ID NO. 5.

[0011] The expressed protein of the mutant gene of PagDELLA08 of Populus, the amino acid sequence of the expressed protein of the loss-of-function mutant gene of PagDELLA08 of Populus is as shown in SEQ ID NO. 4; the amino acid sequence of the expressed protein of the gain-of-function mutant gene of PagDELLA08 of Populus is as shown in SEQ ID NO. 6.

[0012] Application of the loss-of-function mutant gene of PagDELLA08 of Populus in regulating the plant growth rate.

[0013] The regulation of plant growth is to promote the acceleration of plant growth rate.

[0014] Application of the loss-of-function mutant gene of PagDELLA08 of Populus in regulating the plant plant height.

[0015] The regulation of plant plant height is to promote the increase of plant plant height.

[0016] Application of the gain-of-function mutant gene of PagDELLA08 of Populus in regulating plant salt tolerance.

[0017] Application of the gain-of-function mutant gene of PagDELLA08 of Populus in regulating plant growth under salt stress.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1) The CRISPR / Cas9 gene editing vector of the PagDELLA08 gene of Populus was constructed and transformed into Populus, and two homozygous mutant transgenic seedlings of the PagDELLA08 gene were cultivated, screened and obtained. Specifically, for the 2 loss-of-function mutants (pagdella08-1, pagdella08-2), due to the addition of base pairs at the mutation site, the coding frame shifted, and the translation of the DELLA08 protein terminated prematurely, only the first 278 amino acids of the DELLA08 protein were normally translated; for the 2 gain-of-function mutants (pagdella08-DN-1, pagdella08-DN-2), base substitutions and large fragment deletions occurred in the PagDELLA08 gene. The N-terminal and C-terminal sequences of the DELLA08 protein were translated normally, only lacking 66 amino acids near the VHIID domain and having 1 amino acid mutated.

[0020] 2) The growth rates of the loss-of-function mutant lines pagdella08-1 and pagdella08-2 constructed in the present invention were significantly increased, thereby improving the fast growth of Populus.

[0021] 3) After stress in NaCl solution, the leaves of the gain-of-function mutants pagdella08-DN-1 and pagdella08-DN-2 constructed in the present invention remained green, the browning of the leaf margins was weak, and the ROS content in the leaf discs was less. The results showed that the salt tolerance of the leaves of the gain-of-function mutant Populus pagdella08-DN line was enhanced.

[0022] 4) After stress in NaCl solution, the gain-of-function mutant pagdella08-DN-1 constructed in the present invention had more roots and better growth. The results showed that the gain-of-function mutant pagdella08-DN had stronger salt tolerance. Description of the Drawings

[0023] Figure 1 It is the PCR amplification electrophoresis diagram of the CDS fragment of the PagDELLAs gene;

[0024] Figure 2 It is the map of the pYLCRISPR / Cas9P35S-H vector;

[0025] Figure 3 It is the design diagram of the sgRNA target of the PagDELLA08 gene;

[0026] Figure 4 It is the construction process diagram of the sgRNA expression cassette of the PagDELLA08 gene;

[0027] Figure 5Schematic diagram of the PagDELLA08 protein domain in PagDELLA08 gene mutant poplar;

[0028] Figure 6 Figure showing the expression of PagDELLA08 gene in different tissues of poplar;

[0029] Figure 7 Phenotype analysis chart of the growth rate of PagDELLA08 gene mutant poplar (A shows the natural growth states of wild-type and mutant poplar seedlings 1 month and 3 months after transplanting from tissue culture bottles into pots; B is the statistics of the plant heights of the plants in Figure A; C is the statistics of the growth rates of different poplar materials);

[0030] Figure 8 Phenotype chart of PagDELLA08 gene mutant poplar leaf discs under different concentrations of NaCl treatment;

[0031] Figure 9 DAB staining chart of PagDELLA08 gene mutant poplar leaves after treatment with 300 mM NaCl;

[0032] Figure 10 Growth state chart of PagDELLA08 gene mutant poplar tissue culture seedlings after salt stress treatment. Detailed implementation manners

[0033] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, if not otherwise specified in detail, the technical means used are all conventional means well-known to those skilled in the art. For molecular biology experimental methods not specifically described, they can be referred to the methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook or the conventional methods in the art, or carried out according to the kits and product instructions.

[0034] The plant material used in this application is the leaves of 84K poplar, which are stored in the National Key Laboratory of Tree Genetics and Breeding, Nanjing Forestry University (Research Room 60503 for Pine Genetic Improvement).

[0035] Example 1

[0036] 1. Cloning of the poplar PagDELLA08 gene

[0037] Download the CDS sequences of DELLA proteins GAI, RGA, RGL1, RGL2, and RGL3 (AT1G14920, AT2G01570, AT1G66350, AT3G03450, AT5G17490) from the TAIR website of the Arabidopsis thaliana database (https: / / www.arabidopsis.org / ). Use the CDS sequences of Arabidopsis thaliana DELLA to align with the genome of Populus trichocarpa in Plantgenie (https: / / plantgenie.org / ). Finally, four homologous genes of DELLA were found in Populus trichocarpa, including the DELLA gene Potri.008G131700 on chromosome 8 disclosed in this application. Design corresponding primers according to the genomic sequences of the DELLA genes of Populus trichocarpa found and perform cDNA amplification in Populus alba × Populus glandulosa. The amplification primer sequences of the PagDELLA08 gene are shown below:

[0038] PagDEL08-CDS-F: 5’-ATGAAAAGAGAACACCCAAATCTCC-3’,

[0039] PagDEL08-CDS-R: 5’-AGCAGCACCAACTACCG-3’.

[0040] Extract the RNA of Populus alba × Populus glandulosa leaves using a plant total RNA extraction kit (Vazyme Biotech Co., Ltd., RC411-01). Reverse transcribe the above-extracted RNA using a cDNA synthesis kit (Vazyme Biotech Co., Ltd., 7F581J1) to synthesize cDNA. The operation steps and precautions are carried out according to the instructions. Subsequently, use Phanta high-fidelity enzyme (Vazyme Biotech Co., Ltd., P505-d1) to amplify the CDS of the PagDELLA08 gene. PCR reaction system: 11µL ddH2O, 15µL 2×Phanta Max Buffer, 0.6µL dNTP Mix (10mM), 0.6µL Phanta Max Super-Fidelity DNA Polymerase, 1.2µL upstream primer (10µM), 1.2µL downstream primer (10µM), 0.5µL cDNA.

[0041] Perform gene amplification using the PCR reaction program: pre-denaturation, 95℃ for 3min; denaturation, 95℃ for 15s; annealing, 56℃ for 15s; extension, 72℃ for 30s - 60s (33 cycles); final extension, 72℃ for 5min.

[0042] Perform gel electrophoresis detection on the PCR products ( Figure 1)And perform cleavage, glue recovery and sequencing. The CDS nucleotide sequence of the homologous gene PagDELLA08 of Arabidopsis DELLA protein in 84K obtained by sequencing is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2.

[0043] Example 2

[0044] 1. Construction of CRISPR / Cas9 gene editing vector

[0045] The CRISPR / Cas9 system uses the online design platform and vector established by the Liu Yaoguang laboratory ( Figure 2 ). Use the online primer design platform (http: / / skl.scau.edu.cn / ) to screen for targets with lower off-target rates in the 84K poplar DELLA gene, and select Method2 to design sgRNA primers ( Figure 3 ). The target sequences for constructing the PagDELLA08 gene editing vector are as follows:

[0046] sgRNA1: 5’-GGTAGCCACCTACTTCGCCGAGG-3’,

[0047] sgRNA2: 5’-TAATGCCTCGGCGAAGTAGGTGG-3’,

[0048] sgRNA3: 5’-TCCGTGTTATCATGAGCCGGTGG-3’.

[0049] Use the Overlapping PCR amplification method to connect each promoter with the target to construct the sgRNA expression cassette ( Figure 4 ), and finally use the method of cutting and ligating simultaneously with BsaⅠ-HF and T4 DNA ligase to connect each expression cassette to the pYLCRISPR / Cas9-DN vector, and use DH5α competent cells for heat shock transformation in a water bath.

[0050] 2. Transformation of poplar with recombinant pYLCRISPR / Cas9P35S-H vector

[0051] Transform the recombinant pYLCRISPR / Cas9P35S-H vector into poplar by Agrobacterium-mediated method as follows:

[0052] (1) Pick the EHA105 strain containing the constructed vector from the plate and inoculate it into 100 mL of liquid LB medium containing antibiotics (containing 50 mg / L kanamycin and 17 mg / L rifampicin), and culture at 28°C until OD 600 = 0.6 - 0.8.

[0053] (2)Take the leaves of sterile poplar tissue culture seedlings, remove the main veins, cut them into small pieces about 1 cm × 1 cm in size, soak them in the Agrobacterium liquid for 10 - 15 min, and gently shake them 2 - 3 times in the middle.

[0054] (3)Take out the soaked leaves and place them on sterile filter paper. After sucking dry the bacterial liquid adsorbed on their surfaces, transfer them to the differentiation medium and co - culture them for 72 h under dark conditions at 25 °C.

[0055] (4)Wash the materials three times with sterile water and transfer them to the differentiation medium containing 200 mg / L of the antibiotic Timentin, and culture them at 24 °C.

[0056] (5)One week later, transfer the explants to the differentiation medium containing 200 mg / L of Timentin and 10 mg / L of Hyg for screening culture, and change the fresh medium once a week.

[0057] (6)After about 30 days of selective culture, the transformed cells of the leaf explants differentiate into resistant buds. When they grow to 5 - 6 leaves, transfer them to the rooting medium to root. When they grow into complete plants, transfer them from the solid medium to the nutrient solution for open - type culture. After new roots are generated, transfer the seedlings to the greenhouse.

[0058] 3. Identification of gene - edited poplars

[0059] Extract the genomic DNA of each positive transgenic poplar tissue culture seedling, and perform PCR amplification on the fragments near the target site. Send the PCR products detected with gel electrophoresis to the company for sequencing, and obtain two homozygous mutant transgenic seedlings of the PagDELLA08 gene. Specifically: for the 2 loss - of - function mutants (pagdella08 - 1, pagdella08 - 2), due to the addition of bases at the mutation site (the nucleotide sequence of the coding region is SEQ ID NO.3), the coding frame shifts, and the translation of the DELLA08 protein terminates prematurely, only the first 278 amino acids of the DELLA08 protein are normally translated (the amino acid sequence is SEQ ID NO.4); for the 2 gain - of - function mutants (pagdella08 - DN - 1, pagdella08 - DN - 2), base substitution and large - fragment deletion occur in the PagDELLA08 gene (the nucleotide sequence of the coding region is SEQ ID NO.5), the N - terminal and C - terminal sequences of the DELLA08 protein are translated normally, only 66 amino acids near the VHIID domain are missing and 1 amino acid is mutated (the amino acid sequence is SEQ ID NO.6). The specific protein translation situation is as Figure 5 shown.

[0060] 4. Sub - culture of tissue - cultured poplar seedlings

[0061] Culture medium preparation: 2.25 g / L 1 / 2MS, 4 g / L Phytagel, 10 g / L Sucrose. Adjust the pH to 5.7 - 5.8 and then autoclave at 121 °C for 20 min. After sterilization, let it cool to room temperature until it is not hot to the touch. Before it solidifies, add 200 mg / L Timentin and 250 mg / L Cefotaxime antibiotics and mix well. Then dispense the culture medium into tissue culture bottles. Spray the laminar flow hood with 75% alcohol, wipe it clean, turn on the UV lamp for 30 min of ultraviolet sterilization. Turn on the dry heat sterilizer to 300 °C, place the pre-sterilized forceps into the laminar flow hood, sterilize the forceps, scissors using the dry heat sterilizer for at least 30 s, and then use them after cooling.

[0062] Cut off the apical buds about 3 cm long from the vigorously growing tissue culture seedlings, cut off the excess leaves, and vertically insert the apical buds into the prepared culture medium. Place the tissue culture seedlings on the tissue culture rack for light culture. The temperature in the tissue culture room is required to be maintained at 23 - 25 °C, set the photoperiod to 12 h, and the light intensity to 2000 Lux. Subculture the tissue culture seedlings every 4 - 6 weeks.

[0063] Example 3

[0064] 1. Total RNA extraction and cDNA synthesis

[0065] Use a plant total RNA extraction kit (Vazyme Biotech Co., Ltd., RC411-01) to extract RNA from different parts of Populus alba × Populus glandulosa (roots, stems, young leaves, old leaves). The extraction steps and precautions are carried out according to the instructions. Use a cDNA synthesis kit (Vazyme Biotech Co., Ltd., 7F581J1) to reverse transcribe the above-extracted RNA to synthesize cDNA. The operation steps and precautions are carried out according to the instructions.

[0066] 2. Real-time quantitative PCR analysis

[0067] Design qRT-PCR primers according to the CDS sequence of PagDELLA08, and select PagEIF4a as the internal reference gene. The sequences are as follows:

[0068] PagDELLA08-qPCR-F: 5’-TTCCACTGCTACCAGTGCCATG-3’,

[0069] PagDELLA08-qPCR-R: 5’-GTGACCCATGACTTCTTCAAGTTGT-3’;

[0070] PagEIF4a: 5’-TACATTCATCGAATTGGTCGTTCTGGT-3’,

[0071] PagEIF4a: 5’-TTCATAGGCATTTCGTCAATCTGGG-3’.

[0072] The qRT-PCR reaction was carried out using the Novoprotein ChamQ SYBR qPCR Master Mix fluorescence quantitative kit.

[0073] The qPCR reaction system was as follows: 2×ChamQ SYBR qPCR Master Mix 10 μL, forward primer 0.4 μL, reverse primer 0.4 μL, ddH2O 7.7 μL, cDNA 1.5 μL.

[0074] The qPCR reaction program was as follows: pre-denaturation at 95°C for 30 sec; cycling reaction at 95°C for 10 sec, 60°C for 10 sec, for 40 cycles; melting curve at 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec. The relative expression level was calculated using the ΔΔ 2−ΔΔCt method.

[0075] The results were as Figure 6 shown, and the PagDELLA08 gene was expressed in the roots, stems, and leaves of poplar.

[0076] 4. Growth rate of gene-edited poplar

[0077] The obtained gene-edited poplar seedlings and wild-type 84K poplar were subcultured and acclimatized, and then transplanted into nutrient soil (peat soil: perlite: vermiculite mixed at a ratio of 2:1:1) to grow and observe their growth phenotypes. The growth heights and growth rates of the gene-edited seedlings and wild-type 84K poplar were statistically analyzed one month and three months after transplantation.

[0078] The results were as Figure 7 shown. Compared with the wild-type 84K poplar, the growth rates of the gain-of-function mutants pagdella08-DN-1 and pagdella08-DN-2 were lower; while the growth rates of the loss-of-function mutants pagdella08-1 and pagdella08-2 were significantly increased.

[0079] 5. Salt tolerance of gene-edited poplar leaves

[0080] Using the tissue culture seedlings of wild-type 84K poplar and gene-edited poplar (pagdella08-DN-1, pagdella08-DN-2, pagdella08-1, and pagdella08-2), healthy leaves of the same part were taken, and leaf discs of the same size were punched out and placed in different concentrations of NaCl solutions (0 mM, 150 mM, 300 mM, 500 mM) and left at room temperature for 3 days.

[0081] The results are as follows Figure 8 shown. Compared with wild-type poplar, the leaves of the gain-of-function mutants pagdella08-DN-1 and pagdella08-DN-2 still showed green color, and the browning phenomenon at the leaf margins was weak, indicating that this type of gene-edited poplar was insensitive to NaCl treatment; the leaves of the loss-of-function mutants pagdella08-1 and pagdella08-2 showed obvious chlorophyll deficiency and obvious browning at the leaf margins, indicating that this type of gene-edited poplar was more sensitive to NaCl treatment.

[0082] Treat the leaf discs of wild-type 84K poplar and gene-edited poplar with 300 mM NaCl, and observe the accumulation of ROS by DAB staining.

[0083] The results are as follows Figure 9 shown. The ROS content in the leaf discs of the gain-of-function mutants pagdella08-DN-1 and pagdella08-DN-2 was less. In summary, the salt tolerance of the leaves of the gain-of-function mutant poplar pagdella08-DN lines was enhanced, while the salt tolerance of the pagdella08 lines was decreased.

[0084] 6. Salt tolerance of gene-edited poplar tissue culture seedlings

[0085] Select the apical buds of 4-week-old wild-type 84K poplar and gene-edited poplar tissue culture seedlings for subculture in a medium containing 100 mM NaCl, and observe the phenotypes after 50 days.

[0086] The results are as follows Figure 10 shown. Compared with wild-type 84K poplar, the gain-of-function mutant pagdella08-DN-1 had more roots and better growth, while the loss-of-function mutant pagdella08-2 showed a phenotype of no root formation and subsequent death. The results indicated that the pagdella08-DN mutant had stronger salt tolerance.

[0087] The above is only illustrative and not restrictive for the present invention. Those of ordinary skill in the art understand that many modifications, changes or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.

Claims

1. Poplar PagDELLA08 gene, the nucleotide sequence of its coding region is shown in SEQ ID NO.

1.

2. The expressed protein of the Poplar PagDELLA08 gene according to claim 1, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. Poplar PagDELLA08 mutant gene, including Poplar PagDELLA08 loss-of-function mutant gene and / or Poplar PagDELLA08 gain-of-function mutant gene; the nucleotide sequence of the coding region of the Poplar PagDELLA08 loss-of-function mutant gene is shown in SEQ ID NO. 3; the nucleotide sequence of the coding region of the Poplar PagDELLA08 gain-of-function mutant gene is shown in SEQ ID NO.

5.

4. The expressed protein of the Poplar PagDELLA08 mutant gene according to claim 3, the amino acid sequence of the expressed protein of the Poplar PagDELLA08 loss-of-function mutant gene is shown in SEQ ID NO. 4; the amino acid sequence of the expressed protein of the Poplar PagDELLA08 gain-of-function mutant gene is shown in SEQ ID NO.

6.

5. Application of the Poplar PagDELLA08 loss-of-function mutant gene in regulating plant growth rate.

6. The application according to claim 5, wherein The regulation of plant growth is to promote the acceleration of plant growth rate.

7. Application of the Poplar PagDELLA08 loss-of-function mutant gene in regulating plant plant height.

8. The application according to claim 7, wherein The regulation of plant plant height is to promote the increase of plant plant height.

9. Application of the Poplar PagDELLA08 gain-of-function mutant gene in regulating plant salt tolerance.

10. Application of the Poplar PagDELLA08 gain-of-function mutant gene in regulating plant growth under salt stress.

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