Pagmed20 gene and its application in promoting growth of poplar

Abstract

The application discloses a PagMED20 gene and application thereof in promoting growth of poplar, and belongs to the technical field of cross between genetic engineering and genetic improvement of forest trees. The application verifies the function of the gene by cloning and analyzing the PagMED20 gene and overexpressing the PagMED20 gene in 84K poplar by means of agrobacterium-mediated genetic transformation technology. Compared with wild type, the plant overexpressing the PagMED20 gene can significantly promote the plant height, base diameter, net photosynthetic rate, root chlorophyll content and / or root nitrogen content of the poplar, and the important role of the PagMED20 gene in promoting the plant height and secondary growth of the poplar is proved. The application has important practical and theoretical guiding significance for studying the growth stability of trees and cultivating new varieties of forest trees.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of genetic engineering and forest genetic improvement, and more specifically to the PagMED20 gene and its application in promoting poplar growth. Background Technology

[0002] Poplar ( Populus spp. Poplar is an important fast-growing timber and ecological restoration tree species globally. Its economic value and ecological benefits directly depend on two core growth traits: tree height and trunk diameter (i.e., diameter at breast height). Increased tree height directly improves the length of individual trees and the overall biomass accumulation, making it a key indicator for measuring the volume per unit area of ​​forest land; while increased trunk diameter fundamentally determines the timber yield, individual tree volume, and timber quality. Therefore, increasing the tree height and trunk diameter of poplar is a core breeding objective for shortening the forestry production cycle, increasing timber yield and economic benefits, and enhancing carbon sequestration capacity. Since plant growth ultimately stems from the accumulation of photosynthetic products, fundamentally improving photosynthetic efficiency and enhancing carbon and nitrogen metabolism capacity are the key physiological foundations driving rapid growth and high yield in forests.

[0003] Traditional genetic improvement of poplar trees mainly relies on hybridization breeding and phenotypic selection. However, the inherent long generation cycle, highly heterozygous genetic background, and complexity of growth traits being regulated by multiple genes in forest trees make traditional breeding processes slow and inefficient, making it difficult to achieve leapfrog improvements in growth traits. With the development of biotechnology, molecular-assisted breeding and transgenic technology have opened up new pathways for the precise and efficient improvement of forest trees. Among these, analyzing the key genes and their networks that regulate plant growth and development has become the theoretical basis for designing breeding strategies.

[0004] Among numerous regulatory pathways, the mediator complex in eukaryotes, as a universal transcriptional co-regulator connecting gene-specific transcription factors and RNA polymerase II, plays a central role in signal integration and transcriptional program regulation. In plants, it exerts global regulatory effects on light signals, hormone responses (such as auxin and cytokinin), and cell cycle progression. Studies have shown that the loss or alteration of function of specific subunits of the mediator complex (such as certain members in Arabidopsis thaliana) significantly affects plant growth and development. However, existing research has largely focused on annual herbaceous model plants such as Arabidopsis thaliana and rice. For perennial woody plants like poplar, which have complex secondary growth (lignification) processes, research on the function of mediator complex subunits remains extremely limited. This gap in understanding severely restricts our ability to apply this key transcriptional regulatory mechanism to the improvement of forest tree growth traits.

[0005] Although some literature reports on the intermediate subunits in certain species or hybrids of the Populus genus, there is still a lack of clear and complete teachings and technical insights regarding the specific gene described in this invention and its direct and efficient synergistic promotion of Populus tree height and trunk diameter growth. In particular, how to effectively apply this gene to Populus breeding practices to address the urgent industry need for rapid increases in timber production remains a technological gap that has not been fully revealed and realized in the existing technology.

[0006] Therefore, cloning and identifying the mediator gene that plays a key positive regulatory role in poplar growth, elucidating its function, and developing efficient breeding methods based on this gene are of great theoretical and practical significance for breaking through the bottleneck of traditional poplar breeding and realizing targeted genetic improvement of growth traits. Summary of the Invention

[0007] In view of this, the present invention provides the PagMED20 gene and its application in promoting poplar growth. Overexpression of the PagMED20 gene has successfully improved the plant height, trunk diameter, net photosynthetic rate, root chlorophyll content and / or root nitrogen content of poplar.

[0008] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0009] The primary objective of this application is to provide an isolated PagMED20 gene, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0010] Another object of this application is to provide: a PagMED20 protein encoded by the PagMED20 gene, the amino acid sequence of which is shown in SEQ ID NO.4.

[0011] Another object of this application is to provide an expression cassette containing the PagMED20 gene, wherein the expression cassette is capable of driving overexpression of the PagMED20 gene in a host cell.

[0012] Another object of this application is to provide a recombinant vector comprising the aforementioned expression cassette.

[0013] Another object of this application is to provide: a recombinant microorganism comprising the expression cassette or the recombinant vector described herein.

[0014] Another object of this application is to provide: a non-renewable poplar segment comprising the expression cassette, the recombinant vector, or the recombinant microorganism.

[0015] Another object of this application is to provide the application of the PagMED20 gene, the PagMED20 protein, the expression cassette, the recombinant vector, the recombinant microorganism, or the non-renewable poplar part in promoting poplar growth.

[0016] As a preferred technical solution, promoting poplar growth involves increasing the poplar's height, trunk diameter, net photosynthetic rate, root chlorophyll content, and / or root nitrogen content.

[0017] Another object of this application is to provide a method for promoting the growth of poplar trees, including the step of increasing the expression level of the PagMED20 gene in poplar trees, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0018] As a preferred technical solution, the expression level of the PagMED20 gene is increased through transgenic means.

[0019] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention achieves significant and multi-faceted technical effects by increasing the expression of the PagMED20 gene in poplar trees, the core of which is summarized as follows:

[0021] 1. Direct growth phenotypic enhancement

[0022] Synergistic effect: It can simultaneously and significantly improve the plant height, trunk diameter, net photosynthetic rate, root chlorophyll content and / or root nitrogen content of transgenic poplar, breaking through the bottleneck that growth traits often need to be improved separately.

[0023] Rapid growth and increased yield: It directly accelerates the growth rate of poplar trees, providing a key solution for shortening the rotation period and increasing timber production.

[0024] 2. Outstanding value in breeding applications

[0025] Path innovation: It provides a precise and efficient molecular breeding method that overcomes the limitations of traditional forest tree breeding, which has a long cycle and low efficiency.

[0026] The solution is comprehensive: the accompanying expression box, carrier, and transformation method form a complete, operable, and repeatable technical solution that is easy to promote.

[0027] It has great potential: it lays the foundation for further design of synergistic improvements in complex traits such as growth rate, wood quality, and stress resistance.

[0028] In summary, this invention provides a functionally validated key gene and an efficient technical solution for the genetic improvement of rapid growth in poplar trees, and has clear prospects for industrial application. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 The image shows the pCAMBIA2300-35S-GFP vector.

[0031] Figure 2 Image: Nucleic acid gel image after PCR verification of transgenic plants.

[0032] Figure 3 The expression of the PagMED20 gene in different tissues.

[0033] Figure 4 The expression of the PagMED20 gene in wild-type and transgenic plants.

[0034] Figure 5 For: Phenotypic results of plant height of wild-type and transgenic plants.

[0035] Figure 6 For: Quantitative results of plant height of wild-type and transgenic plants.

[0036] Figure 7 For: Quantitative results of the basal diameter of wild-type and transgenic plants.

[0037] Figure 8 For: Quantitative results of net photosynthetic rate of wild-type and transgenic plants.

[0038] Figure 9 The results show the quantitative results of chlorophyll content in wild-type and transgenic plants.

[0039] Figure 10 For: Quantitative results of nitrogen content in wild-type and transgenic plants. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1

[0042] PagMED20 gene cloning and recombinant vector construction

[0043] (1) RNA extraction and reverse transcription: Total RNA from wild-type 84K poplar was isolated using the RNA extraction kit from Beijing Adlai Biotechnology Co., Ltd., and templates were prepared using the EasyScript® First-Strand cDNA Synthetic SuperMix cDNA synthesis kit from Beijing TransGen Biotechnology Co., Ltd.

[0044] (2) Gene amplification system: Specific primers were designed based on the CDS region sequence of the PagMED20 gene, BamHI / KpnI restriction sites were introduced, and PCR amplification was performed using high-fidelity enzymes. The primers and sequences used for gene amplification are shown in Table 1.

[0045] Table 1 Primers and sequences used for gene amplification.

[0046]

[0047] PagMED20 gene CDS sequence:

[0048] ATGCCACTGAAATGGGTTTTGCATTGGCAACCCAACGCAGGAACAACCGTGAACACCCAAATCCTCAATGAAGTAACTCAGTGCGTGGAGAGCATTAACGGCGTTAAAGAAGGCCGATGGAAAGCTACTCTCACCTACTATAAACCCATACTACGAGACCAATCGCAAGCTCCTGAATTGCCGCGAGATTTCTTGGGTATTTCTTTGCCAGAGCAGCCAAATAAGTACTACTTCATAATCCGAGGTCAAAGGATTGTTCTCGAGGCAGATTCCTCCATTCAAACAATAATGGAGAAGCTTCAGTCTTATAAGTCGAGGGTTGCTCTCTACTTTGAGGGTTTTCAGTATCAACTCGGGGACTTTCAGTTGCGAGTTGGGAAAGTTACCCCAACTCATTCGGACAACTTGAGAGGAATTATTATGGAGGTGGAGTATCTTCCTTTGTCATCAATGGACAAATCCAGGCAAGTCATGGAAGAGTTTGTTGATATATGGCAAGAAGCCATCTCAAAAAGATCATTACCAGGCCATTTCATGCATATGGAACCAAACTTTGTGGAGTTCGGTCTCTCGGATCACTATAGCTCGCAACACACAGCTGTGCAGTATGCTACTGTCATGGCTCAACTAATTGCGACTCAGTCAGTGCAAGCCGCGAGAAAC, SEQ ID NO.3.

[0049] PagMED20 protein amino acid sequence: MPLKWVLHWQPNAGTTVNTQILNEVTQCVESINGVKEGRWKATLTYYKPILRDQSQAPELPRDFLGISLPEQPNKYYFIIRGQRIVLEADSSIQTIMEKLQSYKSRVALYFEGFQYQLGDFQLRVGKVTPTHSDNLRGIIMEVEYLPLSSMDKSRQVMEEFVDIWQEAISKRSLPGHFMHMEPNFVEFGLSDHYSSQHTAVQYATVMAQLIATQSVQAARN, SEQ ID NO.4.

[0050] (3) Vector assembly: The pCAMBIA2300-35S-GFP vector (see diagram) was assembled. Figure 1 Linearized by double digestion with BamHI / KpnI, the 663bp PagMED20 fragment was purified by gel electrophoresis and then used to construct the recombinant vector pCAMBIA2300-35S:PagMED20-GFP using Gibson AssemblyMaster Mix (purchased from NEB) for seamless cloning.

[0051] (4) Transformation verification: Transformation of Escherichia coli competent cells TOP 10 (purchased from Beijing Adley Biotechnology Co., Ltd., for specific operation methods and dosages, refer to the instruction manual): Add the reaction product in step (3) to Escherichia coli competent cells for transformation, and then spread evenly on LB plates containing kanamycin and incubate overnight at 37°C with the plates inverted.

[0052] (5) Identification of positive clones: Positive clones were screened by colony PCR (primers JP-MED20-F: 5'-GGTACCATGCCACTGAAATGG-3', SEQ ID NO.5; JP-MED20-R: 5'-AAGTAGTGACAAGTGTTGGCC-3', SEQ ID NO.6). After confirmation by sequencing, plasmids were extracted and transformed into GV3101 Agrobacterium (purchased from Vidi Biotechnology Co., Ltd.).

[0053] Example 2

[0054] Establishment of genetic transformation system

[0055] (1) Explant preparation: Take young leaves of sterile tissue culture seedlings, scratch both sides and culture them in pre-culture medium (pH 5.8, 16h light / 8h dark) for 2 days.

[0056] (2) Agrobacterium infection: Add 500 μL-1 mL of Agrobacterium containing the correctly edited PagMED20 vector to 100 mL of LB broth containing antibiotics (50 mg / L kanamycin, 50 mg / L rifampin), mix well, and incubate overnight at 28°C with shaking at 200 rpm / min. The next day, when the OD600 of Agrobacterium is 0.6-1.0, it is ready. In a sterile laminar flow hood, sterilize the forceps with UV light and turn on the high-temperature sterilizer. After the forceps have cooled, place the leaf from the pre-culture medium into a conical flask containing Agrobacterium and time for 15-20 min, shaking the conical flask evenly for 2-3 min during this period. After the time is up, remove the leaf to filter paper that has been autoclaved, remove the excess bacterial solution from the leaf surface, and then put the leaf back into the original pre-culture medium and incubate at 25°C in the dark for 2-3 days.

[0057] (3) Induction of bud differentiation: After dark culture, the leaves were transferred to a differentiation medium with a kanamycin selection pressure of 20-30 mg / L for induction of differentiation. The medium was changed every 6-8 days. The leaves were cultured at 25℃ under light conditions (16h light / 8h dark) until adventitious buds were generated.

[0058] (4) Induction of rooting in tissue culture bottles: The adventitious buds that have grown to 1-2 cm in length are separated from the leaves with sterilized tweezers and transferred to a rooting medium with kanamycin selection pressure of 15-20 mg / L. They are cultured at 25°C under light conditions (16 h light / 8 h dark) until they root. After about 4-5 weeks, when they grow to the size of normal plants, they are propagated.

[0059] (5) Transplanting: When the tissue culture seedlings have grown for about 45 days and have developed a well-developed root system, after hardening off for 3-5 days, the culture medium on the roots is washed off in the greenhouse and then transplanted.

[0060] The formulation of the culture medium used in this embodiment is shown in Table 2:

[0061] Table 2. Culture medium formulation (1L as an example)

[0062]

[0063] Example 3

[0064] Identification of PagMED20 overexpression transgenic lines

[0065] (1) Genomic PCR detection: The genome of the transgenic plant was obtained using the DNA extraction kit of Jiangsu Kangwei Century Biotechnology Co., Ltd. A specific band of 360 bp was amplified using primers JP-MED20-F / JP-GFP-R (sequences shown in SEQ ID NO.5 and SEQ ID NO.6), confirming that OE was a positive line (see Figure 2 ).

[0066] (2) Expression analysis: The expression of PagMED20 was quantitatively determined using SYBRGreen qPCRMix from Beijing Adlai Biotechnology Co., Ltd. Actin was used as an internal control. The qPCR primers for the PagMED20 gene were qPCR-MED20-F1+ qPCR-MED20-R1, and the internal control gene was Actin. The primers were Actin-F+Actin-R. The primer sequences are shown in Table 3.

[0067] Table 3 Primer sequences used for real-time quantitative PCR

[0068]

[0069] qRT-PCR analysis showed that PagMED20 was expressed at the highest level in leaves of different tissues of wild-type plants (WT) (see [link]). Figure 3 The expression level of OE in transgenic lines was significantly increased compared to wild-type (see [link]). Figure 4 ).

[0070] (3) Phenotypic analysis: Wild-type (WT) and overexpression (OE) lines with uniform growth after rooting were transplanted into soil. Phenotypic observation and statistical analysis after 60 days of soil cultivation revealed that the OE lines exhibited significant growth advantages: OE plant height and stem diameter were significantly higher than WT, with plant height increasing by 20.63% (see [link to relevant documentation]). Figures 5-6 The stem diameter increased by 18.96% (see...). Figure 7 ).

[0071] (4) Using the LI-6800 portable photosynthesis measurement system, from 9:00 AM to 11:00 AM, under constant light intensity (500 µmol photons m -2 s -1 Under conditions of CO2 concentration (400 ppm) and temperature (25°C), the net photosynthetic rate of fully expanded leaves in the middle of the plant was measured: the net photosynthetic rate of the OE line was significantly higher than that of the WT line, with an average increase of 95.51% compared to the WT line (see [reference]). Figure 8 ).

[0072] Non-destructive measurements were performed using a portable SPAD-502 Plus chlorophyll meter (Konica Minolta, Japan). Plants of uniform growth were selected from each treatment group. Measurements were taken in the central region of each leaf, avoiding the midrib. Three measurements were taken on each leaf, and the SPAD values ​​were recorded. The chlorophyll and nitrogen contents of the OE (Oil Expiration) lines were significantly higher than those of the WT (Whole Threcid) lines. The chlorophyll content of the OE lines increased by an average of 101.24% compared to the WT lines, and the nitrogen content increased by an average of 55.76% compared to the WT lines (see [link to relevant documentation]). Figure 9 , 10 ).

[0073] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0074] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of the PagMED20 gene, PagMED20 protein, expression cassette containing the PagMED20 gene, recombinant vector containing the expression cassette, recombinant microorganism containing the expression cassette or the recombinant vector, or non-renewable poplar part containing the expression cassette, the recombinant vector, or the recombinant microorganism in promoting poplar growth. The nucleotide sequence of the PagMED20 gene is shown in SEQ ID NO.3; The amino acid sequence of the PagMED20 protein is shown in SEQ ID NO. 4; The promotion of poplar growth refers to increasing the poplar's height, trunk diameter, net photosynthetic rate, root chlorophyll content, and / or root nitrogen content.

2. A method for promoting poplar tree growth, characterized in that, The method includes the step of increasing the expression level of the PagMED20 gene in poplar, the nucleotide sequence of which is shown in SEQ ID NO.

3.

3. The method according to claim 2, characterized in that, The expression level of the PagMED20 gene was increased through transgenic methods.

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