PagMED20 gene and application thereof in promoting poplar growth

Abstract

The invention discloses a PagMED20 gene and application of the PagMED20 gene in promotion of poplar growth, and belongs to the technical field of genetic engineering and forest genetic improvement crossing. According to the invention, the PagMED20 gene is cloned and analyzed, and the PagMED20 gene is over-expressed in the 84K poplar in combination with an agrobacterium tumefaciens-mediated genetic transformation technology, so that the function of the gene is verified. Compared with a wild type, an overexpressed PagMED20 gene plant can remarkably promote the plant height, the trunk diameter, the net photosynthetic rate, the root chlorophyll content and / or the root nitrogen content of the poplar, and it is proved that the PagMED20 gene plays an important role in promoting the plant height and secondary growth of the poplar. The method has important practical and theoretical guiding significance for researching the growth stability of trees and cultivating new forest species.

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 related researches on the mediator subunit in some species or hybrids of Populus have been reported in the literature, there is still lack of explicit and complete teaching and technical inspiration for the specific genes described in the present application and their specific application in directly and efficiently synergistically promoting the growth of plant height and base diameter of Populus. In particular, how to effectively apply the genes to Populus breeding practice to solve the urgent need of rapid improvement of wood yield in the industry is still a technical gap that has not been fully disclosed and realized in the prior art.

[0006] Therefore, cloning and identifying the mediator genes that play a key positive regulatory role in the growth of Populus, elucidating their functions, and developing efficient breeding methods based on the genes have important theoretical value and practical significance for breaking through the bottleneck of traditional breeding of Populus and realizing the directional genetic improvement of growth traits. SUMMARY

[0007] Therefore, cloning and identifying the mediator genes that play a key positive regulatory role in the growth of Populus, elucidating their functions, and developing efficient breeding methods based on the genes have important theoretical value and practical significance for breaking through the bottleneck of traditional breeding of Populus and realizing the directional genetic improvement of growth traits.

[0008] To solve the above technical problems, the present application adopts the following technical solutions:

[0009] The first object of the present application is to provide an isolated PagMED20 gene, wherein the nucleotide sequence of the PagMED20 gene is shown as SEQ ID NO. 3.

[0010] Another object of the present application is to provide a PagMED20 protein encoded by the PagMED20 gene, wherein the amino acid sequence of the PagMED20 protein is shown as SEQ ID NO. 4.

[0011] Another object of the present application is to provide an expression cassette comprising the PagMED20 gene, and the expression cassette can drive overexpression of the PagMED20 gene in a host cell.

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

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

[0014] Another object of the present application is to provide a non-renewable part of Populus comprising the expression cassette, the recombinant vector or the recombinant microorganism.

[0015] Still another object of the present 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 part of the poplar in promoting the growth of the poplar.

[0016] As a preferred technical solution, the growth of the poplar is improved in terms of the plant height, the base diameter, the net photosynthetic rate, the root chlorophyll content and / or the root nitrogen content.

[0017] Still another object of the present application is to provide a method for promoting the growth of the poplar, which comprises the step of increasing the expression amount of the PagMED20 gene in the poplar, and the nucleotide sequence of the PagMED20 gene is shown in SEQ ID NO. 3.

[0018] As a preferred technical solution, the increase of the expression amount of the PagMED20 gene is realized by a transgenic means.

[0019] According to the above technical solution, compared with the prior art, the present application has the following beneficial effects: The present application achieves significant and multi-level technical effects by increasing the expression of the PagMED20 gene in the poplar, and the core summary is as follows: 1. Direct growth phenotype improvement Synergistic effect: the plant height, the base diameter, the net photosynthetic rate, the root chlorophyll content and / or the root nitrogen content of the transgenic poplar can be simultaneously and significantly improved, which breaks through the bottleneck that the growth traits need to be improved respectively.

[0020] Fast growth and yield increase: the growth rate of the poplar is directly accelerated, which provides a key solution for realizing the shortening of the rotation period and the improvement of the wood yield.

[0021] 2. Outstanding breeding application value Path innovation: a precise and efficient molecular breeding means is provided, which overcomes the limitations of long breeding cycle and low efficiency of traditional forest tree breeding.

[0022] Complete solution: the complete technical solution which is operable and repeatable is formed by the expression cassette, the vector and the transformation method, and is convenient for popularization.

[0023] Great potential: it lays a foundation for further designing the synergistic improvement of complex traits such as growth rate, wood quality and stress resistance in the future.

[0024] In summary, the present application provides a key gene and a set of efficient technical solutions which are verified by functions for the fast-growing genetic improvement of the poplar, and has a clear industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only aim at the embodiments of the present application, and for those skilled in the art, other drawings can be obtained without any creative effort based on the provided drawings.

[0026] Figure 1 Figure 4 is a vector map of pCAMBIA2300-35S-GFP.

[0027] Figure 2 Figure 6 is a nucleic acid gel map after PCR verification of the transgenic plants.

[0028] Figure 3 Figure 8 is the expression of PagMED20 gene in different tissues.

[0029] Figure 4 Figure 10 is the expression of PagMED20 gene in wild type and transgenic plants.

[0030] Figure 5 Figure 12 is the plant height phenotype results of wild type and transgenic plants.

[0031] Figure 6 Figure 14 is the plant height quantitative results of wild type and transgenic plants.

[0032] Figure 7 Figure 16 is the basal diameter quantitative results of wild type and transgenic plants.

[0033] Figure 8 Figure 18 is the net photosynthetic rate quantitative results of wild type and transgenic plants.

[0034] Figure 9 Figure 20 is the chlorophyll content quantitative results of wild type and transgenic plants.

[0035] Figure 10 Figure 22 is the nitrogen content quantitative results of wild type and transgenic plants. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be described clearly and completely with reference to the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without any creative effort belong to the scope of protection of the present application.

[0037] Embodiment 1 Cloning of PagMED20 gene and construction of recombinant vector (1) RNA extraction and reverse transcription: The total RNA of wild type 84K poplar was separated by RNA extraction kit of Beijing Aidley Biotechnology Co., Ltd., and the template was prepared by using EasyScript® First-Strand cDNA Synthetic SuperMix cDNA synthesis kit of Beijing Quansi Gold Biotechnology Co., Ltd.

[0038] (2) Gene amplification system: According to the sequence of PagMED20 gene CDS region, specific primers were designed, BamHI / KpnI enzyme cutting sites were introduced, and high-fidelity enzyme was used for PCR amplification. The primers and sequences used for gene amplification are shown in Table 1.

[0039] Table 1 Primers and sequences used for gene amplification

[0040] PagMED20 gene CDS sequence: ATGCCACTGAAATGGGTTTTGCATTGGCAACCCAACGCAGGAACAACCGTGAACACCCAAATCCTCAATGAAGTAACTCAGTGCGTGGAGAGCATTAACGGCGTTAAAGAAGGCCGATGGAAAGCTACTCTCACCTACTATAAACCCATACTACGAGACCAATCGCAAGCTCCTGAATTGCCGCGAGATTTCTTGGGTATTTCTTTGCCAGAGCAGCCAAATAAGTACTACTTCATAATCCGAGGTCAAAGGATTGTTCTCGAGGCAGATTCCTCCATTCAAACAATAATGGAGAAGCTTCAGTCTTATAAGTCGAGGGTTGCTCTCTACTTTGAGGGTTTTCAGTATCAACTCGGGGACTTTCAGTTGCGAGTTGGGAAAGTTACCCCAACTCATTCGGACAACTTGAGAGGAATTATTATGGAGGTGGAGTATCTTCCTTTGTCATCAATGGACAAATCCAGGCAAGTCATGGAAGAGTTTGTTGATATATGGCAAGAAGCCATCTCAAAAAGATCATTACCAGGCCATTTCATGCATATGGAACCAAACTTTGTGGAGTTCGGTCTCTCGGATCACTATAGCTCGCAACACACAGCTGTGCAGTATGCTACTGTCATGGCTCAACTAATTGCGACTCAGTCAGTGCAAGCCGCGAGAAAC, SEQ ID NO. 3.

[0041] PagMED20 protein amino acid sequence: MPLKWVLHWQPNAGTTVNTQILNEVTQCVESINGVKEGRWKATLTYYKPILRDQSQAPELPRDFLGISLPEQPNKYYFIIRGQRIVLEADSSIQTIMEKLQSYKSRVALYFEGFQYQLGDFQLRVGKVTPTHSDNLRGIIMEVEYLPLSSMDKSRQVMEEFVDIWQEAISKRSLPGHFMHMEPNFVEFGLSDHYSSQHTAVQYATVMAQLIATQSVQAARN, SEQ ID NO. 4.

[0042] (3) Vector assembly: pCAMBIA2300-35S-GFP vector (see Figure for map) Figure 1 ) was linearized by double digestion with BamHI / KpnI, and then the 663 bp PagMED20 fragment was gel purified. The recombined vector pCAMBIA2300-35S:PagMED20-GFP was constructed using Gibson Assembly Master Mix (purchased from NEB) for seamless cloning.

[0043] (4) Transformation verification: The reaction product in step (3) was added to the competent cells of E. coli TOP 10 (purchased from Beijing Aidley Biotechnology Co., Ltd., and the specific operation method and dosage refer to the instruction manual) for transformation, and then uniformly coated on LB plates containing kanamycin and cultured at 37°C overnight.

[0044] (5) Positive clone identification: The 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), and the plasmid was extracted after sequencing to confirm the correctness and then transformed into GV3101 Agrobacterium (purchased from Weidi Biotechnology Co., Ltd.).

[0045] Example 2 Establishment of genetic transformation system (1) Preparation of explants: The young leaves of sterile tissue culture seedlings were cut on both sides, and then cultured in a pre-culture medium (pH 5.8, light for 16 h / dark for 8 h) for 2 days.

[0046] (2) Agrobacterium infection: 500 μL-1 mL of Agrobacterium containing the correctly edited PagMED20 vector was added to 100 mL of LB liquid medium containing antibiotics (50 mg / L kanamycin, 50 mg / L rifampicin), mixed well, and then cultured at 28°C, 200 rpm / min overnight. The next day, when the OD600 of Agrobacterium was 0.6-1.0, it was ready for use. In a sterile clean bench, the forceps were sterilized in advance by ultraviolet sterilization and opening the high-temperature sterilizer. After the forceps cooled down, the leaves on the pre-culture medium were clamped into the conical flask containing Agrobacterium, and the conical flask was shaken evenly for 2-3 min during the 15-20 min timing. After the timing was completed, the leaves were clamped out onto the filter paper sterilized by high-pressure steam, the excess bacterial solution on the surface of the leaves was absorbed, and the leaves were placed back into the original pre-culture medium and cultured at 25°C in the dark for 2-3 days.

[0047] (3) Inducing bud differentiation: the leaf after dark culture was transferred to the differentiation medium with kanamycin screening pressure of 20-30 mg / L for inducing differentiation, the medium was changed every 6-8 days, the culture was carried out at 25°C under light condition (light for 16 h / dark for 8 h) until the adventitious buds were differentiated.

[0048] (4) Rooting induction in tissue culture bottle: the adventitious buds grown to 1-2 cm in length were separated from the leaves by sterilized tweezers and transferred to the rooting medium with kanamycin screening pressure of 15-20 mg / L, the culture was carried out at 25°C under light condition (light for 16 h / dark for 8 h) until rooting, and the normal plant size was obtained after 4-5 weeks, then the propagation was carried out.

[0049] (5) Transplanting: when the tissue culture seedlings grew for about 45 days and the root system was developed, the seedlings were transplanted after washing the root medium in the greenhouse.

[0050] The formula of the medium used in this example is shown in Table 2: Table 2 Formula of the medium (for 1 L)

[0051] Example 3 Identification of PagMED20 overexpression transgenic lines (1) Genomic PCR detection: the transgenic plant genome was obtained by using the DNA extraction kit of Jiangsu Kangweishijishe Biotechnology Co., Ltd., and a 360 bp specific band was amplified by using JP-MED20-F / JP-GFP-R (the sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 6) primers, and the OE was confirmed as a positive line (see Figure 2 ).

[0052] (2) Expression analysis: the SYBR Green qPCR Mix of Beijing Aidley Biotechnology Co., Ltd. was used for reaction system to quantitatively determine the expression of PagMED20, and Actin was used as an internal reference, the qPCR primers of PagMED20 gene were qPCR-MED20-F1+qPCR-MED20-R1, the internal reference gene was Actin, and the primers were Actin-F+Actin-R, and the primer sequences are shown in Table 3.

[0053] Table 3 Primer sequences used for fluorescent real-time quantitative PCR

[0054] The qRT-PCR detection showed that the expression amount of PagMED20 in different tissues of wild type plants (WT) was the highest in leaves (see Figure 3), the transgenic line OE expression was significantly higher than wild type (see Figure 4 ).

[0055] (3) Phenotype analysis: Wild type (WT) and overexpression (OE) lines with consistent growth after rooting were moved to soil for growth. After 60 days of soil culture, phenotype observation and statistical determination were performed, and it was found that the OE lines showed obvious growth advantage: the height and stem diameter of OE lines were significantly higher than WT, among which the height increased by 20.63% (see Figure 5-6 ), and the stem diameter increased by 18.96% (see Figure 7 ).

[0056] (4) Using the LI-6800 portable photosynthesis measurement system, the net photosynthetic rate of the middle fully expanded leaves of the plants was measured under the conditions of constant light intensity (500 µmol photons m -2 s -1 ), CO2 concentration (400 ppm) and temperature (25°C) from 9:00 to 11:00 am: the net photosynthetic rate of the OE lines was significantly higher than that of the WT, and the average growth of the OE lines was 95.51% higher than that of the WT (see Figure 8 ).

[0057] Non-destructive determination was performed using a portable SPAD-502 Plus chlorophyll meter (Konica Minolta, Japan). Plants with consistent growth were selected from each treatment group, and the middle region of each leaf to be measured was measured, avoiding the main vein. Each leaf was measured at 3 points and the SPAD value was recorded: the chlorophyll and nitrogen content of the OE lines was significantly higher than that of the WT, and the average growth of the chlorophyll content of the OE lines was 101.24% higher than that of the WT, and the average growth of the nitrogen content was 55.76% higher than that of the WT (see Figure 9 、 10 ).

[0058] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0059] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An isolated PagMED20 gene, characterized in that, The nucleotide sequence of the PagMED20 gene is shown in SEQ ID NO.

3.

2. The PagMED20 protein encoded by the PagMED20 gene as described in claim 1, characterized in that, The amino acid sequence of the PagMED20 protein is shown in SEQ ID NO.

4.

3. An expression box, characterized in that, The cassette contains the PagMED20 gene as described in claim 1, and the expression cassette is capable of driving overexpression of the PagMED20 gene in host cells.

4. A recombinant vector, characterized in that, It includes the expression box as described in claim 3.

5. A recombinant microorganism, characterized in that, It includes the expression cassette of claim 3 or the recombinant vector of claim 4.

6. A non-renewable poplar segment, characterized in that, It comprises the expression cassette of claim 3, the recombinant vector of claim 4, or the recombinant microorganism of claim 5.

7. The application of the PagMED20 gene of claim 1, the PagMED20 protein of claim 2, the expression cassette of claim 3, the recombinant vector of claim 4, the recombinant microorganism of claim 5, or the non-renewable poplar part of claim 6 in promoting poplar growth.

8. The application according to claim 7, characterized in that, 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.

9. A method for promoting the growth of poplar trees, 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.

10. The method according to claim 9, characterized in that, The expression level of the PagMED20 gene was increased through transgenic methods.

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