PagMED18 gene and its application in promoting poplar growth

Abstract

The present invention discloses the PagMED18 gene and its application in promoting the growth of poplars, and relates to the field of plant genetic engineering technology. The nucleotide sequence of the gene is shown in SEQ ID NO.1. The present invention clones and analyzes the PagMED18 gene, and overexpresses the PagMED18 gene in 84K poplars by combining Agrobacterium-mediated genetic transformation technology to verify the function of the gene. Compared with the wild type, plants overexpressing the PagMED18 gene can significantly promote the plant height, basal diameter and leaf area of ​​poplars, demonstrating the important role of the PagMED18 gene in promoting plant height and secondary growth of poplars. The present invention has important practical and theoretical guiding significance for studying the growth stability of trees and cultivating new forest varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering technology, and more particularly to a PagMED18 gene and its application in promoting the growth of poplars. Background Art

[0002] Poplars, as a typical fast-growing tree species, play an important role in ecological restoration and the timber industry. Characterized by rapid growth, strong adaptability, and a wide range of uses, they are widely used in afforestation, urban greening, and timber production. However, traditional poplar breeding methods are long and inefficient, making it difficult to meet the growing demand for timber. With the advancement of molecular biology and genetic engineering technologies, improving poplar growth traits through genetic engineering has become an effective breeding strategy. However, the number of functional genes associated with secondary growth in poplars is currently limited.

[0003] Therefore, discovering more growth-promoting functional genes is an urgent problem that technicians in this field need to solve. Summary of the Invention

[0004] In view of this, the present invention provides the PagMED18 gene and its application in promoting the growth of poplars.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] PagMED18 gene, the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Another object of the present invention is to provide PagMED18 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] Another object of the present invention is to provide a biomaterial, wherein the biomaterial is any one of the following:

[0009] A: an expression cassette capable of overexpressing the gene with the nucleotide sequence shown in SEQ ID NO.1;

[0010] B: recombinant vector containing the expression cassette described in A;

[0011] C: a recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B;

[0012] D: A non-regenerable poplar part containing the expression cassette described in A, the recombinant vector described in B, or the recombinant microorganism described in C.

[0013] Another object of the present invention is to provide the use of the above-mentioned gene, or the above-mentioned protein, or the above-mentioned biological material in promoting the growth of poplars.

[0014] Preferably, the application is to increase the height of poplar trees and / or increase the trunk diameter of poplar trees and / or increase the leaf area of ​​poplar trees.

[0015] Another object of the present invention is to provide a breeding method for promoting poplar growth, using transgenic means to increase the expression level of the PagMED18 gene, the nucleotide sequence of the PagMED18 gene is shown in SEQ ID NO.1.

[0016] As can be seen from the above technical solutions, compared with the existing technology, the present invention verified the function of the PagMED18 gene by cloning and analyzing it, and combining it with Agrobacterium-mediated genetic transformation to overexpress the PagMED18 gene in 84K poplar trees. Compared with the wild type, plants overexpressing the PagMED18 gene significantly increased plant height, basal diameter, and leaf area. This demonstrates the important role of the PagMED18 gene in promoting plant height and secondary growth in poplar trees. This invention has important practical and theoretical guiding significance for studying tree growth stability and cultivating new forest varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0018] Figure 1 This is a nucleic acid gel image of transgenic plants after PCR verification.

[0019] Figure 2 The expression of PagMED18 gene in different tissues.

[0020] Figure 3 The expression of PagMED18 gene in wild type and transgenic plants.

[0021] Figure 4 The phenotypes of wild type and transgenic plants.

[0022] Figure 5 Quantitative results of plant height of wild type and transgenic plants.

[0023] Figure 6 Quantitative results of the basal diameter of wild-type and transgenic plants.

[0024] Figure 7 Quantitative results of leaf area of ​​wild-type and transgenic plants.

[0025] Figure 8Leaf area phenotypes of wild type and transgenic plants. DETAILED DESCRIPTION

[0026] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the examples, and preferred embodiments of the present invention are provided below. However, the present invention can be implemented in many different forms and is not limited to the examples described herein. The purpose of providing these examples is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. It should be understood that the experimental methods in the following examples, for which specific conditions are not specified, are generally performed according to conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The various commonly used reagents used in the examples are all commercially available products.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] The expression cassette of the present invention refers to DNA capable of expressing the PagMED18 gene in a host cell, and the DNA may include not only a promoter for initiating transcription of the coding gene, but also a terminator for terminating transcription of the coding gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0029] Example 1

[0030] PagMED18 gene cloning and recombinant vector construction

[0031] 1. RNA extraction and reverse transcription: Wild-type 84K poplar total RNA was isolated using the RNA extraction kit from Beijing Adlai Biotechnology Co., Ltd., and the template was prepared using the cDNA synthesis kit EasyScript® First-Strand cDNA Synthetic SuperMix from Beijing Quanshijin Biotechnology Co., Ltd.

[0032] 2. Gene Amplification System: Specific primers were designed based on the PagMED18 gene CDS sequence, introducing BamHI / KpnI restriction sites. Forward primer: 5'-TTTGGAGAGGACAGGGTACCATGGAGTGTGTGGTACAGGGA-3', SEQ ID NO. 3; reverse primer: 5'-CAGAAAATTTCTAGAGGATCCCAAGGTTGTGCCTCCACCATC-3', SEQ ID NO. 4. PCR amplification was performed using a high-fidelity enzyme.

[0033] PagMED18 gene CDS sequence:

[0034] , SEQ ID NO.1.

[0035] Amino acid sequence: MECVVQGIIETQHVEALEILLQGLCGVHKEHLRVHELFLKSGPNLGHVTSEVRLLCNLEHPEPWTVKHVGGALRGAGAEQISVLVRNMVESKASKNVLRLFYALGYK LDHELLRVGSAFHFKRGAWITVTVSSINKMLKLHAIDDTVPVTLGIQVVEVTAPATSENYSEVAAAVSSFCEYLAPLLHLSKPGVSTGVVPTAAAAAASLMSDGGGTTL, SEQ ID NO.2.

[0036] 3. Vector assembly: The pCAMBIA2300-35S-GFP vector was linearized by double enzyme digestion with BamHI / KpnI, and the 648 bp PagMED18 fragment was gel-purified. Then, seamless cloning was performed using NEB's Gibson Assembly Master Mix to construct the recombinant vector pCAMBIA2300-35S:PagMED18-GFP.

[0037] 4. Transformation Verification: After the recombinant vector was transformed into Escherichia coli TOP10 competent cells, positive clones were screened by colony PCR (primers JP-MED18-F: 5'-ATCTTGCACCGCTTCTGCACTT-3', SEQ ID NO. 5; JP-GFP-R: 5'-TTCCCGTCGTCCTTGAAAGAGATGG-3', SEQ ID NO. 6). After confirmation of correctness by sequencing, the plasmid was extracted and transformed into GV3101 Agrobacterium tumefaciens. Colony PCR was performed again to confirm the successful transformation of GV3101 Agrobacterium tumefaciens.

[0038] Example 2

[0039] Establishment of genetic transformation system

[0040] 1. Explant preparation: Take young leaves of sterile tissue culture seedlings, scratch them on both sides, and then culture them in pre-culture medium (pH 5.8, 16h light / 8h dark) for 2 days.

[0041] 2. Agrobacterium infection: 500 μL-1 mL of GV3101 Agrobacterium containing the correctly edited PagMED18 vector was added to 100 mL of LB liquid medium containing antibiotics (50 mg / L kanamycin, 50 mg / L rifampicin) and cultured overnight at 28°C and 200 rpm / min. 600When the concentration reaches 0.6-1.0, the pre-cultured leaves were immersed in the infection for 15-20 minutes. During this period, the conical flask was evenly shaken for 2-3 minutes. Then the leaves were clipped out and placed on sterilized filter paper. After absorbing the excess bacterial liquid on the surface of the leaves, the leaves were cultured in the dark for 3 days to induce transformation.

[0042] 3. Bud differentiation: Transfer the dark-cultured leaves to differentiation medium containing 20-30 mg / L kanamycin and culture at 25°C under light conditions (16 hours of light / 8 hours of darkness), subculture every 7 days until adventitious buds are formed.

[0043] 4. Rooting culture: Cut buds of about 1 cm and transfer them to rooting medium with kanamycin selection pressure of 15-20 mg / L. Culture at 5°C under light conditions (light 16h / dark 8h) until roots form. After 4 weeks, obtain complete plants for propagation.

[0044] 5. Transplantation: When the tissue culture seedlings have grown for about 45 days and the root system is well developed, they can be transplanted after 3-5 days of hardening in the greenhouse and washing away the root culture medium.

[0045] The formula of the culture medium used in this example is shown in Table 1:

[0046] Table 1 Culture medium formula (taking 1L as an example)

[0047]

[0048] Example 3

[0049] Identification of transgenic lines overexpressing PagMED18

[0050] 1. Genomic PCR detection: The transgenic plant genome was obtained using the DNA extraction kit of Jiangsu Kangwei Century Biotechnology Co., Ltd., and a 360bp specific band was amplified using the JP-MED18-F / JP-GFP-R primers to confirm that the OE was a positive strain (see Appendix Figure 1 ).

[0051] 2. Expression Analysis: The expression of PagMED18 was quantitatively determined using SYBR Green qPCR Mix (Beijing Adlai Biotechnology Co., Ltd.). Actin was used as an internal reference. The qPCR primers for the PagMED18 gene used were qPCR-MED18-F1 and qPCR-MED18-R1. The internal reference gene was Actin, and the primers were Actin-F and Actin-R. The primer sequences are shown in Table 2.

[0052] Table 2 Primer sequences used for real-time fluorescence quantitative PCR

[0053]

[0054] qRT-PCR analysis showed that the expression level of PagMED18 was highest in the stem of wild-type plants (see Appendix Figure 2 ), the OE expression level of the transgenic line was significantly higher than that of the wild type (see Appendix Figure 3 ).

[0055] 3. Phenotypic Analysis: The wild-type (WT) and overexpression (OE) lines, which showed similar growth after rooting, were transplanted to soil for growth. Phenotypic observation and statistical analysis after 60 days of soil culture revealed that the OE line exhibited a significant growth advantage: plant height, stem diameter, and leaf area within the same stem internode were significantly higher in the OE line than in the WT line, with plant height increasing by 58.6% (see Appendix). Figure 4 -Attached Figure 5 ), the stem diameter increased by 46.5% (see Appendix Figure 6 ), the leaf area of ​​the same internode expanded by 46.7% (see Appendix Figure 7 -Attached Figure 8 ).

[0056] The various embodiments in this 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 referenced to each other.

[0057] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one 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 present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. PagMED18 The use of a gene, or PagMED18 protein, or a biomaterial in promoting the growth of poplars is characterized in that: The promoting of poplar growth is to increase the plant height and / or the stem diameter and / or the leaf area of ​​the poplar; the poplar is 84K poplar; described PagMED18 The nucleotide sequence of the gene is shown in SEQ ID NO. 1; The amino acid sequence of the PagMED18 protein is shown in SEQ ID NO.2; The biological material is any one of the following: A: an expression cassette capable of overexpressing the gene with the nucleotide sequence shown in SEQ ID NO.1; B: recombinant vector containing the expression cassette described in A; C: a recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B; D: A non-regenerable poplar part containing the expression cassette described in A, the recombinant vector described in B, or the recombinant microorganism described in C.

2. A breeding method for promoting poplar growth, characterized in that: Using genetic modification to improve PagMED18 The expression level of the gene PagMED18 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the promoting poplar growth is to increase the plant height and / or the stem diameter and / or the leaf area of ​​the poplar; the poplar is 84K poplar.

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