PagMED18 gene and application thereof in promoting poplar growth

Abstract

The invention discloses a PagMED18 gene and application thereof in promotion of poplar growth, and relates to the technical field of plant genetic engineering, and the nucleotide sequence of the gene is as shown in SEQ ID NO.1. According to the invention, the PagMED18 gene is cloned and analyzed, and the PagMED18 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, the plant overexpressed with the PagMED18 gene can remarkably promote the plant height, the trunk diameter and the leaf area of the poplar, and it is proved that the PagMED18 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] The present invention relates to the technical field of plant genetic engineering technology, and more specifically to a PagMED18 gene and an application thereof in promoting the growth of poplars. Background Art

[0002] As a typical fast-growing tree species, poplar occupies an important position in ecological restoration and the timber industry. It has the characteristics of fast growth, strong adaptability and wide application. It is widely used in afforestation, urban greening and timber production. However, traditional poplar breeding methods have long cycles and low efficiency, which cannot meet the growing demand for timber. With the development of molecular biology and genetic engineering technology, improving the growth traits of poplar through genetic engineering has become an effective breeding strategy. However, the number of functional genes related to secondary growth of poplar 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 poplar.

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

[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 a gene with a nucleotide sequence as shown in SEQ ID NO.1;

[0010] B: a 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-renewable 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 gene, or the above protein, or the above 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] It can be seen from the above technical scheme that compared with the prior art, the present invention verifies the function of the gene by cloning and analyzing the PagMED18 gene and overexpressing the PagMED18 gene in 84K poplars in combination with Agrobacterium-mediated genetic transformation technology. Compared with the wild type, plants overexpressing the PagMED18 gene can significantly promote the plant height, basal diameter and leaf area of ​​poplars. It proves the important role of the PagMED18 gene in promoting the 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 varieties of forest trees. 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying 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 trunk 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] In order to facilitate the understanding of the present invention, the present invention will be described more fully with reference to the embodiments below, and preferred embodiments of the present invention are given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should be understood that the experimental methods in the following embodiments that do not specify specific conditions are usually carried out according to conventional conditions, such as the conditions described in Sambrook et al. Molecular Cloning: 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 embodiments are all commercially available products.

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

[0028] The expression cassette of the present invention refers to a 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: The 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 according to the CDS sequence of the PagMED18 gene, and BamHI / KpnI restriction sites were introduced. 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: , SEQ ID NO.1.

[0034] Amino acid sequence: MECVVQGIIETQHVEALEILLQGLCGVHKEHLRVHELFLKSGPNLGHVTSEVRLLCNLEHPEPWTVKHVGGALRGAGAEQISVLVRNMVESKASKNVLRLFYALGYK LDHELLRVGSAFHFKRGAWITVTVSSINKMLKLHAIDDTVPVTLGIQVVEVTAPATSENYSEVAAAVSSFCEYLAPLLHLSKPGVSTGVVPTAAAAAASLMSDGGGTTL, SEQ ID NO.2.

[0035] 3. Vector assembly: The pCAMBIA2300-35S-GFP vector was linearized by double restriction digestion with BamHI / KpnI, and the 648 bp PagMED18 fragment was gel purified. Then, the recombinant vector pCAMBIA2300-35S:PagMED18-GFP was constructed by seamless cloning using NEB's Gibson Assembly Master Mix.

[0036] 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 sequencing confirmed that the plasmid was correct, it was extracted and transformed into GV3101 Agrobacterium, and colony PCR was performed again to confirm that the transformation of GV3101 Agrobacterium was successful.

[0037] Example 2

[0038] Establishment of genetic transformation system

[0039] 1. Explant preparation: Take young leaves of sterile tissue culture seedlings, scratch them on both sides, and culture them in pre-culture medium (pH 5.8, 16h light / 8h dark) for 2 days.

[0040] 2. Agrobacterium infection: Add 500 μL-1 mL of GV3101 Agrobacterium containing the correctly edited PagMED18 vector to 100 mL of LB liquid culture medium containing antibiotics (50 mg / L kanamycin, 50 mg / L rifampicin) and culture overnight at 28°C and 200 rpm / min. 600When the concentration reaches 0.6-1.0, the pre-cultured leaves are treated with immersion for 15-20 minutes. During this period, the conical flask is evenly shaken for 2-3 minutes. The leaves are then clipped out and placed on sterilized filter paper. After absorbing the excess bacterial liquid on the surface of the leaves, the leaves are cultured in the dark for 3 days to induce transformation.

[0041] 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 h light / 8 h dark), subculture every 7 days until adventitious buds are formed.

[0042] 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 them at 5°C under light conditions (16h light / 8h dark) until roots form. After 4 weeks, obtain complete plants for propagation.

[0043] 5. Transplantation: When the tissue culture seedlings have grown for about 45 days and have developed root systems, they can be transplanted after 3-5 days of hardening in the greenhouse and washing away the root culture medium.

[0044] The formula of the culture medium used in this example is shown in Table 1:

[0045] Table 1 Culture medium formula (taking 1L as an example)

[0046] Example 3

[0047] Identification of transgenic lines overexpressing PagMED18

[0048] 1. Genome PCR detection: The DNA extraction kit of Jiangsu Kangwei Century Biotechnology Co., Ltd. was used to obtain the genome of the transgenic plant. The JP-MED18-F / JP-GFP-R primers were used to amplify a 360bp specific band to confirm that OE was a positive strain (see Appendix Figure 1 ).

[0049] 2. Expression analysis: The reaction system used SYBR Green qPCR Mix produced by Beijing Aidelai Biotechnology Co., Ltd. to quantitatively measure the expression of PagMED18. Actin was used as an internal reference. The qPCR primers of the PagMED18 gene used were qPCR-MED18-F1 + qPCR-MED18-R1, the internal reference gene was Actin, and the primers were Actin-F + Actin-R. The primer sequences are shown in Table 2.

[0050] Table 2 Primer sequences used for real-time quantitative PCR

[0051] qRT-PCR assay 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 ).

[0052] 3. Phenotypic analysis: The wild type (WT) and overexpression (OE) strains with the same growth after rooting were transferred to soil for growth. Phenotypic observation and statistical determination after 60 days of soil culture revealed that the OE strain showed a significant growth advantage: the plant height, stem diameter and leaf area of ​​the same stem internode of OE were significantly higher than those of WT, among which the plant height increased 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 increased by 46.7% (see Appendix Figure 7 -Attached Figure 8 ).

[0053] 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.

[0054] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. PagMED18 gene, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. PagMED18 protein, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A biomaterial, characterized in that: The biological material is any one of the following: A: an expression cassette capable of overexpressing a gene with a nucleotide sequence as shown in SEQ ID NO.1; B: a 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-renewable poplar part containing the expression cassette described in A, the recombinant vector described in B, or the recombinant microorganism described in C.

4. Use of the gene according to claim 1, or the protein according to claim 2, or the biomaterial according to claim 3 in promoting the growth of poplar.

5. The use according to claim 4, characterized in that: 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.

6. A breeding method for promoting poplar growth, characterized in that: The expression level of the PagMED18 gene is increased by genetic modification, and the nucleotide sequence of the PagMED18 gene is shown in SEQ ID NO.1.

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