Pop_A17G020368 Gene and Its Application in Promoting Plant Growth

Abstract

The present invention discloses the Pop_A17G020368 gene and its application in promoting plant growth, which relates to the technical field of plant genetic engineering. The nucleotide sequence of the gene is shown as SEQ ID NO:1. In the present invention, through the cloning and analysis of the Pop_A17G020368 gene, and by combining the Agrobacterium-mediated genetic transformation technology to overexpress the Pop_A17G020368 gene in 84K poplar, the function of this gene was verified. Compared with the wild type, the plants overexpressing the Pop_A17G020368 gene can significantly promote the growth of the plant height and stem diameter of poplar. It is proved that the Pop_A17G020368 gene plays an important role in promoting the plant height and secondary growth of poplar. The present invention 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] The present invention relates to the technical field of plant genetic engineering, and more specifically to the Pop_A17G020368 gene and its application in promoting plant growth. Background Art

[0002] Poplar is an important forest resource, which is characterized by fast growth, strong adaptability, and wide uses, and is widely used in afforestation, urban greening, and wood production. However, the traditional poplar breeding methods have a long cycle and low efficiency, and it is difficult to meet the increasing wood demand. With the development of molecular biology and genetic engineering technologies, improving the growth traits of poplar by genetic engineering means has become an effective breeding strategy. However, the number of functional genes related to poplar secondary growth is currently limited.

[0003] Therefore, it is an urgent problem for those skilled in the art to discover more growth-promoting functional genes. Summary of the Invention

[0004] In view of this, the present invention provides the Pop_A17G020368 gene and its application in promoting plant growth.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The Pop_A17G020368 gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0007] Another object of the present invention is to provide the Pop_A17G020368 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 biological material, which 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: 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 plant part containing the expression cassette described in A or the recombinant vector described in B or the recombinant microorganism described in C.

[0013] Another object of the present invention is to provide the application of the above gene, or the above protein, or the above biological material in promoting plant growth.

[0014] Preferably, the application is to increase the plant height and / or increase the diameter of the base of the plant stem.

[0015] Another object of the present invention is to provide a breeding method for promoting plant growth. By using transgenic means, the expression level of the Pop_A17G020368 gene is increased, and the nucleotide sequence of the Pop_A17G020368 gene is shown in SEQ ID NO:1.

[0016] Through the above technical solutions, compared with the prior art, the present invention clones and analyzes the Pop_A17G020368 gene, and overexpresses the Pop_A17G020368 gene in 84K poplar by combining the Agrobacterium-mediated genetic transformation technology, verifying the function of this gene. Compared with the wild type, the plants overexpressing the Pop_A17G020368 gene can significantly promote the growth of the plant height and stem diameter of poplar. It proves the important role of the Pop_A17G020368 gene in promoting the plant height and secondary growth of poplar. The present invention has important practical and theoretical guiding significance for studying the growth stability of trees and cultivating new forest tree varieties. Brief Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0018] Figure 1 It is the phenotypes of the wild type and transgenic plants.

[0019] Figure 2 It is the electrophoresis diagram for detecting positive overexpressing plants.

[0020] Figure 3 It is the expression of the Pop_A17G020368 gene in the wild type and transgenic plants.

[0021] Figure 4 It is the expression of the Pop_A17G020368 gene in different tissues of the wild type.

[0022] Figure 5 It is the quantitative result of the plant height of the wild type and transgenic plants.

[0023] Figure 6 It is the quantitative result of the diameter of the base of the plant stem of the wild type and transgenic plants. Detailed Embodiments

[0024] For the convenience of understanding the present invention, the present invention will be described more comprehensively below with reference to embodiments. The following are the preferred embodiments of the present invention. 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 without specific conditions noted in the following embodiments are generally in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer. All common reagents used in the embodiments are commercially available products.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description 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 of the related listed items.

[0026] The expression cassette of the present invention refers to DNA that can express the Pop_A17G020368 gene in a host cell. This DNA may not only include a promoter that initiates the transcription of the coding gene, but also include a terminator that terminates the transcription of the coding gene. Further, the expression cassette may also include enhancer sequences.

[0027] Example 1

[0028] Cloning of Pop_A17G020368 Gene and Vector Construction

[0029] 1. Total RNA extraction: The total RNA of wild-type Populus alba × Populus glandulosa clone 84K was extracted using an RNA extraction kit (purchased from Beijing Aidlab Biotechnologies Co., Ltd.).

[0030] 2. cDNA synthesis: Using the cDNA synthesis kit EasyScript® First-Strand cDNA Synthetic SuperMix (purchased from Beijing TransGen Biotech Co., Ltd.), the total RNA extracted in the previous step was reverse-transcribed into single-stranded cDNA by a one-step method.

[0031] 3. Gene cloning: A high-fidelity enzyme was used to amplify the gene fragment.

[0032] Referring to the CDS sequence of the Pop_A17G020368 gene, the double digestion sites are BamHI and KpnI. Based on this, the forward and reverse primers of pCAMBIA2300:35s:Pop_A17G020368-GFP were designed and entrusted to a biological company for primer synthesis, which were used for the amplification of the Pop_A17G020368 gene.

[0033] CDS sequence of Pop_A17G020368 gene:

[0034]

[0035] Amino acid sequence:

[0036] MPSQTNSHFSLPRWIGLLSHKNSQSETGDNNDNSNLNSTNNIDDSNPISNSIECYACTQVGVPVFHSTSCDQAHQPEWEASAGSSLVPIKNRLGSRKSPASRAQSRRPAGPFGTILDPRSKRVQKWNRAFLLARGMALAVDPLFFYALSIGRNGAPCLYMDGGLAAIVTVLRTSVDAIHLCHLWLQFRLAYVSRESLVVGCGKLVWDARAIASHYVRSLKGFWFDAFVILPVPQAVFWLLVPRLIREEQIKLIMTILLLIFLFQFLPKVYHCICLMKRMQKVTGYIFGTIWWGFGLNLIAYFIASHVAGGCWYVLAIQRVASCLRQSCERRPNCDLSLACSEEVCYQFLLRSGTIGNPCVGNTTHTVRKPMCLDVNGAFNYGIYKWALPVISSNSLSVKILYPIFWGLMTLSTFGNDLEPTSHWLEVIFSICIVLSGLMLFTLLIGNIQVFLHAVMAKKRKMQLRCGDMEWWMRRRQLPSRLRQRVRHYERQRWATMGGEDEIELITDLPEGLRRDIKRYLCLDLIKKVTQMNT, SEQ ID NO.2.

[0037] The primer sequences are as follows:

[0038] 2300-0368-F: 5’-CATTTGGAGAGGACAGGGTACCATGCCTTCCCAGACCAACTCC-3’, SEQ ID NO.3;

[0039] 2300-0368-R: 5’-CAGAAAATTTCTAGAGGATCCAGTGTTCATTTGTGTCACCTTC-3’, SEQ ID NO.4.

[0040] 4. Vector linearization: The vector plasmid of pCAMBIA2300-35S-GFP was double digested with restriction enzymes BamHI and KpnI to obtain a linearized vector fragment.

[0041] 5. Agarose gel electrophoresis detection, purification and recovery: Use 1% agarose gel for electrophoresis detection. The band sizes of the target gene Pop_A17G020368 and the linearized vector are about 1602 bp and 12000 bp respectively. Then use the agarose gel recovery kit (purchased from Beijing Aidlab Biotechnologies Co., Ltd.) to purify and recover the target gene fragment and the linear vector fragment.

[0042] 6. Vector construction: Insert the amplified Pop_A17G020368 gene fragment between the BamHI and KpnI sites of the pCAMBIA2300-35S-GFP vector, and use the premixed solution for seamless cloning, Gibson Assembly Master Mix (purchased from NEB) to construct the recombinant vector pCAMBIA2300:35s:Pop_A17G020368-GFP.

[0043] 7. Transformation of Escherichia coli competent cells TOP 10 (purchased from Beijing Aidlab Biotechnologies Co., Ltd., the specific operation method and dosage refer to the instruction manual): Add the reaction product in step 6 to Escherichia coli competent cells for transformation, and then evenly coat it on the LB plate containing kanamycin. Incubate overnight at 37°C in an inverted position.

[0044] 8. Identification of positive clones: Use JP-0368-F and JP-GFP-R as primers, pick monoclonal colonies for colony PCR identification. Positive clones will obtain an amplified band of 800 bp. Pick several positive clones, streak and preserve them on a new kanamycin plate, and at the same time inoculate them into the LB liquid medium containing kanamycin, and culture overnight at 37°C with shaking at 180 - 230 rpm. Send the bacterial liquid to the company for sequencing the next day. The sequencing primer sequences are as follows:

[0045] JP-0368-F: 5’-GAAAGCCTATGTGCCTGGATG-3’, SEQ ID NO.5;

[0046] P-GFP-R: 5’- TGTTGACGAGGGTGTCTCCCT -3’, SEQ ID NO.6.

[0047] 9. Plasmid extraction: Use the plasmid mini-prep kit (the kit is purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the correctly edited vector plasmid, and the steps refer to the kit instruction manual.

[0048] 10. Transform the competent Agrobacterium tumefaciens GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd., and the specific operation steps refer to the instruction manual). Finally, spread the resuspended bacterial cells on the solid LB medium supplemented with 50 mg / L kanamycin and 50 mg / L rifampicin, and culture at 28 °C for 36 - 48 h. Use primers JP-0368-F and JP-GFP-R for colony PCR detection of positive clones to obtain Agrobacterium tumefaciens containing the correct overexpression vector.

[0049] Example 2

[0050] Agrobacterium-mediated genetic transformation

[0051] 1. Introduce the overexpression vector of Pop_A17G020368 into poplar leaves through the Agrobacterium-mediated genetic transformation system. After leaf pre-culture, Agrobacterium infection and dark culture, induction of meristematic buds, induction of rooting in tissue culture bottles, and acclimatization, transplant them to the greenhouse for soil culture.

[0052] (1) Leaf pre-culture: In a sterile laminar flow hood, turn on the high-temperature sterilizer in advance to sterilize scissors, forceps, and tissue culture scalpels at high temperature. After cooling, cut the leaves of wild-type 84K poplar, and use the scalpel to vertically cut the veins and make 3 - 4 incisions in the middle of the leaves. Then place them on the pre-prepared medium and culture at 25 °C under light conditions (16 h light / 8 h dark) for 2 - 3 days.

[0053] (2) Agrobacterium infection and dark culture: 12 - 16 hours in advance, add 500 μL - 1 mL of Agrobacterium tumefaciens containing the correctly edited Pop_A17G020368 vector to 100 mL of LB liquid medium containing antibiotics (50 mg / L kanamycin, 50 mg / L rifampicin), mix well and place in a shaker at 28 °C, 200 rpm / min for overnight culture. The next day, measure the OD of Agrobacterium tumefaciens. 600 When it reaches 0.6 - 1.0, it is okay. In a sterile laminar flow hood, sterilize the forceps in advance by ultraviolet light and turn on the high-temperature sterilizer. After the forceps cool down, clamp the leaves on the pre-medium into the conical flask containing Agrobacterium tumefaciens, time for 15 - 20 min, and shake the conical flask evenly every 2 - 3 min during this period. After the time is up, clamp out the leaves onto the filter paper sterilized by high-pressure steam, suck off the excess bacterial liquid on the leaf surface, and then place the leaves on the dark medium and culture in the dark at 25 °C for 2 - 3 days.

[0054] (3) Induction of bud differentiation: Transfer the leaves after dark culture to the differentiation medium with a kanamycin selection pressure of 20 - 30 mg / L for induction of differentiation. Change the medium every 6 - 8 days and culture at 25 °C under light conditions (16 h light / 8 h dark) until adventitious buds are differentiated.

[0055] (4)Inducing rooting in tissue culture bottles: The adventitious buds that have grown to a length of 1 - 2 cm are separated from the leaves with sterilized forceps and transferred to a rooting medium with a kanamycin screening pressure of 15 - 20 mg / L. Cultivate at 25°C under light conditions (16 h of light / 8 h of darkness) until rooting. After growing to the size of a normal plant in about 4 - 5 weeks, carry out propagation.

[0056] (5)Hardening off and transplanting to the greenhouse for soil cultivation: When the tissue culture seedlings have grown for about 45 days and have well-developed roots, transplant them after hardening off for 3 - 5 days. Wash the medium from the roots of the poplar with clean water and transplant them into sterilized nutrient soil, and place them in the greenhouse for cultivation.

[0057] The formula of the medium used in this example is shown in Table 1:

[0058] Table 1

[0059]

[0060] Example 3

[0061] 1. Identification of transgenic overexpression plants

[0062] In the laminar flow hood, cut the leaves of different transgenic poplar lines that have rooted after transformation, label each line at the same time, and use a genomic DNA extraction kit (purchased from Nanjing Novoprotein Scientific Inc., and the specific operation steps refer to the instruction manual) to extract genomic DNA.

[0063] Use primers JP-0368-F and JP-GFP-R to identify positive transgenic plants by PCR cloning a fragment of 800 bp containing the fusion expression of Pop_A17G020368 and GFP. Through identification, OE-1 and OE-2 are the overexpression successful lines of the Pop_A17G020368 gene (see Appendix Figure 1 - Appendix Figure 3 ).

[0064] 2. The role of the Pop_A17G020368 gene in regulating tree height and secondary growth

[0065] (1)Detecting the expression level of Pop_A17G020368 in different tissue parts by fluorescence real-time quantitative PCR method:

[0066] Total RNA extraction of Populus alba×P. glandulosa '84K': The root, stem, and leaf parts of wild-type 84K plants were cut to detect the expression levels of target genes in different tissue parts, and the leaves of overexpression plants OE-1 / -2 of different lines and wild-type 84K tissue culture seedlings were cut to identify the expression of target genes in overexpression plants. Using the liquid nitrogen grinding method, according to the EASYspin Plus Plant RNA Rapid Extraction Kit (purchased from Beijing Aidlab Biotechnologies Co., Ltd., and the specific operation steps refer to the instruction manual), a total RNA solution was obtained.

[0067] cDNA synthesis: The total RNA of the above different lines was reverse transcribed into cDNA using a one-step cDNA synthesis kit (purchased from Beijing TransGen Biotech Co., Ltd., and the specific operation steps refer to the instruction manual).

[0068] Fluorescence real-time quantitative PCR detection: SYBR Green qPCR Mix from Beijing Aidlab Biotechnologies Co., Ltd. was used to quantitatively determine the expression of different parts of the Pop_A17G020368 gene. The reaction system and PCR program refer to the instruction manual of the kit. The PCR primers for the Pop_A17G020368 gene used were qPCR-0368-F1 and qPCR-0368-R1, and the internal reference gene was Actin, with primers Actin-F and Actin-R. The results of three biological experiment replicates showed that the tissue expression level of the Pop_A17G020368 gene in 84K was the highest in leaves, followed by stems and roots. The detection results of the expression levels in different tissue parts are shown in the appendix Figure 4 The expression levels of Pop_A17G020368 in OE-1 and OE-2 plants were both higher than those of the wild type. The detection results of the transcriptional expression levels of transgenic poplars are shown in the appendix Figure 3 。

[0069] qPCR-0368-F1: 5’-GAGACAGAGAGTTCGCCATT-3’, SEQ ID NO.7;

[0070] qPCR-0368-R1: 5’-GATAAGATCTAGGCAAAGAT-3’, SEQ ID NO.8.

[0071] Actin-F: 5’-AAACTGTAATGGTCCTCCCTCCG-3’, SEQ ID NO.9;

[0072] Actin-R: 5’-GCATCATCACAATCACTCTCCGA-3’, SEQ ID NO.10.

[0073] (2) Phenotypic observation of transgenic plants: The wild-type (WT) and overexpression (OE-1 / -2) lines with consistent growth after rooting were transplanted into the soil for growth. Phenotypic observation and statistical measurement showed that the plant height of the overexpression lines was significantly higher than that of the wild-type at the same growth stage (see Appendix Figure 5 ); The stem base diameters of the wild-type and OE-1 / -2 plants were statistically measured, and it was found that the stem base diameters of the OE-1 / -2 plants were significantly larger than those of the wild-type (see Appendix Figure 6 ).

[0074] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the various embodiments can be referred to each other.

[0075] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can 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 these embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Pop_A17G020368 A gene characterized by The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. Pop_A17G020368 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 comprising an expression vector pCAMBIA2300-35S-GFP into which a gene with a nucleotide sequence as shown in SEQ ID NO.1 is inserted; 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-regenerable plant 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 diameter of the base of poplar stems.

6. A breeding method for promoting poplar growth, characterized in that: Using genetic modification, Pop_A17G020368 The gene was inserted into the expression vector pCAMBIA2300-35S-GFP to improve Pop_A17G020368 The expression level of the gene Pop_A17G020368 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

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