PopA17G020368 gene and application thereof in promoting plant growth

Abstract

The invention discloses a PopA17G020368 gene and application of the PopA17G020368 gene in promoting plant 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 PopA17G020368 gene is cloned and analyzed, and the PopA17G020368 gene is over-expressed in the 84K poplar in combination with an agrobacterium tumefaciens-mediated genetic transformation technology, so that the function of the PopA17G020368 gene is verified. Compared with a wild type, the plant overexpressed with the PopA17G020368 gene can obviously promote the growth of plant height and stem diameter of the poplar. It is proved that the PopA17G020368 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, and more specifically to Pop_A17G020368 gene and its application in promoting plant growth. Background Art

[0002] Poplar is an important forest resource with 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 Pop_A17G020368 gene and its application in promoting plant growth.

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

[0006] 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 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 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-regenerable plant 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 plant growth.

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

[0015] Another object of the present invention is to provide a breeding method for promoting plant growth, which utilizes transgenic means to increase the expression level of the Pop_A17G020368 gene, wherein the nucleotide sequence of the Pop_A17G020368 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 overexpresses the Pop_A17G020368 gene in 84K poplar by cloning and analyzing the Pop_A17G020368 gene and combining the Agrobacterium-mediated genetic transformation technology to verify the function of the 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 the poplar. It proves the important role of the Pop_A17G020368 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 The phenotypes of wild type and transgenic plants.

[0019] Figure 2 This is the electrophoresis diagram of positive overexpression plant detection.

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

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

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

[0023] Figure 6 Quantitative results of stem base diameter of wild-type and transgenic plants. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] The expression cassette of the present invention refers to a DNA capable of expressing the Pop_A17G020368 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. Further, the expression cassette may also include an enhancer sequence.

[0027] Example 1

[0028] Cloning and vector construction of Pop_A17G020368 gene

[0029] 1. Total RNA extraction: Use RNA extraction kit (purchased from Beijing Adlai Biotechnology Co., Ltd.) to extract total RNA from wild-type silver gland poplar asexual line 84K poplar.

[0030] 2. cDNA synthesis: The total RNA extracted in the previous step was reverse transcribed into single-stranded cDNA in a one-step method using the cDNA synthesis kit EasyScript® First-Strand cDNA SyntheticSuperMix (purchased from Beijing Quanshijin Biotechnology Co., Ltd.).

[0031] 3. Gene cloning: Use high-fidelity enzymes to amplify gene fragments.

[0032] Referring to the CDS sequence of Pop_A17G020368 gene, the double restriction sites are BamHI and KpnI, and the forward and reverse primers of pCAMBIA2300:35s:Pop_A17G020368-GFP are designed. The primers are synthesized by a biological company for the amplification of Pop_A17G020368 gene.

[0033] Pop_A17G020368 gene CDS sequence:

[0034]

[0035] Amino Acid Sequence:

[0036] , SEQ ID NO.2.

[0037] The primer sequences are as follows:

[0038] 2300-0368-F:5'-CATTTGGAGAGGACAGGGTACCATGCCTTCCAGACCAACTCC-3', SEQ IDNO.3;

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

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

[0041] 5. Agarose gel electrophoresis detection and purification and recovery: 1% agarose gel was used for electrophoresis detection. The band size of the target gene Pop_A17G020368 and the band size of the linearized vector were detected to be approximately 1602 bp and 12000 bp, respectively. The target gene fragment and the linear vector fragment were purified and recovered using an agarose gel recovery kit (purchased from Beijing Aidelai Biotechnology Co., Ltd.).

[0042] 6. Vector construction: The amplified Pop_A17G020368 gene fragment was inserted between the BamHI and KpnI sites of the pCAMBIA2300-35S-GFP vector, and the recombinant vector pCAMBIA2300:35s:Pop_A17G020368-GFP was constructed using the seamless cloning premix Gibson Assembly Master Mix (purchased from NEB).

[0043] 7. Transformation of E. coli competent cells TOP 10 (purchased from Beijing Adlai Biotechnology Co., Ltd., refer to the instructions for specific operation methods and dosage): Add the reaction product in step 6 to the E. coli competent cells for transformation, and then evenly spread on the LB plate containing kanamycin. Invert and culture at 37℃ overnight.

[0044] 8. Identification of positive clones: Use JP-0368-F and JP-GFP-R as primers, pick single clones for colony PCR identification, and obtain 800bp amplification bands for positive clones. Pick several positive clones, streak them on new kanamycin plates, and inoculate them into LB liquid culture medium containing kanamycin, and culture them overnight at 37°C and 180-230rpm. The next day, send the bacterial solution to the company for sequencing. The sequencing primer sequences are as follows:

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

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

[0047] 9. Plasmid extraction: Use a plasmid extraction kit (the kit was purchased from Tiangen Biochemical Technology Beijing Co., Ltd.) to extract the correctly edited vector plasmid. Refer to the kit instructions for steps.

[0048] 10. Transform Agrobacterium competent cells GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd., refer to the instructions for specific operation steps), and finally apply the resuspended bacteria to solid LB medium supplemented with 50 mg / L kanamycin and 50 mg / L rifampicin, and culture at 28℃ for 36-48 hours. Use primers JP-0368-F and JP-GFP-R to detect positive clones by bacterial liquid PCR to obtain Agrobacterium containing the correct overexpression vector.

[0049] Example 2

[0050] Agrobacterium-mediated genetic transformation

[0051] 1. The overexpression vector of Pop_A17G020368 was introduced into poplar leaves through the Agrobacterium-mediated genetic transformation system. After leaf pre-culture, Agrobacterium infection and dark culture, induced budding, rooting in tissue culture bottles, and seedling hardening, the seedlings were transplanted to the greenhouse for soil culture.

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

[0053] (2) Agrobacterium infection and dark culture: 12-16 hours in advance, add 500μL-1mL of Agrobacterium containing the correctly edited Pop_A17G020368 vector to 100mL of LB liquid culture medium containing antibiotics (50 mg / L kanamycin, 50 mg / L rifampicin), mix well, and culture overnight at 28℃ and 200 rpm / min. The next day, measure the OD of Agrobacterium. 600 When the value is 0.6-1.0, it is OK. In a sterile clean bench, sterilize with UV in advance and turn on the high-temperature sterilizer to sterilize the tweezers. After the tweezers cool down, clamp the leaves on the pre-culture medium to the conical flask containing Agrobacterium, and time for 15-20 minutes. During this period, shake the conical flask evenly for 2-3 minutes. After the timing is over, clamp the leaves to the filter paper sterilized by high-pressure steam, absorb the excess bacterial liquid on the surface of the leaves, and place the leaves in the dark culture medium at 25°C and culture in the dark for 2-3 days.

[0054] (3) Inducing bud differentiation: After dark culture, the leaves are transferred to a differentiation medium containing 20-30 mg / L of kanamycin for differentiation induction. The medium is changed every 6-8 days and cultured at 25°C under light conditions (16 h light / 8 h dark) until adventitious buds are differentiated.

[0055] (4) Root induction in tissue culture flasks: Use sterilized tweezers to separate adventitious buds that have grown to a length of 1-2 cm from the leaves, and transfer them to rooting medium containing 15-20 mg / L of kanamycin. Culture at 25°C under light conditions (16 h light / 8 h dark) until roots form. After about 4-5 weeks of growth to the normal plant size, propagate.

[0056] (5) After hardening, transplant to greenhouse for soil culture: When the tissue culture seedlings grow for about 45 days and have a well-developed root system, transplant them 3-5 days after hardening. Wash the culture medium at the roots of the poplar with clean water, transplant them to sterilized nutrient soil, and culture them in the greenhouse.

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

[0058] Table 1

[0059] Example 3

[0060] 1. Identification of transgenic overexpressing plants

[0061] Leaves of different poplar strains that had taken root after transformation were cut in a clean bench, and each strain was labeled. Genomic DNA was extracted using a genomic DNA extraction kit (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd., refer to the instruction manual for specific operating steps).

[0062] Using primers JP-0368-F and JP-GFP-R, PCR was used to clone the 800 bp fragment containing the fusion expression of Pop_A17G020368 and GFP to identify the positive plants with successful transgenic success. Through identification, OE-1 and OE-2 were found to be the successful strains of Pop_A17G020368 gene overexpression (see Appendix Figure 1 -Attached Figure 3 ).

[0063] 2. The role of Pop_A17G020368 gene in regulating tree height and secondary growth

[0064] (1) Fluorescence real-time quantitative PCR method was used to detect the expression of Pop_A17G0203688 in different tissues:

[0065] Total RNA extraction from 84K poplar: The root, stem and leaf parts of wild-type 84K plants were cut to detect the expression of the target gene in different tissue parts, and the leaves of overexpression plants OE-1 / -2 and wild-type 84K tissue culture seedlings of different strains were cut to identify the expression of the target gene in the overexpression plants. The total RNA solution was extracted using the liquid nitrogen grinding method according to the EASYspin Plus Plant RNA Rapid Extraction Kit (purchased from Beijing Aidelai Biotechnology Co., Ltd., refer to the instruction manual for specific operation steps).

[0066] cDNA synthesis: The total RNA of the above-mentioned different strains was reverse transcribed and synthesized into cDNA using a one-step cDNA synthesis kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd., refer to the instruction manual for specific operation steps).

[0067] Fluorescence real-time quantitative PCR detection: SYBRGreenqPCRMix of Beijing Aidelai Biotechnology Co., Ltd. was used to quantitatively determine the expression of Pop_A17G020368 in different parts. The reaction system and PCR procedure refer to the instructions of the kit. The PCR primers used for the Pop_A17G020368 gene were qPCR-0368-F1 and qPCR-0368-R1, the internal reference gene was Actin, and the primers were Actin-F and Actin-R. The results of three biological experiments showed that the expression level of Pop_A17G020368 gene in 84K was the highest in leaves, followed by stems and roots. The expression level detection results of different tissue parts are attached. Figure 4 The expression levels of Pop_A17G020368 in OE-1 and OE-2 plants were higher than those in the wild type. The results of the transcriptional expression level detection of transgenic poplars are shown in the attached Figure 3 .

[0068] qPCR-0368-F1: 5'-GAGACAGAGAGTTCGCCATT-3', SEQ ID NO.7;

[0069] qPCR-0368-R1: 5'-GATAAGATCTAGGCAAAGAT-3', SEQ ID NO. 8.

[0070] Actin-F: 5'-AAACTGTAATGGTCCTCCCTCCG-3', SEQ ID NO.9;

[0071] Actin-R: 5'-GCATCATCACAATCACTCTCCGA-3', SEQ ID NO. 10.

[0072] (2) Phenotypic observation of transgenic plants: The wild-type (WT) and overexpression (OE-1 / -2) lines with the same growth after rooting were transferred to soil for growth. Phenotypic observation and statistical determination showed that the plant height of the overexpression line was significantly higher than that of the wild-type line at the same growth period (see Appendix Figure 5 ); The stem base diameters of wild-type and OE-1 / -2 plants were statistically analyzed and it was found that the stem base diameters of OE-1 / -2 plants were significantly larger than those of wild-type plants (see Appendix Figure 6 ).

[0073] 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.

[0074] 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. Pop_A17G020368 gene, characterized in that 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 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-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 biological material according to claim 3 in promoting plant growth.

5. The use according to claim 4, characterized in that: The application is to increase the height of the plant and / or increase the diameter of the base of the plant stem.

6. A breeding method for promoting plant growth, characterized in that: The expression level of the Pop_A17G020368 gene is increased by genetic modification, and the nucleotide sequence of the Pop_A17G020368 gene is shown in SEQ ID NO:1.

Images