PtH4 gene and application thereof

Abstract

The invention discloses a PtH4 gene and application thereof, and relates to the technical field of biology, and the nucleotide sequence of the PtH4 gene is as shown in SEQ ID NO. 1. The function of the gene is verified by cloning and analyzing the PtH4 gene and overexpressing the PtH4 gene in arabidopsis thaliana in combination with an agrobacterium tumefaciens-mediated genetic transformation technology. Compared with a wild type, the plant overexpressed with the Pth4 gene can remarkably promote vegetative growth of organs such as roots, leaves and rachis. The invention has important practical and theoretical guidance significance for researching plant growth and cultivating new plant varieties.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to a PtH4 gene and its application. Background Art

[0002] Chinese pine (Pinus tabulaeformis Carr.) is an evergreen coniferous tree of the genus Pinus in the family Pinaceae. It is a unique tree species in China and is widely distributed in Northeast China, Central Plains, Northwest China and Southwest China. Chinese pine is not only an essential key tree species for barren mountain greening, afforestation and urban greening, but also widely used in industries such as construction, bridges and railways.

[0003] Vegetative growth and reproductive growth are two important life processes in the growth and development of plants. Vegetative growth plays a crucial role in aspects such as the overall growth, development, reproduction and stress resistance of plants. Well-developed roots can stabilize the plant and improve the plant's ability to absorb water and nutrients in the soil, ensuring the normal growth of the plant body. An increase in leaf area can increase the photosensitive area of the plant, thereby improving the photosynthesis efficiency and achieving a large accumulation of organic matter in the plant body. Improving the plant's ability to absorb water and nutrients through well-developed vegetative organs of the plant is more conducive to storing the resources required for plant survival, thereby enhancing its adaptability to adversity.

[0004] However, traditional Chinese pine breeding methods have a long cycle and low efficiency, and it is difficult to meet the growing demand for Chinese pine wood. By comparing and analyzing the phenotypes of wild-type Chinese pine and natural female sterile mutant lines, it was found that the sterile line showed a significantly more vigorous vegetative growth than the wild-type. If the key genes can be locked and transgenic plants can have more developed leaves and roots under the same resource conditions, it may be possible to achieve the cultivation of highly growing plants, which has extremely high significance for exploring the internal regulatory mechanism of plant vegetative growth. Summary of the Invention

[0005] In view of this, the present invention provides a PtH4 gene and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] The PtH4 gene, the nucleotide sequence of the gene is as shown in SEQ ID NO.1.

[0008] Another object of the present invention is to provide a PtH4 protein, the coding gene sequence of the protein is as shown in SEQ ID NO.1.

[0009] Preferably, the amino acid sequence of the protein is as shown in SEQ ID NO.2.

[0010] Another object of the present invention is to provide a biological material, which is any one of the following:

[0011] A: An expression cassette capable of overexpressing a gene with a nucleotide sequence as shown in SEQ ID NO.1;

[0012] B: A recombinant vector containing the expression cassette described in A;

[0013] C: A recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B;

[0014] D: A non-renewable plant part containing the expression cassette described in A, the recombinant vector described in B, or the recombinant microorganism described in C.

[0015] Another object of the present invention is to provide the application of the above-mentioned gene, or the above-mentioned protein, or the above-mentioned biological material in promoting vegetative growth of plants.

[0016] Preferably, the application is to promote the growth of plant roots, leaves and / or inflorescence axes.

[0017] Another object of the present invention is to provide a breeding method for promoting plant growth, which uses transgenic means to increase the expression level of the PtH4 gene, and the nucleotide sequence of the PtH4 gene is as shown in SEQ ID NO.1.

[0018] Beneficial effects:

[0019] The present invention provides the PtH4 gene, the PtH4 protein encoded by it, and their applications. By cloning and analyzing the PtH4 gene and overexpressing the PtH4 gene in Arabidopsis thaliana using the Agrobacterium-mediated genetic transformation technology, the function of this gene was verified. Compared with the wild type, the plants overexpressing the PtH4 gene can significantly promote the vegetative growth of organs such as roots, leaves and inflorescence axes. The present invention has important practical and theoretical guiding significance for studying plant growth and cultivating new plant varieties. Description of the drawings

[0020] 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.

[0021] Figure 1 It is the electrophoresis diagram for the transformation identification of PtH4 transgenic plants, where M is the 2000 molecular weight standard reference, WT is wild-type Arabidopsis thaliana, and PtH4 OE is the PtH4 transgenic overexpressing plant.

[0022] Figure 2 Results of RT-qPCR expression levels in PtH4 transgenic plants, where WT is wild-type Arabidopsis thaliana and PtH4 OE is the PtH4 transgenic overexpression plant.

[0023] Figure 3 Comparison of roots of wild-type and transgenic plants grown for 7 days, where WT is wild-type Arabidopsis thaliana and PtH4OE is the PtH4 transgenic overexpression plant.

[0024] Figure 4 Histogram of root length statistics of wild-type and transgenic plants grown for 7 days, where WT is wild-type Arabidopsis thaliana and PtH4 OE is the PtH4 transgenic overexpression plant.

[0025] Figure 5 Comparison of plants in pots of wild-type and transgenic plants grown for 21 days, where WT is wild-type Arabidopsis thaliana and PtH4 OE is the PtH4 transgenic overexpression plant.

[0026] Figure 6 Comparison of leaves of wild-type and transgenic plants grown for 21 days, where WT is wild-type Arabidopsis thaliana and PtH4OE is the PtH4 transgenic overexpression plant.

[0027] Figure 7 Histogram of leaf width statistics of wild-type and transgenic plants grown for 21 days, where WT is wild-type Arabidopsis thaliana and PtH4 OE is the PtH4 transgenic overexpression plant.

[0028] Figure 8 Comparison of inflorescence axes of wild-type and transgenic plants grown for 35 days, where WT is wild-type Arabidopsis thaliana and PtH4 OE is the PtH4 transgenic overexpression plant.

[0029] Figure 9 Histogram of inflorescence axis length statistics of wild-type and transgenic plants grown for 35 days, where WT is wild-type Arabidopsis thaliana and PtH4 OE is the PtH4 transgenic overexpression plant. Detailed implementation

[0030] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the 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.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the related listed items.

[0032] The expression cassette of the present invention refers to DNA that can express the PtH4 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.

[0033] Example 1

[0034] 1. Cloning of the PtH4 gene

[0035] Specific primers were designed according to the PtH4 gene sequence, and the CDS sequence of PtH4 was amplified using the cDNA obtained by reverse transcription of total RNA from Pinus tabuliformis ovules as a template. The upstream primer used was CGCGGATCCATGGCTCCAAAGGCAGAG, SEQ ID NO.3, and the downstream primer was ACGCGTCGACGGCACTGGTGAACTTAG, SEQ ID NO.4.

[0036] The amplification system is as follows: Table 1

[0037] CDS sequence of the PtH4 gene: ATGGCTCCAAAGGCAGAGAAGAAACCAGCTGAAAAGAAGCCTGTTGCAGAGAAGAAGCCCGCTCCTAAGGCCGAGAAGAAGCTTCCAAAAGAAGGCGGAGACAAGAAGGGCAAGAAGAAGCACAAGAAGAGCGTCGAGACGTACAAGATCTATATCTTTAAGGTTTTGAAGCAGGTTCACCCAGATATCGGCATTTCTAGCAAGGCCATGGGGATAATGAACAGCTTCATCAACGATATCTTTGAGAAGTTAGCGAGTGAGTCATCGAAACTTGCCCGCTACAACAAGAAGCCCACCATTACATCTCGCGAGATTCAGACCGCTGTAAGGCTGGCGCTTCCAGGAGAGCTCGCCAAGCACGCTGTCTCGGAGGGTACCAAGGCGGTGACTAAGTTCACCAGTGCCTGA, SEQ ID NO.1.

[0038] PtH4 protein sequence: MAPKAEKKPAEKKPVAEKKPAPKAEKKLPKEGGDKKGKKKHKKSVETYKIYIFKVLKQVHPDIGISSKAMGIMNSFINDIFEKLASESSKLARYNKKPTITSREIQTAVRLALPGELAKHAVSEGTKAVTKFTSA, SEQ IDNO.2.

[0039] 2. Vector construction

[0040] Take 2 μL of PtH4 obtained in the above step and ligate it with the T-vector. Select restriction enzymes (BamH I and SalI) to cut the target fragment from the T-vector and recover the target fragment. Then use the same restriction enzymes to digest the vector plasmid (pCAMBIA2300-35S-GFP) and recover the vector fragment. Ligate the two recovered fragments to obtain the pCAMBIA2300-35S-PtH4-GFP plasmid.

[0041] 3. Arabidopsis transformation and screening of transgenic plants

[0042] The Arabidopsis variety used in this part is the wild-type Arabidopsis 'Columbia'.

[0043] In this part, the Arabidopsis thaliana was genetically transformed by the floral dipping method, and the specific operation is as follows: First, the pCAMBIA2300-35S-PtH4-GFP plasmid was transformed into competent cells of Agrobacterium tumefaciens GV3101, and then the Agrobacterium was inoculated into LB liquid medium supplemented with antibiotics (kanamycin 50 μg / mL, rifampicin 25 μg / mL). After culturing with shaking at 28 °C for 12 h, the cells were collected by centrifugation at 6500 rpm for 10 min; then the cells were resuspended with 50 mL of 5% sucrose solution, and 10 μL of Silwet L-77 was added; the inflorescences of Arabidopsis thaliana grown for 3-4 weeks were immersed in the infection solution for 15 s; finally, the Arabidopsis thaliana after floral dipping was cultured in the dark for 24 h, and then the growth conditions were changed to normal growth conditions until the seeds matured, and the T0 generation seeds were harvested.

[0044] The T0 generation seeds were disinfected and then sown on 1 / 2 MS medium containing kanamycin and vernalized at 4 °C for 2-4 days. Then, they were cultured at 22 °C for one week. Arabidopsis thaliana plants with normal root and hypocotyl growth and non-yellowing leaf color were initially determined as positive seedlings. After transferring the positive seedlings to the soil for cultivation, the DNA of the positive seedlings was extracted, and positive verification was carried out by PCR, and then the expression level of PtH4 in the transgenic plants was detected by RT-qPCR.

[0045] The results showed that the expression level of the PtH4 gene in the transgenic plants was significantly higher than that in the wild type, indicating that the PtH4 gene had been successfully transferred into Arabidopsis thaliana and was expressed in the transgenic plants (see Figure 1 and Figure 2 ).

[0046] 4. Phenotypic analysis

[0047] The wild-type Arabidopsis thaliana seeds and transgenic Arabidopsis thaliana seeds were sown in the same substrate. It was found that after 7 days of cultivation, the root length of the transgenic plants was significantly longer than that of the wild type (see Figure 3 and Figure 4 ); when cultured for 21 days, it was found that the leaf width of the transgenic positive plants was significantly larger than that of the wild type (see Figures 5 - 7 ); when cultured for 35 days, it was visible that the inflorescence axis length of the transgenic plants was significantly longer than that of the wild-type Arabidopsis thaliana plants (see Figure 8 and Figure 9 ).

[0048] It can be seen that the technical solution provided by the present invention can accurately reflect that the PtH4 gene promotes the growth of plant roots, leaves and inflorescence axes. Applying it to plant breeding can significantly promote the vegetative growth of plants.

[0049] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. The same or similar parts among the embodiments can be referred to each other.

[0050] The foregoing description of the disclosed embodiments enables those skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those 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. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The PtH4 gene, characterized in that, The nucleotide sequence of the said gene is as shown in SEQ ID NO.

1.

2. The PtH4 protein, characterized in that, The coding gene sequence of the said protein is as shown in SEQ ID NO.

1.

3. The PtH4 protein according to claim 2, characterized in that, The amino acid sequence of the said protein is as shown in SEQ ID NO.

2.

4. A biological material, characterized in that, The said biological material is any one of the following: A: An expression cassette capable of overexpressing the gene with the 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 plant part containing the expression cassette described in A, or the recombinant vector described in B, or the recombinant microorganism described in C.

5. The application of the gene described in claim 1, or the protein described in claim 2 or 3, or the biological material described in claim 4 in promoting vegetative growth of plants.

6. The application according to claim 5, wherein The said application is to promote the growth of roots, leaves and / or inflorescence axes of plants.

7. A breeding method for promoting plant growth, characterized in that By means of transgenic technology, the expression level of the PtH4 gene is increased, and the nucleotide sequence of the PtH4 gene is as shown in SEQ ID NO.1.

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