Plant constitutive expression promoter and application thereof

Abstract

The invention relates to the field of gene engineering, in particular to a plant constitutive expression promoter and application thereof. The plant constitutive expression promoter provided by the invention comprises a DNA (Deoxyribose Nucleic Acid) sequence as shown in SEQ ID NO. 1. The plant constitutive expression promoter provided by the invention can regulate and control the expression of genes in plants, and has significant value in practical application. Gene modification is carried out on a crop variety through the promoter, for example, expression of a target gene in a plant is regulated and controlled through the promoter, so that an ideal transgenic plant variety is cultivated.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a plant constitutive expression promoter and application thereof. Background Art

[0002] As one of the most important food crops in the world, the production efficiency and quality of rice are directly related to global food security. With the rapid development of plant genetic engineering technology, improving rice varieties through transgenic means to improve their stress resistance, yield and quality has become a research hotspot. In plant genetic engineering, promoters are one of the important elements for regulating gene expression. The function of promoters is to control the transcription level of downstream genes by binding to transcription factors, thus determining the expression characteristics of target genes in plants.

[0003] According to the expression characteristics of promoters under different conditions, promoters are usually divided into three categories: inducible promoters, constitutive promoters and tissue-specific promoters. Inducible promoters initiate gene expression under the action of specific environmental stimuli or activating factors, constitutive promoters can maintain stable gene expression in all growth stages and different tissues of plants, and tissue-specific promoters refer to promoters under which the expression of exogenous genes generally only occurs in cells or tissues of certain specific sources.

[0004] Constitutive promoters can continuously and stably drive the efficient expression of target genes in multiple tissues, different developmental stages and different environmental conditions of plants. This feature makes constitutive promoters an important tool in plant genetic engineering research, especially in gene function research and the construction of transgenic crops, where constitutive promoters can ensure the effectiveness and stability of transgenic expression systems.

[0005] At present, the constitutive promoters used in plant genetic engineering mainly include the cauliflower mosaic virus (CaMV 35S) promoter and the ubiquitin promoter. Among them, the CaMV 35S promoter can drive efficient gene expression in most plants, especially in crops such as rice, corn, soybeans, etc. However, in some cases, it may lead to overexpression or unstable expression in specific plants, thus affecting its application effect. The Ubiquitin promoter can stably express the target gene in multiple tissues of most plants by participating in the basic metabolic processes of cells, and its expression is generally relatively stable. However, the Ubiquitin promoter may also be affected by differences in plant species and expression systems, resulting in different expression effects.

[0006] Although existing constitutive promoters are widely used in plant transgenic research, there are still some limitations and challenges. When the CaMV 35S promoter is used in some transgenic rice, overexpression of the target gene may cause transgene silencing. Although constitutive promoters should theoretically maintain stable expression, in addition, existing constitutive promoters may limit their application in commercial breeding due to patent protection. Therefore, it is of great significance to develop an efficient, broad-spectrum, and constitutive expression promoter that is suitable for a variety of plants. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a plant constitutive expression promoter and its application, which can regulate the strong expression of genes in various tissues in the plant. The promoter is derived from the natural genome of the plant and has high-efficiency and stable transcriptional activity, and is suitable for a variety of species and tissues.

[0008] To this end, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a plant constitutive expression promoter in an optional embodiment, wherein the plant constitutive expression promoter comprises a DNA sequence as shown in SEQ ID NO.1.

[0010] Furthermore, the plant constitutive expression promoter comprises a DNA sequence as shown in SEQ ID NO. 2, which is referred to as PosHi1 or promoter PosHi1 in the present invention.

[0011] Preferably, the DNA sequence of the plant constitutive expression promoter has at least 80% homology with the DNA sequence shown in SEQ ID NO.1 or SEQ ID NO.2; or, the plant constitutive expression promoter is a mutant, allele or derivative generated by adding, substituting, inserting or deleting one or more nucleotides in the DNA sequence shown in SEQ ID NO.1 or SEQ ID NO.2; or, the plant constitutive expression promoter has a product that hybridizes with the DNA sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0012] In the present invention, these plant constitutive promoters have the same function as the DNA sequence shown in SEQ ID NO.1 or SEQ ID NO.2, namely, driving the target gene to be expressed in the plant.

[0013] In a second aspect, the present invention provides, in an optional embodiment, an expression cassette comprising the above-mentioned plant constitutive expression promoter.

[0014] In a third aspect, the present invention provides a recombinant expression vector in an optional embodiment, the recombinant expression vector comprises the above-mentioned plant constitutive expression promoter; in the recombinant expression vector, the plant constitutive expression promoter is connected to the upstream of the gene sequence to be expressed in the vector. Preferably, the gene to be expressed is the GUS gene, the vector is pCAMBIA1305, and the recombinant expression vector is constructed by constructing the promoter PosHi1 in pCAMBIA1305, and the promoter PosHi1 is connected to the upstream of the GUS gene to obtain a recombinant expression vector, which is recorded as pCAMBIA1305-PosHi1.

[0015] In a fourth aspect, the present invention provides a host bacterium in an optional embodiment, comprising the plant constitutive expression promoter, the expression cassette or the recombinant expression vector. Preferably, the host bacterium is Agrobacterium tumefaciens.

[0016] In a fifth aspect, the present invention provides a transformant in an optional embodiment, comprising the plant constitutive expression promoter, the expression cassette, the recombinant expression vector or the host bacteria. The transformant is preferably a transgenic cell, callus or plant.

[0017] In a sixth aspect, the present invention provides, in an optional embodiment, an application of the above-mentioned plant constitutive expression promoter in cultivating transgenic plants, the application comprising: connecting the above-mentioned plant constitutive expression promoter to the upstream of the gene sequence to be expressed in the vector, constructing a recombinant expression vector, and then transforming the recombinant expression vector into plant cells, tissues or organs for cultivation; the plant is a monocotyledonous plant, and the monocotyledonous plant includes rice, wheat, corn, barley, sorghum or oats, preferably rice.

[0018] In addition, the DNA sequence of the plant constitutive expression promoter provided by the present invention can be connected to a plant binary expression vector to replace the constitutive promoter. Moreover, the DNA sequence of the plant constitutive expression promoter can be linked to a desired target gene to construct a recombinant plant expression vector. After transformation, the constitutive promoter can drive the target gene to be expressed in the plant.

[0019]

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of constructing the promoter PosHi1 into the pCAMBIA1305 vector plasmid in Example 1-2 of the present invention, wherein: Figure 1 A is the gel electrophoresis diagram of promoter PosHi1 amplification. Figure 1 B is the gel electrophoresis image of double restriction enzyme digestion of pCAMBIA1305-PosHi1, Figure 1 C is a schematic diagram of the pCAMBIA1305-Pos Hi1 vector, showing the use of the PosHi1 promoter to drive the expression of the Gus gene located downstream thereof;

[0022] Figure 2 Schematic diagram of Gus staining results of various parts of transgenic rice driven by PosHi1 promoter to express Gus gene in Example 3 of the present invention. Figure 2 A), stem ( Figure 2 B) Leaves Figure 2 C) Leaf sheath ( Figure 2 D) Occipital Figure 2 E) Ligule ( Figure 2 F) Glumes Figure 2 G) is expressed in, scale bar = 2 mm;

[0023] Figure 3 This is a schematic diagram of the results of detecting the PosHi1-driven GUS gene by real-time fluorescence quantitative PCR in Example 4 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] Example 1

[0028] This embodiment provides a method for preparing a promoter PosHi1 containing a restriction site, comprising the following steps:

[0029] According to the whole genome sequence of rice variety Nipponbare provided by NCBI, amplification primers were designed according to the promoter sequence of PosHi1 gene, and the restriction sites of primers were designed according to the characteristics of the selected vector and target gene.

[0030] In this embodiment, the rice binary expression vector pCAMBIA1305 is used, the marker gene is the GUS gene, and the specifically designed primers are: the forward primer, i.e. the upstream primer, has EcoRI at its 5' end and a restriction site (GAATTC), and the reverse primer, i.e. the downstream primer, has NcoI at its 5' end and a restriction site (CCATGC).

[0031] The forward primer and reverse primer were synthesized by Hefei Youkang Biological Company.

[0032] Using the DNA of the rice variety Nipponbare as a template, the promoter PosHi1 was amplified using forward primers and reverse primers. According to the conventional PCR system, the following amplification procedure was used:

[0033] Pre-denaturation at 95°C for 5 min, denaturation at 90°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, 35 cycles from pre-denaturation at 95°C to extension at 72°C, and finally extension at 72°C for 5 min.

[0034] The target fragment amplified by PCR was recovered, and the length of the target fragment was 2000 bp. Figure 1 As shown in A, it was connected to the blunT-SimpleT vector (purchased from Transgene, mixed according to the ratio in the vector manual), and transformed into Escherichia coli XL-Blue competent cells according to the heat shock method, and the competent cells were activated, and then the target fragment was transferred into the activated competent cells, and then the positive clones were screened by colony PCR, and the single clone was picked to extract the plasmid, and double restriction enzyme digestion was performed with EcoRⅠ and NcoⅠ for verification, as shown in FIG. Figure 1 As shown in B, the identified positive clones are sequenced to verify that the correct positive clone is the promoter PosHi1 to be obtained, and its nucleotide sequence is shown in SEQ ID NO.2.

[0035] Example 2

[0036] This embodiment provides a method for constructing a plant expression vector and transforming Agrobacterium, specifically:

[0037] Plasmids were extracted from the positive clones prepared in Example 1, and double digested with EcoRI and NcoI to recover the promoter PosHi1 fragment. At the same time, pCAMBIA1305 was linearized with EcoRI and NcoI to recover pCAMBIA1305. The PosHi1 fragment and the pCAMBIA1305 fragment were connected with Quick Linker (purchased from NEB) to obtain the plant expression vector pCAMBIA1305-PosHi1 (see Figure 1 C), the plant expression vector pCAMBIA1305-PosHi1 was transferred into Agrobacterium tumefaciens EHA105 (stored by the Rice Group of the Supervision and Inspection Center for Transgenic Biological Products of the Ministry of Agriculture, Anhui Academy of Agricultural Sciences) by freeze-thaw method, and the positive plasmid was extracted from the product obtained by freeze-thaw method, and the enzyme digestion was verified by EcoRⅠ and NcoⅠ. The verification results are as follows Figure 1 As shown in B.

[0038] Example 3

[0039] This embodiment provides a method of using the promoter PosHi1 to drive the expression of the GUS reporter gene in rice, specifically:

[0040] (1) After removing the husk from mature seeds, soak the seeds in 70% alcohol for 1 minute, pour out the alcohol, and then soak the seeds in 50% sodium hypochlorite solution (the effective chlorine concentration of the original solution is greater than 4%) containing 1 drop of Tween 20 for 40 minutes (150r / min), then pour out the sodium hypochlorite, wash with sterile water 5 times until the solution is clear and there is no smell of sodium hypochlorite. Soak the seeds in sterile water overnight, peel off the endosperm of the pretreated seeds with a scalpel, peel off the embryo, and inoculate the embryo on the callus induction medium. After dark culture at 30°C for 11 days, separate the callus from the endosperm and embryo, and pre-culture the primary callus tissue with good bud removal and vigorous division for 3-5 days before using it for Agrobacterium transformation. Agrobacterium tumefaciens transformed with the recombinant expression vector prepared in Example 2 was used for Agrobacterium-mediated genetic transformation. The genetic transformation, transformant screening and transgenic plant regeneration were carried out according to the method proposed by Yongbo Duan (Yongbo Duan, Chenguang Zhai, et al. An efficient and high-throu ghput protocol for Agrobacterium mediated transformation based on phosphomannose isomerase positive selection in Japonica rice (Oryza sativa L.) [J]. Plant Cell Report, 2012. DOI 10.1007 / s00299-012-1275-3.). A total of 10 pCAMBIA1305-PosHi1 plants (PosHi1::GUS transgenic rice plants) were obtained.

[0041] (2) Referring to the method proposed by Jefferson et al. (Jefferson RA et al. GUS fusion: β-Glucuronidases as a sensitive and versatile gene fusion marker in higher plants [J]. EMBO J., 1987, 6: 3901-3907), the tissue to be stained was evacuated, then immersed in the staining solution and stained at 37°C for 24 hours. When decolorizing, 95% ethanol was used at 37°C until the negative control material turned white. After staining of the PosHi1::GUS transgenic plant tissue, the transgenic root ( Figure 2 A), stem ( Figure 2 B) Leaves Figure 2 C) Leaf sheath ( Figure 2 D) Occipital Figure 2 E) Ligule ( Figure 2 F) Glumes Figure 2 G) and GUS staining results are shown in Figure 2 .

[0042] Example 4

[0043] The results of GUS staining qualitatively showed that PosHi1 is a constitutive promoter. To verify the activity of PosHi1, RNA was extracted from 10-day-old transgenic seedlings and then reverse transcribed into cDNA. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression changes of the PosHi1-driven GUS gene, and transgenic plants driven by CaMV 35S for GUS expression were used as controls.

[0044] The plant total RNA extraction kit (TIANGEN, centrifugal column type, DP432) was used. The obtained RNA was reverse transcribed into cDNA according to the following procedure: 5 μL RNase-Free ddH 2 O, 2 μL 5×gDNA buffer, 3 μL RNA, incubate at 42°C for 3 min, and then place on ice; 5 μL RNase-Free ddHO were added to the above reaction solution. 2 O, 2μL FQ-RT Primer Mix, 2μL 10×Fast RT Buffer, and 1μL RT Enzyme Mix, mix thoroughly, incubate at 42℃ for 15min; incubate at 95℃ for 3min, and then put on ice to obtain cDNA.

[0045] RT-qPCR was performed using the SuperReal fluorescent quantitative premix kit (TIANGEN, SYBRGreen, FP205) from Tiangen (Beijing). The rice ACTIN gene was used as an internal reference gene to quantify the amount of RNA template used. – ΔΔCT (ΔCT = CT target gene - CT reference gene; ΔΔCT = ΔCT after treatment - ΔCT control) The obtained signals and data were processed. Each gene was repeated 3 times. The quantitative primers for the genes used in this example are:

[0046] Actin-FP, 5′-CCTGACGGAGCGTGGTTAC-3′; and Actin-RP, 5′-CCAGGGCGATGTAGGAAAGC-3′ were used for amplification of ACTIN;

[0047] GUS-FP, 5′-TACGGCAAAGTGTGGGTCAATAATCA-3′, and GUS-RP, 5′-CAGGTGTTCGGCGTGGTGTAGAG-3′ were used for the amplification of GUS.

[0048] The quantitative PCR results were as follows Figure 3 As shown in the figure, the expression level of GUS gene driven by CaMV 35S was taken as 1, and the expression level of GUS gene in each tissue of PosHi1::GUS transgenic plants was detected. The results showed that the expression level of transgenic plants in roots, stems, leaves, leaf sheaths, leaf pillows, leaf tongues and husks was equivalent to that of CaMV 35S::GUS. This shows that PosHi1::GUS is a constitutively expressed promoter.

[0049] Although the principles of the present invention are described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely explanations of the exemplary implementations of the present invention, and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute limitations on the scope of the present invention, and any obvious changes such as equivalent transformations, simple replacements, etc. based on the technical solution of the present invention, without departing from the spirit and scope of the present invention, fall within the protection scope of the present invention.

Claims

1. A plant constitutive expression promoter, characterized in that: The plant constitutive expression promoter comprises the DNA sequence shown as SEQ ID NO.

1.

2. The plant constitutive expression promoter according to claim 1, characterized in that The plant constitutive expression promoter comprises the DNA sequence shown as SEQ ID NO.

2.

3. The plant constitutive expression promoter according to claim 1, characterized in that The DNA sequence of the plant constitutive expression promoter has at least 80% homology with the DNA sequence shown in SEQ ID NO.1 or SEQ ID NO.2; or, The plant constitutive expression promoter is a mutant, allele or derivative generated by adding, replacing, inserting or deleting one or more nucleotides in the DNA sequence shown in SEQ ID NO.1 or SEQ ID NO.2; or, The plant constitutive expression promoter has a product that hybridizes with the DNA sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

4. An expression cassette, characterized in that Contains the plant constitutive expression promoter according to any one of claims 1 to 3.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the plant constitutive expression promoter according to any one of claims 1 to 3; In the recombinant expression vector, the plant constitutive expression promoter is connected to the upstream of the gene sequence to be expressed in the vector.

6. A host bacterium, characterized in that It comprises the plant constitutive expression promoter according to any one of claims 1 to 3, the expression cassette according to claim 4 or the recombinant expression vector according to claim 5.

7. A transformant, characterized in that: It comprises the plant constitutive expression promoter according to any one of claims 1 to 3, the expression cassette according to claim 4, the recombinant expression vector according to claim 5 or the host bacteria according to claim 6.

8. Use of the plant constitutive expression promoter according to any one of claims 1 to 3 in cultivating transgenic plants, characterized in that: The application comprises: connecting the plant constitutive expression promoter described in any one of claims 1 to 3 to the upstream of the gene sequence to be expressed in the vector, constructing a recombinant expression vector, and then transforming the recombinant expression vector into plant cells, tissues or organs for cultivation.

9. The use according to claim 8, characterized in that: The plant is a monocotyledonous plant, which includes rice, wheat, corn, barley, sorghum or oats.

10. Use of the plant constitutive expression promoter according to any one of claims 1 to 3 in improving plant growth characteristics.

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