A promoter and its use in orchidaceae plants

Abstract

The present application relates to the technical field of plant genetic engineering, and particularly relates to a promoter and application thereof in orchid plants. The present application provides an endogenous promoter of orchid plants, the promoter is a strong constitutive promoter, and can drive strong expression of a target gene in each tissue of the orchid plants such as roots, stems, leaves and flowers; compared with the CaMV 35S promoter, the promoter greatly improves the driving efficiency of the gene in the orchid plants, provides an important tool for molecular breeding of plants, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, in particular to a promoter and its application in orchid plants. Background Art

[0002] Orchidaceae ( Cymbidium ) plants and Phalaenopsis ( Phalaenopsis Orchids are the most culturally and economically valuable group in the orchid family, prized for their unique flower shapes, rich colors, and captivating fragrance. Despite significant progress in orchid breeding over the past few decades, many challenges remain. Breeding of Cymbidium and Phalaenopsis species primarily relies on traditional hybridization, selective breeding, and mutagenesis. While these methods have contributed to improved varieties to some extent, they also present challenges such as high genetic complexity and long reproductive cycles. In contrast, transgenic and gene-editing technologies can effectively address the pain points of traditional breeding and provide new breakthroughs in plant breeding. The selection and utilization of promoters is crucial in developing orchid-specific molecular breeding systems. As key regulatory elements for gene expression, promoters play a crucial role in ensuring efficient and stable expression of target genes in plants. Currently, the tobacco cauliflower virus (CaMV) 35S promoter (hereafter referred to as 35S) is the most commonly used constitutive promoter in plants, capable of driving gene expression. However, this promoter suffers from low efficiency in driving gene expression in monocots, limiting its application in orchid breeding. Orchids possess unique mechanisms for regulating gene expression, and their endogenous promoters may be highly efficient, but currently, there is a lack of potent endogenous promoters in orchids. Therefore, developing promoters derived from orchids is of great significance. Summary of the Invention

[0003] The invention provides a promoter and application thereof in orchid plants.

[0004] During research on orchid promoters, researchers successfully identified a promoter for the ubiquitin gene (UBI). This promoter efficiently drives gene expression in various tissues of orchids, demonstrating significant regulatory function. The gene expression levels driven by this promoter are significantly higher than those of the commonly used 35S promoter.

[0005] Specifically, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a promoter, the nucleotide sequence of the promoter is shown as SEQ ID NO.1.

[0007] The promoter, whose nucleotide sequence is shown in SEQ ID NO. 1, was cloned from the Cymbidium orchidense plant. Located upstream of the ubiquitin gene, it represents the endogenous ubiquitin promoter of Cymbidium orchidense and is designated as ProCsUBI in this invention. This promoter has been shown to be a strong constitutive promoter capable of driving efficient expression of the target gene in various orchidaceae plants and in diverse plant tissues.

[0008] In a second aspect, the present invention provides a recombinant DNA construct comprising the promoter described above.

[0009] Preferably, the recombinant DNA construct further comprises a DNA molecule of interest operably linked to the promoter.

[0010] The above-mentioned target DNA molecules include but are not limited to protein-encoding genes, RNA-encoding DNA molecules, gene transcription or translation regulatory sequences, etc.

[0011] In a third aspect, the present invention provides an expression cassette, comprising the promoter or the recombinant DNA construct described above.

[0012] Preferably, the expression cassette comprises the above-mentioned promoter and a target gene operably linked thereto.

[0013] The expression cassette may further comprise other gene transcription or translation regulatory sequences, such as a terminator.

[0014] In a fourth aspect, the present invention provides a recombinant vector comprising the above-mentioned promoter, the recombinant DNA construct or the expression cassette.

[0015] In the present invention, the vector includes but is not limited to a plasmid vector, a transposon, a viral vector, an artificial chromosome vector, etc. Among them, the plasmid vector can be a cloning vector, an expression vector or an integrating vector.

[0016] In some embodiments of the present invention, the recombinant vector is an expression plasmid. Exemplarily, the recombinant vector is a pCAMBIA3301 expression plasmid containing the above-mentioned promoter.

[0017] In some embodiments of the present invention, the promoter was replaced with the 35S promoter in the pCAMBIA3301 vector to successfully construct an efficient expression vector suitable for orchids. In addition, the promoter was connected to the target gene and integrated into the pCAMBIA3301 expression vector, which can effectively drive the expression of the target gene in orchid cells. Exemplary target genes are eGFP genes or RUBY genes. In the eGFP gene expression verification, the Bar gene driven by the 35S promoter in the pCAMBIA3301 vector was used as an internal reference gene. The eGFP expression level driven by the promoter shown in SEQ ID NO.1 was 60 times higher than the Bar gene expression level in Mo orchid leaves and 30 times higher than the Bar gene expression level in Jian orchid leaves. In the RUBY gene expression verification, in Phalaenopsis orchid petals, the RUBY gene driven by the promoter shown in SEQ ID NO.1 made the petals deeper red compared to the 35S promoter.

[0018] In a fifth aspect, the present invention provides a host cell, which comprises the promoter, the recombinant DNA construct, the expression cassette, or the recombinant vector described above.

[0019] The host cells include microbial cells or plant cells. The microbial cells include Escherichia coli, Agrobacterium, yeast, etc. The plant cells are cells that will not develop into plant individuals.

[0020] In some embodiments of the present invention, the host cell is Escherichia coli.

[0021] In a sixth aspect, the present invention provides any of the following uses of the above-mentioned promoter, the recombinant DNA construct, the expression cassette, the recombinant vector, or the host cell:

[0022] (1) Expressing the target nucleic acid molecule in plants;

[0023] (2) Construction of transgenic plants;

[0024] (3) Plant gene editing;

[0025] (4) Plant genetic breeding.

[0026] The applications described in (1) to (4) above include: driving the transcription of the target nucleic acid molecule with a promoter having a nucleotide sequence such as that shown in SEQ ID NO.1, or including: introducing a vector containing a promoter having a nucleotide sequence such as that shown in SEQ ID NO.1 into the plant.

[0027] The promoter provided by the present invention, such as the nucleotide sequence shown in SEQ ID NO.1, can be used not only to drive the transcription of protein-encoding genes, but also to drive the transcription of RNA-encoding DNA molecules. The nucleic acid molecule driven by the promoter can be an endogenous nucleic acid molecule of the plant (for example, for overexpression of endogenous genes) or a heterologous nucleic acid molecule (for expression of heterologous genes). Therefore, the above-mentioned promoter, the recombinant DNA construct, the expression cassette, the recombinant vector, or the host cell can be used to construct transgenic plants (for example, for overexpression of endogenous genes or expression of heterologous genes), plant gene editing (for example, using the CRISPR / Cas gene editing system driven by the promoter to drive Cas genes or sgRNA for plant gene editing), or plant genetic breeding.

[0028] Preferably, the plant is a monocotyledonous plant, more preferably an orchid plant. The orchid plant includes orchids and phalaenopsis plants. The orchid plant can be any species under the genus, including but not limited to black orchid ( Cymbidium sinense )、Jianlan( Cymbidium ensifolium )、Cymbidium ( Cymbidium hybridum )、Chunlan( Cymbidium goeringii )、Cold orchid( Cymbidium kanran )、Rabbit Ear Orchid( Cymbidium lancifolium ) etc. Phalaenopsis plants may be any species under the genus, including but not limited to large white flowered Phalaenopsis ( Phalaenopsis amabilis )、Pink Phalaenopsis( Phalaenopsis equestris )、variegated Phalaenopsis ( Phalaenopsis schilleriana )、Yellow Phalaenopsis ( Phalaenopsis cornu-cervi )、Night-scented Phalaenopsis( Phalaenopsis bellina )wait.

[0029] In a seventh aspect, the present invention provides a method for expressing a target nucleic acid molecule in a plant, the method comprising: in the plant, driving the transcription of the target nucleic acid molecule with a promoter having a nucleotide sequence as shown in SEQ ID NO.1.

[0030] Preferably, the method comprises: operably linking the promoter to the target nucleic acid molecule and then introducing the resultant into the plant; or comprises: operably linking the promoter to the target nucleic acid molecule and then linking the resultant to a vector to obtain a recombinant vector, and then introducing the recombinant vector into the plant.

[0031] Preferably, the plant is a monocotyledonous plant, more preferably an orchidaceae plant. The orchidaceae plant includes orchidaceae plants and phalaenopsis plants.

[0032] After the promoter is connected to the target gene and introduced into the plant, the promoter can drive the target gene to be constitutively and strongly expressed in various tissues of the plant, such as roots, stems, leaves, and flowers.

[0033] In an eighth aspect, the present invention provides a method for constructing a transgenic plant, the method comprising: using a promoter having a nucleotide sequence such as SEQ ID NO.1 to drive the transcription of a target nucleic acid molecule in the plant.

[0034] Preferably, the method comprises: operably linking the promoter to the target nucleic acid molecule and then introducing the resultant into the plant; or comprises: operably linking the promoter to the target nucleic acid molecule and then linking the resultant to a vector to obtain a recombinant vector, and then introducing the recombinant vector into the plant.

[0035] Preferably, the plant is a monocotyledonous plant, more preferably an orchidaceae plant. The orchidaceae plant includes orchidaceae plants and phalaenopsis plants.

[0036] In a ninth aspect, the present invention provides a transgenic plant constructed by the above method.

[0037] In a tenth aspect, the present invention provides a method for obtaining processed agricultural products, comprising: processing the transgenic plants or a portion of the harvested products thereof as described above to obtain processed agricultural products.

[0038] The beneficial effects of the present invention include at least: the present invention provides an endogenous promoter of orchid plants, which is a strong constitutive promoter that can drive the strong expression of target genes in various tissues such as roots, stems, leaves, and flowers of orchid plants or Phalaenopsis plants; compared with the 35S promoter, this promoter greatly improves the driving efficiency of genes in orchid plants and Phalaenopsis plants, provides an important tool for molecular breeding of plants, and lays the foundation for further analysis of the gene functions of orchid plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0040] Figure 1 is the blue blue ubiquitin gene in Example 1 of the present invention ( CsUBIs ) expression analysis in various tissues.

[0041] Figure 2 This is a map of the Cs3301-eGFP expression vector in Example 2 of the present invention.

[0042] Figure 3 This is the expression analysis of eGFP driven by the ProCsUBI promoter in the leaves of Cymbidium orchid and Cymbidium orchid in Example 2 of the present invention.

[0043] Figure 4 This is a map of the 35S3301-RUBY expression vector in Example 3 of the present invention.

[0044] Figure 5 This is a map of the Cs3301-RUBY expression vector in Example 3 of the present invention.

[0045] Figure 6 This is the transient expression phenotype of the RUBY gene driven by the ProCsUBI and 35S promoters in Phalaenopsis petals in Example 3 of the present invention. The left side shows RUBY driven by the 35S promoter, and the right side shows RUBY driven by the ProCsUBI promoter. DETAILED DESCRIPTION

[0046] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0047] The nucleotide sequence (CDS sequence) of the eGFP gene involved in the following examples is shown in SEQ ID NO.2; the nucleotide sequence of the Bar gene is shown in SEQ ID NO.3; and the nucleotide sequence of the RUBY gene is shown in SEQ ID NO.4.

[0048] Example 1 Obtaining the Cymbidium orchid ubiquitin promoter

[0049] Using the amino acid sequence of maize UBI as the parent, we conducted homologous sequence analysis and found five highly homologous UBI genes in Cymbidium sinense ( CsUBIa 、 CsUBIb 、 CsUBIc 、 CsUBId 、 CsUBIe ), downloaded transcriptome data of roots, stems, leaves, and flowers of Cymbidium orchid, and analyzed the expression levels of the above five ubiquitin genes in various Cymbidium orchid tissues using Fragments Per Kilobase of exon model per Million mapped fragments (FPKM). The results showed that CsUBIb Highly expressed in all tissues ( Figure 1 ), so choose CsUBIb promoter sequence.

[0050] Example 2 Transient expression of eGFP gene driven by Cymbidium ubiquitin promoter in Cymbidium and Cymbidium ensifolium leaves

[0051] (1) Obtaining of Cymbidium ubiquitin promoter and construction of expression vector

[0052] Amplification of Cymbidium gene CsUBIb The sequence of the first 1500 bp (SEQ ID NO. 1) was used as the Cymbidium ProCsUBI promoter sequence. The ProCsUBI promoter and the green fluorescent protein gene eGFP were integrated into the pCAMBIA3301 vector to drive the expression of the eGFP gene with the Cymbidium ubiquitin promoter ProCsUBI, and the recombinant vector was named Cs3301-eGFP (Fig. 2). Figure 2 ).

[0053] (2) Preparation of Agrobacterium and injection of leaves

[0054] The Cs3301-eGFP was transformed into competent Agrobacterium by liquid nitrogen method, and positive Agrobacterium monoclonal was selected by PCR method. After expansion of the positive Agrobacterium, Agrobacterium infection solution was prepared, and Agrobacterium infection solution was injected into the leaves of Cymbidium and Cymbidium ensifolium by needle injection method, and cultured overnight at 28°C in the dark. Four days later, the photographs were taken.

[0055] (3) Bar test strip detection and eGFP expression analysis

[0056] The leaf area injected with Agrobacterium infection solution in the above (2) was cut and stored in liquid nitrogen, 200 mg of explant was ground thoroughly, and Bar test strip (Youlong Biological) was used for detection, and the result was positive. Another 200 mg sample was ground thoroughly under liquid nitrogen, and RNA was extracted by Trizol method, and reverse transcribed into cDNA. The Bar gene driven by the 35S enhanced promoter (i.e. 35S enhance) contained in the pCAMBIA3301 vector was used as the internal reference gene, and the expression amount of eGFP was analyzed by real-time fluorescent quantitative PCR method. The results showed that the ratio of the gene expression amount driven by ProCsUBI promoter to the gene expression amount driven by 35S enhanced promoter in Cymbidium ensifolium was more than 30 times; the ratio of the gene expression amount driven by ProCsUBI promoter to the gene expression amount driven by 35S enhanced promoter in Cymbidium was more than 60 times (Fig. 3). Figure 3 ).

[0057] Example 3 Transient expression of RUBY gene driven by Cymbidium ubiquitin promoter in Cymbidium

[0058] (1) Construction of overexpression vector of RUBY gene driven by Cymbidium ubiquitin promoter

[0059] The RUBY system can efficiently synthesize betaine in plant tissues, making plants appear reddish purple. It is a commonly used plant indicator gene. The 35S promoter and ProCsUBI promoter were respectively integrated into the pCAMBIA3301 vector with the RUBY expression system to construct the vector 35S3301-RUBY (RUBY driven by the 35S promoter, the vector map is shown in Figure 4 ) and Cs3301-RUBY (RUBY driven by ProCsUBI promoter, vector map see Figure 5 ).

[0060] (2) Agrobacterium preparation and explant infection

[0061] Cs3301-RUBY and 35S3301-RUBY were respectively transferred into competent Agrobacterium by liquid nitrogen method, and positive Agrobacterium single clones were selected by PCR method. After the positive Agrobacterium was propagated, an Agrobacterium infection solution was prepared. The Phalaenopsis orchid variety "A-Ma" is a white-flowered Phalaenopsis orchid, and instantaneous infection of petals can effectively remove background noise. The prepared Agrobacterium infection solution was injected into the petals of "A-Ma" using the pinhole injection method, cultured overnight at 28°C in the dark, and observed and photographed after four days. The results showed that the petals injected with the Cs3301-RUBY vector were more obviously red than those injected with the 35S3301-RUBY vector ( Figure 6 ).

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

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

1.

2. A recombinant DNA construct, characterized in that The recombinant DNA construct comprises the promoter of claim 1.

3. The recombinant DNA construct according to claim 2, characterized in that The recombinant DNA construct further comprises a target DNA molecule operably linked to the promoter.

4. An expression cassette, characterized in that The expression cassette comprises the promoter of claim 1 or the recombinant DNA construct of claim 2 or 3.

5. A recombinant vector, characterized in that The recombinant vector comprises the promoter according to claim 1 or the recombinant DNA construct according to claim 2 or 3 or the expression cassette according to claim 4.

6. A host cell, characterized in that The host cell comprises the promoter according to claim 1, the recombinant DNA construct according to claim 2 or 3, the expression cassette according to claim 4, or the recombinant vector according to claim 5; and the host cell is not a plant cell.

7. Use of any of the following: the promoter according to claim 1, the recombinant DNA construct according to claim 2 or 3, the expression cassette according to claim 4, the recombinant vector according to claim 5, or the host cell according to claim 6: (1) Expressing the target nucleic acid molecule in plants; (2) Construction of transgenic plants; (3) Plant gene editing; (4) Plant genetics and breeding; The plant is an orchid plant.

8. A method for expressing a target nucleic acid molecule in a plant, characterized in that: The method comprises: driving transcription of a target nucleic acid molecule in a plant using the promoter of claim 1; The plant is an orchid plant.

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