Promoter and application thereof in orchid

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a promoter and application thereof in orchid plants. The invention provides an orchid endogenous promoter which is a powerful constitutive promoter and can drive a target gene to be powerfully expressed in various tissues such as roots, stems, leaves and flowers of an orchid. Compared with a CaMV 35S promoter, the promoter has the advantages that the driving efficiency of the gene in orchid plants is greatly improved, an important tool is provided for molecular breeding of plants, and the promoter has a relatively good application prospect.

Description

Technical Field

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

[0002] Cymbidium ( Cymbidium ) plants and Phalaenopsis ( Phalaenopsis ) plants are the most culturally and economically valuable groups in Orchidaceae. Their unique flower shapes, rich colors and charming aromas are highly favored. Although significant progress has been made in the breeding of Orchidaceae plants in the past few decades, many challenges still remain. The breeding of Cymbidium plants and Phalaenopsis plants mainly relies on traditional cross-breeding, selection breeding and mutagenesis breeding. Although these methods have promoted variety improvement to a certain extent, there are also problems such as high genetic complexity and long breeding cycles. In contrast, transgenic breeding and gene editing breeding technologies can effectively solve the pain points in traditional breeding and provide a new breakthrough for plant breeding. In the construction of a specific molecular breeding system for Orchidaceae plants, the screening and utilization of promoters are crucial steps. As a key element for regulating gene expression, promoters play an important role in regulating the efficient and stable expression of target genes in plants. At present, in plants, the cauliflower mosaic virus promoter CaMV 35S (hereinafter referred to as 35S) of tobacco is the most commonly used constitutive promoter, which can drive gene expression in plants. However, this promoter has the problem of low gene expression driving efficiency in monocotyledonous plants, which limits its application in the breeding of Orchidaceae plants. Orchidaceae plants have a unique gene expression regulation mechanism, and their endogenous promoters may have high efficiency, but there is still a lack of endogenous strong promoters in Orchidaceae plants. Therefore, it is of great significance to develop promoters derived from Orchidaceae plants. Summary of the Invention

[0003] The present invention provides a promoter and its application in Orchidaceae plants.

[0004] In the process of studying the promoters of Orchidaceae plants, the present invention successfully identified a promoter of a ubiquitin gene (Ubiquitin, UBI). This promoter can efficiently drive gene expression in various tissues of Orchidaceae plants, showing significant regulatory functions, and the expression level of the gene it drives is significantly higher than that 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, and the nucleotide sequence of the promoter is as shown in SEQ ID NO.1.

[0007] The promoter with the nucleotide sequence shown in SEQ ID NO.1 was cloned from Cymbidium sinense, an orchid plant, which is located upstream of the ubiquitin gene and is an endogenous ubiquitin promoter of Cymbidium sinense. In the present invention, it is named promoter ProCsUBI. It has been verified that this promoter is a strong constitutive promoter, which can drive the efficient expression of target genes in different orchid plants and can be expressed in different plant tissues.

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

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

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

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

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

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

[0014] In a fourth aspect, the present invention provides a recombinant vector, which comprises the promoter or the recombinant DNA construct or the expression cassette described above.

[0015] In the present invention, the vectors include, but are not limited to, plasmid vectors, transposons, viral vectors, artificial chromosome vectors, etc. Among them, the plasmid vector can be a cloning vector, an expression vector or an integrative vector.

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

[0017] In some embodiments of the present invention, the promoter is replaced with the 35S promoter in the pCAMBIA3301 vector, and an efficient expression vector suitable for orchids is successfully constructed. In addition, the promoter is 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 of the pCAMBIA3301 vector is used as the internal reference gene. The eGFP expression driven by the promoter shown in SEQ ID NO.1 is 60 times higher than the Bar gene expression in the leaves of Molan and 30 times higher than the Bar gene expression in the leaves of Jianlan. In the RUBY gene expression verification, in the petals of Phalaenopsis, compared with the 35S promoter, the RUBY gene driven by the promoter shown in SEQ ID NO.1 makes the petals redder.

[0018] In a fifth aspect, the present invention provides a host cell, wherein the host cell comprises the promoter or the recombinant DNA construct or 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: (1) Expressing the target nucleic acid molecule in plants; (2) Construction of transgenic plants; (3) Plant gene editing; (4) Plant genetics and breeding.

[0022] The applications described in (1) to (4) above include: using a promoter with a nucleotide sequence such as SEQ ID NO.1 to drive the transcription of the target nucleic acid molecule, or include: introducing a vector containing a promoter with a nucleotide sequence such as SEQ ID NO.1 into the plant.

[0023] The promoter with the nucleotide sequence shown in SEQ ID NO.1 provided by the present invention can not only be used to drive the transcription of genes encoding proteins, but also be used to drive the transcription of DNA molecules encoding RNAs. The nucleic acid molecule driven by the promoter can be a nucleic acid molecule endogenous to the plant (for example, for overexpression of endogenous genes), or a heterologous nucleic acid molecule (for example, for expression of heterologous genes). Therefore, the above-mentioned promoter or the recombinant DNA construct or the expression cassette or the recombinant vector or the host cell can be used for constructing 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 the Cas gene or sgRNA for plant gene editing), or plant genetic breeding.

[0024] Preferably, the plant is a monocotyledonous plant, more preferably an orchid plant. The orchid plants include plants of the genus Cymbidium and plants of the genus Phalaenopsis. Among them, the plants of the genus Cymbidium can be any species under this genus, including but not limited to Cymbidium sinense ( Cymbidium sinense ), Cymbidium ensifolium ( Cymbidium ensifolium ), Cymbidium hybridum ( Cymbidium hybridum ), Cymbidium goeringii ( Cymbidium goeringii ), Cymbidium kanran ( Cymbidium kanran ), Cymbidium lancifolium ( Cymbidium lancifolium ), etc. The plants of the genus Phalaenopsis can be any species under this genus, including but not limited to Phalaenopsis amabilis ( Phalaenopsis amabilis ), Phalaenopsis equestris ( Phalaenopsis equestris ), Phalaenopsis aphrodite ( Phalaenopsis schilleriana ), Phalaenopsis amboinensis ( Phalaenopsis cornu-cervi ), Phalaenopsis nocturna ( Phalaenopsis bellina ), etc.

[0025] In a seventh aspect, the present invention provides a method for expressing a target nucleic acid molecule in a plant, the method comprising: driving the transcription of the target nucleic acid molecule in the plant with a promoter having a nucleotide sequence as shown in SEQ ID NO.1.

[0026] Preferably, the method comprises: operably connecting the promoter to the target nucleic acid molecule and then introducing it into the plant; or comprises: operably connecting the promoter to the target nucleic acid molecule, then ligating it to a vector to obtain a recombinant vector, and introducing the recombinant vector into the plant.

[0027] Preferably, the plant is a monocotyledonous plant, more preferably an orchid plant. The orchid plants include plants of the genus Cymbidium and plants of the genus Phalaenopsis.

[0028] After connecting the promoter to the target gene and transferring it into a plant, the promoter can drive the constitutive and strong expression of the target gene in various tissues of the plant, such as roots, stems, leaves, and flowers.

[0029] In an eighth aspect, the present invention provides a method for constructing a transgenic plant, the method comprising: transcribing a target nucleic acid molecule in a plant driven by a promoter having a nucleotide sequence as shown in SEQ ID NO.1.

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

[0031] Preferably, the plant is a monocotyledonous plant, more preferably an orchid plant. The orchid plants include plants of the genus Cymbidium and plants of the genus Phalaenopsis.

[0032] In a ninth aspect, the present invention provides a transgenic plant constructed by the above method.

[0033] In a tenth aspect, the present invention provides a method for obtaining processed agricultural products, comprising: processing the above-mentioned transgenic plant or its harvested parts to obtain processed agricultural products.

[0034] The beneficial effects of the present invention at least include: the present invention provides an endogenous promoter of an orchid plant, which is a strong constitutive promoter and can drive the strong expression of a target gene in various tissues of plants of the genus Cymbidium or plants of the genus Phalaenopsis; compared with the 35S promoter, this promoter greatly improves the driving efficiency of genes in plants of the genus Cymbidium and plants of the genus Phalaenopsis, provides an important tool for plant molecular breeding, and also lays a foundation for further analyzing the gene functions of orchid plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0036] Figure 1 Expression analysis of the ubiquitin gene of Cymbidium sinense in various tissues in Example 1 of the present invention ( CsUBIs )

[0037] Figure 2 Cs3301-eGFP expression vector map in Example 2 of the present invention.

[0038] Figure 3 Expression analysis of eGFP driven by the ProCsUBI promoter in Cymbidium sinense and Cymbidium ensifolium leaves in Example 2 of the present invention.

[0039] Figure 4 Map of the 35S3301-RUBY expression vector in Example 3 of the present invention.

[0040] Figure 5 Map of the Cs3301-RUBY expression vector in Example 3 of the present invention.

[0041] Figure 6 Phenotype of transient expression of the RUBY gene driven by the ProCsUBI and 35S promoters in Phalaenopsis petals in Example 3 of the present invention. The left side is RUBY driven by the 35S promoter, and the right side is RUBY driven by the ProCsUBI promoter. Detailed implementation manners

[0042] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0043] 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; the nucleotide sequence of the RUBY gene is shown in SEQ ID NO.4.

[0044] Example 1 Obtaining of the Cymbidium sinense ubiquitin promoter Homologous sequence analysis was performed using the amino acid sequence of maize UBI as the parent. A total of 5 UBI genes with relatively high homology were found in Cymbidium sinense ( CsUBIa , CsUBIb , CsUBIc , CsUBId , CsUBIe ). The transcriptome data of Cymbidium sinense root, stem, leaf, and flower tissues were downloaded, and the expression levels of the above 5 ubiquitin genes in various tissues of Cymbidium sinense were analyzed by Fragments Per Kilobase of exon model per Million mapped fragments (FPKM). The results showed that CsUBIb was highly expressed in all tissues ( Figure 1 ), so the promoter sequence of CsUBIb was selected.

[0045] Example 2 Transient expression of eGFP gene driven by the Cymbidium orchid ubiquitin promoter in Cymbidium orchid and Cymbidium orchid leaves (1) Obtaining the ubiquitin promoter of Cymbidium orchid and constructing its expression vector Intercepting the Molan gene CsUBIb The first 1500 bp sequence (SEQ ID NO.1) was used as the ProCsUBI promoter sequence of Cymbidium orchid. The ProCsUBI promoter and the green fluorescent protein gene eGFP were co-integrated into the pCAMBIA3301 vector, and the expression of the eGFP gene was driven by the Cymbidium orchid ubiquitin promoter ProCsUBI. The recombinant vector was named Cs3301-eGFP ( Figure 2 ).

[0046] (2) Agrobacterium preparation and leaf injection Cs3301-eGFP was transferred into competent Agrobacterium by liquid nitrogen method, and positive Agrobacterium single clone was selected by PCR method. After the positive Agrobacterium was propagated, Agrobacterium infection solution was prepared, and the Agrobacterium infection solution was injected into the leaves of Molan and Jianlan by pinhole injection method, respectively, and cultured overnight at 28℃ in the dark, and observed and photographed after four days.

[0047] (3) Bar test strip detection and eGFP expression analysis The leaf area injected with Agrobacterium infection solution in (2) above was cut off and stored in liquid nitrogen. 200 mg of the explant was fully ground and tested using Bar test paper (Youlong Biotechnology), and the result showed positive. Another 200 mg sample was fully ground under liquid nitrogen, RNA was extracted using the 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 itself was used as the internal reference gene, and the expression level of eGFP was analyzed by real-time fluorescence quantitative PCR. The results showed that in Jianlan, the ratio of the gene expression level driven by the ProCsUBI promoter to the gene expression level driven by the 35S enhanced promoter was more than 30 times; in Molan, the ratio of the gene expression level driven by the ProCsUBI promoter to the gene expression level driven by the 35S enhanced promoter was more than 60 times ( Figure 3 ).

[0048] Example 3 Transient expression of RUBY gene driven by the Ubiquitin promoter in Phalaenopsis orchids (1) Construction of an overexpression vector of the RUBY gene driven by the Ubiquitin promoter of Cymbidium orchid The RUBY system can efficiently synthesize betalain in plant tissues, making the plants show red-purple color, and it is a commonly used plant indicator gene. The 35S promoter and the ProCsUBI promoter were respectively integrated with the RUBY expression system into the pCAMBIA3301 vector to construct the vector 35S3301-RUBY (driving RUBY by the 35S promoter, the vector map is shown in Figure 4 ), and Cs3301-RUBY (driving RUBY by the ProCsUBI promoter, the vector map is shown in Figure 5 ).

[0049] (2) Agrobacterium preparation and explant infection Cs3301-RUBY and 35S3301-RUBY were respectively transferred into competent Agrobacterium by the liquid nitrogen method, and positive Agrobacterium monoclonal colonies were selected by the PCR method. After the positive Agrobacterium was propagated, an Agrobacterium infection solution was prepared. The Phalaenopsis variety "Ama" is a white Phalaenopsis, and transiently infecting the petals can effectively remove background noise. The prepared Agrobacterium infection solution was injected into the petals of "Ama" by the pinhole injection method, and cultured overnight at 28 °C in the dark. After four days, observations and photographs were taken. The results showed that the petals injected with the Cs3301-RUBY vector were more obviously red than those injected with 35S3301-RUBY ( Figure 6 ).

[0050] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the 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 also 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 or the recombinant DNA construct according to claim 2 or 3 or the expression cassette according to claim 4 or the recombinant vector according to claim 5.

7. Any of the following uses of 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.

8. The use according to claim 7, characterized in that: The plant is an orchidaceae plant.

9. A method for expressing a target nucleic acid molecule in a plant, characterized in that: The method comprises: driving the transcription of a target nucleic acid molecule in a plant with the promoter of claim 1.

10. The method according to claim 9, characterized in that The plant is an orchidaceae plant.

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