Oil palm ubq promoter and use thereof

Abstract

The present application relates to an oil palm UBQ promoter and application thereof, and belongs to the technical field of biology.The nucleic acid sequence of the promoter is shown as SEQ ID NO.1, the present application firstly aligns and intercepts a sequence of 2000bp upstream of a start codon in an oil palm genome, and synthesizes an oil palm RNA polymerase II type promoter, i.e., an oil palm endogenous ubiquitin promoter EgUBQ.The promoter has transcription activity, and can drive expression of downstream green fluorescent protein NeonGreen.The present application provides an oil palm endogenous UBQ promoter for genetic transformation and gene editing of oil palm.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an oil palm UBQ promoter and its applications. Background Technology

[0002] Oil palm (scientific name: Elaeis guineensis Oil palm is an important tropical economic palm crop. Currently, the breeding of new oil palm varieties in my country still mainly relies on traditional hybridization breeding, which suffers from low efficiency and long cycles, seriously hindering the progress of high-yield and high-quality oil palm breeding. With the development of biotechnology, the application of molecular breeding technologies, represented by gene editing technology, in various crops has accelerated the breeding of new varieties, but related research on oil palm still needs to be carried out.

[0003] Genome editing technology is an important tool for studying gene function. It can precisely modify genes at specific sites on the chromosomes of recipient cells, efficiently generating functionally inactive mutants of specific genes and providing high-quality genetic material for functional genomics research. The application of gene editing technology highly relies on transcriptional regulatory elements capable of precisely controlling gene expression. Whether it's the expression of small RNA molecules (such as various sgRNAs edited by CRISPR / Cas), the expression of nucleases like CAS9, or the expression of reporter genes like fluorescent protein genes, all require promoters. Ubiquitin (UBQ) is a constitutively expressed gene in plants. Due to its stable and high expression levels in various tissues and at different developmental stages, its promoter is often used to drive coding genes that require stable and high-level expression. The UBQ promoter is a type II RNA polymerase promoter and has been widely used in CRISPR / Cas9 systems in many species, such as maize.

[0004] Although CRISPR / Cas9 genome editing technology has been widely used in many species, gene editing techniques for oil palm are rarely reported. This is mainly because, while UBQ promoters have been extensively reported in many species, exogenous UBQ promoters are often not suitable. Therefore, the lack of suitable UBQ promoters has become a limiting factor for current CRISPR / Cas9 gene editing systems in oil palm. Thus, screening for functionally active UBQ promoters in oil palm is of positive significance for the development of gene breeding technology in oil palm. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an oil palm UBQ promoter and its application, so as to solve the problem that there is no suitable UBQ promoter in the existing oil palm gene editing system.

[0006] The technical solution of this invention mainly includes the following: To achieve the above objectives, this invention provides an oil palm UBQ promoter, EgUBQ-1, the nucleotide sequence of which is shown in SEQ ID NO.1. This promoter belongs to the type II RNA polymerase promoter of the oil palm Ubiquitin gene and is derived from African oil palm (…). Elaeis guineensis ).

[0007] Meanwhile, the present invention also provides biological materials containing the oil palm UBQ promoter EgUBQ-1, including a recombinant vector, an expression cassette, and recombinant bacteria, wherein the recombinant bacteria are preferably Escherichia coli.

[0008] Furthermore, this invention also discloses the application of the oil palm UBQ promoter and the aforementioned biological materials in driving fluorescent protein genes in the transformation of oil palm protoplasts. The fluorescent protein specifically includes NeonGreen fluorescent protein.

[0009] The present invention also constructed an oil palm transient transformation vector containing the promoter and a NeonGreen fluorescent protein expression vector, which were obtained by linking the UBQ promoter EgUBQ-1 with the NeonGreen gene and a plant expression vector.

[0010] Furthermore, this invention provides a method for transforming a fluorescent protein in plant protoplasts using this promoter. The method includes constructing an expression vector, transforming *E. coli* to propagate the plasmid, and introducing the plasmid into plant protoplasts via PEG-mediated protoplast transformation. Specifically, it includes the following steps: ligating the UBQ promoter EgUBQ-1 with the NeonGreen gene and a plant expression vector to obtain the pEgUBQ-NeonGreen expression vector; transforming the pEgUBQ-NeonGreen expression vector into *E. coli* DH5α for propagation to obtain a plasmid; and using the plasmid for plant protoplast transformation. The plant protoplast includes oil palm protoplasts.

[0011] Specifically, the PEG-mediated protoplast transformation method mainly includes the following steps: (1) Preparation of PEG instantaneous conversion solution: Dissolve 4g of PEG in 10mL of deionized water (ddH2O). The solution contains 0.8M mannitol and different concentrations of calcium chloride (0.1M, 0.2M, 0.3M, 0.4M, 0.5M).

[0012] (2) The isolated protoplasts were first incubated in an ice bath for 30 minutes, followed by centrifugation to remove the supernatant. The protoplasts were then resuspended in an equal volume of mannitol-magnesium-glucose (MMG) solution (0.4 M mannitol, 15 mM magnesium chloride, 4 mM MES, pH 5.8). Different concentrations of high-concentration plasmid DNA (10 µg, 15 µg, 20 µg, 30 µg, and 40 µg) were aliquoted into 2 mL centrifuge tubes, followed by the addition of 100 µL of resuspended protoplasts. Gently mix to ensure uniform distribution. An equal volume of freshly prepared PEG transient transformation solution was then added to the protoplast suspension, and the tubes were gently inverted to mix thoroughly. The mixture was then heat-shocked (at 42 °C) for 1 minute. Additionally, the protoplasts and plasmids were incubated in the dark at 25 ± 1 °C for 30 minutes. Transient transformation was terminated by diluting the reaction mixture with 600 µL of W5 solution. After centrifuging the suspension at 150 g for 2 minutes, the resulting protoplast precipitate was gently resuspended in 2 mL of W5 solution. Incubate in the dark at 25±1℃ for 16 hours. After incubation, centrifuge at 150 g for 2 minutes and carefully remove approximately 75% of the supernatant. To minimize mechanical damage, the protoplast precipitate was gently resuspended.

[0013] The beneficial effects of this invention are: This invention is the first to obtain the type II promoter of oil palm RNA polymerase, namely the oil palm endogenous UBQ promoter EgUBQ, in oil palm.

[0014] This invention is the first to link a cloned oil palm endogenous RNA polymerase type II promoter to drive NeonGreen fluorescent protein. The feasibility of this promoter driving fluorescent protein expression was verified by transiently transforming oil palm leaf protoplasts, providing a rapid and effective method for subsequent screening and verification of other oil palm endogenous UBQ promoters. Attached Figure Description

[0015] Figure 1 : Simplified structure diagram of NeonGreen expression vector.

[0016] Figure 2 : Simplified structure diagram of pEgUBQ-NeonGreen expression vector.

[0017] Figure 3 : Results of transient transformation of oil palm protoplasts (empty vector control: NeonGreen expression vector, pEgUBQ-1-NeonGreen: pEgUBQ-NeonGreen expression vector). Detailed Implementation

[0018] To facilitate a clearer understanding of the technical content of this invention by those skilled in the art, the invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0020] The carriers used in the following examples were purchased from Wemi Biotechnology Co., Ltd. The above-mentioned biological materials are only used to repeat the relevant experiments of the present invention and should not be used for other purposes.

[0021] Example 1: Obtaining and analyzing the transcriptional activity of the oil palm UBQ gene promoter EgUBQ-1. Using the mRNA sequence of the sorghum polyubiquitin (LOC8063786) gene (Genebank accession number: XM_002438859.2) as a reference, the TBtools software was used to align the sequence with the African oil palm genome (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_000442705.2 / ). Homology analysis identified a candidate EgUBQ gene on oil palm chromosome 2. The 2000bp sequence upstream of the start codon of this gene (located at 96854779bp-96856778bp on African oil palm chromosome 2) was obtained as the EgUBQ promoter sequence of oil palm (SEQ ID NO.1).

[0022] The 2000bp oil palm EgUBQ promoter sequence was directly synthesized, and the empty vector NeonGreen and the oil palm pEgUBQ-NeonGreen expression vector were constructed by Weimi Biotechnology Co., Ltd. A schematic diagram of the vectors is shown below. Figure 1 and Figure 2 .

[0023] After isolating oil palm leaf protoplasts, the above-mentioned vector was transformed into Escherichia coli DH5α for propagation to obtain high-concentration plasmids for PEG-mediated transient transformation of oil palm protoplasts.

[0024] The transient transformation procedure using oil palm protoplasts was as follows: The isolated protoplasts were first incubated on ice for 30 minutes, followed by centrifugation to remove the supernatant. The protoplasts were then resuspended in an equal volume of mannitol-magnesium-glucose (MMG) solution (0.4 M mannitol, 15 mM magnesium chloride, 4 mM MES, pH 5.8). 40 µg of plasmid DNA was aliquoted into 2 mL centrifuge tubes, followed by the addition of 100 µL of the resuspended protoplasts. The mixture was gently mixed to ensure uniform distribution. An equal volume of freshly prepared PEG transient transformation solution (containing 4 g PEG, 10 mL deionized water, 0.8 M mannitol, and 0.4 M calcium chloride) was then added to the protoplast suspension, and the tube was gently inverted to mix thoroughly. A heat shock treatment was then performed at 42 °C for 1 minute. Additionally, the protoplasts and plasmids were incubated in the dark at 25 ± 1 °C for 30 minutes. The transient transformation was terminated by diluting the reaction mixture with 600 µL of W5 solution. After centrifuging the suspension at 150 g for 2 minutes, the resulting protoplast precipitate was gently resuspended in 2 mL of W5 solution (containing 2 mmol / L 2-morpholinoethanesulfonic acid, 154 mmol / L NaCl, 125 mmol / L CaCl2, and 5 mmol / L KCl). The protoplasts were incubated in the dark at 25 ± 1 °C for 16 hours. After incubation, the protoplasts were centrifuged at 150 g for 2 minutes, and approximately 75% of the supernatant was carefully removed. To minimize mechanical damage, the protoplast precipitate was gently resuspended.

[0025] The isolated protoplasts were observed using a fluorescence stereomicroscope (Nikon SMZ18) with an excitation wavelength of 488 nm. The fluorescence intensity of the protoplasts was assessed and images were acquired using the NeonGreen channel of an inverted fluorescence microscope (excitation wavelength 488 nm, Thermo Fisher Scientific EVOS M5000).

[0026] After transformation of oil palm protoplasts, fluorescence detection results are shown in the figure. Figure 3 The empty vector NeonGreen served as a negative control, and no green fluorescence signal was detected. The oil palm pEgUBQ-NeonGreen expression vector showed green fluorescence, indicating that EgUBQ has transcriptional activity.

[0027] Therefore, the endogenous UBQ promoter of oil palm in this invention can effectively drive the expression of exogenous genes in oil palm protoplasts, providing a reliable endogenous promoter tool for oil palm gene function research. The successful application of this promoter provides support for constructing gene editing vectors that drive Cas9 and establishing an oil palm gene editing technology system.

[0028] The above description is only a part of the embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. The application of the oil palm UBQ promoter EgUBQ-1, characterized in that, The nucleotide sequence of the UBQ promoter EgUBQ-1 is shown in SEQ ID NO.

1. The application is to drive the transient conversion of the NeonGreen fluorescent protein gene in oil palm protoplasts.

2. A NeonGreen fluorescent protein expression vector, characterized in that, The expression vector was obtained by linking the UBQ promoter EgUBQ-1 as described in claim 1 with the NeonGreen gene and a plant expression vector, wherein the plant is an oil palm.

3. A method for driving the transformation of fluorescent proteins in plant protoplasts, characterized in that, The process includes the following steps: ligating the UBQ promoter EgUBQ-1 described in claim 1 with the NeonGreen gene and a plant expression vector to obtain the pEgUBQ-NeonGreen expression vector; transforming the pEgUBQ-NeonGreen expression vector into Escherichia coli DH5α for propagation to obtain a plasmid; and using the plasmid for transient protoplast transformation of a plant, wherein the plant is an oil palm.

4. The method according to claim 3, characterized in that, The transient transformation of plant protoplasts was carried out via PEG-mediated protoplast transformation.

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