Coconut CnUBQ11 promoter, expression vector and application
By constructing a highly efficient transient expression system based on the CnUBQ11 promoter in coconut callus, the problem of low genetic transformation efficiency in coconut plants has been solved, achieving rapid gene function verification and progress in molecular breeding.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HAINAN UNIVERSITY SANYA NANFAN RESEARCH INSTITUTE
- Filing Date
- 2026-03-27
- Publication Date
- 2026-07-10
AI Technical Summary
The low efficiency of genetic transformation in coconut plants, the long and difficult-to-achieve stable genetic transformation technology, and the lack of suitable and efficient promoters limit the process of gene function analysis and genetic improvement.
A highly efficient transient expression system based on coconut callus was constructed. The expression of eGFP and GUS reporter genes was driven by the coconut CnUBQ11 promoter. Exogenous genes were introduced through Agrobacterium infection or gene gun bombardment to achieve rapid and efficient gene expression.
This study achieved efficient and rapid expression of the target gene in coconut callus, filling the gap in rapid gene function analysis platforms, breaking through the bottleneck of stable genetic transformation, promoting the process of molecular breeding, and providing technical reference for other palm trees.
Smart Images
Figure CN121915039B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a coconut CnUBQ11 promoter, expression vector, and its applications. Background Technology
[0002] coconut( Cocos nucifera Coconut (L.) is an important tropical economic crop, hailed as the "tree of life," and plays a pivotal role in the agricultural economy and ecosystems of tropical coastal regions worldwide. As a typical tropical woody monocotyledonous plant, the coconut's unique biological traits (such as fruit development, lipid synthesis, and stress resistance mechanisms) are difficult to fully mimic in herbaceous model plants. Furthermore, traditional stable genetic transformation techniques in coconut face bottlenecks such as long cycles, difficult regeneration, and extremely low transformation efficiency, severely restricting its functional genomics research and genetic improvement progress.
[0003] Plant transgenic technology is one of the core methods of modern agricultural biotechnology, providing important technical support for crop trait genetic improvement, functional gene analysis, and the development of plant bioreactors. Among these, transient expression systems have become a commonly used technical platform for gene function verification and recombinant protein production due to their advantages such as ease of operation, short experimental cycle, no need to obtain stable genetically transformed lines, and rapid assessment of gene function and protein expression levels. For functional gene research in species with low genetic transformation efficiency, such as coconut, there is an urgent need to establish a rapid, efficient, and more physiologically accurate transient expression system. Plant callus tissue, a thin-walled cell mass formed through dedifferentiation, is characterized by vigorous division, homogeneous physiological state, and easy uptake of exogenous genetic material, making it an ideal recipient material for various plant genetic manipulations. Constructing a transient expression system based on callus tissue can circumvent the technical bottleneck of difficult plant regeneration and provide a cellular background and metabolic environment highly similar to the target species, thus providing a more reliable research platform for gene function analysis and signaling pathway research.
[0004] However, one of the key aspects of transgenic technology application lies in the precise regulation of exogenous or endogenous gene expression. Promoters are the core components determining the spatiotemporal expression patterns and intensity of genes. Currently, the types and number of highly efficient promoters suitable for plant genetic transformation are limited, and promoters for coconut callus genetic transformation are even rarer. Therefore, establishing a highly efficient transient expression system specifically for coconut, based on callus tissue, can not only fill the gap in rapid gene function analysis technology platforms for this species and effectively overcome the bottleneck of its difficult stable genetic transformation, but also provide a valuable technical reference and methodological paradigm for other palm trees and even difficult-to-transform woody plants. Summary of the Invention
[0005] The purpose of this invention is to provide a coconut CnUBQ11 promoter, expression vector, and application, which is beneficial to accelerate the identification and verification of genes related to important agronomic traits of coconut and promote the process of molecular breeding.
[0006] This invention provides a coconut CnUBQ11 promoter, the nucleotide sequence of which is shown in SEQ ID NO. 1.
[0007] This invention provides a plant expression vector containing the coconut CnUBQ11 promoter described in the above technical solution.
[0008] Preferably, the plant expression vector comprises: pc1300-UBQ11pro-eGFP and / or pc1300-UBQ11pro-GUS.
[0009] This invention provides the coconut CnUBQ11 promoter according to the above technical solution, or the application of the plant expression vector described above, wherein the application is to drive the expression of a target gene in coconut callus; the plant is coconut.
[0010] Application in driving the expression of target genes.
[0011] Preferably, the target gene includes: eGFP reporter gene or GUS reporter gene.
[0012] This invention provides a method for constructing the plant expression vector, comprising the following steps:
[0013] The coconut CnUBQ11 promoter sequence described in the above technical solution was amplified to obtain the amplified fragment;
[0014] The amplified fragment and the linearized vector were connected to obtain a plant expression vector; the plant was coconut.
[0015] Preferably, when the plant expression vector is used to drive the eGFP reporter gene, the coconut CnUBQ11 promoter sequence is amplified with UBQ11pro-GFP-F and UBQ11pro-GFP-R;
[0016] The nucleotide sequence of the UBQ11pro-GFP-F is shown in SEQ ID NO. 2;
[0017] The nucleotide sequence of the UBQ11pro-GFP-R is shown in SEQ ID NO. 3.
[0018] Preferably, when the plant expression vector is used to drive the eGFP reporter gene, it is through... Hind III and SalThe pc1300-eGFP vector was digested with enzyme I to obtain a linearized vector.
[0019] Preferably, when the plant expression vector is used to drive the GUS reporter gene, the coconut CnUBQ11 promoter sequence is amplified with UBQ11pro-GUS-F and UBQ11pro-GUS-R.
[0020] The nucleotide sequence of the UBQ11pro-GUS-F is shown in SEQ ID NO. 4;
[0021] The nucleotide sequence of the UBQ11pro-GUS-R is shown in SEQ ID NO. 5.
[0022] Preferably, when the plant expression vector is used to drive the GUS reporter gene, it is through... Hind III and Bgl The pc1300-GUS vector was digested with enzyme II to obtain a linearized vector.
[0023] Beneficial effects:
[0024] This invention provides a coconut CnUBQ11 promoter, the nucleotide sequence of which is shown in SEQ ID NO. 1. This promoter can strongly drive the efficient expression of eGFP reporter genes or GUS reporter genes.
[0025] This invention also provides a plant expression vector containing the coconut CnUBQ11 promoter and its applications. Using callus tissue induced from immature or zygotic coconut embryos as a recipient, exogenous genes are introduced via methods such as Agrobacterium infection or gene gun bombardment, achieving rapid, efficient, and transient expression of the target gene in callus cells. This is applicable to various fields such as functional gene verification, rapid protein production, and gene editing system testing.
[0026] In summary, the technical solution of this invention is a method specifically designed for coconut, based on the construction of a highly efficient transient expression system using callus tissue. This method not only fills the gap in rapid gene function analysis technology platforms for this species and overcomes the obstacle of difficult stable transformation, but also provides a referable technical solution for other palm families and even difficult-to-transform woody plants. It can greatly accelerate the identification and verification of genes related to important agronomic traits in coconut, promote its molecular breeding process, and is expected to expand into the field of rapid expression and production of high-value plant-derived proteins (such as enzymes and medicinal proteins), possessing significant theoretical and practical value. Attached Figure Description
[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0028] Figure 1 Electrophoresis diagrams for detecting the linkage of pc1300-eGFP and pc1300-GUS vectors to the CnUBQ11 promoter;
[0029] Figure 2 Image showing the detection of eGFP signal in coconut callus transiently transformed by CnUBQ11-eGFP plasmid;
[0030] Figure 3 The results of GUS staining of coconut callus after transient transformation with CnUBQ11-GUS plasmid. Detailed Implementation
[0031] Unless otherwise specified, all equipment, reagents and methods used in this invention are conventionally purchased.
[0032] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0033] Example 1
[0034] 1. Construction of plant overexpression + reporter gene vector
[0035] The expression of the reporter genes eGFP and GUS is driven by the promoter of the coconut housekeeper gene CnUBQ11.
[0036]
[0037] The eGFP reporter gene expression vector was designed and ligated. Primers for amplification of the CnUBQ11 promoter (SEQ ID NO. 1) were UBQ11pro-GFP-F and UBQ11pro-GFP-R. The pc1300-eGFP vector digested with HindIII and SalI was ligated using homologous recombination. The successfully constructed vector was named pc1300-UBQ11pro-eGFP, where:
[0038] The nucleotide sequence of UBQ11pro-GFP-F (SEQ ID NO. 2) is as follows: 5'-GTAAAACGACGGCCAGTGCCAAGCTTCTAAATAGGGCAAAACAAGG-3';
[0039] The nucleotide sequence of UBQ11pro-GFP-R (SEQ ID NO. 3) is as follows: 5'-GGTACCAGATCTACTAGTGGTACCCTGTAATAAGAGATCCAAGAGATAA-3'.
[0040] The design of the ligation-driven GUS reporter gene expression vector, using primers UBQ11pro-GUS-F and UBQ11pro-GUS-R for amplification of the CnUBQ11 promoter (SEQ ID NO. 1), was followed by ligation of the pc1300-GUS vector digested with HindIII and BglII using homologous recombination. The successfully constructed vector was named pc1300-UBQ11pro-GUS, where:
[0041] The nucleotide sequence of UBQ11pro-GUS-F (SEQ ID NO. 4) is as follows: 5'-GTCGACCTGCAGGCATGCAAGCTTCTAAATAGGGCAAAACAAGG-3';
[0042] The nucleotide sequence of UBQ11pro-GUS-R (SEQ ID NO. 5) is as follows: 5'-AAAACTAGAAATTTACCCTCAGATCTACCATCTGTAATAAGAGATCCAAGAGATAA-3'.
[0043] The promoter sequence was amplified using Novizan high-fidelity enzyme C112, and the vector was constructed using the Uniclone One Step Seamless Cloning Kit from Jinsha Biotechnology Co., Ltd.
[0044] 2. Instantaneous transformation of coconut callus tissue
[0045] The constructed pc1300-UBQ11pro-eGFP, pc1300-UBQ11pro-GUS and control empty vector plasmids pc1300-eGFP and pc1300-GUS were heat-shocked and transformed into competent Agrobacterium tumefaciens cells of EHA105. After single colony selection, the cells were cultured in a shaker at 28°C for 1-2 days. PCR verification was performed using promoter primers (SEQ ID NO. 2-SEQ ID NO. 5), and positive clones were selected for amplification culture.
[0046] Positive cells were collected using a rotation speed of 3500 rpm, then resuspended in Y3 medium (EEUWENS Y3 Basal medium, Phytotechnology laboratory) containing 200 µM acetylsyringone, and the OD was adjusted. 600 =0.2. Embryogenic callus induced from coconut embryos was used as explants. The explants were immersed in a resuspension and vacuum-sealed for 5 min, then infected at room temperature for 30 min at 45 rpm. After infection, the explants were placed on sterile paper to absorb the liquid and co-cultured in the dark at 28°C for 3 days on Y3 medium containing 100 µM acetylsylcholine. After co-culture, the explants were washed 3-4 times with liquid Y3 medium containing 500 mg / L carbenicillin, and then subjected to recovery culture for 1 week (28°C, 16 / 8 h) on Y3 medium containing 250 mg / L carbenicillin. Subsequently, selection culture was performed on Y3 medium containing hygromycin resistance (30 μg / mL).
[0047] Positive transformation materials and control materials were selected for reporter gene detection. GFP signal was detected by taking pictures using a handheld confocal microscope. GUS staining was performed using the Cooler Master SL7160 staining kit.
[0048] 3. Experimental Results
[0049] (1) Construction of GFP and GUS expression vectors driven by CnUBQ11 promoter
[0050] The target sequence was amplified using CnUBQ11 promoter amplification primers. After linearization of the pc1300-eGFP and pc1300-GUS vectors with their respective enzymes, ligation of the target sequence was performed using a 5 µL homologous recombination reaction system. Positive clones were detected using flanking primers at the vector insertion site and promoter amplification primers. Results are shown in [Figure number missing]. Figure 1 The correct clone was preserved after sequencing verification.
[0051] (2) Detection of eGFP signal in coconut callus transiently transformed
[0052] The correct clone was transformed into EHA105, and then transiently transformed into coconut callus (denoted as UBQ11pro::eGFP) using the above procedures. An empty vector was used as a control. Callus samples were then subjected to confocal scanning. Results are shown below. Figure 2 (exist Figure 2 In the diagram, Nos is the terminator used to terminate transcription; GFP is the green fluorescence channel image; Bright field is the bright field image; Merge is the overlay of the fluorescence channel and bright field images; UBQ11pro::eGFP is the transformed sample with pCnUBQ11 promoter-driven eGFP expression; Control is the transformed sample with empty vector.
[0053] Combination Figure 2 The results showed that coconut callus had GFP signal, which was much higher than that of the empty vector control, indicating that the exogenous plasmid was successfully expressed in the callus.
[0054] (3) Detection of GUS signal in coconut instantaneous callus transformation
[0055] The correct clone was transformed into EHA105, and then transiently transformed into coconut callus (denoted as CnUBQ11pro::GUS) using the above procedures. An empty vector was used as a control. Callus samples were then subjected to GUS staining. Results are shown below. Figure 3 (exist Figure 3 In the text, Nos is a terminator used to terminate transcription; CnUBQ11pro::GUS is a transformed sample with CnUBQ11 promoter driving GUS reporter gene expression; Control is a transformed sample with an empty vector.
[0056] Combination Figure 3 The results showed that the CnUBQ11pro::GUS sample exhibited a distinct blue color, indicating that pCnUBQ11 could efficiently drive downstream gene expression; the control sample showed only a very light background color, excluding non-specific staining or autofluorescence interference. It is evident that the coconut callus showed a much higher GUS staining signal than the empty vector control, indicating that the exogenous plasmid was successfully expressed in the callus.
[0057] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A coconut CnUBQ11 promoter, characterized in that, The nucleotide sequence of the coconut CnUBQ11 promoter is shown in SEQ ID NO.
1.
2. A plant expression vector containing the coconut CnUBQ11 promoter as described in claim 1.
3. The plant expression vector according to claim 2, characterized in that, The plant expression vectors include: pc1300-UBQ11pro-eGFP and / or pc1300-UBQ11pro-GUS; The pc1300-UBQ11pro-eGFP is obtained by amplifying the CnUBQ11 promoter with the nucleotide sequence shown in SEQ ID NO. 1 and then ligating it into the pc1300-eGFP vector using homologous recombination. The pc1300-UBQ11pro-GUS is obtained by amplifying the CnUBQ11 promoter with the nucleotide sequence shown in SEQ ID NO. 1 and then ligating it into the pc1300-GUS vector using homologous recombination.
4. The application of the coconut CnUBQ11 promoter according to claim 1 or the plant expression vector according to claim 2 or 3, characterized in that, The application is to drive the expression of the target gene in coconut callus tissue; The plant in question is a coconut.
5. The application according to claim 4, characterized in that, The target gene includes: eGFP reporter gene or GUS reporter gene.
6. The method for constructing the plant expression vector according to claim 2 or 3, characterized in that, Includes the following steps: Amplify the coconut CnUBQ11 promoter sequence as described in claim 1 to obtain the amplified fragment; The amplified fragment and the linearized vector were connected to obtain the plant expression vector; The plant in question is a coconut.
7. The construction method according to claim 6, characterized in that, When the plant expression vector is used to drive the eGFP reporter gene, the coconut CnUBQ11 promoter sequence is amplified with UBQ11pro-GFP-F and UBQ11pro-GFP-R; The nucleotide sequence of the UBQ11pro-GFP-F is shown in SEQ ID NO. 2; The nucleotide sequence of the UBQ11pro-GFP-R is shown in SEQ ID NO.
3.
8. The construction method according to claim 6, characterized in that, When the plant expression vector is used to drive the eGFP reporter gene, the pc1300-eGFP vector is linearized by digesting it with HindIII and SalI enzymes.
9. The construction method according to claim 6, characterized in that, When the plant expression vector is used to drive the GUS reporter gene, the coconut CnUBQ11 promoter sequence is amplified with UBQ11pro-GUS-F and UBQ11pro-GUS-R. The nucleotide sequence of UBQ11pro-GUS-F is shown in SEQ ID NO. 4; The nucleotide sequence of the UBQ11pro-GUS-R is shown in SEQ ID NO.
5.
10. The construction method according to claim 6, characterized in that, When the plant expression vector is used to drive the GUS reporter gene, the pc1300-GUS vector is linearized by digesting it with HindIII and BglII enzymes.