A coconut U6 promoter and its application

Abstract

The invention relates to a coconut U6 promoter and an application thereof, and belongs to the field of biotechnology. The nucleotide sequence of the promoter is shown in SEQ ID NO.1. The invention clones and obtains a coconut RNA polymerase III type promoter, a coconut endogenous U6 promoter CnU6, in a coconut genome for the first time. The promoter has high-efficiency transcription activity, can drive the expression of downstream fluorescent proteins, and verifies the activity of the promoter by transiently transforming coconut protoplasts. The method can be used to detect candidate coconut endogenous promoters, and can also be further used to truncate and screen core U6 promoters.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a coconut U6 promoter and application thereof. Background Art

[0002] Coconut (scientific name: Cocos nucifera L.) is an important tropical economic palm crop. At present, the selection and breeding of new coconut varieties in my country is still mainly based on traditional hybrid breeding, which has problems of low efficiency and long cycle, seriously hindering the process of high-yield and high-quality coconut breeding. With the development of biotechnology, the application of molecular breeding technology represented by gene editing technology in a variety of crops has accelerated the cultivation of new varieties, but relevant research on coconut has yet to be carried out.

[0003] Genome editing technology is an important tool for studying gene function. It can accurately modify genes at specific sites on the chromosomes of recipient cells, efficiently produce functionally inactive mutants of specific genes, and provide high-quality genetic materials for biological functional genomic research. sgRNA and Cas9 are two necessary components of this technology system, among which sgRNA plays a role in targeting and binding to the target gene site during the CRISPR / Cas9 genome editing process. Studies have shown that in the CRISPR / Cas9 gene editing system, the content level of sgRNA in recipient cells is one of the important factors affecting the editing efficiency. Therefore, polymerase III type promoters that can accurately initiate sgRNA transcription in vivo have received widespread attention. U6RNA is a non-coding RNA involved in the splicing of pre-mRNA, and its corresponding U6 promoter is a type of RNA polymerase III promoter, which has been widely used in the CRISPR / Cas9 system of many species.

[0004] Although CRISPR / Cas9 genome editing technology has been widely used in many species, there is no report on gene editing technology for coconut. This is mainly because although there are a lot of reports on U6 promoters in many species, exogenous U6 promoters are usually not applicable. It can be seen that the lack of applicable U6 promoters has become a limiting factor in the current coconut CRISPR / Cas9 gene editing system. Therefore, screening out U6 promoters with functional activity in coconut has positive significance for the development of coconut genetic breeding technology. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a coconut U6 promoter and application.

[0006] In order to solve the above technical problems, the present invention provides a coconut U6 gene promoter CnU6, comprising a DNA nucleotide sequence as shown in SEQ ID No. 1. The coconut U6 gene promoter CnU6 belongs to the RNA polymerase III type promoter of the coconut U6snRNA gene, and is derived from coconut ( Cocos nucifera L.).

[0007] The invention also discloses a coconut transient transformation vector, which contains the coconut U6 gene promoter CnU6.

[0008] Specifically, the transient transformation vector is the recombinant plasmid pCnU6-mNeonGreen.

[0009] The present invention also discloses a method for cloning the coconut U6 gene promoter CnU6, comprising the following steps:

[0010] (1) Using the genomic DNA of the leaves of the coconut Hainan high variety as a template, the following specific primers containing the CnU6 sequence (underlined) that can be used for seamless cloning were designed:

[0011] CnU6-F:

[0012] GTAAAACGACGGCCAGTGCCAAGCTCTTTTTTTTTTTTTTTTTTTTTGGT;

[0013] CnU6-R:

[0014] ccttgctcaccatcAAACCCCATCCTTTAGCTTTC;

[0015] (2) Perform PCR amplification in a 20 μl reaction system using KOD enzyme;

[0016] (3) The amplified product was subjected to agarose electrophoresis and then cut and recovered according to the size of the target band, thereby obtaining a 301 bp coconut U6 gene promoter DNA fragment CnU6.

[0017] Specifically, in step (2), the reaction procedure of the PCR amplification step is: pre-denaturation at 95°C for 6 min, denaturation at 95°C for 15 s, annealing at 57°C for 15 s, extension at 72°C for 5 s, 35 cycles, and final extension at 72°C for 7 min.

[0018] The present invention also discloses a method for constructing the coconut protoplast transient transformation vector, comprising the following steps:

[0019] (a) Prepare CnU6 fragment using steps

[0011] to

[0016] .

[0020] (b) Design the following specific primers containing the mNeonGreen fluorescent protein sequence that can be used for seamless cloning of the fragment (underlined):

[0021] CnU6-Neon F:

[0022] AGGATGGGGTTTgatggtgagcaagggcgagga;

[0023] CnU6-Neon R:

[0024] AATTCGAGCTCGGTACCCGGGGATCttacttgtacagctcgtccatgcc;

[0025] The mNeonGreen fragment was amplified by PCR in a 20 μl reaction system using KOD enzyme;

[0026] The amplified product was subjected to agarose electrophoresis and then cut and recovered according to the target band size to obtain a mNeonGreen gene fragment containing 711 bp.

[0027] Specifically, in step

[0025] , the reaction procedure of the PCR amplification step is: pre-denaturation at 95°C for 6 min, denaturation at 95°C for 15 s, annealing at 57°C for 15 s, extension at 72°C for 10 s, 35 cycles, and final extension at 72°C for 7 min.

[0028] (c) The pUN1301 vector was double-digested with HindⅢ and BamHI to recover a 12088 bp vector backbone fragment.

[0029] (d) The fragments CnU6, mNeonGreen, and pUN1301 in steps (a), (b), and (c) were connected by seamless cloning to obtain the expression vector pCnU6-mNeonGreen containing the coconut CnU6 promoter-driven fluorescent protein.

[0030] The above vector was transformed into Escherichia coli DH5α and propagated to obtain a high-concentration plasmid for coconut protoplast transformation.

[0031] The invention clones and obtains the coconut RNA polymerase III type promoter, namely the coconut endogenous U6 promoter CnU6, in coconut for the first time.

[0032] The present invention connects the cloned coconut endogenous RNA polymerase III type promoter to drive mNeonGreen fluorescent protein for the first time, verifies the feasibility of driving fluorescent protein expression by the promoter through transient transformation of coconut leaf protoplasts, and provides a rapid and effective method for subsequent screening and verification of other coconut endogenous U6 promoters. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the pCnU6-mNeonGreen expression vector structure.

[0034] Figure 2 Isolation and microscopic examination of protoplasts from leaves of coconut embryo culture seedlings (A: coconut embryo culture seedlings, B: cutting off the white tender part for later use, C: cutting into slices for easy enzymatic hydrolysis, D: microscopic examination of protoplasts).

[0035] Figure 3 Fluorescence detection of untransformed coconut protoplasts (control).

[0036] Figure 4 Results of transient transformation of coconut protoplasts (CnU6-mNeon expression vector).

[0037] Figure 5 Results of transient transformation of coconut protoplasts (35s-GFP expression vector). DETAILED DESCRIPTION

[0038] In the following examples of the present invention, unless otherwise specified, conventional methods are used. The vector pUN1301 in the following examples was donated by Li Wenqiang's research group at Northwest Agriculture and Forestry University, and the pmNeonGreen vector was purchased from Miaoling Plasmid Platform. The above biological materials are only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0039] Example 1 Obtaining the Coconut U6 Gene Promoter CnU6

[0040] Using the DNA sequences of Arabidopsis thaliana AtU6-26 gene (Genebank accession number: X52528.1) and cotton GhU6-9 gene (Genebank accession number: XR_001680717.1) as references, the coconut genome database was searched, and a coconut candidate CnU6 gene was found by homology alignment, and the upstream reference sequence of this gene was obtained.

[0041] Using the genomic DNA of the leaves of the coconut Hainan high variety (preserved by the Coconut Research Institute of the Chinese Academy of Tropical Agricultural Sciences) as a template, the following CnU6 (301 bp) specific primers were designed to clone the promoter region DNA fragment:

[0042] CnU6 F:

[0043] GTAAAACGACGGCCAGTGCCAAGCTCTTTTTTTTTTTTTTTTTTTTTGGT;

[0044] CnU6 R:

[0045] ccttgctcaccatcAAACCCCATCCTTTAGCTTTC;

[0046] PCR amplification was performed using KOD enzyme (TOYOBO) in a 20 μl reaction system. The specific reaction procedure was: 95°C pre-denaturation for 6 min, 95°C denaturation for 15 s, 57°C annealing for 15 s, 72°C extension for 5 s, 35 cycles, and 72°C final extension for 7 min. The target band was obtained by agarose electrophoresis and recovered for later use.

[0047] Using the plasmid containing the mNeonGreen gene as a template, specific primers were designed for constructing the CnU6-Neon expression cassette to clone the fluorescent protein gene fragment:

[0048] CnU6-Neon F:

[0049] AGGATGGGGTTTgatggtgagcaagggcgagga;

[0050] CnU6-Neon R:

[0051] AATTCGAGCTCGGTACCCGGGGATCttacttgtacagctcgtccatgcc;

[0052] PCR amplification was performed using KOD enzyme (TOYOBO) in a 20 μl reaction system. The specific reaction procedure was as follows: pre-denaturation at 95°C for 6 min, denaturation at 95°C for 15 s, annealing at 57°C for 15 s, extension at 72°C for 10 s, 35 cycles, and final extension at 72°C for 7 min.

[0053] The target band was obtained by agarose electrophoresis and recovered for later use.

[0054] The pUN1301 vector was double-digested with HindⅢ and BamHI, and the 12088bp vector backbone fragment was recovered. The target band was obtained by agarose electrophoresis and recovered for later use.

[0055] The prepared fragments CnU6, mNeonGreen, and pUN1301 were connected by seamless cloning to obtain the expression vector pCnU6-mNeonGreen containing the coconut CnU6 promoter-driven fluorescent protein. The vector expression structure is shown in Figure 1 .

[0056] The above vector was transformed into Escherichia coli DH5α and propagated to obtain a high-concentration plasmid for coconut protoplast transformation.

[0057] A brief procedure for isolating protoplasts from young coconut leaves is given in Figure 2 .

[0058] The fluorescence detection results are shown in Figures 3 to 5 ,in Figure 3 represents untransformed protoplasts, serving as negative control; Figure 4 It indicates that fluorescence is detected in CnU6, indicating that CnU6 is transcriptionally active; Figure 5 The 35s promoter drives GFP expression as a positive control. Therefore, the present invention can be used to screen the coconut endogenous U6 promoter, thereby providing support for the subsequent establishment of a coconut gene editing technology system.

[0059] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

[0060] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by ordinary technicians in this technical field within the essential scope of the present invention should fall within the protection scope of the present invention.

Claims

1. A coconut U6 promoter CnU6, characterized in that The nucleic acid sequence of the promoter is shown in SEQ ID NO.

1.

2. Application of the coconut U6 promoter CnU6 according to claim 1 in driving the mNeonGreen green fluorescent protein gene in coconut protoplast expression.

3. A coconut transient transformation vector comprising the coconut U6 promoter CnU6 according to claim 1.

4. a recombinant vector, an expression cassette or a recombinant bacterium containing the coconut U6 promoter CnU6 according to claim 1.

5. Application of the recombinant vector, expression cassette and recombinant bacteria described in claim 4 in driving the mNeonGreen green fluorescent protein gene in coconut protoplast transformation.

6. A mNeonGreen green fluorescent protein expression vector, characterized in that: The expression vector is prepared by connecting the coconut U6 promoter CnU6 described in claim 1 with the mNeonGreen gene and cloning the resultant into a plant expression vector.

7. The mNeonGreen green fluorescent protein expression vector according to claim 6, characterized in that: The plant expression vector is pUN1301.

8. Use of the mNeonGreen green fluorescent protein expression vector according to claim 6 or 7 in driving the mNeonGreen gene in screening U6 promoter based on coconut protoplast transient transformation.

9. A method for cloning the coconut U6 promoter CnU6 according to claim 1, comprising the steps of: (1) Using the genomic DNA of the leaves of coconut Hainan high variety as a template, the following specific primers containing the CnU6 sequence that can be used for seamless cloning were designed: CnU6-F: GTAAAACGACGGCCAGTGCCAAGCTCTTTTTTTTTTTTTTTTTTTTTGGT; CnU6-R: ccttgctcaccatcAAACCCCATCCTTTAGCTTTC; (2) Perform PCR amplification using KOD enzyme in a 20 μl reaction system; (3) The amplified product was subjected to agarose electrophoresis and then cut and recovered according to the target band size, thereby obtaining a 301 bp coconut U6 gene promoter DNA fragment CnU6.

10. A method for constructing a coconut protoplast transient transformation vector, comprising the steps of: (a) preparing a CnU6 fragment; the nucleic acid sequence of the CnU6 fragment is shown in SEQ ID NO.1; (b) Design the following specific primers containing the mNeonGreen fluorescent protein sequence that can be used for seamless cloning: CnU6-Neon F: AGGATGGGGTTTgatggtgagcaagggcgagga; CnU6-Neon R: AATTCGAGCTCGGTACCCGGGGATCttacttgtacagctcgtccatgcc; The mNeonGreen fragment was amplified by PCR in a 20 μl reaction system using KOD enzyme; The amplified product was subjected to agarose electrophoresis and then cut and recovered according to the target band size to obtain a 711 bp mNeonGreen gene fragment; (c) pUN1301 vector was double-digested with HindIII and BamHI to recover a 12088 bp vector backbone fragment; (d) The fragments CnU6, mNeonGreen, and pUN1301 in steps (a), (b), and (c) were connected by seamless cloning to obtain the expression vector pCnU6-mNeonGreen containing the coconut CnU6 promoter-driven fluorescent protein.

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