CN119020364B

CN119020364B - Coconut U6 promoter and application thereof

Info

Publication number: CN119020364B
Application number: CN202411535161.0A
Authority: CN
Inventors: 张大鹏; 石鹏; 王永; 李志瑛; 尹敏慧
Original assignee: Sanya Research Institute Chinese Academy Of Tropical Agricultural Sciences; Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Current assignee: Sanya Research Institute Chinese Academy Of Tropical Agricultural Sciences; Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Priority date: 2024-10-31
Filing date: 2024-10-31
Publication date: 2025-03-14
Anticipated expiration: 2044-10-31
Also published as: CN119020364A

Abstract

The invention relates to a coconut U6 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, and the coconut RNA polymerase III-type promoter-coconut endogenous U6 promoter CnU is cloned in a coconut genome for the first time, so that the promoter has high-efficiency transcriptional activity, can drive the expression of downstream fluorescent protein, and the activity of the promoter is verified by transient transformation of coconut protoplast. The method can be used for detecting candidate coconut endogenous promoters, and can also be further used for truncating and screening a core U6 promoter.

Description

Coconut U6 promoter and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a coconut U6 promoter and application thereof.

Background

Coconut (academic name: cocos nucifera L.) is an important tropical economic palm crop, and at present, the breeding of new coconuts in China is still mainly based on traditional hybridization breeding, and the problems of low efficiency and long period exist, so that the progress of high-yield and high-quality coconuts breeding is seriously hindered. With the development of biotechnology, the application of molecular breeding techniques represented by gene editing techniques to various crops has accelerated the cultivation of new varieties, but related studies of coconuts have yet to be developed.

The genome editing technology is an important tool for researching gene functions, can accurately modify genes at specific sites of receptor cell chromosomes, can efficiently generate functional inactivation mutants of specific genes, and can provide high-quality genetic materials for biofunctional genome research. sgrnas and Cas9 are two essential elements of this technical system, where the sgrnas function to target binding to the gene locus of interest during CRISPR/Cas9 genome editing. Studies show that in CRISPR/Cas9 gene editing system, the content level of sgRNA in receptor cells is one of important factors affecting editing efficiency, so that polymerase III type promoter capable of accurately starting in-vivo transcription of sgRNA has received extensive attention. U6RNA is a non-coding RNA involved in pre-mRNA splicing, and its corresponding U6 promoter is a class III RNA polymerase promoter and has found great use in CRISPR/Cas9 systems in many species.

Although CRISPR/Cas9 genome editing techniques have been widely used in a number of species at present, gene editing techniques for coconuts have not yet been reported. This is mainly due to the fact that although U6 promoters have been reported in a large number of species, exogenous U6 promoters are not generally suitable. It can be seen that the lack of a suitable U6 promoter has become the limiting factor for the current coconut CRISPR/Cas9 gene editing system. Therefore, screening of U6 promoters functionally active in coconut is of positive interest for the development of genetic breeding technology for coconut.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a coconut U6 promoter and application thereof.

In order to solve the technical problems, the coconut U6 gene promoter CnU provided by the invention comprises a DNA nucleotide sequence shown as SEQ ID No. 1. The coconut U6 gene promoter CnU is an RNA polymerase type III promoter of the coconut U6snRNA gene and is derived from coconut (Cocos nucifera l.).

The invention also discloses a coconut transient transformation vector, namely a coconut U6 gene promoter CnU.

Specifically, the transient transformation vector is recombinant plasmid pCnU-mNeonGreen.

The invention also discloses a method for cloning the coconut U6 gene promoter CnU, which comprises the following steps:

(1) Using the coconut Hainan higher leaf genomic DNA as a template, specific primers containing the sequence CnU for the seamless cloning fragment (underlined) were designed as follows:

CnU6-F:

GTAAAACGACGGCCAGTGCCAAGCTCTTTTTTTTTTTTTTTTTTTTTGGT;

CnU6-R:

ccttgctcaccatcAAACCCCATCCTTTAGCTTTC;

(2) PCR amplification was performed in 20. Mu.l of the reaction system using KOD enzyme;

(3) And (3) subjecting the amplified product to agarose electrophoresis, and then cutting and recovering according to the size of a target band to obtain the DNA fragment CnU containing the 301bp coconut U6 gene promoter.

Specifically, in the step (2), the reaction program of the PCR amplification step is that the reaction is pre-denatured at 95 ℃ for 6min, denatured at 95 ℃ for 15s, annealed at 57 ℃ for 15s, extended at 72 ℃ for 5s,35 cycles, and finally extended at 72 ℃ for 7min.

The invention also discloses a method for constructing the coconut protoplast transient transformation vector, which comprises the following steps:

(a) The CnU fragment was prepared by the method of steps [0011] to [0016 ].

(B) Specific primers containing fluorescent protein sequences useful for seamless cloning of fragments (underlined) mNeonGreen were designed as follows:

CnU6-Neon F:

AGGATGGGGTTTgatggtgagcaagggcgagga;

CnU6-Neon R:

AATTCGAGCTCGGTACCCGGGGATCttacttgtacagctcgtccatgcc;

PCR amplification of mNeonGreen fragments was performed in 20. Mu.l reaction system using KOD enzyme;

and (3) subjecting the amplified product to agarose electrophoresis, and then cutting and recovering according to the size of a target band to obtain mNeonGreen gene fragments containing 711 bp.

Specifically, in the step [0025], the reaction procedure of the PCR amplification step is that the reaction is performed for 6min at 95 ℃, 15s at 57 ℃ annealing, 10s at 72 ℃ extension, 35 cycles, and 7min at 72 ℃.

(C) pUN1301 vector was digested with HindIII and BamHI, and 12088bp vector backbone fragment was recovered.

(D) And (3) connecting the fragments CnU, mNeonGreen, pUN1301 in the steps (a), b) and (c) by adopting a seamless cloning method to obtain the expression vector pCnU-mNeonGreen containing the coconut CnU promoter-driven fluorescent protein.

The vector is transformed into escherichia coli DH5 alpha to obtain high-concentration plasmid for transformation of coconut protoplast.

The invention clones coconut RNA polymerase III type promoter, namely coconut endogenous U6 promoter CnU, in coconut for the first time.

The invention connects 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 of the promoter by transiently transforming coconut leaf protoplast, and provides a rapid and effective method for subsequent screening and verification of other coconut endogenous U6 promoters.

Drawings

FIG. 1 shows a schematic diagram of pCnU6-mNeonGreen expression vector.

FIG. 2 separation and microscopic examination of leaf protoplasts of coconut embryo seedlings (A: coconut embryo seedlings, B: cutting off white tender parts for later use, C: slicing for enzymolysis, D: protoplast microscopic examination).

FIG. 3 fluorescence detection of coconut protoplast untransformed group (control).

FIG. 4 results of transient transformation of coconut protoplasts (CnU-mNeon expression vector).

FIG. 5 transient transformation results of coconut protoplasts (35 s-GFP expression vector).

Detailed Description

In the following examples of the present invention, conventional methods are used unless otherwise specified. The vector pUN1301 in the following examples was purchased from vast plasmid platform, which was offered by the group of the subject of the North-China agricultural and forestry university Li Wenjiang, and pmNeonGreen vector, and the above-mentioned biological materials were used only for repeated experiments related to the present invention, and were not used for other purposes.

Example 1 obtaining coconut U6 Gene promoter CnU6

The DNA sequences of Arabidopsis AtU-26 genes (Genebank accession number: X52528.1) and cotton GhU6-9 genes (Genebank accession number: XR_ 001680717.1) are used as references, a coconut genome database is searched, and 1 coconut candidate CnU6 gene is found by a homology alignment mode, so that an upstream reference sequence of the gene is obtained.

The following CnU6 (301 bp) specific primer was designed using the leaf genomic DNA of the Cocois Hainan high species (saved by Cocois of the national academy of sciences of Tropical agriculture) as a template to clone the DNA fragment of the promoter region:

CnU6 F:

GTAAAACGACGGCCAGTGCCAAGCTCTTTTTTTTTTTTTTTTTTTTTGGT;

CnU6 R:

ccttgctcaccatcAAACCCCATCCTTTAGCTTTC;

PCR amplification was performed using KOD enzyme (TOYOBO) in 20. Mu.l reaction system with the specific reaction procedure of 95℃pre-denaturation for 6min,95℃denaturation for 15s,57℃annealing for 15s,72℃extension for 5s,35 cycles, and 72℃final extension for 7min. And obtaining a target strip through agarose electrophoresis, and recovering the target strip for later use.

Specific primers for constructing CnU6-Neon expression cassette were designed using a plasmid containing mNeonGreen genes as a template to clone fluorescent protein gene fragments:

CnU6-Neon F:

AGGATGGGGTTTgatggtgagcaagggcgagga;

CnU6-Neon R:

AATTCGAGCTCGGTACCCGGGGATCttacttgtacagctcgtccatgcc;

PCR amplification was performed using KOD enzyme (TOYOBO) in 20. Mu.l reaction system with the specific reaction procedure of 95℃pre-denaturation for 6min,95℃denaturation for 15s,57℃annealing for 15s,72℃extension for 10s,35 cycles, and 72℃final extension for 7min.

And obtaining a target strip through agarose electrophoresis, and recovering the target strip for later use.

PUN1301 vector was digested with HindIII and BamHI, 12088bp vector backbone fragment was recovered, and the target band was obtained by agarose electrophoresis, and recovered for use.

And (3) connecting the prepared fragments CnU and mNeonGreen, pUN1301 by adopting a seamless cloning method to obtain expression vectors pCnU-mNeonGreen containing coconut CnU promoter-driven fluorescent protein, wherein the expression structure of the vectors is shown in figure 1.

The brief procedure for protoplast isolation of tender leaves of coconut is shown in FIG. 2.

The fluorescence detection results are shown in FIGS. 3 to 5, wherein FIG. 3 shows untransformed protoplasts as a negative control, FIG. 4 shows that CnU6 detects fluorescence, indicating that CnU6 has transcriptional activity, and FIG. 5 shows that 35s promoter drives GFP expression as a positive control. Therefore, the invention can be used for screening coconut endogenous U6 promoter, thereby providing support for the subsequent establishment of coconut gene editing technology system.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

It should be understood that the above description is not intended to limit the invention to the particular embodiments disclosed, but to limit the invention to the particular embodiments disclosed, and that various changes, modifications, additions and substitutions can be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims

1. A coconut U6 promoter CnU6, characterized in that the nucleotide 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. An 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 , wherein 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 in a 20 μl reaction system using KOD enzyme;

(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.

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.