CN119193599B - Coconut strong promoter, plant expression vector and application - Google Patents

Abstract

The present invention discloses a coconut strong promoter, a plant expression vector and an application thereof, wherein the nucleotide sequence of the promoter is shown in SEQ ID NO. 1. Compared with known promoters, the promoter provided by the present invention can drive the efficient expression of related genes and is a strong promoter. The present invention also relates to a plant expression vector containing the strong promoter and an application thereof in initiating the expression of a target gene.

Description

Coconut strong promoter, plant expression vector and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a coconut strong promoter, a plant expression vector and application thereof.

Background

Coconut (Cocos nucifera l.) belongs to the palmaceae monocotyledonous plant and is an important economic forest in the hot zone. The coconut life cycle is long, researchers gradually perfects the technology of tissue culture by taking embryo, anther, ovary and other tissues as explants, and a coconut transgenic technology system is also constructed. Transgenic technology achieves crop improvement by introducing specific genes or reducing endogenous gene expression, while the main challenge of transgenic technology is to precisely regulate exogenous gene or endogenous gene expression. Suitable promoters are critical to achieving the desired expression pattern, but the number and types of promoters currently suitable for genetic transformation of plants are still limited. The cauliflower mosaic virus 35S promoter is one of the most commonly used universal promoters, which is capable of efficiently driving gene expression in many monocotyledonous and dicotyledonous plants, but whose expression efficiency varies among different plants. There are some differences between promoters of monocotyledonous and dicotyledonous plants in gene expression control, and it is necessary to effectively promote construction of transgenic systems of coconuts, clone, identify and utilize strong promoters of coconuts themselves.

Promoter sequences of monocots generally contain regions of higher GC content, whereas promoters of dicots are more prone to higher AT content. The variety and distribution of these elements may vary from plant to plant. The cis-acting elements common in monocots and dicots, although overlapping in some way, differ in their specific function and binding properties. Cloning of constitutive promoters of plants themselves, such as promoters of housekeeping genes such as actin, ubiquitin, etc., in place of the cauliflower mosaic virus 35S promoter, can more effectively drive transcription of foreign genes in monocots. In coconut, screening and identifying the stably expressed gene can lay a foundation for cloning and utilizing the promoter with stable transcriptional activity.

Disclosure of Invention

In view of the defects in the prior art, the invention provides a coconut strong promoter, a plant expression vector and application.

The technical scheme of the invention mainly comprises the following contents:

the invention provides a coconut strong promoter, and the nucleotide sequence of the coconut strong promoter is shown as SEQ ID NO. 1.

The invention further relates to a plant expression vector containing the strong promoter, which contains the coconut strong promoter.

Further, the construction method of the plant expression vector comprises the following steps:

(1) Amplifying the coconut strong promoter fragment;

(2) And connecting the coconut strong promoter fragment with a linearized vector by a homologous recombination method to obtain the plant expression vector.

Further, the linearized vector is obtained by using KpnI and HindIII to cleave pGreen II LUC vector or using HindIII and SalI to cleave pCAMBIA1300-eGFP vector.

The construction method of the plant expression vector further comprises the following steps:

(1) Amplifying the coconut strong promoter fragment of claim 1 using the primer sequences shown in SEQ ID No.2 and SEQ ID No.3, or using the primer sequences shown in SEQ ID No.4 and SEQ ID No. 5;

(2) Ligating the coconut strong promoter fragment of claim 1 to a linearized vector by means of homologous recombination to obtain the plant expression vector;

The linearized vector is obtained by using KpnI and HindIII to cleave pGreen II 0800 LUC vector or using HindIII and SalI to cleave pCAMBIA1300-eGFP vector.

In another aspect, the invention also relates to the use of said coconut strong promoter for driving expression of a gene of interest.

Further, the gene includes a LUC reporter gene or a GFP reporter gene.

The invention has the beneficial effects that:

The invention discloses a coconut strong promoter, and the nucleotide sequence of the coconut strong promoter is shown as SEQ ID NO. 1. Compared with the promoters known in the prior art, the coconut promoter provided by the invention can forcefully drive the high-efficiency expression of related genes and has the characteristic of a strong promoter.

The invention also relates to application of the promoter, for example, the promoter can be used for promoting the expression of a reporter gene, thereby facilitating the visual tracking and quantitative analysis of the space-time specificity of the gene expression by researchers. By connecting a reporter gene (such as green fluorescent protein gene GFP and the like) with a coconut strong promoter to construct a plant expression vector, after plant cells are transformed, researchers can intuitively observe the expression position and intensity of the reporter gene in plant tissues by utilizing equipment such as a fluorescent microscope and the like by means of the high-efficiency driving capability of the strong promoter, and a powerful basis is provided for understanding a gene expression regulation mechanism.

In the process of cultivating transgenic plants with excellent properties, the target genes can be accurately and efficiently introduced into plant cells through the plant expression vector provided by the invention, and the coconut strong promoter is utilized to drive the target genes to express, so that the plants obtain the desired excellent properties such as insect disease resistance, stress resistance enhancement, quality improvement and the like, and a powerful tool is provided for agricultural production and plant scientific research.

Drawings

FIG. 1 shows the detection electrophoresis pattern of the CnMAC promoter linked to pCAMBIA1300-eGFP (A) and pGreen II 0800-LUC (B) vectors. pC1300-proF/R and pGreen II 0800-LUC-F/R represent the results of electrophoresis after ligation of the promoter with the vector, respectively, and MACpro-F/R represents the results of electrophoresis of the promoter.

FIG. 2-pCnMAC schematic representation of control plasmids and transient transformed tobacco LUC signal profile. A) Schematic diagrams of pCnMAC, pmiR156-7, pmiR-5 and 35S driven LUC reporter genes, B) to D) pCnMAC LUC signal detection results of LUC and control plasmids.

FIG. 3 schematic representation of pCnMAC and control plasmids and transient transformed tobacco GFP signal profile. Schematic of A) pCnMAC and 35S driven eGFP reporter, B) pCnMAC: GFP signal from tobacco transiently transformed with eGFP, C) pCnMAC:: GFP signal detection results from eGFP and 35S: eGFP.

Detailed Description

For a better understanding of the technical content of the present invention, the present invention will be further described with reference to the following specific examples and the accompanying drawings.

1. Determination of coconut Strong promoter

By analyzing the coconut genome sequence information, the following CnMAC promoter sequence (SEQ ID NO. 1) was obtained:

AACGGAACGCTATGGTGAACCTGTTATAATGGCGTCTCTTCCATGCGGTGGGATCTGCCTCCTACCTACCAGCAGATGGAGCCTAGGCAAACAGAGAGGCCTCAAGCCATATTGCCTCCCAGCGGCATTTCAGGACCACACAAAACCCTTCCTCTACTAGCCAAGATGCTCCATTCCAAGGGCTTCTTCATCACCTTTTTCCGCTACGAAATGCGCCGTGTTAGATGGCCTCTCACCATCCATGCTCTTGTAATCAAGCTTAACAGTGCATTATCGTCGGAGATTCCCTGAATCAGTCGTGGTTTGGGATGGCATCACGAGCTTCGAATGGCGGGCAGCGGCAGAGCTTGGCATCCCTGACATCATGTTCCGGATTGCCAGCACCCGTGGTTTGATGGTCAACCTCCATTACCTGCAGCTCATTGATAAGGGTTGGCAAATAACGTACGCAGGCATAGAATAGATCGCCTTATGATGGAAAGATGTTCTTTTATGCATATTCCAGATGCATACCATATGAGAGTGTCAGCGAATAGATAATTATTGTCAAAACTCCACAACCGAGAAGTGAAAGGTTCTTCAGTTGCCTGTGATTTTTGTCGATGTCAAGTGGTTCAATCCTCTTGTAGATTGCTTTAATCAATTATTGGTTGAGAGTGTACGTGGATTGGAACCACCTACACATGCCATGAACCACACACGTATAGCCCTGATTTTATGGACCTTATTGGCTGTTTGCTCCAATACCCGCCCAAAGTTAATGCCATGCAAATGGCTTTTAGCCTATGTACATGTACGTGAATGTCCAATAATAAGTCTCTAAATGGGCAGATAATGTTGTATCATATTTAAGCATGTCTAATATGGTTGAAAGTCAGGATGCTTTGCGGTGCATGTTTTGCACCATCAAGGATGGTATCGTCCAGCCATGTCATCTATTTTAAAAATGGATAACTATCAAATTTATCCCAGGCATACTTTGTGTTTGGTACCGAGCACGACCACTCGGATTCTCAAGGCAAGCAATCGGAGATGGATGACATGATCGATATCTAGAGATTTACAGCTTTTTATGGTGCAAAATATATACCACAGAAGATCCAAACTTCTATAGTTGCAACCATAGCAAAGAGAAACAAATAAAGTGGACCAACCAAGAGCATGATAATTGTCCCTAATGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAGTAAAACCAAAATGACATGGACCCTAAGCCCATATAATGATGGGCCAGTTATGTGAACGATAGGCCCAATATCTGGACCCGAAGCCTGTTAGGCCATCCAGCAGCCCATAACAAGATATCATATAAGAGGGACAGCTGGGGGATTCCTCGCCCTCTTTCTTCCATTCTTCCGTCGTCAGGA

2. cloning of the coconut CnMAC promoter to construct a plant expression vector

(1) Construction of recombinant vector pCnMAC-LUC

Primers pLUC-MAC-F and pLUC-MAC-R (5' -end is introduced into the homologous sequence at the end of the carrier) are designed, and CnMAC promoter fragments (homologous sequences corresponding to the end of the carrier are added at both ends) are obtained through amplification. The vector pGreenII to 0800 LUC (commercially available vector) was digested with KpnI and HindIII, and the CnMAC promoter fragment was ligated into the digested vector by homologous recombination to obtain recombinant vector pCnMAC-LUC.

pLUC-MAC-F:

TCACTATAGGGCGAATTGGGTACCAACGGAACGCTATGGTGAA(SEQ ID NO.2)

pLUC-MAC-R:

CTGCAGGAATTCGATATCAAGCTTTCCTGACGACGGAAGAATG(SEQ ID NO.3)

(2) Construction of recombinant vector pc1300-CnMACpro-eGFP

The primers pGFP-MAC-F and pGFP-MAC-R (5' end introduced into the homologous sequence at the end of the vector) were designed and amplified to give CnMAC promoter fragment (both ends added with homologous sequences corresponding to the end of the vector). The pCAMBIA1300-eGFP vector (commercial vector) is subjected to enzyme digestion by using HindIII and SalI enzymes, and a promoter fragment is connected to the digested vector by a homologous recombination method to obtain a recombinant vector pc1300-CnMACpro-eGFP which can be used for the subsequent construction of a coconut transgenic expression vector.

pGFP-MAC-F(SEQ ID NO.4):

GTAAAACGACGGCCAGTGCCAAGCTTAACGGAACGCTATGGTGAA

pGFP-MAC-R(SEQ ID NO.5):

GGTACCAGATCTACTAGTGTCGACTCCTGACGACGGAAGAATG promoter sequence was amplified using the Norflu Hi-Fi enzyme C112 and vector construction used the Uniclone One STEP SEAMLESS Cloning Kit from Jinsha Bio Inc.

(3) Construction of control plasmid

The promoter fragment (with the addition of homologous sequences corresponding to the ends of the vector) was amplified using the corresponding pmiR-156-5-LUC-F/R, pmiR-156-7-LUC-F/R, pLUC-35S-F/R (5' end introduced into the homologous sequence of the ends of the vector). The recombinant vectors pmiR-5-LUC, pmiR156-7-LUC and 35S-LUC were obtained by ligating the promoter fragment into the digested vector by homologous recombination using KpnI and HindIII digested pGreen II 0800 LUC vector. The promoter fragment (both ends of which are added with homologous sequences corresponding to the ends of the vector) was amplified using pc1300-35S-eGFP-F/R primer (5' -end introduced into the homologous sequence at the ends of the vector). The pCAMBIA1300-eGFP vector is digested with KpnI and HindIII, and the promoter fragment is connected to the digested vector by homologous recombination to obtain a recombinant vector pc1300-35S-eGFP.

1) PmiR156 the amplification primer and promoter sequences of 156-5 are as follows:

pmiR156-5-LUC-F(SEQ ID NO.6):TCACTATAGGGCGAATTGGGTACCAGGGAAAAACCGAACAAAAGG

pmiR156-5-LUC-R(SEQ ID NO.7):CTGCAGGAATTCGATATCAAGCTTTTGGCCCCAACGGAATAC

pmiR156 promoter sequence 156-5 (SEQ ID NO. 8):

AGGGAAAAACCGAACAAAAGGTTATGCATGGTCATAGCAAGCAGACTTTTCCTTGAAAGAGAAGTATACATTATTTGTTTTGGTACAAAGTTACACAAATGGATATATATGACATTTGCACAAGCAATCTAGAGAGAGATATTTAGCAAACTAAGAAAGTGACTACTCCAAAACATTGCCCTAGATCAGTCCTGTTGTTAATGCATAGGGTCGTCCGAAACCTTTTGGACTATGCAGCTCATTTCGCTTGTAATATCCTAAGAAAGTTATAAACCTTTTTATTTTCTAATAAATTTGATTGGGGTTACGTCATCGACTTCCCTTTAGTTCAATAAATAAATATGATATTCTCTCACCAAAAGCATGCTACAGATACTAAATAATTTTACATCCACTATTTTAAAAAAGGAATAAATATGATATTCTTGCACCAAGACCGTGCTACACATATTGAACAACCTTACATCCGTGATTGTATTAGTGTCGACACCCCATTCAGTAGCCTACATAGCTTGTCTAGAAGAATGTACAATTGAACTCTTCCTTGCAGTGATTTGAATAAAAAACGAAACTTACTACTGGAATTAAAAACTTAATATACACGTACAAGTTTATCAAGCTGCAAAATTTTCATTATGTATCAATCATTTACATGTGTTGGTAGATTTATTCCTAAATCACAATTTTTATTATAAAAAGATGGAACTTGGAGCAATATAAATCTTCCATGAACATTTGGTTGAAGAGCCGAACCAAAAGGAAGTTAGACTTCTTGATGCACATGTAACCTATTACCTATCGGCCTTTACGCCGTTTGTTTGACTTGGTCATACCATGTTCAAATTGTATATGTGAATTCCGAATAATTCAAATTAGCTAAATGGTAGATTTGCTCGAACTTGTTACAATTATTCCAATCATCATAAAAGACCGGTCACATAATCACAATAGCATTCAATTAATTTCATTCATGCCGGATTTGTTCTTGACATGTAAATCAGATAACTATCTCTCTACTTGATATTTTTTAAGGATGGAATATATTAAGAAAAGCCACCTCTAATTGATAGGTCTCATCCCGATTAGGATTCATGGTTGACACGTGTACAGGTTTGCTATATTCCTACCTCACTTATCAATGTGACATTTCCTTAGAGAACCTAAACTTGGGTTGGTATAAGATAATTTTATTTTATAATTCCCACAAAGGCATCATCTGGAACAAACGTGGTCCCCACGGCCGGTGGGGTCCACCCCAACTAGCTCCACTCCGGTTCGGGCGGTGTCCTTCACACCAAGGATCGATCGATCTTCTTTTTCTGATCTCAACCGTTGGATCGTTTTTTGCACAGATCTCAAAATTTTTTTTTTTTTTTTGCGCGAAAGGTACAAACCGAATCCCTCCTCTCTTCACCGACAGCCGAGGCGAGTTCAGAAATTAAAATTAAATCGAATCAATAACTAAGCGACTAGAGTCGCCCACGCTGCACCTGGCAGTCCCATCACTGCAGCGGCCCCCACGGACAAAGATCAACGGACGATATCAAATCAGAGGGAGGGAGGACGTACAGAATGCCACGAAGATAGACAGCGTCCGGAGATCCCCCTTCTTGTTATTCTTCTTCCCATCGTGCGGTTGCGGTACAAAGGACCGACCCGTATCATTCATTTCCGGGCCATCATTAATCAACAACGGTAAAGGGAAGTTTGAAAGCCGGGTGGGAGTCGGGTGACCTCTTCCGCTCGAATATCATCAGTACCTTTCCTTCCCTCAGCTGGCGACACGTGCGTCGGGTACCATCGCATCATAGAGAGAGAGAGAGAGAGAGAGAGAGAGGAGGTATTCCGTTGGGGCCAA

2) pmiR156 the amplification primer and promoter sequences of 156-7 are as follows:

pmiR156-7-LUC-F(SEQ ID NO.9):TCACTATAGGGCGAATTGGGTACCACATCTGATGCAGCCGCTAT

pmiR156-7-LUC-R(SEQ ID NO.10):CTGCAGGAATTCGATATCAAGCTTAAAGCCTCTTGATGCTTGTGTA

pmiR156 promoter sequence 156-7 (SEQ ID NO. 11):

CACATCTGATGCAGCCGCTATTTTAAAGGGCTGTATCCATCCCCCCTCTGATCTTTGCCAAAGGCGGAATACCCGAAAACTGCCAATAATGTCTACGTCCATGAATAGGGTGGCGACACGTAAACAGTGGACCATCACCAATATTATGCGGGCTAAGCTCATGGTAAAGGTGGCAAAACATCATTACAATGATTAAAATAATAGAAAGAGCCATCAGGGAGCATGGTCACTACGTTGGTTCAGTTGATAAAAAGATTCTTGTCCCCACCCCCCCACACCTAGCTTTCCGAGTCCATGCCCTAAAAGGCAAGATTCCATGAAACGCTACAGTCCAAATTACATGAAAATGCTCTCTCATTTTCCCCAAAGAGAAAAGAAAAAAGGACCCTGCCATCTCCGGTGGCAGGCATGGAAATTCTTTACGCTAGTGCAGTGTAGTGTTTCCTTTCCCCCGGGTCCTTGGGTTGCCACTTGCCACCCCTTTCCTCATGTACATGGTCCCACCACCATGATGGCAATAGAGTTTTCTCAAAGCATGCTTCACGATGAAACTTTGGCATCCAATCCAGTCTATATACCATCCATTATATTGCATGTAGTCTGCACAAAAATATTATATAGCTATGTATGAGTTTCCTGTTGTACCTATGCATACTGAGGTACTGACCATCACTTGGAGCCAAAAGAGAAGGGGCATAAAAAACAAAAAACTACAGCGGTCATGTAACTCTCTTGCTTGAAATTTTTCATATTACTTTGGCGTTCACAGTAAGATTCCCTTAATCATAAGACGAGAACGCTTGACCAATAAATGAGCCGAGTTTGAACACTTAAGATCGATCGATGCAGTCTCCTTTAATTTCAATTGGATAACATGCATGTTCTACTGGAAAATGAGCCAAACCTGAATATTTTATCAATTAGCTCGGATGAGACGCTTCTATAAGCAGCCGATGAGCAACCCAAAGAGGATGCATGGGTAGTCACTTCTATGTGCATGTCCGGAGGAAAGCCTGCTTCATGAGACTACTAAATACTAGCTTTGGGGTTCCAGGTTCCATAAGTCAGGTGGATTAAGATATAGGATAAATGAAAGGGTCCATCGTTCGTCCAACGGCTGTAAAAGATGCACTTGCACCGTGTGACAAGTAGGCTTGGGGTCTGGACACGAGGATCAGCTGGGGCCACGCCCCATTATTCTTCTGAAATGGACGGATGAAAGAGTGGGAGATAAGATTGCAGGATAAGGGGTGGGTGGGAAGCTTTTAATTCAGCCCTGTATCGTCTCCATTTTTTGTCTCTCTTTAATCTCCTCCTATTCGTATCGTGACTGACAGTAAGAATAATGAAAGAACGAAAAAAAAAAAGGAGCACAAAAATATTTATTAAGTTTGATCCACTAACCTACGTTCATGGATAAAGATGATACAAAAGTTTCACCATAAATAACTAGACATTACAGAACTAATACAAGATTTCTTCCACACGCTTCACTCACTCGCTCCGGAAAAATTAAAGATCCTATTTTGCTTTTCTAGAACATATAAATTAACTCAAGATATACTCAACAGTTCCTCTCCATGCTTCTAGCCTTCCTTTTTTTTTTTTTTTTAAAAATTTCTTATTTATTAGGGTGGGGACATGTTTGTGGGCTAATTGCAAGAACGTCCACATCCCTTACCCAATCCTCCTAACCCGTTGTCACCATGTGGGCCCACGATACTGTCTTCCTTTAGCTGTCCGATCCACATCAAGCTTTTGCAAGGCGGTGCACCATTACCTAATCATGGTGGAATCAACGCAAACCAAGGAAATGTACGCAAGATTGCACTCCGATCACCATTTTATGTCACTCCCTTCTATAAATAGAAGGGTGTGGAAGAGTCGTTTCGGTTCTTACAC

2) The amplification primer and promoter sequences of 35S are as follows:

pLUC-35S-F(SEQ ID NO.12):TCACTATAGGGCGAATTGGGTACCACCCCCCTACTCCAAAAATG

pLUC-35S-R(SEQ ID NO.13):CTGCAGGAATTCGATATCAAGCTTAGCTTGGGCTGTCCTCTCC

pc1300-35S-eGFP-F(SEQ ID NO.14):

AAAACGACGGCCAGTGCCAAGCTTACCCCCCTACTCCAAAAATG

pc1300-35S-eGFP-R(SEQ ID NO.15):CTTGCTCACCATGGATCCGGTACCAGCTTGGGCTGTCCTCTCC

35S promoter sequence (SEQ ID NO. 16):

ACCCCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACAGCCCA

3. coconut CnMAC promoter transcriptional Activity assay

The constructed pCnMAC-LUC/pc1300-CnMACpro-eGFP and control plasmids pmiR156-5-LUC, pmiR156-7-LUC and 35S-LUC/pc1300-35S-eGFP are subjected to heat shock transformation to form GV3101 (pSoup-p 19) and are subjected to competent Agrobacterium, single colony is selected, a shaking table at 28 ℃ is used for continuous culture of 1-2 d, PCR verification is carried out by using a promoter primer, and positive clones are selected for expansion culture. Positive bacteria after overnight shaking were adjusted for OD value with infection solution (10 mM MgCl ₂, 10mM mes,150 μm acetosyringone) to prepare OD ₆₀₀ =1.0. Then, the cells were activated by shaking them slowly in a shaking table at room temperature for 3 h hours. Selecting tobacco growing for 3-4 weeks, injecting activated bacterial liquid into leaves by a 1ml needleless injector, culturing in darkness 24h, culturing in light 24h, and detecting signals of a reporter gene LUC and fluorescent protein GFP.

And the LUC signal detection process comprises the steps of cutting tobacco leaves injected with bacterial liquid, uniformly smearing a D-fluorescein substrate on the back surfaces of the leaves, reacting in the dark for 3-5 min, observing fluorescent signals by using a plant living body image system, and photographing. Detection of GFP signal was observed and photographed using a hand-held uv lamp 3260 RB.

Note that MES is 2-morpholinoethanesulfonic acid

Experimental results

(1) CnMAC promoter-driven LUC and GFP expression vector construction

Amplification of the target sequence was performed using the CnMAC promoter amplification primers, and after linearization of pGreen II 0800 LUC vector and pCAMBIA1300-eGFP vector using the corresponding enzymes, ligation of the target sequence was performed using a 5ul homologous recombination reaction system, and positive clones were detected using the flanking primers and promoter amplification primers at the vector insertion position (FIG. 1). Correct clones were saved after verification by sequencing.

(2) MAC promoter transcriptional Activity assay

The correct clone was transformed into GV3101, transiently expressed by tobacco injection, and the CnMAC promoter was higher in transcriptional activity than the 35S promoter, and far higher than the other control promoters, as detected by LUC signal (fig. 2).

The correct clone was transformed into GV3101, transiently expressed by tobacco injection, and the CnMAC promoter was very Gao Zhuailu active and higher than the 35S promoter in transcriptional activity, as a strong promoter, as detected by GFP signal (fig. 3).

(3) CnMAC promoter-driven plant expression vector

The plant binary expression plasmid of pc1300-CnMACpro-eGFP is successfully constructed, pCAMBIA1300 is taken as a skeleton plasmid, and eGFP is taken as a reporter gene.

The above embodiments are only some of the embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention are all within the scope of the present invention.

Claims (7)

1. A coconut strong promoter is characterized in that the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.

2. A plant expression vector comprising the coconut strong promoter according to claim 1.

3. The method for constructing a plant expression vector according to claim 2, comprising the steps of:

(1) Amplifying the coconut strong promoter fragment of claim 1;

(2) And (3) connecting the coconut strong promoter fragment in the step (1) with a linearized vector to obtain the plant expression vector.

4. The method of constructing a plant expression vector according to claim 3, wherein the linearized vector is obtained by using KpnI and HindIII to cleave pGreen II 0800 LUC vector or by using HindIII and SalI to cleave pCAMBIA1300-eGFP vector.

5. A method of constructing a plant expression vector according to claim 3, comprising the steps of:

(1) Amplifying the coconut strong promoter fragment of claim 1 using the primer sequences shown in SEQ ID No.2 and SEQ ID No.3, or using the primer sequences shown in SEQ ID No.4 and SEQ ID No. 5;

(2) Connecting the coconut strong promoter fragment in the step (1) with a linearized vector by a homologous recombination method to obtain the plant expression vector;

The linearized vector is obtained by using KpnI and HindIII to cleave pGreen II 0800 LUC vector or using HindIII and SalI to cleave pCAMBIA1300-eGFP vector.

6. Use of the strong coconut promoter of claim 1, wherein the use is to drive expression of a gene of interest.

7. The use according to claim 6, wherein the gene comprises a LUC reporter gene or a GFP reporter gene.