Mint McHD-Zip1 gene promoter and application thereof

Abstract

The invention discloses a mint McHD-Zip1 gene promoter and application thereof, and relates to the technical field of gene engineering. According to the mint McHD-Zip1 gene promoter disclosed by the invention, the nucleotide sequence of the mint McHD-Zip1 gene promoter is as shown in SEQ ID NO. 1. When the promoter is used for constructing a plant expression vector pGC-GUS-McHD-Zip1pro, the result of the embodiment shows that the promoter can drive a target gene to be predominantly expressed in stem tips, leaf axils and secretory glandular hairs of a plant instead of being widely expressed in various tissues and organs of the whole plant, so that the adverse effect on normal growth and development of the plant can be reduced, and the plant expression vector pGC-GUS-McHD-Zip1pro can be used for preparing the plant expression vector pGC-GUS-McHD-Zip1pro. The metabolism or development process of the specific part of the plant can be more accurately regulated and controlled; compared with a constitutive promoter, the specific promoter avoids interference of whole plant expression on plant metabolic balance, and provides a precise tool for studying plant glandular hair development and metabolic engineering.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and more particularly relates to a Mentha McHD-Zip1 gene promoter and application thereof. BACKGROUND

[0002] Mentha L. plants are perennial or rarely annual herbaceous plants of Labiatae, the dry aboveground parts of which can be used as medicine and have the effects of clearing the head, dispelling wind-heat, etc. At the same time, Mentha plants are widely used as aromatic plants at home and abroad. Mentha stems and leaves contain rich essential oils, which are applied in food industry, biological medicine, cosmetics and other industrial fields, and have important economic and medicinal values. Mentha essential oil is a mixture mainly composed of alcohols, ketones and terpenes, including menthone, menthol, lauric aldehyde, limonene and menthofuran, etc. Mentha essential oil is mainly synthesized, secreted and stored in the shield gland hairs of Mentha. Gland hair is a special epidermal tissue with secretory function on the surface of plants, and is an important place for synthesizing various important natural metabolites, such as artemisinin in gland hair of Artemisia annua. Plant gland hair development usually develops with the formation of leaves, and the genes involved in gland hair development are generally preferentially expressed in plant meristems (such as shoot tips and leaf axils) and young leaves.

[0003] The promoter is located in the 5' region of the gene, composed of specific nucleotide sequences, and controls the expression of DNA through cis-acting elements and trans-acting factors. In plant research, promoters responsible for the transcription of mRNA are divided into three types, including constitutive promoters, inducible promoters and specific promoters. Constitutive promoters are mainly used for plant transformation, and have the effect of promoting the continuous expression of different target genes in all tissues and organs of plants, among which the 35S promoter from cauliflower mosaic virus (CaMV) (CaMV35S) is the most popular constitutive promoter in transgenic crops. Since the target gene is continuously expressed in all tissues and organs of plants driven by the constitutive promoter, it will disturb the original metabolic balance in the plant body and cause adverse effects on the plant. The specific promoter can regulate the expression of genes in specific tissues, organs or developmental stages, which is beneficial to the targeted metabolic or developmental regulation of plants.

[0004] Gland hair is a "biological factory" for producing important secondary metabolites, and enzymes for synthesizing secondary metabolites are preferentially expressed in gland hair. Using the promoter preferentially expressed in gland hair can not only modify the metabolic system of gland hair, but also avoid the disadvantages brought by constitutive promoters, which has important significance for the study of plant gland hair development and metabolic engineering. SUMMARY

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present application is to provide a Mentha haplocalyx McHD-Zip1 gene promoter.

[0006] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0007] A Mentha haplocalyx McHD-Zip1 gene promoter, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0008] An amplification primer of the Mentha haplocalyx McHD-Zip1 gene promoter, the sequence of which is shown as follows:

[0009] McHD-Zip1 pro-F: 5'-TCTTTGCACGCACATCCT-3',

[0010] McHD-Zip1 pro-R: 5'-TTCTCTTCCTTTCTTTTCTTC-3'.

[0011] A vector containing the Mentha haplocalyx McHD-Zip1 gene promoter, the vector being a plant expression vector pGC-GUS-McHD-Zip1 pro recombinant vector.

[0012] Application of the Mentha haplocalyx McHD-Zip1 gene promoter in driving a target gene to be expressed dominantly in a plant stem tip.

[0013] Application of the Mentha haplocalyx McHD-Zip1 gene promoter in driving a target gene to be expressed dominantly in a plant leaf axil.

[0014] Application of the Mentha haplocalyx McHD-Zip1 gene promoter in driving a target gene to be expressed dominantly in a plant secretory trichome.

[0015] Further, the target gene comprises a GUS gene.

[0016] Further, the plant comprises a Nicotiana plant.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] 1) The mint McHD-Zip1 gene promoter disclosed in the present application, the nucleotide sequence of which is shown as SEQ ID NO. 1. The cis-acting elements on the promoter include A-box, AAGAA-motif, ABRE, AE-box, ATC-motif, AT-TATA-box, Box 4, CAAT-box, CARE, CCGTCC motif, F-box, G-Box, GARE-motif, MYB, MYC, STRE, TATA-box, TC-rich repeats, TCA-element, TGA-element, WRE3 and WUN-motif.

[0019] 2) The mint McHD-Zip1 gene promoter disclosed in the present application can drive the target gene to be expressed dominantly in the plant shoot tip, leaf axil and secretory gland hair, but not widely expressed in all tissues and organs of the whole plant, which can reduce the adverse effects on the normal growth and development of the plant and more accurately regulate the metabolism or development process of specific parts of the plant. The results of the examples show that the top of the shoot tip, leaf axil, young leaf and secretory gland hair of the mature leaf of the transgenic tobacco present obvious blue color, while the roots and other parts are almost not stained, which embodies high tissue specificity. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Structure diagram of pGC-GUS-McHD-Zip1 pro;

[0021] Figure 2 GUS tissue staining diagram of the promoter McHD-Zip1 pro after stable transformation of tobacco (A is GUS staining of the shoot tip of the transgenic tobacco; B is GUS staining of the young leaf of the transgenic tobacco; C is GUS staining of the stem of the transgenic tobacco; D is GUS staining of the gland hair of the young leaf of the transgenic tobacco; E is GUS staining of the mature leaf of the transgenic tobacco; F is GUS staining of the root of the transgenic tobacco);

[0022] Figure 3 GUS tissue staining diagram of wild type (control) tobacco (A is GUS staining of the shoot tip of the wild type tobacco; B is GUS staining of the young leaf of the wild type tobacco; C is GUS staining of the stem of the wild type tobacco; D is GUS staining of the gland hair of the young leaf of the wild type tobacco; E is GUS staining of the mature leaf of the wild type tobacco; F is GUS staining of the root of the transgenic tobacco). DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present application more clear, the present application will be further described below in combination with specific examples. In the following examples, if not specified in detail, the technical means used are all conventional means well known to those skilled in the art.

[0024] The plant material used in the following examples is Mentha haplocalyx Briq. preserved in the Jiangsu Province Chinese Academy of Sciences Institute of Botany Germplasm Resource Garden.

[0025] Example 1

[0026] 1. Cloning of Mentha haplocalyx Briq. McHD-Zip1 gene promoter McHD-Zip1 pro

[0027] Total DNA of Mentha haplocalyx Briq. was extracted using CTAB method with Mentha haplocalyx Briq. leaves as material. The sequence of Mentha haplocalyx Briq. McHD-Zip1 gene was compared in the Mentha haplocalyx Briq. genome database (http: / / langelabtools.wsu.edu / mgr / ), and the homologous gene was searched. The upstream promoter sequence of the homologous gene in Mentha haplocalyx Briq. was used as a reference to design amplification primers, and the primer sequences are as follows:

[0028] McHD-Zip1 pro-F: 5'-TCTTTGCACGCACATCCT-3',

[0029] McHD-Zip1 pro-R: 5'-TTCTCTTCCTTTCTTTTCTTC-3'.

[0030] Mentha haplocalyx Briq. DNA was used as a template, and McHD-Zip1 pro-F and McHD-Zip1 pro-R were used as primers to perform PCR amplification of McHD-Zip1 pro using PhantaMax Super-Fidelity DNA Polymerase (Vazyme, Nanjing).

[0031] The PCR reaction program was as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 2.5 min, 35 cycles; 72°C extension for 5 min. After agarose gel electrophoresis detection, the company (Shanghai, China) sequenced to obtain the nucleotide sequence of the promoter McHD-Zip1 pro as shown in SEQ ID NO. 1, and the length was 1612 bp.

[0032] 2. Analysis of Mentha haplocalyx Briq. McHD-Zip1 gene promoter cis-acting elements

[0033] Plantcare (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) was used to analyze the cis-acting elements on the promoter McHD-Zip1 pro.

[0034] The results are shown in Table 1. The cis-acting elements on the promoter include A-box, AAGAA-motif, ABRE, AE-box, ATC-motif, AT-TATA-box, Box 4, CAAT-box, CARE, CCGTCC motif, F-box, G-Box, GARE-motif, MYB, MYC, STRE, TATA-box, TC-rich repeats, TCA-element, TGA-element, WRE3 and WUN-motif.

[0035] Table 1 Analysis of regulatory elements of the McHD-Zip1 pro promoter

[0036] Element name Sequence Element name Sequence A-box CCGTCC G-Box CACGTG AAGAA-motif AAGAAAG GARE-motif TCTGTTG ABRE CACGTG MYB CAACAG AE-box AGAAACTT MYC CATTTG ATC-motif AGTAATCT STRE AGGGG AT ~ TATA-box TATATA TATA-box ATATAA Box 4 ATTAAT TC-rich repeats GTTTTCTTAC CAAT-box CAAT TCA-element CCATCTTTTT CARE CAACTCAC TGA-element AACGAC CCGTCC motif CCGTCC WRE3 CCACCT F-box CTATTCTCATT WUN-motif AAATTTCCT

[0037] Example 2

[0038] 1. Construction of a plant expression vector containing the McHD-Zip1 pro promoter

[0039] The McHD-Zip1 pro promoter was ligated to the pGC-GUS (G5) vector, which was cleaved with Kpn I as the cleavage site. Primers containing homologous arms were designed, and the cloned McHD-Zip1 pro sequence was used as a template for PCR amplification. After recovering the band, the pGC-GUS-McHD-Zip1 pro recombinant vector was constructed by homologous recombination to drive the expression of the downstream GUS gene. Figure 1 The primer sequences are as follows:

[0040] McHD-Zip1 pro-G5-F:

[0041] 5'-CGCGTTGGGAGCTCCTCGAGGGTACCTCTTTGCACGCACATCCT-3', McHD-Zip1 pro-G5-R:

[0042] 5'-ACTCATTCTAGAGAATTCAAGCTTGGTACCTTCTCTTCCTTTCTTTT CTTC-3'.

[0043] 2. Transformation of tobacco with the McHD-Zip1 pro promoter

[0044] The constructed plant expression vector was transformed into Agrobacterium EHA105, and after screening and verifying positive strains, Agrobacterium-mediated genetic transformation was performed using the leaf disc method. The specific steps are as follows:

[0045] 1) Preparation of bacterial solution

[0046] Picked positive monoclonal colonies into YEB medium containing antibiotics, 28℃, 200rpm shaker culture to turbidity, 1ml bacterial liquid was taken to 25ml new YEB medium, expanded culture to OD 600 0.6-0.8, centrifuged, discarded the supernatant, suspended and precipitated with MS salt solution according to 1:1, and added 50mg / L acetosyringone.

[0047] 2) Transformation and identification

[0048] Tobacco tissue culture seedlings were cut into 0.5cm x 0.5cm small pieces, and the cut tobacco leaves were placed in the suspended Agrobacterium in MS salt solution and shaken for 10min; then the leaves were transferred to co-culture medium (MS+2mg / L 6-BA+0.1mg / L NAA+3% sucrose+0.8% agar+50mg / L acetosyringone) and grown at 25℃ in the dark for two days, and then transferred to screening medium (MS+2mg / L 6-BA+0.1mg / L NAA+3% sucrose+0.8% agar+200mg / L cephalothin+50mg / L kanamycin) and cultured at 25℃ with 16h light / 8h dark. The medium was replaced every 10-15 days, and when the leaf edges grew tender buds, a sterile scalpel was used to cut off the buds at the base and transfer them to rooting screening medium (MS+3% sucrose+0.8% agar+200mg / L cephalothin+50mg / L kanamycin), and after antibiotic screening and rooting, the plants were transplanted. DNA was extracted from the initially screened tobacco leaves and subjected to PCR detection to obtain positive transgenic plants.

[0049] 3) GUS staining

[0050] The stem tips, stems, leaves, mature leaves, and roots of wild-type plants and screened positive transgenic plants were stained with β-glucuronidase (GUS), and the staining results were observed after ethanol decolorization.

[0051] The results are shown in Figure 2 and Figure 3 The young leaves, stem tips, and leaf axils of McHD-Zip1 pro transgenic plants were obviously stained blue, the heads of the secretory trichomes of young leaves and mature leaves were stained blue, and the roots were almost not blue Figure 2 , while the young leaves, stem tips, leaf axils, trichomes, and roots of wild-type plants were not stained blue Figure 3 , indicating that the promoter McHD-Zip1 pro drives the target gene to be expressed dominantly in the stem tips, leaf axils, and secretory trichomes.

[0052] The above description is only illustrative and is not restrictive of the present application and modifications will occur to those skilled in the art upon reading the foregoing description. It is therefore intended that the scope of the present application be given by the appended claims, rather than by the foregoing description, and that the application be interpreted to embrace all equivalents.

Claims

1. A peppermint McHD-Zip1 gene promoter, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The amplification primers for the peppermint McHD-Zip1 gene promoter as described in claim 1, characterized in that, The primer sequences are shown below: McHD-Zip1 pro-F: 5'-TCTTTGCACGCACATCCT-3', McHD-Zip1 pro-R: 5'-TTTCCTTCCTTCTTTTCTTC-3'.

3. A vector containing the peppermint McHD-Zip1 gene promoter as described in claim 1.

4. The carrier according to claim 3, characterized in that, The vector is a plant expression vector.

5. The carrier according to claim 4, characterized in that, The plant expression vector is the pGC-GUS-McHD-Zip1pro recombinant vector.

6. The application of the peppermint McHD-Zip1 gene promoter as described in claim 1 in driving the dominant expression of the target gene in the shoot tip of a plant.

7. The application of the peppermint McHD-Zip1 gene promoter as described in claim 1 in driving the dominant expression of the target gene in the leaf axil of plants.

8. The application of the peppermint McHD-Zip1 gene promoter as described in claim 1 in driving the dominant expression of the target gene in plant secretory glandular trichomes.

9. The application according to any one of claims 6-8, characterized in that, The target gene includes the GUS gene.

10. The application according to any one of claims 6-8, characterized in that, The plants mentioned include plants of the genus *Nicotiana*.

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