CN115927323A - Strong promoter CP05 specifically expressed in late development stage of plant anther and application thereof - Google Patents

Abstract

The invention discloses a strong promoter CP05 specifically expressed in the later development stage of plant anther, a gene expression cassette containing the CP05, a recombinant expression vector, a recombinant bacterium and a transgenic plant cell line, wherein the strong promoter CP05 specifically expressed in the later development stage of plant anther, the gene expression cassette, the recombinant expression vector, the recombinant bacterium and the transgenic plant cell line can efficiently drive exogenous genes to specifically express in the later development stage of plant anther, the expression level is accurate, the adverse effect caused by the continuous expression of target genes in other tissues of plants can be avoided, the method can be used for creating a plant male sterile line, and the method has good application prospects in plant genetic engineering and the utilization of heterosis. In addition, the strong promoter CP05 specifically expressed in the later development stage of the plant anther disclosed by the invention has a short nucleotide sequence, the full length is only 999bp, the cloning is easy, the cloning difficulty and the cost of the promoter can be effectively reduced, the construction and the transformation efficiency of a recombinant expression vector are improved, and the rapid acquisition of a transgenic plant is facilitated.

Description

Strong promoter CP05 specifically expressed in late development stage of plant anther and application thereof

Technical Field

The invention relates to the field of plant genetic engineering and molecular biology, in particular to a strong promoter CP05 specifically expressed in the later development stage of plant anthers and application thereof.

Background

Plant gene expression regulation is mainly the regulation of transcription level, and is coordinately controlled by various cis-acting elements and trans-acting factors. Promoters are important cis-acting elements that play a key role in transcriptional regulation. The tissue-specific promoter can drive gene expression only in certain specific tissues or organs, can accurately regulate and control the expression of the exogenous gene in a plant body in a timed and quantitative manner, can effectively reduce the adverse effects of energy loss or excessive accumulation of exogenous protein on the growth and development of the plant caused by continuous expression of the constitutive promoter at different parts, and has wide application value in the field of plant genetic engineering. Although various plant promoters have been disclosed, the development of tissue-specific promoters is relatively slow, and the demand for the industrialization of transgenes is not yet expanded.

Anthers are important organs in the sexual reproduction stage of plants, and anther-specific promoters are usually used for creation of germplasm resources of male sterile lines, restoration of male fertility, research of certain metabolic processes or gene regulation pathways in the development process of anthers, and the like. On one hand, the plant anther specific promoter and the cytotoxin gene can be embedded to construct an expression vector, and the development of pollen is blocked by transforming plants to create male sterile plants; fertility can also be restored by driving the specific expression of foreign genes in anthers. On the other hand, when some metabolic processes or gene regulation ways in the anther are researched, the over-expression or gene silencing of the gene can be driven by a certain anther specific promoter, and the effect of the gene in the anther development process can be researched in a targeted manner; sometimes, in order to improve the efficiency and research speed of transgenosis, different anther-specific promoters with low homology are also used for being embedded with different genes in the same expression vector, so that gene silencing possibly caused by high-homology promoter sequences is effectively prevented. However, there are very few anther-specific promoters having strong driving activity and good specificity that have been disclosed so far, and it is difficult to satisfy the above-mentioned demands.

In addition, the nucleotide sequence of the disclosed plant anther-specific promoter is generally long and is 1000-3000bp at most, for example, CN108486112B, a promoter with anther tissue specificity, is disclosed as 2147bp long; CN108753777B a promoter with anther tissue specificity and the application thereof disclose that the length of the promoter is 2384bp; CN106480026B an anther early development specific expression promoter and its application disclose that the promoter length is 3007bp, although these promoters show good anther specificity, the promoter nucleotide sequence is longer, increasing the cloning difficulty and cost of the promoter, reducing the expression vector construction and transformation efficiency, and not beneficial to rapid cloning and obtaining transgenic plants.

Therefore, the development of the plant anther specific promoter with short nucleotide sequence and strong driving activity is beneficial to the functional analysis and identification of plant anther development related genes and the creation of plant male sterile line germplasm resources, and has good application prospect in plant genetic engineering and heterosis utilization.

Disclosure of Invention

In view of the above, the present invention aims to provide a plant anther-specific promoter with a short nucleotide sequence and strong driving activity in the late development stage of plant anthers, and a method for cloning and applying the promoter, which can promote the functional analysis and identification of genes related to plant anther development, thereby providing a powerful tool for the creation of plant male sterile line germplasm resources and the utilization of heterosis.

In order to achieve the purpose, the invention provides a strong promoter CP05 specifically expressed in the later development stage of plant anther, which has a nucleotide sequence shown as SEQ ID No. 1.

The invention also provides a gene expression cassette containing the strong promoter CP05 specifically expressed in the later development stage of the plant anther, wherein the gene expression cassette is formed by connecting a strong promoter CP05 sequence specifically expressed in the later development stage of the plant anther, an exogenous target gene and a transcription termination sequence in a specific direction; the exogenous target gene includes but is not limited to a structural gene, a regulatory gene, an antisense gene of the structural gene, an antisense gene of the regulatory gene or a small RNA gene, a long non-coding RNA (lncRNA) gene, a circular RNA (circRNA) gene capable of interfering with the expression of the endogenous gene; in one embodiment of the invention, the exogenous gene of interest is specifically a β -glucuronidase Gene (GUS).

The invention also provides a recombinant expression vector containing the gene expression cassette, wherein the recombinant expression vector is any vector known in the prior art and capable of being expressed in plants, and includes but is not limited to pCAMBIA3301, pCAMBIA1300, pBI101, pBI121, pRI201, pBin19, pCAMBIA2301 and pCAMBIA1301 plant expression vectors; the recombinant expression vectors may also contain selectable marker genes, typically including genes that provide antibiotic resistance or herbicide resistance, such as: hygromycin resistance gene, glyphosate resistance gene or glufosinate resistance gene, etc.; in one embodiment of the invention, the recombinant expression vector is specifically pBI101.

The invention also provides a recombinant bacterium and a transgenic plant cell line containing the gene expression cassette.

The invention also provides the strong promoter CP05 specifically expressed in the later development stage of the plant anther, and an application of the gene expression cassette, the recombinant expression vector, the transgenic plant cell line and the recombinant bacteria containing the strong promoter CP05 specifically expressed in the later development stage of the plant anther in driving the specific expression of the exogenous target gene in the plant anther.

The invention also provides the strong promoter CP05 specifically expressed in the later development stage of the plant anther, a gene expression cassette containing the strong promoter CP05 specifically expressed in the later development stage of the plant anther, a recombinant expression vector, a transgenic plant cell line and application of recombinant bacteria in preparation of transgenic plants.

The transgenic plant of the present invention can be obtained by any known transgenic technique capable of introducing an exogenous gene into a plant cell or plant tissue; in one embodiment of the invention, the transgenic technology is agrobacterium-mediated transformation.

The transgenic plant is a transgenic plant with an exogenous target gene specifically expressed in anthers, preferably a transgenic plant with enhanced or weakened pollination or fertilization capability, and more preferably a male sterile transgenic plant.

Plants of the present invention include, but are not limited to, arabidopsis, bok choy, cabbage, canola, capsicum, sunflower, musk, tobacco, sweet potato, lupin, tomato, potato, grape, soybean; preferably Chinese cabbage, rape, arabidopsis thaliana; in one embodiment of the invention, the plant is arabidopsis thaliana.

The invention also provides a primer pair for PCR amplification of the strong promoter CP05 specifically expressed in the later development stage of the plant anther, and the nucleotide sequences of the primer pair are shown as SEQ ID No.2 and SEQ ID No. 3.

The invention also provides a method for separating or identifying the strong promoter CP05 specifically expressed at the later development stage of the plant anther, which comprises the step of obtaining the nucleotide sequence of the strong promoter CP05 specifically expressed at the later development stage of the plant anther by carrying out PCR amplification on genome DNA of Columbia arabidopsis through using primer pairs of SEQ ID No.2 and SEQ ID No. 3.

The DNA molecule complementary to the strong promoter CP05 specifically expressed in the late developmental stage of plant anther also belongs to the content of the present invention, and the same object as the strong promoter CP05 specifically expressed in the late developmental stage of plant anther can be achieved by using the DNA molecule.

The beneficial effects of the invention are as follows:

1) The invention discloses a strong promoter CP05 specifically expressed in the later development stage of plant anther for the first time, and provides a novel method for efficiently driving the specific expression of an exogenous gene in the later development stage of plant anther.

2) The strong promoter CP05 specifically expressed in the later development stage of the plant anther, provided by the invention, is expressed in the later development stage of the plant anther, has strong driving activity and tissue specificity, the CP05 can efficiently drive the specific expression of an exogenous gene in the later development stage of the plant anther, the expression level is accurate, the adverse effect caused by the continuous expression of a target gene in other tissues of the plant can be avoided, and the strong promoter CP05 has wide application value in the field of plant genetic engineering.

3) The strong promoter CP05 specifically expressed at the later development stage of the plant anther provided by the invention has a shorter nucleotide sequence, the full length of the promoter CP05 is only 999bp, the cloning is easy, the cloning difficulty and the cost of the promoter can be effectively reduced, the construction and the transformation efficiency of a recombinant expression vector can be improved, and the transgenic plant can be rapidly obtained.

4) The strong promoter CP05 specifically expressed at the later development stage of the plant anther can be used for functional analysis and identification of genes related to the development of the plant anther and is helpful for understanding the molecular mechanism of the development regulation of the plant anther.

5) The strong promoter CP05 specifically expressed in the later development stage of plant anther can be used for creating plant male sterile line germplasm resources, and has good application prospect in plant genetic engineering and heterosis utilization.

Drawings

FIG. 1 is a schematic diagram of construction of a recombinant expression vector pCP05-GUS of a strong promoter CP05 specifically expressed in a late stage of plant anther development;

FIG. 2 is a picture of the GUS staining of pCP05-GUS transgenic seedlings and tissues, A: young seedlings; b: stems and mature leaves; c: siliques and seeds; d: inflorescence; e: and (4) flower.

Detailed Description

The present invention will now be described more fully hereinafter with reference to the accompanying specific embodiments, in which some, but not all embodiments of the invention are shown. Modifications or substitutions to methods, steps or conditions of the invention by those skilled in the art without departing from the spirit and substance of the invention are within the scope of the invention.

The experimental procedures in the following examples, unless otherwise specified, are all conventional procedures; materials, reagents and instruments in the following examples are all commercially available; in the following examples, the 1 st position and the last position of each nucleotide sequence in the sequence listing are both 5 'terminal nucleotides of the corresponding DNA and 3' terminal nucleotides of the corresponding DNA, unless otherwise specified.

Example 1: obtaining of strong promoter CP05 specifically expressed in late development of plant anther

1) Preparation of Columbia type Arabidopsis thaliana genome DNA

FN-C cell lysate (CN 114426966A, a cell lysate used for rapid non-toxic extraction of animal and plant or microorganism genome DNA, a kit and a method) is used for extracting genome DNA from Columbia arabidopsis leaves, and the genome DNA is stored at the temperature of 20 ℃ below zero for later use.

2) Cloning of strong promoter CP05 specifically expressed in late development stage of plant anther

Obtaining an arabidopsis thaliana reference genome DNA sequence from an http:// www.gramene.org/microsat database, designing a specific PCR amplification primer of the CP05 by utilizing SnapGene software, wherein the sequences of a forward primer and a reverse primer are respectively shown as SEQ ID No.2 and SEQ ID No.3, carrying out PCR amplification by using the prepared arabidopsis thaliana genome DNA as a template, cloning an amplification product to a pMD19-T vector, and measuring that the sequence of the promoter CP05 is shown as SEQ ID No. 1. The plasmid with the correct sequencing is named as pMD19-CP05 and is used for constructing PCR amplification templates of various recombinant expression vectors of a promoter CP 05. The PCR reaction system and the PCR reaction program are specifically as follows:

a) PCR reaction (50. Mu.L):

b) PCR reaction procedure:

example 2: construction of recombinant expression vector pCP05-GUS for promoter CP05 (construction scheme shown in FIG. 1)

1) The same primer design method as that of example 1 is used to obtain PCR amplification primer pair of promoter CP05, hindIII and BamHI restriction sites are added to the 5 'ends of the forward primer and reverse primer, respectively, and further 15bp sequence overlapping with the corresponding connection position of pBI101 vector is added to the 5' ends of the forward primer and reverse primer, respectively, to obtain PCR amplification primer pair of promoter CP05 for connecting to pBI101 vector, wherein the sequences of the forward primer and reverse primer are shown as SEQ ID No.4 and SEQ ID No.5, respectively.

2) The obtained promoter CP05 amplification primer pair (shown in SEQ ID No.4 and SEQ ID No. 5) is utilized to carry out PCR amplification by taking pMD19-CP05 plasmid as a template, the PCR product is subjected to gel recovery to obtain a target fragment, and the PCR reaction system and the procedure are the same as those in the example 1.

3) The pBI101 vector was digested with HindIII and BamHI, and the digested linear vector was recovered.

4) And (2) carrying out high-efficiency connection on the target fragment in the step 2) and the linear vector in the step 3) by using a seamless connection recombinase, transforming escherichia coli competent cells, screening monoclonal sequencing verification, wherein sequencing primer sequences are shown as SEQ ID No.6 and SEQ ID No.7, and the vector with correct sequencing is named as pCP05-GUS.

Example 3: the recombinant expression vector pCP05-GUS is used for transforming arabidopsis thaliana

1) Transferring the recombinant expression vector pCP05-GUS into agrobacterium

Mu.g-2. Mu.g of the recombinant expression vector pCP05-GUS plasmid obtained in example 2 was taken, transformed into competent cells of Agrobacterium GV3101 by freeze-thaw method, single clones were screened by culturing on sterile YEB solid plates containing rifampicin (25. Mu.g/mL) and kanamycin (50. Mu.g/mL), PCR-verified on pCP05-GUS recombinant bacteria using specific primers SEQ ID No.6 and SEQ ID No.7 to determine positive clones, and the PCR reaction system and procedure were the same as in example 1.

2) Agrobacterium-mediated genetic transformation of Arabidopsis

Activating pCP05-GUS agrobacterium liquid at 28 ℃, transforming wild arabidopsis thaliana by an inflorescence dip-dyeing method, culturing in a normal culture environment, harvesting transgenic seeds, putting the transgenic seeds in a 1.5mL centrifuge tube, adding 3-8 pieces of allochroic silica gel drying agent, sealing by a sealing film, and storing at 4 ℃ for a long time.

Example 4: screening and identification of pCP05-GUS transgenic arabidopsis

The transgenic seeds obtained in example 3, which were subjected to cryopreservation, were sterilized and disinfected, and then seeded in a sterile 1/2CS solid plant tissue culture medium (CN 110754366B, a eurytopic plant tissue culture medium and 1/2 medium) containing kanamycin (50. Mu.g/mL), and cultured under conditions of a temperature of 22 to 24 ℃, a humidity of 50 to 70%, a light intensity of 1500 to 2000Lux, and alternation of light and darkness (a light time of 16h and a dark time of 8 h) for 10 to 15 days, where dark green resistant seedlings are visible, while yellow or white seedlings are non-resistant wild-type seedlings, and the resistant seedlings were transplanted into a nutrient medium (vermiculite: nutrient soil =1, 2V/V) to continue culturing. Selecting young tissues of the screened T1 generation positive transgenic plants, extracting genome DNA for PCR detection (primer sequences are shown as SEQ ID No.6 and SEQ ID No. 7), obtaining T2 generation plants and seeds by repeated screening if the positive transgenic plants contain target strips, continuously screening the T2 generation seeds, and obtaining pCP05-GUS transgenic homozygous strains if the descendants are dark green resistant seedlings.

Example 5: histochemical detection of GUS gene expression in various tissues and organs of transgenic arabidopsis plant

The method comprises the steps of soaking seedlings and different tissue organs of a pCP05-GUS transgenic homozygous line in a substrate X-Gluc staining reaction solution, vacuumizing for 5min, keeping the temperature at 37 ℃ overnight, removing the staining solution, decoloring with 70% ethanol for 2-3 times, observing and photographing under a stereoscope and a microscope, wherein the result is shown in figure 2, no blue color is detected in the seedlings (including tissue organs such as roots, hypocotyls and young leaves), stems, mature leaves, horn fruits, seeds, sepals, petals, filaments, pistils and early anthers of the pCP05-GUS transgenic arabidopsis thaliana, and the deep blue color is detected only in late anthers, so that the promoter CP05 can efficiently drive the GUS gene to be specifically expressed in the later development stage of the arabidopsis thaliana anthers.

The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. Of course, the derived nucleotide sequence with one or more nucleotides substituted, deleted or added in the nucleotide sequence shown in SEQ ID No.1 and the equivalent pollen-specific promoter function is also within the protection scope of the application, in particular the derived nucleotide sequence with over 75 percent homology with the nucleotide sequence shown in SEQ ID No.1 and the equivalent pollen-specific promoter function.

Claims (10)

1. A strong promoter CP05 specifically expressed in the late development stage of plant anther has a nucleotide sequence shown in SEQ ID No. 1.

2. A gene expression cassette comprising the strong promoter CP05 of claim 1 specifically expressed in the late developmental stage of plant anthers.

3. A recombinant expression vector comprising the gene expression cassette of claim 2.

4. A recombinant bacterium comprising the gene expression cassette of claim 2.

5. A transgenic plant cell line comprising the gene expression cassette of claim 2.

6. Use of the strong promoter CP05 of claim 1 specifically expressed late in the development of plant anthers, or the gene expression cassette of claim 2, or the recombinant expression vector of claim 3, or the recombinant bacterium of claim 4, or the transgenic plant cell line of claim 5 to drive the specific expression of an exogenous gene of interest late in the development of plant anthers.

7. Use of the strong promoter CP05 of claim 1 specifically expressed late in the development of plant anthers, or the gene expression cassette of claim 2, or the recombinant expression vector of claim 3, or the recombinant bacterium of claim 4, or the transgenic plant cell line of claim 5 for the production of transgenic plants.

The primer pair of the strong promoter CP05 specifically expressed in the late development stage of the plant anther, which is disclosed by claim 1, is amplified by PCR, and the nucleotide sequences of the primer pair are shown as SEQ ID No.2 and SEQ ID No. 3.

9. A method for isolating or identifying the strong promoter CP05 specifically expressed in the late developmental stage of plant anthers according to claim 1, comprising obtaining the nucleotide sequence of the strong promoter CP05 specifically expressed in the late developmental stage of plant anthers according to claim 1 from Columbia arabidopsis genomic DNA by PCR amplification using primer pairs of SEQ ID No.2 and SEQ ID No. 3.

10. A DNA molecule complementary to the strong promoter CP05 of claim 1 specifically expressed late in plant anther development.

Images