CN109750037B - Promoter PCHF40 specifically expressed in rice pollen and application thereof - Google Patents

Abstract

The invention discloses a promoter PCHF40 specifically expressed in rice pollen and application thereof, and relates to the fields of genetic engineering and molecular biology. The promoter PCHF40 has a nucleotide sequence shown in SEQ ID NO.1, and the invention further provides an expression vector, a gene expression cassette, an engineering bacterium or a cell line containing the promoter PCHF 40. The invention also provides a primer pair for amplifying the pollen specific promoter. The pollen specific promoter of the invention is a rice endogenous gene, is very beneficial to rice genetic engineering, can drive the specific expression of an exogenous gene in pollen and has accurate expression level, and provides a new method for driving the specific expression of the exogenous gene in rice pollen.

Description

Promoter PCHF40 specifically expressed in rice pollen and application thereof

Technical Field

The invention relates to the field of genetic engineering and molecular biology, in particular to a rice pollen specific promoter PCHF40 and application thereof.

Background

Transcriptional regulation is one of the major forms of plant gene expression regulation and is coordinated by cis-acting elements and trans-acting factors. The promoter is one of the most important cis-elements in plant gene transcription regulation, is generally located in the upstream region of the 5' end of a gene, and is a recognition and binding site for RNA polymerase and some trans-acting factors. The promoter mainly comprises two functional regions, namely a core promoter region and a transcription regulation region. The core promoter region is the shortest promoter fragment for transcription initiation, typically 40nt, a DNA sequence recognized and bound by RNA polymerase families I, II and III. This region contains several important functional elements that can accurately locate the transcription start point and direction, which is the basis of gene expression regulation. The transcription regulation region is located at the upstream (or downstream) of the core promoter, and can be combined with a specific transcription factor to play a role in regulating the space-time and strength of transcription, such as an enhancer, a silencer and the like. The deep research on the expression mode of the promoter is not only beneficial to understanding the expression regulation mechanism and biological function of the gene, but also beneficial to controlling the expression of the exogenous gene.

Promoters can be classified into constitutive promoters, inducible promoters, and space-time specific promoters according to their expression modes. Constitutive promoters are capable of initiating gene transcription in all or most tissues, resulting in spatiotemporal persistence and constancy of expression. The 35S promoter of the tobacco mosaic virus, the Actin promoter of rice and the Ubiquitin promoter of corn all belong to constitutive promoters. Constitutive promoters are widely used in genetic engineering research of plants for overexpression of target genes, such as insect-and herbicide-resistant genes. Inducible promoters can initiate or greatly increase gene expression upon stimulation by certain physical or chemical signals. They have sequence structures with enhancer, silencer or similar functions and have obvious specificity. The inducible promoters can be classified into light-inducible promoters, heat-inducible promoters, low-temperature inducible promoters, drought-inducible promoters, wound-inducible promoters, hormone-inducible promoters and the like according to different inducing signals. Spatio-temporal specific promoters only initiate gene expression in specific growth stages or sites. A tissue-specific promoter is one of the spatio-temporal specific promoters, which only promotes expression in a specific cell, tissue or organ. The expression of a target gene is controlled by using a promoter with tissue specific expression in the genetic transformation of plants, so that potential side effects caused by using a constitutive promoter can be avoided more effectively, such as reduction of metabolic burden increased by constitutive expression, reduction of safety risk of transgenic food and adverse effect on environment, gene silencing caused by repeated use of the same promoter, and the like. There are various types of rice tissue-specific promoters developed so far, and promoters having tissue-specific expression have been found in almost various tissues such as roots, stems, leaves, seeds, and fruits.

Male sterility is the basis of heterosis utilization, is the core technology of the hybrid rice industry, and has important theoretical and practical significance in researching the rice fertility regulation and control mechanism. Anthers are the organs of rice that produce mature male gametophytes (i.e., pollen or microspores). In the early stages of anther development, there is a primary sporogenic cell which undergoes a series of divisions and differentiations to produce tapetum and pollen mother cells. The tapetum provides nutrition for the development of the male gametophyte, includes various enzymatic and non-enzymatic proteins, and is involved in the synthesis of the outer wall of the pollen. Pollen mother cells undergo meiosis to produce tetrads. The single cells in the tetrad are separated to generate haploid microsporidia under the action of callase secreted by tapetum. The haploid microsporidia further develop to the late stage forming a central large vacuole. The large central vacuole displaces the nucleus to the edge of the cell (called the mononuclear-side phase), creating cell polarity. This cellular polarity promotes the microsporidian to undergo an unequal mitosis, producing a large vegetative cell and a small germ cell. Small germ cells are contained entirely within large vegetative cells, creating a cell-specific phenomenon within the cell. The germ cells then undergo a second mitosis, producing two sperm cells, forming a mature pollen grain. Therefore, screening and determining the rice pollen specific promoter provides a new choice for the genetic engineering of rice, particularly in the aspects of fertility regulation, transgene drift prevention and control and the like.

Disclosure of Invention

The invention aims to provide a plant pollen specific promoter and application thereof.

The invention provides a plant pollen specific promoter PCHF40, which comprises the following components:

1) SEQ ID No:1, or a nucleotide sequence shown in the specification,

or 2) in SEQ ID No:1, the nucleotide sequence which is derived from the nucleotide sequence 1) and has the same specific pollen starting function by substituting, deleting or adding one or more nucleotides in the nucleotide sequence shown in 1;

or 3) with SEQ ID No:1, or a sequence complementary to the nucleotide sequence shown in the figure.

Wherein, the nucleotide sequence derived from 1) in 2) has more than 70 percent of homology, more than 80 percent of homology, more than 85 percent of homology, more than 90 percent of homology, more than 95 percent of homology, more than 98 percent of homology or more than 99 percent of homology with the nucleotide sequence in 1) and has the same function of a pollen specific promoter.

The DNA molecule complementary to the nucleotide sequence of the plant pollen specific promoter PCHF40 can be easily identified and utilized by those skilled in the art for the same purpose, and therefore, a DNA sequence having promoter activity and capable of hybridizing to the promoter sequence of the present invention or a fragment thereof under stringent conditions is included in the present invention. Wherein, the nucleotide sequence is complementary, which means that the nucleotide sequence can be hybridized with PCHF40 under strict conditions.

Stringent conditions refer to conditions under which a probe will hybridize to a detectable degree to its target sequence over other sequences (e.g., at least 2 times background). Stringent conditions are sequence dependent and will vary with the other conditions of the experiment. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified that are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some sequence mismatches so that a lower degree of similarity is detected (heterologous detection). Generally, probes are no longer than 1000 nucleotides in length, preferably shorter than 500 nucleotides.

Typically, stringent conditions are those in which the salt concentration is less than about 1.5M Na ion, typically about 0.01-1.0M Na ion concentration (or other salts) at a pH of 7.0-8.3, and the temperature is at least about 30 ℃ for short probes (e.g., 10-50 nucleotides) and at least about 60 ℃ for long probes (e.g., more than 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. Low stringency conditions, for example, include hybridization at 37 ℃ in a buffer solution of 30-35% formamide, 1M NaCl, l% SDS (sodium dodecyl sulfate), washing at 50-55 ℃ in 1 × to 2 × SSC (20 × SSC =3.0M NaCl/0.3M trisodium citrate). Moderately stringent conditions, for example, comprise hybridization at 37 ℃ in a buffer solution of 40-45% formamide, 1.0M NaCl, l% SDS, washing at 55-60 ℃ in 0.5X to 1 XSSC. Highly stringent conditions, for example, include hybridization at 37 ℃ in a buffer solution of 50% formamide, 1M NaCl, l% SDS, and washing at 60-65 ℃ in 0.1 XSSC. Optionally, the wash buffer may contain about 0.1% to 1% SDS. Hybridization times are generally less than about 24 hours, usually about 4-12 hours.

Particularly typically as a function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, tm can be estimated from the equation of Meinkoth and Wahl (Anal Biochem,1984, 138, 267-284) Tm =81.5 ℃ +16.6 (logM) +0.41 (% GC) -0.61 (% form) -500/L; where M is the molar concentration of monovalent cations,% GC is the percentage of guanine and cytosine nucleotides in DNA,% form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in a base pair. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm needs to be lowered by about l ℃ per 1% mismatch; thus, tm hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if the sought sequence has 90% identity or greater, the Tm can be lowered by 10 ℃. Generally, stringent conditions are selected to be about 5 ℃ below the thermal melting point (Tm) for the specific sequence, and which are complementary at a defined ionic strength and pH. However, highly stringent conditions may employ hybridization and/or washing at 1, 2, 3, or 4 ℃ below the thermal melting point (Tm); moderately stringent conditions can employ a hybridization and/or wash at 6, 7, 8, 9, or 10 ℃ below the thermal melting point (Tm); low stringency conditions can employ hybridization and/or washing at 11, 12, 13, 14, 15, or 20 ℃ below the thermal melting point (Tm). One of ordinary skill in the art will appreciate that conditions for hybridization and/or wash solutions vary with stringency, and that this equation can be used to calculate hybridization and wash compositions and desired Tm. If the desired degree of mismatch is such that the Tm is below 45 deg.C (aqueous solution) or 32 deg.C (formamide solution), it is preferred to increase the concentration of SSC to enable the use of higher temperatures. Guidelines for nucleic acid hybridization are found in Tijssen (1993) biochemical and molecular biology laboratory techniques using nucleic acid probe hybridization, part I, chapter 2 (Elsevier, new York); and Ausubel et al, edited (1995) Chapter 2, a modern method of molecular biology (Greene Publishing and Wiley-Interscience, new York). See Sambrook et al (1989) molecular cloning-A Laboratory Manual, second edition, cold Spring Harbor Laboratory Press, plainview, new York.

The stringent conditions are preferably selected from hybridization at 65 ℃ in a solution of 6 XSSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate), followed by washing the membrane 1 times each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

The invention provides a gene expression cassette containing the plant pollen specific promoter PCHF40, an expression vector and a host cell containing the expression vector.

The gene expression cassette is an expression cassette which is connected with 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 capable of interfering the expression of an endogenous gene at the downstream of a pollen specific promoter PCHF 40.

The present invention further provides a plant comprising the gene expression cassette for the pollen-specific promoter PCHF 40.

The invention provides application of the rice pollen specific promoter PCHF40 or an expression cassette, an expression vector or a host cell containing the same in driving specific expression of an exogenous gene in plant pollen.

The invention provides application of a rice pollen specific promoter PCHF40 or an expression cassette, an expression vector or a host cell containing the same in preparation of transgenic plants.

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

Such plants include, but are not limited to, rice, corn, sorghum, barley, oats, wheat, millet, sugarcane, soybean, brassica species, cotton, safflower, tobacco, alfalfa, and sunflower.

The invention also provides a primer pair for amplifying the pollen specific promoter PCHF40, wherein the nucleotide sequence of the primer pair is SEQ ID NO:2-3.

The invention provides a method for separating a pollen specific promoter PCHF40, which is a method for separating a pollen specific promoter PCHF40 by using SEQ ID NO:2-3 primer pair PCR amplifies the nucleotide sequence of pollen specific promoter PCHF 40.

The invention also provides a method for driving the specific expression of the exogenous gene in the pollen, which comprises the following steps:

the rice pollen specific promoter PCHF40 and the target exogenous gene are cloned into a vector to obtain a recombinant expression vector of an expression cassette containing the PCHF40 and the target exogenous gene, and the recombinant expression vector is introduced into a plant genome to obtain a transgenic plant of which the exogenous gene is specifically expressed in pollen.

The pollen specific promoter PCHF40 provided by the invention has the following advantages:

1) The PCHF40 is an endogenous DNA sequence of rice, and the transgenic safety risk is extremely low.

2) The PCHF40 can drive the specific expression of the exogenous gene in the pollen, and the expression level is accurate.

3) The invention provides a novel method for driving exogenous genes to be specifically expressed in pollen.

Drawings

FIG. 1 is a flow chart of construction of a recombinant expression vector 1300gus-PCHF40 of the anther-specific promoter PCHF40 in example 2.

FIG. 2 is a photograph of anthers of non-transgenic mid-flower 11 rice stained with pollen GUS.

FIG. 3 is a photograph of 1300GUS-PCHF40 transgenic rice anthers and pollen GUS staining in example 4.

FIG. 4 is a photograph showing GUS staining of pistil (A), glume (B), root (C), leaf (D) and stem (E) of 1300GUS-PCHF40 transgenic rice in example 4.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 acquisition of a Rice pollen-specific promoter PCHF40

1. Extraction of genomic DNA from rice

The genomic DNA of rice was extracted using a plant DNA isolation kit (Chengdu Fuji Biotechnology Co., ltd.). The genome is derived from fresh leaves of Nipponbare of a rice variety. The extracted genome DNA is subpackaged and stored at-20 ℃ for later use.

PCR primer design and amplification of PCHF40

Primer design Using the Gibson Assembly method, the amplification product was inserted into the Nco I and Hind III cleavage sites of the 1300GUSPlus vector (obtained by inserting GUSPlus elements into the pC1300 multiple cloning site). The sequence of a primer for amplifying PCHF40 is shown as SEQ ID NO:2 and SEQ ID NO:3, respectively. Wherein the 5' ends of the upstream and downstream primers have 15 nucleotide sequences overlapping with the corresponding ligation sites in the vector for ligation by Gibson Assembly.

PCR reaction (100. Mu.L): DNA template: 3 μ L (50 ng), KOD polymerase (from Toyo Fang): 2 μ L,10 × buffer:10 μ L,10 μ M forward primer: 3 μ L,10 μ M reverse primer: 3 μ L,10 μ M dNTP:10 μ L, mgSO ₄ ：4μL，1/10DMSO：20μL，ddH ₂ O：45μL。

PCR procedure: pre-denaturation at 95 ℃ for 4min. Denaturation at 94 ℃ for 30s; annealing at 50 ℃ for 30s; extending at 68 deg.C for 2min;35 cycles. Extension was 68 ℃ for 10min.

The amplification product contains 2034bp of pollen-specific promoter PCHF40 (the sequence is shown as SEQ ID NO: 1).

Example 2 construction of recombinant expression vector p1300gus-PCHF40 for promoter PCHF40

The PCR product obtained in example 1 was electrophoresed on 1% agarose gel, and a band of about 2034bp in size was collected. The vector p1300GUSPlus was double digested with Nco I and Hind III to recover the linearized vector.

Ligation of the PCR-recovered product and linearized p1300GUSPLUS empty vector was performed using the Lighting Cloning Kit (Bio-technology, inc., beijing) in a 10. Mu.L system as follows: mu.L of the recovered product (50 ng/. Mu.L), 0.5. Mu.L of the digestion vector (100 ng/. Mu.L), and 2.5. Mu.L of the Ligation Mix. And (3) connecting procedures: 50 ℃ for 60min.

5. Mu.L of the ligation product was used to transform E.coli competent cells by electroporation. Primers SEQ ID NO:4 and SEQ ID NO:5, carrying out colony PCR, selecting positive clone, sequencing and verifying. The vector with the correct sequencing was named p1300gus-PCHF40 (FIG. 1). The p1300GUSPlus vector contains the GUS gene. The tissue expressing GUS gene is blue after being stained, and can be used for indicating the expression position and the strength of the promoter.

EXAMPLE 3 obtaining of P1300gus-PCHF 40-transgenic Rice

Agrobacterium EHA105 stored at-70 ℃ was streaked on a plate containing 50. Mu.g/mL rifampicin and cultured at 28 ℃. Single colonies were picked and inoculated into 50mL YEB broth, and shake-cultured at 220rpm at 28 ℃ for 12-16hr. 2mL of the suspension was transferred to 100mL of YEB broth (containing antibiotics) and cultured at 28 ℃ with shaking at 220rpm until OD600=0.5. Precooled on ice for 10 minutes and centrifuged at 5000rpm for 10min (refrigerated centrifuge precooled to 4 ℃). The solution was washed 2 times with sterile deionized water (10 mL each) and 1 time with 10% glycerol in 3mL of 10% glycerol. mu.L of competent cells was added with 1. Mu.L of the p1300gus-PCHF40 plasmid obtained in example 1, and transformed with 2.5KV shock. Positive clones were selected by culturing on YEB plates containing kanamycin and rifampicin at 28 ℃, and cloned with p1300GUSplus vector-specific primers SEQ ID NO:4 and SEQ ID NO:5, performing PCR verification.

The correct clones were verified and rice medium flower 11 was infected by Agrobacterium-mediated genetic transformation (Hiei Y Ohta S, komari T, kumashiro T (1994) efficacy transformation of rice (Oryza sativa L.) -mediated by Agrobacterium and sequence analysis of the genome of the T-DNA. The Plant Journal 6, 271-282). Obtaining T through co-culture, screening, differentiation, rooting and other links ₀ Transgenic seedlings are generated. Extracting total DNA of leaves of transformed plants, using primersSEQ ID NO:6 and SEQ ID NO:7 carrying out PCR positive detection, selecting positive plants verified by PCR for cultivation, selfing and fructifying to obtain T ₁ And (4) generation. Get T ₀ Or T ₁ And carrying out subsequent analysis on the generation plants.

Example 4 GUS staining analysis of transgenic Rice

GUS staining solution X-Gluc reaction solution (50 mM sodium phosphate buffer, pH 7.0,0.5mM potassium ferricyanide, 0.5mM potassium ferrocyanide, 0.5mg/ml X-Gluc, 20% by volume methanol, 0.1% Triton X-100) was prepared, 5 or more transgenic positive lines obtained in example 3 were randomly selected, tissue samples such as anthers, pistils, glumes, roots, leaves, stems were collected, immersed in the X-Gluc reaction solution at 37 ℃ for 2 hours or overnight, and then the color of chloroplasts of the tissues was removed with 75% by volume of ethanol, followed by photography. As shown in FIGS. 2 to 4, the pollen of flower 11 in the wild type was not stained (FIG. 2), while half of the pollen of the transgenic rice was stained blue (FIG. 3), and none of the other tissues such as pistil (A in FIG. 4), glume (B in FIG. 4), root (C in FIG. 4), leaf (D in FIG. 4), stem (E in FIG. 4) was stained, indicating that the PCHF40 promoter can drive the GUS gene to be expressed specifically in the pollen of rice.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims (9)

1. The rice pollen specific promoter PCHF40 is characterized in that the nucleotide sequence is shown in SEQ ID No. 1.

2. A gene expression cassette comprising the rice pollen-specific promoter PCHF40 according to claim 1.

3. An expression vector comprising the rice pollen-specific promoter PCHF40 according to claim 1.

4. The use of the rice pollen-specific promoter PCHF40 of claim 1, or an expression cassette, expression vector or host cell comprising the same, to drive the specific expression of a foreign gene in plant pollen.

5. The rice pollen specific promoter PCHF40 or an expression cassette, an expression vector or a host cell containing the same as claimed in claim 1 is used for preparing transgenic plants.

6. The use of the rice pollen-specific promoter PCHF40 of claim 1, or an expression cassette, expression vector or host cell comprising the same in the preparation of transgenic rice.

7. The primer pair for amplifying the rice pollen specific promoter PCHF40 as claimed in claim 1, wherein the nucleotide sequence of the primer pair is SEQ ID NO:2-3.

8. The use of the rice pollen specific promoter PCHF40 of claim 1 or an expression cassette, an expression vector or a host cell comprising the same or the primer pair of claim 7 in the preparation of transgenic rice in which an exogenous gene is specifically expressed in rice pollen.

9. A method for driving the specific expression of a foreign gene in plant pollen, comprising the steps of: the rice pollen specific promoter PCHF40 and the target exogenous gene of claim 1 are introduced into a vector to obtain a recombinant expression vector containing an expression cassette of the PCHF40 and the target exogenous gene, and the recombinant expression vector is introduced into a plant genome to obtain a transgenic plant of which the exogenous gene is specifically expressed in pollen.

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