CN110055252B

CN110055252B - Promoter PCHF45 specifically expressed in rice pollen and application thereof

Info

Publication number: CN110055252B
Application number: CN201910343747.XA
Authority: CN
Inventors: 黄培劲; 龙湍; 吴春瑜; 张维; 刘昊; 吴永忠; 曾翔; 李新鹏; 安保光
Original assignee: Hainan Bolian Rice Gene Technology Co ltd
Current assignee: Hainan Bolian Rice Gene Technology Co ltd
Priority date: 2019-04-26
Filing date: 2019-04-26
Publication date: 2022-11-01
Anticipated expiration: 2039-04-26
Also published as: CN110055252A

Abstract

The invention discloses a promoter PCHF45 specifically expressed in rice pollen and application thereof, and relates to the fields of genetic engineering and molecular biology. The promoter PCHF45 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 PCHF45. 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 PCHF45 specifically expressed in rice pollen and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a rice pollen specific promoter PCHF45 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 to initiate transcription, typically 40nt, and is 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 combine with a specific transcription factor to regulate 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 tobacco mosaic virus, the Actin promoter of rice and the Ubiquitin promoter of corn 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. 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 the difference of induction 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 that 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. The types of rice tissue-specific promoters developed at present are various, and promoters having tissue-specific expression are 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 for 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 the 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 develop further, forming a central large vacuole by the late stage. 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 microsporidia 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 PCHF45, 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) derived from the nucleotide sequence 1) which is substituted, deleted or added with one or more nucleotides and has the same pollen specificity starting function;

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.

A DNA molecule complementary to the nucleotide sequence of the plant pollen specific promoter PCHF45 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 it can hybridize with PCHF45 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 with the addition of destabilizing agents such as formamide. Low stringency conditions, for example, include hybridization in buffer solutions of 30-35% formamide, 1M NaCl, l% SDS (sodium dodecyl sulfate) at 37 ℃ and washing in 1X to 2 XSSC (20 XSSC =3.0M NaCl/0.3M trisodium citrate) at 50-55 ℃. 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, the Tm can be lowered by 10 ℃. Generally, stringent conditions are selected to be about 5 ℃ below the thermal melting point (Tm) for the particular 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 utilize a hybridization and/or wash 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-hybridization with nucleic acid probes, 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 PCHF45, 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 PCHF45.

The present invention further provides an expression vector containing the gene expression cassette for the pollen-specific promoter PCHF45.

The invention provides application of the rice pollen specific promoter PCHF45 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 PCHF45 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 exogenous genes specifically expressed in pollen, preferably a transgenic plant with enhanced or weakened pollination and 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 PCHF45, wherein the nucleotide sequence of the primer pair is SEQ ID NO:2-3 or SEQ ID NO:6-7.

The invention provides a method for separating a pollen specific promoter PCHF45, which is a method for separating a pollen specific promoter PCHF45 by using SEQ ID NO:2-3 or SEQ ID NO: the 6-7 primer pair PCR amplifies the nucleotide sequence of the pollen-specific promoter PCHF45.

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 PCHF45 and the target exogenous gene are cloned into a vector to obtain a recombinant expression vector of an expression cassette containing the PCHF45 and the target exogenous gene, and the recombinant expression vector is introduced into a plant genome to obtain a transgenic plant with the exogenous gene specifically expressed in pollen.

The pollen specific promoter PCHF45 provided by the invention has the following advantages:

1) PCHF45 is an endogenous DNA sequence of rice, and the transgenic safety risk is extremely low.

2) The PCHF45 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 vector map of the recombinant expression vector 1300gus-PCHF45 of the pollen-specific promoter PCHF45 in example 2.

FIG. 2 is a map of the recombinant expression vector DX2182-PCHF45 of the pollen-specific promoter PCHF45 in example 3.

FIG. 3 is a photograph of anther and pollen GUS staining of non-transgenic medium flower 11 rice, in which no pollen was stained blue.

FIG. 4 is a photograph of 1300GUS-PCHF45 transgenic rice anthers and pollen GUS staining in example 6.

FIG. 5 is a photograph of gynoecium GUS staining of 1300GUS-PCHF45 transgenic rice in example 6.

FIG. 6 is a photograph of GUS staining of glume of 1300GUS-PCHF45 transgenic rice in example 6.

FIG. 7 is a photograph of GUS staining of 1300GUS-PCHF45 transgenic rice roots in example 6.

FIG. 8 is a photograph showing GUS staining of stem of 1300GUS-PCHF45 transgenic rice in example 6.

FIG. 9 is a photograph of GUS staining of 1300GUS-PCHF45 transgenic rice leaves in example 6.

FIG. 10 is a photograph of non-transgenic mid-flower 11 rice pollen under a green fluorescence microscope, where no pollen fluoresces.

FIG. 11 is a photograph of the pollen of the Actin promoter transgenic rice in example 7 under a green fluorescence microscope, wherein most of the pollen fluoresces.

FIG. 12 is a photograph of the pollen of DX2182-PCHF45 transgenic rice in example 7 under a green fluorescence microscope, wherein half of the pollen fluoresces.

FIG. 13 is a photograph under a green fluorescence microscope of the DX2182-PCHF45 transgenic rice anthers in example 7.

FIG. 14 is a photograph of the transgenic rice glumes of DX2182-PCHF45 in example 7 under a green fluorescence microscope.

FIG. 15 is a photograph of DX2182-PCHF45 transgenic rice leaves under a green fluorescence microscope in example 7.

FIG. 16 is a photograph under a green fluorescence microscope of the pistils of DX2182-PCHF45 transgenic rice in example 7.

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 Rice pollen-specific promoter PCHF45

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 standby.

PCR primer design and amplification of PCHF45

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) and into the Xma I and Sal I cleavage sites of the DX2182 vector. The sequence of a primer for amplifying PCHF45 is shown as SEQ ID NO:2-3 and SEQ ID NO:6-7. Primer sequences SEQ ID NO:2-3 upstream and downstream primers have 15 nucleotide sequences (cgacggccagtgcca) at their 5' ends that overlap with the corresponding ligation sites in the vector for Gibson Assembly ligation. Primer sequence SEQ ID NO:6-7, the 5 'end of the upstream primer contains an Xma I enzyme cutting site sequence cccggg, and the 5' end of the downstream primer contains a Sal I enzyme cutting site sequence gtcgac.

PCR reaction (100. Mu.L): rice genomic DNADNA 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, mgSO₄：4μL，1/10 DMSO：20μL，ddH₂O：45μL。

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

The amplification product comprises a 2051bp pollen-specific promoter PCHF45 (the sequence is shown as SEQ ID NO. 1).

Example 2 construction of recombinant expression vector p1300gusplus-PCHF45 for promoter PCHF45

The PCR product obtained in example 1 was electrophoresed on 1% agarose gel, and a band of approximately 2051bp in size was collected. The vector p1300gusplus was double digested with Nco I and Hind III to recover the linear digested vector.

The PCR-recovered product and the linearized p1300gusplus empty vector were ligated using the lightning Cloning Kit (King Biotech Co., ltd., 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 is taken to transform the competent cells of the Escherichia coli by electric shock. Primers SEQ ID NO:4 and SEQ ID NO:5, carrying out colony PCR, selecting positive clone, sequencing and verifying. The correctly sequenced vector was named p1300gusplus-PCHF45 and the map is shown in 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 construction of recombinant expression vector DX2182-PCHF45 containing PCHF45 promoter sequence

1. Construction of recombinant cloning vector pGEM-PCHF45 containing PCHF45 promoter sequence

With reference to the method of example 1, the primer pair shown in SEQ ID NO:6 and SEQ ID NO:7 was used in place of the primer pair shown in SEQ ID NO:2 and SEQ ID NO:3 in example 1 for amplification, and the resulting amplification product was subjected to A-tailing (3. Mu.L of dNTP and 0.5. Mu.L of Taq enzyme were added to the unpurified product (50. Mu.L), and the reaction was continued at 72 ℃ for 20 min). And (4) purifying and recovering the product after adding the A tail, connecting the product with a pGEM-T vector, and performing the operation steps according to the instruction of the pGEM-T vector of the Promega company to obtain a recombinant cloning vector pGEM-PCHF45. And transforming escherichia coli competent cells, further selecting positive clones, and sequencing. The correctly sequenced clones were used to extract plasmids.

2. Construction of recombinant expression vector DX2182-PCHF45 containing PCHF45 promoter sequence

The recombinant cloning vector pGEM-PCHF45 and the expression vector DX2182 obtained in the example 3 are subjected to double digestion by restriction enzymes XmaI and SalI, and the cut fragment with the size of about 2051bp containing the PCHF45 promoter is inserted into the Xma I and SalI digestion sites of the linear digestion vector DX 2182.

Ligation of the PCR recovery product with linearized DX2182 empty vector using T4 ligase, 10. Mu.L system was as follows: mu.L of the recovered product (50 ng/. Mu.L), 4. Mu.L of the digestion vector (100 ng/. Mu.L), and 1. Mu.L of T4 ligase. And (3) connecting procedures: 22 ℃ for 2 hours.

5 mu L of the ligation product is taken to transform the competent cells of the Escherichia coli by electric shock. Primers SEQ ID NO:8 and SEQ ID NO:9, carrying out colony PCR, selecting positive clone, sequencing and verifying. The vector with correct sequencing was named DX2182-PCHF45 and the map is shown in figure 2. The DX2182 vector contains the EGFP gene (an enhanced GFP gene). The tissues expressing the EGFP gene emit fluorescence under a green fluorescence microscope and can be used for indicating the expression site and the strength of the promoter.

Example 4 obtaining of p1300gusplus-PCHF45 transgenic Rice

Agrobacterium EHA105 stored at-70 ℃ was streaked on a rifampicin-containing plate of 50. Mu.g/mL and cultured at 28 ℃. Single colonies were picked and inoculated into 50mL YEB broth, and shake-cultured at 220rpm for 12-16hr at 28 ℃. 2mL of the cell suspension was transferred to 100mL of YEB liquid medium (containing antibiotics), and the cell suspension was cultured at 28 ℃ and 220rpm with shaking 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 the p1300gusplus-PCHF45 plasmid obtained in example 2 and example 3 was added to 100. Mu.L of competent cells in two tubes, respectively, and transformed with 2.5 KV 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:4-5 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) efficiency transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the nucleic acids of the T-DNA. The Plant Journal6: 271-282). Obtaining T through links of co-culture, screening, differentiation, rooting and the like₀Transgenic seedlings are generated. Extracting total DNA of leaves of the transformed plants, and performing DNA amplification by using primers SEQ ID NO:10 and SEQ ID NO:11 carrying out PCR positive detection, selecting positive plants verified by PCR for cultivation, selfing and fructifying to obtain T₁And (4) generation. Get T₀Or T₁And carrying out subsequent analysis on the generation plants.

Example 5 acquisition of DX2182-PCHF45 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 for 12-16hr at 28 ℃. 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 ℃). Washed 2 times with sterile deionized water (10 mL each) and 1 time with 10% glycerol dissolved in 3mL10% glycerol. mu.L of DX2182-PCHF45 plasmid obtained in example 3 was added to 100. Mu.L of competent cells, and transformed with 2.5 KV shock. Positive clones were selected by culturing on YEB plates containing kanamycin and rifampicin at 28 ℃, and amplified with DX2182 vector-specific primers SEQ ID NO:8-9 PCR validation was performed.

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) efficiency transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the nucleic acids of the T-DNA. The Plant Journal6: 271-282). Obtaining T through links of co-culture, screening, differentiation, rooting and the like₀Transgenic seedlings are generated. Extracting total DNA of leaves of the transformed plants, and performing DNA amplification by using primers SEQ ID NO:10 and SEQ ID NO:11 PCR positive detection, selecting positive plant verified by PCR, cultivating, selfing and obtaining T₁And (4) generation. Get T₀Or T₁And carrying out subsequent analysis on the generation plants.

Example 6 GUS staining analysis of transgenic Rice

Preparing GUS staining solution X-Gluc reaction liquid (50 mM sodium phosphate buffer solution, pH value 7.0,0.5mM potassium ferricyanide, 0.5mM potassium ferrocyanide, 0.5mg/ml X-Gluc, 20% by volume of methanol, 0.1% Triton X-100), randomly selecting more than 5 1300GUS-PCHF45 transgenic positive strains obtained in example 4, collecting anther, pistil, glume, root, leaf, stem and other tissue samples, soaking in the X-Gluc reaction liquid at 37 ℃ for 2h or overnight, removing the chloroplast color of the tissues by using 75% by volume of ethanol, and observing and photographing. The results showed that pollen from flower 11 in the wild type could not be stained (FIG. 3), while half of the pollen from transgenic rice could not be stained blue (FIG. 4), and none of the other tissues such as pistil (FIG. 5), glume (FIG. 6), root (FIG. 7), stem (FIG. 8), leaf (FIG. 9) could be stained, indicating that the PCHF45 promoter could drive the GUS gene to be expressed specifically in rice pollen, and none of the tissues such as rice anther, glume, root, stem, leaf, pistil could drive the expression of the foreign gene.

Example 7 transgenic Rice pollen EGFP Green fluorescence assay

More than 5 DX2182 transgenic positive lines obtained in example 5 were randomly selected to collect anthers and pollen slides in the maturation stage, and the slides were analyzed under a green fluorescence microscope. The results showed that pollen from flower 11 did not fluoresce in the wild type (FIG. 10), whereas transgenic pollen from the positive control Actin promoter (also constructed upstream of the DX2182 vector EGFP reporter gene) fluoresced (FIG. 11). Referring to the positive control result, the transgenic pollen of the PCHF45 promoter can emit obvious fluorescence (figure 12), and tissues such as other anthers (figure 13), glumes (figure 14), leaves (figure 15), pistils (figure 16) and the like can not emit fluorescence, which shows that the PCHF45 promoter can drive the specific expression of EGFP gene in rice pollen, but can not drive the expression of exogenous genes in the tissues such as rice anthers, glumes, leaves, pistils and the like.

Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Sequence listing

<110> Hainan Borax Rice Gene science and technology Co., ltd

<120> promoter PCHF45 specifically expressed in rice pollen and application thereof

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aatccatcgc cagttttttt ttagcaaaaa ccgaaatatc aataaaccta aacagaaaac 1800

tgcatttctc tcaaccatgt ccaaaacgtg ggtcccatgc atcttctcca actcgattcc 1860

cgccaaaaat agcgcgccaa accaccggaa cacgcgcgcc gccgcacggc catggccacc 1920

gccgctgccc tatatctccc ctctctcacc accctccata taggcaggca ggcaccgtcg 1980

ccggtggtgg cccacctcgt gcctcctccc ccaccccccg gccctctcct cccccacctc 2040

cagccatgga g 2051

<210> 2

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgacggccag tgccacctag ctctatgtac ttcccata 38

<210> 3

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gaaatttacc ctcagatcta ccatctccat ggctggaggt gg 42

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gatcagttta aagaaagatc aaagctc 27

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctgcaaggcg attaagttgg gtaac 25

<210> 6

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cccgggccta gctctatgta cttcccataa gcc 33

<210> 7

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtcgacggtg cctgcctgcc tatatg 26

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aactgcctgg cacagcaatt g 21

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gacgttgtgg ctgttgtagt tgtac 25

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cttagccaga cgagcgggtt c 21

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gcttctgcgg gcgatttgt 19

Claims

1. The rice pollen specific promoter PCHF45 is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. A gene expression cassette comprising the rice pollen-specific promoter PCHF45 according to claim 1.

3. An expression vector comprising the rice pollen-specific promoter PCHF45 of claim 1.

4. The use of the rice pollen-specific promoter PCHF45 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 PCHF45 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 rice pollen specific promoter PCHF45 or an expression cassette, an expression vector or a host cell containing the same as claimed in claim 1 is used for preparing transgenic rice.

7. The primer pair for amplifying the rice pollen specific promoter PCHF45 as claimed in claim 1, wherein the nucleotide sequence of the primer pair is SEQ ID NO:2-3 or SEQ ID NO:6-7.

8. The use of the rice pollen specific promoter PCHF45 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: introducing the rice pollen specific promoter PCHF45 and the target exogenous gene into a vector to obtain a recombinant expression vector of an expression cassette containing the PCHF45 and the target exogenous gene, and introducing the recombinant expression vector into a plant genome to obtain a transgenic plant of which the exogenous gene is specifically expressed in pollen; the nucleotide sequence of the rice pollen specific promoter PCHF45 is shown in SEQ ID NO. 1.