CN110982819B - Promoter PCHF7 specifically expressed in rice anther and pollen and application thereof - Google Patents
Promoter PCHF7 specifically expressed in rice anther and pollen and application thereof Download PDFInfo
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Abstract
The invention discloses a promoter PCHF7 specifically expressed in anthers and pollen of rice and application thereof, and relates to the fields of genetic engineering and molecular biology. The promoter PCHF7 has a nucleotide sequence shown as SEQ ID NO.1, and the invention further provides an expression vector, a gene expression cassette, engineering bacteria or a cell line containing the promoter PCHF 7. The invention also provides a primer pair for amplifying anther and pollen specific promoters. The anther and pollen specific promoter is a rice endogenous gene, is very beneficial to rice genetic engineering, can drive the specific expression of exogenous genes in the anther and pollen and has accurate expression level, and a novel method for driving the specific expression of exogenous genes in the anther and pollen of rice is provided.
Description
Technical Field
The invention relates to the fields of genetic engineering and molecular biology, in particular to a rice anther and pollen specific promoter PCHF7 and application thereof.
Background
Transcriptional regulation is one of the major forms of plant gene expression regulation, coordinated by cis-acting elements and trans-acting factors. Promoters are one of the most important cis-elements in the transcriptional regulation of plant genes, and are generally located in the 5' -end upstream region of genes, and are recognition and binding sites for RNA polymerase and some trans-acting factors. The promoter mainly comprises a core promoter region and a transcription regulatory region. The core promoter region is the shortest promoter fragment, typically 40nt, that initiates transcription and is a DNA sequence recognized and bound by RNA polymerase families I, II and III. The region contains some important functional elements, can accurately position transcription start points and directions, and is the basis of gene expression regulation. The transcription regulatory region is located upstream (or downstream) of the core promoter, and can be combined with a specific transcription factor to regulate the time and intensity of transcription, such as enhancers and silencers. The expression mode of the promoter is studied deeply, which is not only beneficial to understanding the expression regulation mechanism and biological function of the gene, but also beneficial to controlling the expression of exogenous genes.
Promoters can be classified into three types of constitutive promoters, inducible promoters and space-time specific promoters according to their expression patterns. Constitutive promoters can initiate gene transcription in all or most tissues, allowing for time-space persistence and constancy of expression levels in gene expression. The 35S promoter of tobacco mosaic virus, the action promoter of rice and the Ubiquitin promoter of corn all belong to constitutive promoters. The constitutive promoter is widely applied to genetic engineering research of plants and is used for overexpression of target genes, such as insect-resistant and herbicide-resistant genes. Inducible promoters can initiate or greatly enhance gene expression under the stimulation of certain physical or chemical signals. They have sequence structures with enhancer, silencer or similar functions and have obvious specificity. The inducible promoters may be classified into light-inducible promoters, heat-inducible promoters, low-temperature-inducible promoters, drought-inducible promoters, wound-inducible promoters, hormone-inducible promoters, etc., according to the difference of the induction signals. Spatiotemporal specific promoters only initiate gene expression in a particular growth stage or site. Tissue-specific promoters are one type of spatiotemporal-specific promoters that only promote expression in a particular cell, tissue or organ. The use of a promoter for tissue specific expression in genetic transformation of plants to control the expression of a target gene can more effectively avoid potential side effects caused by the use of a constitutive promoter, such as reduction of metabolic burden increased by constitutive expression, reduction of transgenic food safety risks and adverse effects on the environment, and gene silencing caused by repeated use of the same promoter. The variety of rice tissue-specific promoters developed so far is varied, and promoters for tissue-specific expression have been found in almost all tissues such as roots, stems, leaves, seeds and fruits.
Male sterility is the basis of heterosis utilization, is the core technology of hybrid rice industry, and has important theoretical and practical significance for researching the mechanism of rice fertility regulation. Anthers are organs of rice that produce mature male gametophytes (i.e., pollen or microspores). In the early stages of anther development, there is a protozoan spore forming cell that undergoes a series of divisions and differentiation, producing the tapetum and pollen mother cells. The tapetum provides nutrition for the development of male gametophytes, including various enzymatic and non-enzymatic proteins, and is involved in the synthesis of the pollen outer wall. Pollen mother cells undergo meiosis, resulting in tetrads. Single cells in tetrads are separated by the action of callase secreted by the tapetum to produce haploid microsporosomes. Haploid small sporophytes develop further, forming central large vacuoles by late stages. The central large vacuole forces the nucleus to the cell edge (called the mononuclear borderline), creating cell polarity. This cell polarity promotes the small sporophytes to undergo an unequal mitosis, producing a large vegetative cell and a small germ cell. The small germ cells are completely contained within the large vegetative cells, forming a cell-specific phenomenon in the cells. The germ cells then undergo a second mitosis, producing two sperm cells, forming mature pollen grains. Therefore, screening and determining rice anther and pollen specific promoters provides new choices for genetic engineering of rice, in particular in fertility regulation, transgenic drift prevention and control and the like.
Disclosure of Invention
The invention aims to provide a promoter specifically expressed in plant anthers and pollen and application thereof.
The invention provides a plant anther and pollen specific promoter PCHF7, which comprises the following components:
1) The nucleotide sequence shown in SEQ ID NO.1, or
2) A nucleotide sequence derived from 1) which is substituted, deleted or added with one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.1 and has the same anther and pollen specific initiation function; or (b)
3) A sequence complementary to the nucleotide sequence shown in SEQ ID NO. 1.
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 compared with 1) and has the same anther and pollen specific promoter function as the nucleotide sequence in 1).
DNA molecules complementary to the plant anther and pollen-specific promoter PCHF7 nucleotide sequence can be readily identified and utilized by those skilled in the art for the same purposes, and thus DNA sequences which have promoter activity and which hybridize under stringent conditions to the promoter sequences of the present invention or fragments thereof are included in the present invention. Wherein the nucleotide sequence is complementary, meaning that it hybridizes to PCHF7 under stringent conditions.
Stringent conditions are those under which the probe will hybridize to its target sequence to a detectable extent that hybridizes to other sequences (e.g., at least 2 times background). Stringent conditions are sequence-dependent and will be different depending on the other conditions of the experiment. By controlling the stringency of hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homology detection). Alternatively, stringent conditions may be adjusted to allow some sequence mismatches so that a lower degree of similarity is detected (heterologous probing). Typically, the probe is no more than 1000 nucleotides in length, preferably less than 500 nucleotides in length.
Typically, stringent conditions will be 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 salt) at pH 7.0-8.3, at a temperature of 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 30-35% formamide, 1M NaCl, l% SDS (sodium dodecyl sulfate) buffer at 37℃and washing in 1 XSSC to 2 XSSC (20 XSSC=3.0M NaCl/0.3M trisodium citrate) at 50-55 ℃. Moderately stringent conditions, for example, include hybridization in a buffer solution of 40-45% formamide, 1.0M NaCl, l% SDS at 37℃and washing in 0.5 XSSC to 1 XSSC at 55-60 ℃. High stringency conditions, for example, include hybridization in 50% formamide, 1M NaCl, l% SDS buffer at 37℃and washing in 0.1 XSSC at 60-65 ℃. Optionally, the wash buffer may contain about 0.1% -1% SDS. Hybridization times are generally less than about 24 hours, typically about 4-12 hours.
Particularly typically as a function of post-hybridization washes, the key 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=81.5deg.C+16.6 (log M) +0.41 (% GC) -0.61 (% form) -500/L; where M is the molar concentration of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is the temperature (at a defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. The Tm is reduced by about l ℃ for every 1% mismatch; thus, tm hybridization and/or wash conditions can be adjusted to hybridize to sequences of desired identity. For example, if the sequences sought have an identity of 90% or more, the Tm can be reduced by 10 ℃. Generally, stringent conditions are selected to be about 5 ℃ below the thermal melting point (Tm) for a particular sequence and to be complementary at the specified ionic strength and pH. However, highly stringent conditions can be used for hybridization and/or washing at temperatures below the thermal melting point (Tm) of 1, 2, 3, or 4 ℃; 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 apply hybridization and/or washing at 11, 12, 13, 14, 15, or 20℃below the thermal melting point (Tm). Those of ordinary skill in the art will appreciate that the conditions of the hybridization and/or wash solutions vary with the stringency and that this equation is used to calculate the hybridization and wash compositions and desired Tm. If the degree of mismatch desired is such that Tm is below 45 ℃ (aqueous solution) or 32 ℃ (formamide solution), the SSC concentration is preferably increased to enable the use of higher temperatures. Nucleic acid hybridization guidance is found in Tijssen (1993) biochemistry and molecular biology laboratory techniques using nucleic acid probes for hybridization, part I, chapter 2 (Elsevier, new York); and Ausubel et al, editions (1995) modern methods of molecular biology chapter 2 (Greene Publishing and Wiley-Interscience, new York). See Sambrook et al (1989) molecular cloning, a laboratory Manual (second edition, cold Spring Harbor Laboratory Press, planview, new York).
The stringent conditions are preferably hybridization in a solution of 6 XSSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate), at 65℃and then washing the membrane 1 time with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
The invention provides a gene expression cassette containing the plant anther and pollen specific promoter PCHF7, an expression vector and a host cell containing the expression vector.
The gene expression cassette is an expression cassette in which a structural gene, a regulating gene, an antisense gene of the structural gene, an antisense gene of the regulating gene or a small RNA gene capable of interfering the expression of an endogenous gene is connected to the downstream of an anther and pollen specific promoter PCHF 7.
The invention further provides plants containing the gene expression cassette for the anther-and pollen-specific promoter PCHF 7.
The invention provides application of the rice anther and pollen specific promoter PCHF7 or an expression cassette, an expression vector or a host cell containing the rice anther and pollen specific promoter PCHF7 in driving exogenous genes to specifically express in plant pollen.
The invention provides application of rice anther and pollen specific promoter PCHF7 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 anthers and 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, maize, sorghum, barley, oats, wheat, millet, sugarcane, cotton, safflower, tobacco, alfalfa, sunflower, brassica species, leguminous plants.
The invention also provides a primer pair for amplifying the anther and pollen specific promoter PCHF7, wherein the nucleotide sequences of the primer pair are SEQ ID NO.2 and SEQ ID NO.3.
The invention provides a method for separating anther from pollen specific promoter PCHF7, which uses SEQ ID NO.2 and SEQ ID NO.3 primer pair to PCR amplify nucleotide sequences of anther and pollen specific promoter PCHF 7.
The invention also provides a method for driving the specific expression of exogenous genes in anthers and pollen, which comprises the following steps:
cloning the rice anther and pollen specific promoter PCHF7 and the target exogenous gene into a vector to obtain a recombinant expression vector containing the expression cassette of the PCHF7 and the target exogenous gene, and introducing the recombinant expression vector into a plant genome to obtain a transgenic plant with the exogenous gene specifically expressed in the anther and pollen.
The anther and pollen specific promoter PCHF7 provided by the invention has the following advantages:
1) PCHF7 is an endogenous DNA sequence of rice, and the transgenic safety risk is extremely low.
2) PCHF7 can drive the specific expression of exogenous gene in anther and pollen, and the expression level is accurate.
3) The invention provides a novel method for driving exogenous genes to specifically express in anthers and pollen.
Drawings
FIG. 1 is a diagram showing the element distribution required for anther and pollen expression in example 1.
FIG. 2 is a flow chart showing the construction of the recombinant expression vector pc1300gusplus-PCHF7 of the anther-and pollen-specific promoter PCHF7 of example 2.
FIG. 3 is a photograph of non-transgenic medium flower 11 rice anther and pollen GUS staining.
FIG. 4 is a photograph of GUS staining of the anther of pc1300gusplus-PCHF7 transgenic rice in example 4.
FIG. 5 is a photograph of GUS staining of pc1300gusplus-PCHF7 transgenic rice pollen in example 4.
FIG. 6 is a photograph of GUS staining of the pistil (A), glume (B), leaf (C), root (D), stem (E) of pc1300 guslus-PCHF 7 transgenic rice in example 4.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
EXAMPLE 1 acquisition of the anther-and pollen-specific promoter PCHF7 of Rice
1. Extraction of genomic DNA from rice
Rice genomic DNA was extracted using a plant DNA isolation kit (Chengdu Fuji Biotechnology Co., ltd.). The genome is derived from fresh leaves of the rice variety Nipponbare. The extracted genome DNA is split-packed and stored at-20 ℃ for standby.
PCR primer design and amplification of PCHF7
The promoter element predicted website New PLACE (https:// www.dna.affrc.go.jp/PLACE/. The DNA fragments containing different numbers of the POLLEN1LELAT52 and GTGANTG10 elements with different lengths are respectively intercepted for carrying out promoter activity identification, and the result shows that the expression effect of the DNA fragments containing 3 POLLEN1LELAT52 elements and 3 GTGANTG10 elements is best, so that the-1985 to-1 bp fragment (SEQ ID NO. 1) containing 3 POLLEN1LELAT52 elements and 3 GTGANTG10 elements is finally determined as a promoter sequence, and the DNA fragment has the excellent function of driving the expression of genes in anthers and POLLEN, and can be used as a promoter for driving the specific expression of exogenous genes or rice endogenous genes in anthers or POLLEN.
Primers were designed according to the nucleotide sequence of PCHF7 (shown as SEQ ID NO. 1) using Primer Premier software, and the Primer sequences are shown as SEQ ID NO.2 and SEQ ID NO.3.
Wherein the 5' ends of the upstream and downstream primers are overlapped with the corresponding connection positions of the vector by 15 nucleotide sequences so as to carry out Gibson Assembly connection.
When the primers shown in SEQ ID No.2 and SEQ ID No.3 are used for amplifying the PCHF7 promoter, the following components are added into a 100 mu L PCR reaction system: 3. Mu.L of DNA template (containing about 50ng of total genomic DNA), 2. Mu.L of KOD polymerase (available from Toyoba Corp.), 10. Mu.L of 10 Xbuffer, 3. Mu.L of 10. Mu.M forward primer, 3. Mu.L of 10. Mu.M reverse primer, 10. Mu.L of 10. Mu.M dNTP, 4. Mu.L of MgSO 4 ，20μL 1/10DMSO，45μL ddH 2 O。
PCR procedure: pre-denaturation at 95℃for 4min. Denaturation at 94℃for 30s; annealing at 50 ℃ for 30s; extending at 68deg.C for 2min;35 cycles. Extending at 68deg.C for 10min.
The amplified product contains 1988bp anther and pollen specific promoter PCHF7 (sequence shown as SEQ ID NO. 1).
EXAMPLE 2 construction of the recombinant expression vector pc1300 guspus-PCHF 7 for the promoter PCHF7
The PCR product obtained in example 1 was electrophoresed in 1% agarose gel, and a 1988 bp-sized band was recovered. The linearized vector was recovered by double digestion of the vector pc1300gusplus with Nco I and Hind III.
The PCR recovery product was ligated with linearized pc1300gusplus empty vector using Lightening Cloning Kit (Jin Fusai (Beijing) Biotech Co., ltd.) and 10. Mu.L of the system was as follows: 2.5. Mu.L of recovered product (50 ng/. Mu.L), 0.5. Mu.L of digested carrier (100 ng/. Mu.L), 2.5. Mu.L of Ligation Mix. And (3) connection procedure: 50℃for 60min.
E.coli competent cells were transformed with 5. Mu.L of the ligation product. Using the primer SEQ ID NO:4 and SEQ ID NO:5, colony PCR is carried out, and positive clone sequencing verification is selected. The correct sequencing vector was designated pc1300gusplus-PCHF7 (FIG. 2). The pc1300gusplus vector contains the GUS gene. PCHF7 is inserted in the upstream of GUS gene to drive GUS gene expression, and the tissue expressing GUS gene is dyed to blue for indicating the expression site and strength of the promoter.
Example 3 obtaining of pc1300gusplus-PCHF7 transformed rice
Agrobacterium EHA105, stored at-70℃was streaked onto plates containing 50. Mu.g/mL rifampicin and incubated at 28 ℃. Single colonies were picked and inoculated into 50mL of YEB liquid medium and shake-cultured at 220rpm at 28℃for 12-16hr. 2mL of the bacterial liquid was transferred to 100mL (containing antibiotics) of YEB liquid medium, and cultured with shaking at 28℃and 220rpm until OD600 = 0.5. Pre-cooling on ice for 10min, centrifuging at 5000rpm for 10min (pre-cooling to 4 ℃ C. In a refrigerated centrifuge). 2 washes (10 mL each) with sterile deionized water, 1 wash with 10% glycerol in 3mL 10% glycerol. 100. Mu.L of competent cells were transformed with 1. Mu.L of the pc1300gusplus-PCHF7 plasmid obtained in example 2 by electric shock at 2.5 KV. Positive clones were selected for PCR verification with pc1300gusplus vector specific primers SEQ ID NO.4 and SEQ ID NO.5 by incubation at 28℃on YEB plates containing kanamycin and rifampicin.
The correct clones were verified and flowers 11 (Hiei Y Ohta S, komari T, kumashro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal 6:271-282) were infected in rice by Agrobacterium-mediated genetic transformation. Obtaining T through co-culture, screening, differentiation, rooting and other links 0 Transgenic seedlings were generated. Extracting total DNA of leaves of the transformed plant, carrying out PCR positive detection by using primers SEQ ID NO.6 and SEQ ID NO.7, selecting positive plant cultivation verified by PCR, and obtaining T by selfing and setting 1 And (3) replacing. Taking T 0 Or T 1 The plants were subsequently analysed.
EXAMPLE 4 GUS staining analysis of transgenic Rice
Preparation of 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% methanol by volume, 0.1% Triton X-100), random selection of more than 5 tissue samples such as anthers, pistils, glumes, roots, leaves, stems obtained in example 3, and the like were collected, immersed in the X-Gluc reaction solution at 37℃for 2 hours or overnight, and then observed for photography after removal of the chloroplast color of the tissue with 75% ethanol by volume. As a result, as shown in FIGS. 3 to 6, pollen of flower 11 in the wild type could not be stained (FIG. 3), whereas anthers (FIG. 4) and pollen (FIG. 5) of transgenic rice were stained blue, and tissues such as other pistils (A of FIG. 6), glumes (B of FIG. 6), leaves (C of FIG. 6), roots (D of FIG. 6), stems (E of FIG. 6) could not be stained, indicating that the PCHF7 promoter could drive GUS gene expression specifically in rice anthers and pollen.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
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gaaatttacc ctcagatcta ccatgggagg aggtagaaca agg 43
<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> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
cttagccaga cgagcgggtt c 21
<210> 7
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gcttctgcgg gcgatttgt 19
Claims (9)
1. The rice anther and pollen specific promoter PCHF7 is characterized in that the nucleotide sequence is as follows: the nucleotide sequence shown in SEQ ID NO. 1.
2. A gene expression cassette comprising the rice anther and pollen specific promoter PCHF7 of claim 1.
3. An expression vector comprising the rice anther of claim 1 together with a pollen-specific promoter PCHF 7.
4. Use of the rice anther and pollen specific promoter PCHF7 of claim 1 or an expression cassette, expression vector or host cell containing it to drive the specific expression of exogenous genes in plant anthers and pollen.
5. Use of the rice anther and pollen specific promoter PCHF7 of claim 1 or an expression cassette, expression vector or host cell containing it for the preparation of transgenic plants.
6. The use according to claim 4 or 5, wherein the plant is rice, maize, sorghum, barley, oat, wheat, millet, sugarcane, cotton, safflower, tobacco, alfalfa, sunflower, brassica species, legumes.
7. A primer pair for amplifying the rice anther and pollen specific promoter PCHF7 according to claim 1, wherein the nucleotide sequence of the primer pair is the nucleotide sequence shown in SEQ ID No.2 and SEQ ID No.3.
8. The use of the rice anther and pollen specific promoter PCHF7 of claim 1 or an expression cassette, expression vector or host cell containing the same or the primer pair of claim 7 for preparing transgenic rice with exogenous genes specifically expressed in the rice anther and pollen.
9. The method for driving the specific expression of the exogenous gene in the plant anther and pollen is characterized by comprising the following steps: the rice anther and pollen specific promoter PCHF7 and a target exogenous gene are introduced into a vector to obtain a recombinant expression vector containing an expression cassette of the PCHF7 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 the anther and pollen.
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