Plant anther and pistil-specific expression promoter and use thereof

Abstract

The application discloses a plant anther and pistil specific expression promoter and application thereof, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the promoter is shown as SEQ ID NO. 1, and the promoter is an anther and pistil specific expression promoter. The promoter has an important role in plant genetic engineering research and application, the application constructs a biological spontaneous light system module without a screening marker and a gene editing element by using the promoter, uses an FBP biological light emitting system to efficiently identify a transgenic plant, and cuts off a screening marker gene and a gene editing Cas9 element in a sexual reproduction process of a transgenic T0 generation, so that a transgenic plant with better biological safety is obtained. In addition, the plant after the screening marker gene is cut off can continue to be transformed by a carrier carrying a screening marker in the next round, a large number of target genes are transformed, and the crop genome is modified for multiple rounds.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to an anther and pistil-specific promoter (APSP) and its applications. Background Technology

[0002] Gene editing is an emerging and relatively precise genetic engineering technology that can modify specific target genes in an organism's genome. The advent of the CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system has accelerated the development of gene editing technology and has been widely applied in the life sciences. To improve the performance of these systems, researchers have designed and developed a variety of CRISPR-Cas tools with a wider target range, higher efficiency and specificity, and higher precision (Liu et al. 2022). How to precisely modify the genes of plants and animals using gene editing technology, and then remove the exogenous CRISPR-Cas system to obtain gene-edited plants and animals without any exogenous genes, is a common concern in biosafety, clinical safety, and food safety.

[0003] Hygromycin B is a commonly used resistance selection drug. It works by interfering with 70S ribosome translocation, leading to translational errors, thereby inhibiting protein synthesis and ultimately killing cells. Its resistance gene encodes hygromycin phosphotransferase (Hpt). Phosphorylation of hygromycin B inactivates it, thus enabling the selection of transgenic positive plants using hygromycin. However, how to remove the Hpt gene after obtaining transgenic positive plants is a biosafety issue.

[0004] The Cre-loxP recombination system is a site-specific recombinase technology that can perform deletion, insertion, translocation, and inversion at specific sites on DNA. The Cre / LoxP system mainly consists of two components: Cre recombinase and a DNA sequence called the LoxP site. Cre recombinase is a 38kD protein encoded by bacteriophage P1, which recognizes two 13bp recombination-binding elements (RBEs) on the loxP site and exerts a cleaving effect (McLellan et al. 2017). This technology is characterized by high efficiency, high specificity, and wide applicability, playing an important role in plant and animal genetic engineering. However, how to regulate the specific expression of Cre recombinase to better achieve its effects is a problem that needs to be solved by those skilled in the art.

[0005] Plant gene promoters are DNA sequences that RNA polymerase recognizes, binds to, and initiates transcription. They contain conserved sequences required for RNA polymerase-specific binding and transcription initiation. These promoters regulate the expression of downstream genes, determining the orderly expression of genes in time and space, which is crucial for plant morphogenesis and maintaining normal growth cycles. Tissue-specific or conditionally induced promoters are also major factors in the rational allocation of energy flow and environmental adaptability.

[0006] Currently, promoters used in genetic engineering include constitutive promoters, inducible promoters, and tissue-specific promoters. Constitutive promoters can drive the continuous expression of target genes in various plant tissues, but they excessively consume substances and energy within the recipient cells, thus having certain limitations in application. Inducible promoters, under the induction of exogenous physical and chemical factors, can rapidly induce the "on" and "off" of gene transcription, thus allowing for the regulation of transgene expression in plants according to experimental needs. Tissue-specific promoters can only initiate the expression of downstream genes in specific organs or tissues, thereby overcoming the waste caused by the non-specific, continuous, and efficient expression of exogenous genes initiated by constitutive promoters in the recipient, increasing the effectiveness of transgenes. Therefore, they have received increasing attention in plant genetic modification; however, very few useful tissue-specific promoters have been identified.

[0007] Therefore, it is very important to find tissue-specific promoters in plants and animals. Driving Cre recombinase-specific expression through tissue-specific promoters and removing genes or editing elements that are no longer necessary during transgenic processes is an important research topic involving biosafety and life safety. Summary of the Invention

[0008] The purpose of this invention is to provide a promoter that is specifically expressed in anthers and pistils, and to apply it to transgenic engineering.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention, through analysis of tobacco transcriptome data, screened a candidate anther- and pistil-specific expression promoter, APSP, whose nucleotide sequence is shown in SEQ ID NO.1. Studies have shown that, compared to growth during tobacco transgenic processes, the expression level of downstream genes initiated by this promoter in the anthers and pistils is more than 10 times higher than in other tested tissues.

[0011] This invention provides a method for obtaining the DNA molecule shown in SEQ ID NO.1, comprising: firstly extracting genomic DNA from tobacco (N. tabacum cultivar ZY100), and then cloning the DNA molecule fragment using PCR technology; the primers used for PCR amplification are: F primer: 5'-gagaaaaactagaaatttacgacatAATTTCTCCTAACTTTCACTTTGATTTTC-3', R primer: 5'-cagtaggatagaggtggctcCCATTAATCAACATACAATG-3'.

[0012] This invention provides the application of the plant anther and pistil-specific expression promoter APSP in the construction of gene expression cassettes or gene expression vectors that specifically express genes in anthers and pistils.

[0013] By integrating the aforementioned promoter upstream of the target gene, which cannot induce or induces the expression of the downstream target gene at a low level during the plant's growth period, the target gene is specifically activated to be highly expressed in the anthers and pistils during the flowering period.

[0014] Furthermore, the present invention provides a gene expression cassette specifically expressed in anthers and pistils, comprising the aforementioned anther and pistil-specific expression promoters.

[0015] Furthermore, the present invention provides a gene expression vector specifically expressed in anthers and pistils, comprising the aforementioned gene expression cassette.

[0016] Preferably, the gene expression vector is a marker-free transformation vector.

[0017] Preferably, the marker-free transformation vector is a plant Cre / loxP recombinant expression vector, wherein the T-DNA region of the plant Cre / loxP recombinant expression vector contains two or more unidirectional loxP sequences, and between the two unidirectional loxP sequences are a Cre recombinase expression cassette and a marker gene expression cassette; the promoter of the Cre recombinase expression cassette is the above-mentioned anther and pistil-specific expression promoter.

[0018] After the plant Cre / loxP recombinant expression vector is transferred into a plant, during the development of the anther and pistil, the promoter drives the expression of Cre recombinase, which deletes the gene expression cassette between the two loxP sequences, thereby removing the marker gene and the Cre gene itself.

[0019] This invention demonstrates that the pistil and anther-specific expression promoter APSP driving Cre recombinase expression can excise multiple target sites, providing technical support for multi-gene, multi-system modification in plant genetic engineering. Furthermore, multiple loxP sequences are introduced into the T-DNA region of the plant Cre / loxP recombinant expression vector, and Cre recombinase expression cassettes, marker gene expression cassettes, or gene editing element expression cassettes are inserted between the loxP sequences. The marker gene expression cassettes or gene editing element expression cassettes use promoters different from those used for the Cre recombinase expression cassette, such as the CaMV 35S promoter.

[0020] Furthermore, the present invention provides a recombinant engineered bacterium, wherein the engineered bacterium contains the aforementioned gene expression vector.

[0021] This invention provides a method for expressing a target nucleotide sequence in the anthers and pistils of plants, comprising: constructing an expression vector containing the promoter APSP and the target nucleotide sequence linked to the promoter; introducing the expression vector into a plant; and screening and cultivating transgenic plants.

[0022] Furthermore, the target nucleotide sequence can be a structural gene, a regulatory gene, or a small RNA that can interfere with the expression of endogenous genes. Its specific expression during pollen development can regulate pollen fertility and pollen germination.

[0023] The plant is a dicotyledonous plant, and more specifically, the plant is tobacco.

[0024] The present invention also provides a method for cultivating transgenic plants without selection markers, comprising:

[0025] (1) Construct a plant Cre / loxP recombinant expression vector, wherein the T-DNA region of the plant Cre / loxP recombinant expression vector contains two or more loxP sequences, and between the two loxP sequences therein is a Cre recombinase expression cassette and a marker gene expression cassette; the promoter for initiating Cre recombinase expression in the Cre recombinase expression cassette is the above-mentioned plant anther and pistil-specific expression promoter APSP.

[0026] (2) The T-DNA region of the plant Cre / loxP recombinant expression vector was introduced into the recipient plant using transgenic technology, and the plant was cultured to obtain transgenic plants without selection markers.

[0027] In the above method, the APSP promoter drives the expression of the Cre enzyme. This promoter cannot induce or induces low-level expression of the downstream target gene Cre in callus, leaves, stems, and roots during the transgenic selection and differentiation stages. However, it induces high expression of the downstream Cre enzyme gene in anthers and pistils during flowering. The Cre enzyme recognizes recombination-binding elements on loxP and exerts a cleaving effect. The selection marker gene is excised during the sexual reproduction of the transgenic T0 generation without affecting the acquisition of the target trait.

[0028] Furthermore, the plant Cre / loxP recombinant expression vector also contains a multiple cloning site or an expression cassette for the exogenous target gene; the multiple cloning site or the expression cassette for the exogenous target gene is located within the T-DNA region, outside the two loxP sequence regions.

[0029] This invention provides a method for cultivating self-luminescent transgenic plants without selection markers and gene editing elements, comprising:

[0030] 1) Using multi-gene assembly technology, the Hpt gene, Cas9 gene, Cre gene, Hisps gene, CPH gene, H3H gene, and Luz gene are integrated into a receptor vector to construct a multi-gene vector. The multi-gene vector contains two or more lox-P elements. The expression modules of the Hpt gene, Cas9 gene, and Cre gene are located between two lox-P elements. The promoter APSP is integrated upstream of the Cre gene. The expression modules of the Hisps gene, CPH gene, H3H gene, and Luz gene are located outside the two lox-P sequence regions.

[0031] 2) Using transgenic technology, the T-DNA region fragment from the multi-gene vector is introduced into the recipient plant, cultivated, and T0 generation transgenic plants are obtained. During the T1 generation seedling stage, the transgenic plants are screened to obtain transgenic plants with Hpt and Cas9 cut off.

[0032] In the above method, the constructed multi-gene fragment is introduced into recipient plants, where it is expressed. The expressed proteases participate in the caffeic acid cycle, causing the plant to glow, and serving as a reporter system for convenient screening of transgenic positive plants. The Cre gene is expressed only in the reproductive organs during the flowering stage of T0 generation transgenic plants, and the target genes Hpt and Cas9 are removed from the loxP site. Finally, transgenic plants with the selection marker Hpt gene and the Cas9 gene removed during gene editing are obtained in the T1 generation.

[0033] The sequence information for Hisps can be found in accession number QJQ48095.1; the sequence information for CPH can be found in accession number QJQ48093.1; the sequence information for H3H can be found in accession number QJQ48094.1; and the sequence information for Luz can be found in accession number QJQ48096.1. The sequence information for the Hpt gene can be found in accession number MH752994.1 of the vector pEASY-tub / hptII; the sequence information for the Cas9 gene can be found in accession number MG719602.1 of the vector pYLCRISPR / Cas9Pubi-N; and the sequence information for the Cre gene can be found in accession number MK854762.1.

[0034] Preferably, in step 1), the TransGene Stacking II system is used for multi-genome assembly. pYL322d1 is used as donor vector I, pYL322d2 as donor vector II, pYLTAC380GW as recipient vector, and pYLMF-H as the selection marker Hpt self-deletion supply vector, wherein the promoter driving Cre recombinase expression is replaced with promoter APSP.

[0035] Specifically, the method for constructing the multi-gene vector includes the following steps:

[0036] a. Insert the Hisps and H3H gene fragments into the multiple cloning site of pYL322d1 to obtain pYL322d1-35S-Hisps and pYL322d1-35S-H3H, respectively.

[0037] The CPH and Luz gene fragments were inserted into the multiple cloning site of pYL322d2 to obtain pYL322d2-35S-CPH and pYL322d2-35S-Luz, respectively.

[0038] The PV4 promoter in the pYLMF-H basic vector was replaced with the APSP promoter using homologous recombination technology, and the P35S-Cas9-T35S module was added to the XbaⅠ restriction site of the pYLMF-H vector to construct pYLMF-H-APSP-Cre-35S-Cas9-35S-HPT.

[0039] b. Mix the donor vector pYL322d1-35S-Hisps and the recipient vector pYLTAC380GW at a ratio of 1:1 to 2:1, and co-transfer them into Escherichia coli NS3529 competent cells. Spread the mixture on a double-antibiotic medium containing kanamycin and chloramphenicol and culture it. Extract plasmids from positive strains.

[0040] c. The plasmid extracted in step b was digested with homing enzyme I-Sce I, then transformed into Escherichia coli strain NEB10-β, cultured, screened, and plasmid extracted to obtain a positive clone pYLTAC380GW-Hisps containing the target gene Hisps.

[0041] d. Mix the donor vector pYL322d2-35S-CPH and the recipient vector pYLTAC380GW-Hisps prepared in step c at a ratio of 1:1 to 2:1, and co-transfer them into Escherichia coli NS3529 competent cells. Spread the mixture on a double-antibiotic medium containing kanamycin and ampicillin and culture it. Extract plasmids from positive strains.

[0042] e. The plasmid extracted in step d was digested with the homing enzyme PI-Sce I, then transformed into Escherichia coli strain NEB10-β, cultured, screened, and plasmid extracted to obtain a positive clone pYLTAC380GW-Hisps-CPH containing the target genes Hisps and CPH.

[0043] f. Repeat step be, using the new plasmid containing the target gene obtained in the previous step as the recipient vector, and cross-recombining it with d1 and d2 donor vectors containing different genes until the Hisps, CPH, H3H, and Luz genomes are loaded into the recipient vector. In the final step, a BP recombination reaction is performed with pYLMF-H-APSP-Cre-35S-Cas9-35S-HPT to ligate the Hpt and Cas9 gene expression cassette elements that can be removed to construct a multi-gene vector.

[0044] The beneficial effects of this invention are as follows:

[0045] (1) This invention is the first to identify a promoter sequence as shown in SEQ ID NO.1 from the tobacco genome, which is a promoter specifically expressed in anthers and pistils. This promoter plays an important role in plant genetic engineering research and application, and its application prospects are very broad.

[0046] (2) This invention utilizes the promoter to construct a bioluminescent system module without selection markers and gene editing elements. The FBP bioluminescent system is used for efficient identification of transgenic plants. During the sexual reproduction of the T0 generation of transgenic plants, the selection marker gene and the Cas9 gene editing element are removed, resulting in transgenic plants with better biosafety. Furthermore, the plants after the selection marker gene has been removed can be transformed again in the next round using vectors carrying the selection marker, achieving multiple rounds of introduction of a large number of target genes and enabling multiple rounds of modification of the crop genome. Attached Figure Description

[0047] Figure 1This study aimed to provide tissue-specific analysis of downstream target genes driven by promoters specifically expressed in tobacco anthers and pistils using qPCR technology.

[0048] Figure 2 This is a schematic diagram of the transgenic vector design used to verify the effect of the promoter.

[0049] Figure 3 Diagrams showing the callus screening stage (A) and the differentiation stage (B) of transgenic tobacco.

[0050] Figure 4 The purpose of this study is to screen transgenic tobacco for T0 positive results using a chemiluminescence detection instrument. (A) is under bright field conditions, and (B) is under chemiluminescence conditions.

[0051] Figure 5 The target gene was detected in the transgenic tobacco T1 plants. Plants 1-3 were three different plants from which Clean-FBP was obtained, and plants 4-6 were negative controls (the target gene was not removed).

[0052] Figure 6 The plants are transgenic positive plants in which the Hpt and Cas9 genes have been cut, where (A) represents light conditions and (B) represents dark conditions.

[0053] Figure 7 The percentage of transgenic positive plants in which the Hpt and Cas9 genes were cut. Detailed Implementation

[0054] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0056] Example 1: Identification of anther- and pistil-specific expression promoters

[0057] To identify promoters specifically expressed in reproductive organs, drive the unnecessary exogenous gene excision in transgenic vectors after obtaining positive plants, create biosafe transgenic plants, and conduct synthetic biology research based on tobacco, we analyzed tissue-specific transcriptome data from tobacco genes and screened for anther and pistil-specific promoter APSP (Anther and Pistil Specific Promoter), whose nucleotide sequence is shown in SEQ ID NO. 1.

[0058] We designed qPCR primers for its downstream gene SLP8: F primer: 5'-GGGTTGAATTTGGGCGGAAG-3'; R primer: 5'-TCGGTGGTAGTGCTTTGCAT-3'.

[0059] For tobacco detached leaf tissues used in the transgenic engineering tissue culture process, we selected tobacco leaves during the transgenic screening process, tobacco leaves during the differentiation process, roots, stems and other tissues, extracted RNA, and used qPCR to verify the tissue specificity of downstream genes driven by the APSP promoter.

[0060] The results are as follows Figure 1 As shown, experimental data indicate that downstream genes driven by the APSP promoter are mainly expressed in the anthers and pistils.

[0061] Example 2 Construction of the plant transgenic vector Clean-FBP

[0062] Based on the results of Example 1, the promoter sequence was amplified using PCR technology. The F primer was 5'-gagaaaaactagaaatttacgacatAATTTCTCCTAACTTTCACTTTGATTTTC-3', and the R primer was 5'-cagtaggatagaggtggctcCCATTAATCAACATACAATG-3'. Genomic DNA was extracted from tobacco (N. tabacum cultivar ZY100) as a template.

[0063] The amplified promoter elements were linked upstream of the Cre gene. This operation used the TransGene Stacking II system developed by Academician Liu Yaoguang's research group at South China Agricultural University for multi-genome assembly. See Chinese Patent Application No. 2017103841977. pYL322d1, pYL322d2, pYLMF-H, and pYLTAC380GW were donated by Professor Liu Yaoguang's laboratory at South China Agricultural University.

[0064] To verify the effectiveness of the experimental design, we introduced the fungal bioluminescent pathway (FBP) into the transgenic vector. This fungal luminescent system integrates fungal luciferase (Luz), hispidin synthase (HispS), hispidin 3-hydroxylase (H3H), and caffeoylpyruvate hydrolase (CPH) (Mitiouchkina et al. 2020), emitting green light with a wavelength of approximately 520 nm. The luminescent substrate is caffeic acid. By coupling with the plant's endogenous caffeic acid metabolism, the plant can continuously emit green light visible to the naked eye. Using the FBP luminescent system as a marker for positive selection of transgenic plants allows for efficient and non-destructive screening of transgenic plants in vivo.

[0065] The construction method is as follows: The H3H (hispidin-3-hydroxylase) gene, Hisps (hispidin synthase) gene, CPH (caffeoyl pyruvate hydrolase) gene, and Luz (fungal luciferase) gene from the *Neonothopanus nambi* genome were integrated into the pYLTAC380GW plasmid, where H3H, Hisps, CPH, and Luz are all driven by the 35S promoter. Further, the Cre, Cas9, and Hpt gene expression cassette elements linked to the APSP promoter were integrated into the above plasmid, with Cas9 and Hpt driven by the 35S promoter, thus constructing the Clean-FBP vector. Figure 2 ).

[0066] The coding sequences of the Hisps gene are shown in SEQ ID NO.2, the CPH gene in SEQ ID NO.3, the H3H gene in SEQ ID NO.4, the Luz gene in SEQ ID NO.5, the Cre gene in SEQ ID NO.6, the Cas9 gene in SEQ ID NO.7, the Hpt gene in SEQ ID NO.8, and the loxP sequence in SEQ ID NO.9.

[0067] The specific process is as follows:

[0068] (1) Constructing donor vectors: pYL322d1-35S-Hisps, pYL322d2-35S-CPH, pYL322d1-35S-H3H, pYL322d2-35S-Luz;

[0069] Selection markers and gene editing element self-deletion feed plasmid: pYLMF-H-APSP-Cre-35S-Cas9-35S-HPT.

[0070] Each target gene fragment was synthesized by a biotechnology company. The Hisps and H3H gene fragments were inserted into the multiple cloning site of pYL322d1 to obtain pYL322d1-35S-Hisps and pYL322d1-35S-H3H, respectively; the CPH and Luz gene fragments were inserted into the multiple cloning site of pYL322d2 to obtain pYL322d2-35S-CPH and pYL322d2-35S-Luz, respectively.

[0071] The PV4 promoter in the pYLMF-H basic vector was replaced with the P::APSP promoter using homologous recombination technology. Then, a P35S-Cas9-T35S module was added to the XbaⅠ restriction site of the pYLMF-H vector, with a loxp site introduced upstream of the Cas9 gene fragment, thus constructing pYLMF-H-APSP-Cre-35S-Cas9-35S-HPT.

[0072] (2) The donor vector pYL322d1-35S-Hisps and the recipient vector pYLTAC380GW (at a ratio of 1:1 to 2:1) were mixed in NS3529 competent cells for cotransformation. The heat shock method was used: ice bath for 30 min, heat shock for 90 s, ice bath for 2-3 min, and revive in LB without antibiotics at 37°C and 200 rpm for 2 h. The revival was then spread on LA plates containing kanamycin (Km, 25 mg / L) and chloramphenicol (Chl, 15 mg / L). After about 18 h, single clones grew. All single clones were washed into tubes with ddH2O and the mixed plasmid was extracted.

[0073] (3) Take 100-200 ng of mixed plasmid and digest it with 0.5 uL I-Sce I (NEB) in a 10 uL system for 4-5 h. Transform it into Escherichia coli strain NEB10-β (Bomaide), plate it on LA plate containing kanamycin (Km, 25 mg / L), incubate at 37℃ for 15 h, then pick single clones and culture them in LB (containing 25 mg / L Km and 0.5 mM IPTG). Perform bacterial PCR identification. Use Green Taq Mix to extract plasmids that can amplify bright bands and sequence them for verification.

[0074] (4) The donor vector pYL322d2-35S-CPH and the recipient vector pYLTAC380GW-Hisps from (3) (at a ratio of 1:1 to 2:1) were mixed in NS3529 competent cells for cotransformation. The transformation was carried out according to the method in (2), and the mixture was plated on LA plates containing kanamycin (Km, 25 mg / L) and ampicillin (Amp, 70 mg / L). After about 18 hours, single colonies grew. All single colonies were washed into tubes with ddH2O and the mixed plasmid was extracted.

[0075] (5) Take 100-200ng of mixed plasmid and digest it with 0.5uL PI-Sce I (NEB) and 0.5uL BSA in a 10uL system for 4-5h. Then transform and verify according to the method in (3). If six bands appear, the positive clone pYLTAC380GW-Hisps-CPH is the band containing the target gene 1.7kbp CPH and 6.2kbp Hisps.

[0076] (6) Multiple rounds of recombination were performed, using d1 and d2 donor vectors containing different genes in cross-transformation with the recipient vector constructed in the previous round to construct the pYLTAC380GW-G4 vector (containing four gene fragments: Hisps, CPH, H3H, and Luz). Finally, the APSP-Cre gene expression cassette, Cas9 gene expression cassette, and Hpt gene expression cassette elements from pYLMF-H-APSP-Cre-35S-Cas9-35S-HPT were ligated into the pYLTAC380GW-G4 vector through a BP recombination reaction to construct Clean-FBP. Figure 2 Transform the strain into NEB10-β (Bomaide) competent cells, and select single clones for identification. Confirm the correct strain for subsequent transgenic experiments.

[0077] Example 3: Obtaining transgenic plants with target gene excision

[0078] 1. Streak the EHA105 bacterial culture containing the validated vector plasmid Clean-FBP onto LA+Rif+Kana plates at 28°C for 36 hours. Pick single colonies and transfer them to 3-5 ml of LB medium at 200 rpm for 36 hours at 28°C. Expand the culture to 50 ml at a ratio of 1:100-1:50 and culture for 3-5 hours until OD = 0.6. Then centrifuge the bacterial culture and resuspend the cells in MS0 liquid medium (MS + 3% Sucrose + pH 5.8, 50 ml) until OD = 0.6 for infection.

[0079] 2. Select wild-type tobacco ZY100 seeds and plant healthy tobacco leaves that have fully expanded for 4-5 weeks on sterile MS medium. Cut the leaves into 0.5cm square pieces with a scalpel (cut off the leaf edges and avoid the midrib). Place the leaves with the upper surface facing down on MS1 ​​solid medium (MS + 0.5mg / L IAA + 2.0mg / L BA + 3% sucrose + 0.6-0.8% Phytagel, pH=5.8) and incubate in the dark at 25℃ for 2-3 days.

[0080] 3. Add the pre-cultured tobacco leaves to the bacterial solution, vortex to ensure the leaf cut is submerged, let stand for 5-30 minutes, and then blot away the adhering bacterial solution with sterile filter paper; place the infected leaves, top surface down, on MS1 ​​solid medium and incubate in the dark at 28°C for 2 days; then place the leaves, top surface up, on MS1 ​​selection medium containing Timentin and Hygromycin B and incubate in the light at 25°C. Figure 3 A); When buds emerge from the leaf margins and can be separated (over 1 cm), cut off the buds and transfer them to MS2 (MS + 0.5 mg / L IAA + 3% sucrose + 0.6-0.8% Phytagel, pH = 5.8) solid medium containing antibiotics (TM + Hygromycin B). Roots will emerge after two weeks. Figure 3 B), after opening the seedling tray lid and hardening off for one week, transfer the seedlings to planting soil for cultivation as the T0 generation. Take leaves from the T0 generation transgenic plants and use a Tianneng 4600 imaging device to photograph and identify positive plants. Figure 4 A total of 47 transgenic tobacco seedlings were obtained, including 45 positive luminescent tobacco seedlings, with a positive rate of 95.7%. This indicates that the APSP promoter did not initiate the expression of the Cre gene or cleave the Hpt gene during the transgenic process, thus allowing the positive plants to maintain resistance to the hygromycin selection reagent during the transgenic process.

[0081] 4. Seeds from the above-mentioned positive T0 generation transgenic plants were used as the T1 generation. Three independent families (Clean-FBP-3, Clean-FBP-8, Clean-FBP-26) were germinated from T1 generation seeds and grown in MS medium containing 3 μg / mL Hygromycin B. Weaker seedlings were selected and grown in hygromycin-free MS medium. At the 4-5 leaf stage, leaf DNA was extracted, and PCR detection of the three target genes was performed. It was found that the plant's Hpt and Cas9 genes were successfully excised in the tested leaves, while the Luz gene remained in the FBP module. Figure 5 ).

[0082] The obtained Clean-FBP plants (with Hpt and Cas9 genes removed) were transplanted into nutrient soil and grown in a greenhouse (25℃, 12h light / 12h dark, 6000 lux) for 50 days. Afterward, photos were taken with a Nikon 700 camera under light conditions with an exposure time of 1 / 60s and in darkness with an exposure time of 3 minutes. It was found that the tobacco could emit light. Figure 6 ).

[0083] Statistical analysis of the three transgenic families tested revealed that the proportion of clean transgenic plants was between 6% and 10%. Figure 7 This demonstrates that the APSP promoter we identified can be used for the effective excision of the target gene in the transgenic T1 generation, creating clean transgenic plants.

[0084] The above results indicate that the APSP promoter shows promising application potential in creating clean transgenic plants. Furthermore, tobacco plants with the selection marker Hpt gene removed can be transformed again in subsequent rounds using vectors carrying the Hpt gene, achieving the requirement of multiple rounds of introduction of large amounts of the target gene. This has significant application value for tobacco design breeding and synthetic biology research and applications based on tobacco.

Claims

1. A plant anther- and pistil-specific expression promoter, characterized in that, The nucleotide sequence of the promoter is shown in SEQ ID NO.

1.

2. The application of the plant anther and pistil-specific expression promoter as described in claim 1 in the construction of gene expression cassettes or gene expression vectors specifically expressed in anthers and pistils.

3. A gene expression cassette specifically expressed in anthers and pistils, characterized in that, It includes the plant anther and pistil-specific expression promoter as described in claim 1.

4. A gene expression vector specifically expressed in anthers and pistils, characterized in that, It includes the gene expression cassette as described in claim 3.

5. A recombinant engineered bacterium, characterized in that, The engineered bacteria contain the gene expression vector as described in claim 4.

6. A method for expressing a target nucleotide sequence in the anther and pistil of a plant, characterized in that, include: An expression vector containing the promoter as described in claim 1 and a target nucleotide sequence linked to the promoter is constructed, and then the expression vector is introduced into a plant. Transgenic plants are obtained by screening and cultivation. The plant is tobacco.

7. A method for cultivating transgenic plants without selection markers, characterized in that, include: (1) Construct a plant Cre / loxP recombinant expression vector, wherein the T-DNA region of the plant Cre / loxP recombinant expression vector contains two or more loxP sequences, and between the two loxP sequences therein is a Cre recombinase expression cassette and a marker gene expression cassette; the promoter in the Cre recombinase expression cassette that initiates Cre recombinase expression is the plant anther and pistil-specific expression promoter as described in claim 1; (2) The T-DNA region of the plant Cre / loxP recombinant expression vector was introduced into the recipient plant using transgenic technology, and the plant was cultured to obtain a transgenic plant without selection markers; the recipient plant was tobacco.

8. The method for cultivating transgenic plants without selection markers as described in claim 7, characterized in that, The plant Cre / loxP recombinant expression vector further contains a multiple cloning site or an expression cassette for the exogenous target gene; the multiple cloning site or the expression cassette for the exogenous target gene is located within the T-DNA region, outside the two loxP sequence regions.

9. A method for cultivating a self-luminescent transgenic plant without selection markers and gene editing elements, characterized in that: include: 1) Utilizing multi-genome assembly technology to... Hpt Gene, Cas9 Gene, Cre Gene, Hisps Gene, CPH Gene, H3H Genes and Luz Genes are integrated into a receptor vector to construct a multi-gene vector containing two or more lox-P elements. Hpt Gene, Cas9 Gene, Cre The gene expression module is located between two co-directional lox-P elements. Cre The upstream integration of the gene has the promoter as described in claim 1; Hisps Gene, CPH Gene, H3H Genes and Luz The gene expression module is located outside the two unidirectional loxP sequence regions; 2) Using transgenic technology, the T-DNA region fragment from a multi-gene vector was introduced into recipient plants, and the plants were cultured to obtain T0 generation transgenic plants. The transgenic plants were then screened during the T1 generation seedling stage to obtain... Hpt and Cas9 The cut-off transgenic plant; the recipient plant is tobacco.

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