Cotton vegetative tissue-specific promoter pCAB and application thereof

Abstract

The application belongs to the technical field of plant genetic engineering, and discloses a cotton vegetative tissue-specific promoter and application thereof. In view of the technical problems of lack of cotton vegetative tissue-specific promoter resources and lack of endogenous promoters with both vegetative organ broad-spectrum expression and seed silencing characteristics, the application clones a promoter upstream of GH_D10G0420 based on a Gossypium hirsutum transcriptome. The recombinant expression vector is constructed, and Arabidopsis thaliana is transformed to verify that the promoter is expressed in root, stem, leaf and other vegetative organs in a broad spectrum and high efficiency, and is not expressed in seeds. The application can be used to drive the directional expression of pest-resistant, disease-resistant, photosynthetic and stress-resistant genes in cotton vegetative tissues, realize the directional improvement of gossypol, achieve the goal of plant gossypol preservation and seed gossypol removal, and provide a promoter resource for cotton precision molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a cotton vegetative tissue-specific promoter and its application. Background Technology

[0002] Cotton (Gossypium hirsutum L.) is an important natural fiber crop and economic crop. With climate change, constraints on arable land resources, and increasing pressure from pests and diseases, cotton production faces higher demands for improved traits such as high yield, high quality, stress resistance, and disease and pest resistance. Traditional breeding methods are time-consuming and have limited efficiency in targeted improvement of target traits. Genetic engineering and molecular breeding technologies can target and regulate genes, providing an effective approach to improving important cotton traits.

[0003] Promoters are important cis-regulatory sequences located upstream of structural genes that regulate gene transcription initiation and expression levels. They are key elements in constructing plant expression vectors and regulating the expression of target genes. Commonly used constitutive promoters such as CaMV 35S can continuously drive gene expression in various tissues, but may lead to the accumulation of exogenous proteins or metabolites in non-target tissues, increasing the metabolic burden on plants and adversely affecting normal growth and development. Tissue-specific promoters enable the expression of target genes in specific tissues or organs, thereby improving the spatial precision of gene expression and reducing ineffective expression in non-target tissues.

[0004] Roots, stems, and leaves, among other vegetative tissues, are involved in water and nutrient absorption, substance transport, photosynthesis, and stress response, respectively, and are crucial functional sites for cotton's resistance to stress, diseases, and pests, as well as yield formation. Vegetative tissue-specific or dominant expression promoters can drive the expression of genes related to stress resistance, disease resistance, pest resistance, photosynthetic efficiency improvement, and metabolic regulation in their respective tissues, and have significant application value. However, current research on cotton tissue-specific promoters largely focuses on reproductive tissues such as fibers, seeds, and floral organs; promoter resources derived from cotton endogenous genes and suitable for targeted expression in vegetative tissues such as roots, stems, and leaves remain insufficient.

[0005] Cottonseed is rich in oils and proteins, making it valuable for agricultural use. However, the accumulation of defensive metabolites such as gossypol in the seeds limits its safety for edible and feed use. Simultaneously, gossypol in the plant's vegetative tissues helps resist insect and pathogen attacks. Therefore, obtaining an endogenous promoter that can drive the expression of a target gene in cotton vegetative tissues but is expressed at low levels or not at all in seed tissues is of great significance for improving cotton's insect resistance, disease resistance, and stress tolerance, as well as for the targeted regulation of gossypol.

[0006] Therefore, there is still a need to develop a tissue-specific promoter derived from cotton endogenous genes that can drive the expression of target genes in vegetative tissues such as roots, stems, and leaves, and be expressed at low levels or not at all in seed tissues, in order to meet the needs of precise genetic improvement, molecular breeding, and functional gene research in cotton. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide a vegetative tissue-specific promoter derived from cotton (Gossypium hirsutum L.) and its application, so as to solve the problems of the scarcity of cotton endogenous vegetative tissue-specific promoter resources and the lack of promoters that have both vegetative organ expression activity and low or no expression in seeds in the prior art.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: a cotton vegetative tissue-specific promoter, named pCAB, whose nucleotide sequence is shown in SEQ ID NO:1; or a nucleotide sequence having at least 90% to at least 95% sequence identity with the sequence shown in SEQ ID NO:1, and having the function of driving gene expression in plant vegetative tissues and low expression or no expression in seed tissues.

[0009]

[0010] The promoter pCAB is derived from the upstream regulatory region of the cotton gene GH_D10G0420, and the length of the upstream regulatory region is 1398 bp.

[0011] The promoter pCAB exhibits tissue-specific expression characteristics, showing strong expression activity in vegetative tissues such as plant roots, stems, and leaves, weak expression in flowers and pods, and virtually no expression in seeds.

[0012] The present invention also provides an expression cassette or recombinant expression vector containing the above-mentioned promoter pCAB, wherein the promoter pCAB is operatively linked upstream of the target gene to drive the expression of the target gene in plant vegetative tissues.

[0013] Preferably, the recombinant expression vector uses pBWA(V)BII-Eco31I-GUS as the backbone vector, and pCAB is inserted upstream of the GUS reporter gene through homologous recombination to construct the pCAB::GUS plant expression vector.

[0014] The present invention also provides a host cell containing the above-mentioned promoter pCAB or the above-mentioned recombinant expression vector, wherein the host cell includes Escherichia coli Trans5α, Agrobacterium tumefaciens GV3101 or plant cells.

[0015] The present invention also provides the application of the above-mentioned promoter pCAB in plant genetic improvement, the application of which includes driving the expression of genes related to insect resistance, disease resistance, stress resistance, photosynthetic efficiency improvement or gossypol metabolism regulation in plant vegetative tissues, so as to improve the expression efficiency of target genes in target tissues and reduce their unnecessary expression in seeds.

[0016] Preferably, the insect-resistant genes include Bt toxin protein genes or chitinase genes; the disease-resistant genes include chitinase genes, glucanase genes, or disease-related protein genes; the photosynthetic efficiency-improving genes include Rubisco activator genes or photosynthetic electron transport chain-related genes; and the stress-resistant genes include SOD genes, POD genes, CAT genes, LEA protein genes, or osmotic regulator synthesis-related genes.

[0017] The present invention also provides a method for cultivating transgenic plants, comprising transforming the above-mentioned recombinant expression vector into plant cells, tissues or explants, and obtaining transgenic plants through screening, cultivation and regeneration.

[0018] Preferably, the plant includes cotton, Arabidopsis thaliana, tobacco, rice, wheat, corn, or soybean; the transformation method includes Agrobacterium-mediated transformation, gene gun transformation, pollen tube pathway transformation, or protoplast transformation.

[0019] The beneficial effects of this invention are as follows: the promoter pCAB is derived from an endogenous gene in cotton, possessing good species adaptability and expression stability, thus avoiding the problem of altered expression patterns in cotton caused by heterologous promoters; simultaneously, this promoter exhibits strong activity in vegetative tissues such as roots, stems, and leaves, while being largely unexpressed in seeds, making it suitable for limiting the accumulation of exogenous gene products in seeds; furthermore, this promoter can be used to drive the targeted expression of genes related to insect resistance, disease resistance, stress resistance, and photosynthetic improvement in key vegetative organs of cotton, and can also be used for gossypol metabolism regulation and cotton functional genomics research, providing a new regulatory element for precision molecular breeding of cotton. Attached Figure Description

[0020] Figure 1 This is a graph showing the expression level of the GH_D10G0420 gene in different tissues of upland cotton. The horizontal axis represents different tissues (root, stem, stamen, pistil, ovary, leaf, flower, and seed), and the vertical axis represents the relative gene expression level. The expression differences between different tissues were statistically analyzed using one-way ANOVA, and the Tukey method was used for comparisons between groups. P < 0.05 indicates a significant difference.

[0021] Figure 2 This is an electrophoresis diagram of PCR amplification of the pCAB promoter sequence; lane 1: DNA Marker; lanes 2-4: pCAB amplification products; lane 5: negative control.

[0022] Figure 3 The diagrams show the construction and PCR verification of the pCAB::GUS recombinant expression vector. (A) shows the vector construction diagram, with pBWA(V)BII-Eco31I-GUS as the backbone and the pCAB promoter sequence inserted upstream of the GUS reporter gene. (B) shows the PCR verification of E. coli positive clones, with lane 1: DNA Marker 3; lanes 2-4: positive clone amplification products; lane 5: negative control. (C) shows the PCR verification of Agrobacterium positive clones, with lane 1: DNA Marker 3; lanes 2-4: positive clone amplification products; lane 5: negative control.

[0023] Figure 4 The image shows the PCR molecular detection results of transgenic Arabidopsis plants; lane 1: DS2000 DNA Marker; lanes 2-6: transgenic Arabidopsis lines to be tested (L1-L5); lane 7: wild-type Arabidopsis; lane 8: negative control; the expected amplified band size is 123 bp.

[0024] Figure 5The image shows the results of GUS histochemical staining in different tissues of transgenic Arabidopsis thaliana; from left to right, there are 5 positive transgenic lines (L1-L5) and the wild-type control (WT); from top to bottom, there are roots, stems, leaves, flowers, pods and seeds; blue precipitates indicate GUS positive expression signals.

[0025] Figure 6 This is a graph showing the quantitative analysis of GUS enzyme activity in transgenic Arabidopsis leaves; the horizontal axis represents the five positive transgenic lines (L1-L5) and the wild type (WT), and the vertical axis represents β-GUS activity (μmol·h⁻¹). -1 ·g -1 Data are expressed as mean ± standard deviation (n=3); different letters indicate significant differences between groups (P<0.05). Detailed Implementation

[0026] The present invention will now be described in detail through specific embodiments. These embodiments are provided so that this invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0027] Unless otherwise specified, the techniques used in the embodiments are conventional methods well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used are commercially available.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0029] Unless otherwise stated, the present invention will be implemented using conventional botanical techniques, tissue culture, molecular biology, chemistry, biochemistry, and DNA recombination techniques that are readily apparent to those skilled in the art. These techniques have been fully explained in the published literature. The methods used in this invention for vector construction, transient expression of plants, etc., except for those used in the examples below, can all be achieved using methods already disclosed in the existing literature.

[0030] The Biorun Pfu PCR Mix used in this invention was purchased from Beijing Biorun Biotechnology Co., Ltd.; the EASY spinPlus Plant DNA Kit was purchased from Aidlab (Beijing); the NanoDrop ND-1000 micro-spectrophotometer was purchased from Thermo Fisher Scientific, USA; the general plasmid mini-prep kit was purchased from TIANGEN (Beijing); the Biorun 2×EasyClone Mix was purchased from Beijing Biorun Biotechnology Co., Ltd.; the plant genomic DNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; the GUS staining kit was purchased from Beijing Coollab Technology Co., Ltd.; and the β-glucuronidase kit was purchased from Suzhou Greens Biotechnology Co., Ltd. The culture media used for plant transformation and tissue culture were purchased from Beijing Coollab Technology Co., Ltd. Primers and sequencing were performed at Xinjiang Youkang Biotechnology Co., Ltd.

[0031] Example 1: Screening of candidate promoters

[0032] Based on whole-genome transcriptome data of upland cotton (Gossypium hirsutum L.), gene expression patterns in different tissues were analyzed to screen candidate genes that exhibit dominant expression in vegetative tissues but low or no expression in seeds.

[0033] The analysis results show (see) Figure 1 The gene GH_D10G0420 is highly expressed in the vegetative tissues of cotton, such as roots, stems, and leaves, but its expression level is low or absent in seed tissues. This gene encodes a chlorophyll a / b-binding protein (CAB), which belongs to the photosynthetic antenna protein family.

[0034] Based on the above expression pattern, the 1398 bp upstream sequence of the GH_D10G0420 gene was selected as a candidate promoter fragment, named pCAB, and its nucleotide sequence is shown in SEQ ID NO: 1, which will be used for subsequent cloning and functional verification.

[0035] Example 2: Cloning of the pCAB promoter fragment

[0036] Using leaves of the upland cotton variety “Xinluzao 33” as material, cotton genomic DNA was extracted using a plant genomic DNA extraction kit. Specific amplification primers were designed based on the 1398 bp upstream sequence of the GH_D10G0420 gene. The primer sequences are as follows: pCAB-F: 5′-CGCCTGCAGGTCTAGATAGActgtgactggtcagaaattgcctgtg-3′ (SEQ ID NO: 2); pCAB-R: 5′-CCCTCAGATCTACCATCGCAtgtgaagcaactgattttgttggtgatggg-3′ (SEQ ID NO: 3).

[0037] PCR amplification was performed using genomic DNA from the cotton variety “Xinluzao 33” as a template. The PCR reaction volume was 50 μL, including 25 μL Biorun Pfu PCR Mix, 2 μL each of pCAB-F and pCAB-R, 1 μL cotton genomic DNA, and 20 μL Nuclease-free Water. The PCR amplification program was as follows: pre-denaturation at 94℃ for 5 min; 30 cycles of 94℃ for 30 s, 50℃ for 45 s, and 72℃ for 84 s; extension at 72℃ for 10 min; and storage at 16℃.

[0038] The PCR products were detected by 1% agarose gel electrophoresis, and a specific amplified band was obtained at approximately 1400 bp (see [link to PCR product]). Figure 2 The target band was consistent with the expected fragment size of 1398 bp. The target band was excised and recovered to obtain the pCAB promoter fragment. Sequencing confirmed that the nucleotide sequence of the obtained pCAB promoter fragment is shown in SEQ ID NO:1.

[0039] Example 3: Construction of pCAB::GUS recombinant expression vector

[0040] The pCAB::GUS plant expression vector was constructed using homologous recombination. Using pBWA(V)BII-Eco31I-GUS(2021) as the backbone vector, the linearized vector fragment was recovered after Eco31I digestion. The pCAB promoter fragment obtained in Example 2 was homologously recombinated with the linearized vector using Biorun 2×EasyClone Mix to obtain the pCAB::GUS plant expression vector (see [link to example]). Figure 3 (A)

[0041] The ligation product was transformed into *E. coli* Trans5α competent cells, plated on LB agar containing kanamycin, and incubated overnight at 37°C. Single clones were picked for colony PCR identification using the following primers: pCAB-JD-F: 5′-gctccaccatgttggcaagc-3′ (SEQ ID NO: 4); pCAB-JD-R: 5′-ggtgttggtctagtttataccaagt-3′ (SEQ ID NO: 5).

[0042] PCR identification results showed that positive clones amplified a specific band of approximately 690 bp, while the negative control did not amplify the corresponding band (see...). Figure 3 (Section B) Positive clones were selected for Sanger sequencing. Sequencing results showed that the inserted fragment sequence was consistent with SEQ ID NO:1, and the pCAB promoter fragment was inserted upstream of the GUS reporter gene in the correct orientation. The recombinant plasmid with correct sequencing was named pBII-GUS-pCAB, i.e., pCAB::GUS plant expression vector.

[0043] The pCAB::GUS recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 competent cells using electroporation. After transformation, the cells were plated on LB agar containing kanamycin and rifampin and incubated at 28°C. Single clones were picked for PCR verification to obtain the correct bands (see...). Figure 3 The C), that is, the Agrobacterium engineered strain containing the pCAB::GUS recombinant plasmid.

[0044] Example 4: Obtaining and identifying pCAB::GUS transgenic Arabidopsis thaliana

[0045] Wild-type Arabidopsis thaliana Columbia-0 was transformed using the Agrobacterium-mediated flower dip method. Agrobacterium GV3101 containing the pCAB::GUS recombinant plasmid was cultured, centrifuged to collect the cells, and resuspended in flower dip buffer to adjust the OD of the culture. 600 To 0.6-0.8. Arabidopsis inflorescences were immersed in Agrobacterium suspension for infection, cultured in the dark, and then transferred to normal conditions for continued growth. Seeds of the T0 generation were harvested after the plants matured.

[0046] After drying and vernalization, T0 generation seeds were sown in a solution containing 20 mg L... -1 The plants were screened on 1 / 2 MS Basta medium. After obtaining resistant plants, they were transplanted and self-crossed. Through continuous screening and identification, T3 generation homozygous pCAB::GUS transgenic Arabidopsis lines were obtained.

[0047] Genomic DNA was extracted from leaves of T3 generation transgenic Arabidopsis thaliana, and PCR identification was performed using the GUS gene as the detection target. The GUS detection primers are as follows: GUS-F: 5′-CGGCGACGAGCCAGGGATA-3′ (SEQ ID No: 6); GUS-R: 5′-GCACCATCGTCAACCACTACAT-3′ (SEQ ID No: 7).

[0048] The PCR amplification system consisted of 25 μL, including 2.5 μL of 10×Taq Buffer, 2 μL of 2.5 mM dNTPs, 1 μL each of 10 μM GUS-F and GUS-R, 2 μL of Arabidopsis thaliana genomic DNA, 0.25 μL of Taq DNA polymerase, and ultrapure water to a final volume of 25 μL. The PCR program was as follows: 94℃ for 3 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min, for a total of 35 cycles; 72℃ for 10 min.

[0049] PCR results showed that five independent T3 generation homozygous transgenic lines (L1, L2, L3, L4, and L5) all amplified a GUS-specific band of approximately 123 bp, while the wild-type control and water control did not amplify the corresponding band (see [link to PCR results]). Figure 4 This indicates that the GUS reporter gene has been integrated into the Arabidopsis genome.

[0050] Example 5: Tissue Expression Characterization Analysis of pCAB Promoter

[0051] To detect the tissue expression characteristics of the pCAB promoter, GUS histochemical staining analysis was performed on roots, stems, leaves, flowers, pods, and seeds from T3 generation pCAB::GUS transgenic Arabidopsis plants. Corresponding tissues from wild-type Arabidopsis served as negative controls.

[0052] Fresh plant tissues were placed in GUS staining working solution, ensuring complete immersion, and incubated at 37°C in the dark. After staining, chlorophyll-containing tissues such as leaves were destained in 70% ethanol until the negative control material was mostly destained. The destained material was stored in 70% ethanol and observed and photographed under a stereomicroscope.

[0053] GUS histochemical staining results showed that blue staining signals were observed in the roots, stems, and leaves of pCAB::GUS transgenic Arabidopsis plants, with the staining signal being more pronounced in the leaves; weaker GUS staining signals were detected in the flowers and pods; and no obvious GUS staining signal was observed in the seeds. No blue staining signals were observed in any tissue of wild-type Arabidopsis (see...). Figure 5 ).

[0054] The above results indicate that the pCAB promoter can drive the expression of downstream genes in the vegetative tissues of Arabidopsis thaliana, with weaker expression in flowers and pods, and low or no expression in seeds, exhibiting the promoter characteristics of vegetative tissue-predominant expression.

[0055] Example 6: Quantitative analysis of GUS enzyme activity of pCAB promoter

[0056] To further verify the driving activity of the pCAB promoter, leaf tissues from T3 generation pCAB::GUS transgenic Arabidopsis thaliana lines and wild-type Arabidopsis thaliana were used to determine GUS enzyme activity. Approximately 0.1 g of fresh leaf was taken from each sample, added to the extraction buffer, ground in an ice bath, centrifuged, and the supernatant was collected. The GUS enzyme activity was measured according to the β-glucuronidase kit instructions. GUS enzyme activity was expressed as μmol·h⁻¹. -1 ·g -1 express.

[0057] The results of GUS enzyme activity assays showed that the GUS enzyme activities in the leaves of pCAB::GUS transgenic Arabidopsis lines L1, L2, L3, L4, and L5 were significantly higher than those in the wild-type control. Compared with the wild-type, the GUS enzyme activities of the L1, L2, L3, L4, and L5 lines increased by 4428.6%, 4360.0%, 2141.0%, 1928.1%, and 1122.0%, respectively, indicating that the pCAB promoter has strong driving activity in the leaves of transgenic plants (see...). Figure 6 ).

[0058] In summary, the cotton endogenous pCAB promoter of this invention can effectively drive the expression of target genes in vegetative tissues such as roots, stems, and leaves, while exhibiting low or no expression in seeds, providing a reliable vegetative tissue-dominant expression promoter tool for plant gene function research. The application of this promoter can support the construction of plant vegetative tissue-dominant expression vectors, and facilitate plant genetic improvement efforts such as insect resistance, disease resistance, photosynthetic efficiency enhancement, and targeted regulation of gossypol metabolism.

[0059] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible embodiments of the present invention. Any obvious modifications, equivalent substitutions, improvements, or combinations made to the present invention within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A cotton vegetative tissue-specific promoter, characterized in that, The promoter comprises a nucleotide sequence as shown in SEQ ID NO:1, or a functional variant thereof having at least 95% and at least 99% sequence identity and having the function of driving gene expression in plant vegetative tissues, and is derived from cotton.

2. An expression box, characterized in that, It contains the cotton vegetative tissue-specific promoter as described in claim 1, and the promoter is operatively linked to the target gene.

3. A recombinant expression vector, characterized in that, It contains the cotton vegetative tissue-specific promoter as described in claim 1, and the promoter is operatively linked to the target gene.

4. A host cell, characterized in that, It contains the expression cassette as described in claim 2 or the recombinant expression vector as described in claim 3.

5. The host cell as described in claim 4, characterized in that, The host cell is Escherichia coli, Agrobacterium, or a plant cell.

6. The application of the cotton vegetative tissue-specific promoter of claim 1, the expression cassette of claim 2, the recombinant expression vector of claim 3, or the host cell of claim 4 or 5 in driving the expression of the target gene in plant vegetative tissues, plant genetic transformation, plant molecular breeding, or plant gene function research.

7. A method for cultivating transgenic plants, characterized in that, The process includes the following steps: operatively linking the cotton vegetative tissue-specific promoter of claim 1 with the target gene to construct an expression cassette or recombinant expression vector; introducing the expression cassette or recombinant expression vector into plant cells, tissues or explants, and obtaining transgenic plants through screening, culture and regeneration.

8. The method as described in claim 7, characterized in that, The plants are selected from cotton, Arabidopsis thaliana, tobacco, rice, wheat, corn, or soybean; the transformation method is selected from Agrobacterium-mediated transformation, gene gun transformation, pollen tube pathway transformation, or protoplast transformation.

9. A transgenic plant, characterized in that, It is introduced by the promoter as described in claim 1, or obtained by the method as described in claim 7 or 8.

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