A promoter for regulating expression of a plant development regulatory factor and use thereof

Abstract

The application discloses a plant development regulation factor expression regulating promoter and application thereof, and belongs to the technical field of biotechnology and genetic engineering. The application provides a plant development regulation factor expression regulating promoter, wherein the nucleotide sequence of the promoter is shown as SEQ ID NO. 5 or SEQ ID NO. 6. The promoter has transcription activity in the somatic embryogenesis period, can be specifically expressed in the somatic embryo development process, and has significant value in actual production practice. The promoter is expected to be used as a candidate promoter for precisely regulating development regulation factor expression, and can be applied in an island cotton transgenic new technology mediated by a development regulation factor promoting plant regeneration system.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and genetic engineering, and in particular to a promoter for regulating the expression of plant development regulatory factors and its applications. Background Technology

[0002] Agrobacterium-mediated cotton transgenic technology is a cotton molecular breeding technique with advantages such as being unrestricted by seasons, high efficiency, and directionality. This technology relies on the establishment of a cotton plant regeneration tissue culture system. Plant regeneration is a complex and dynamic process of plant cell type conversion, involving the differentiation and generation of various plant cells and tissues. In these complex processes, the specific expression of certain "developmental regulatory key genes," such as WUSCHEL (WUS) and BABY BOOM (BBM), plays a decisive role, similar to a molecular switch. Ectopic expression of "developmental regulatory factors" can transform plant somatic cells into totipotent cells with embryogenetic potential, thereby enhancing the plant's regeneration capacity. However, overexpression of these genes also has certain drawbacks, such as causing deformities, sterility, poor function, and thickened roots in transformed plants, thus affecting the normal growth and development of regenerated plants. This greatly limits the application of developmental regulatory factor-mediated plant regeneration technology in improving plant transformation efficiency. Studies have shown that tissue-specific promoters driving developmental regulatory genes can achieve a normal plant regeneration rate. Therefore, finding promoters that can drive gene expression in young leaves, embryos, and callus tissues, while inhibiting gene expression in other plant tissues and cells, to control the appropriate expression of "developmental regulators," is an effective method to obtain normal transgenic offspring while promoting plant regeneration. Tissue-specific promoters can initiate the targeted expression of target genes in a specific tissue or organ of the plant. The LEC2 (LEAFY COTYLEDON2) gene plays an important role in maintaining stalk morphology, cotyledon development, the synthesis of storage proteins during embryo maturation, and the inhibition of premature seed germination. In most plants, including Arabidopsis thaliana, castor bean (Ricinus communis), cacao (Theobroma cacao), and wheat (Triticum aestivum), the LEC2 gene exhibits the same expression pattern: specifically high expression only in developing seeds. Therefore, developing a promoter that regulates the expression of developmental regulatory factors based on the promoter fragment of the LEC2 gene (GbLEC2) of sea island cotton, thereby achieving precise expression of plant regulatory factors, is of great significance for the application of novel transgenic technology of sea island cotton mediated by the developmental regulatory factor-driven plant regeneration system. Summary of the Invention

[0003] The purpose of this invention is to provide a promoter for regulating the expression of plant developmental regulatory factors and its application, in order to solve the problems existing in the prior art. This promoter can drive the expression of developmental regulatory factors, and thus can be applied in the novel transgenic technology of island cotton mediated by developmental regulatory factors.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides a promoter for regulating the expression of plant development regulatory factors, the nucleotide sequence of which is shown in SEQ ID NO.5 or SEQ ID NO.6.

[0006] The present invention also provides a recombinant vector, including the promoter.

[0007] The present invention also provides a recombinant bacterium, comprising the aforementioned recombinant vector.

[0008] The present invention also provides a method for constructing the promoter, comprising the following steps:

[0009] The promoter was obtained by PCR amplification using primer pairs, with the whole genome of sea island cotton as a template.

[0010] When the nucleotide sequence of the promoter is as shown in SEQ ID NO.5, the nucleotide sequence of the primer pair is as shown in SEQ ID NO.11-12;

[0011] When the nucleotide sequence of the promoter is as shown in SEQ ID NO.6, the nucleotide sequences of the primer pair are as shown in SEQ ID NO.13-14.

[0012] The present invention also provides the application of the promoter, the recombinant vector, or the recombinant bacteria described herein in enhancing the synthesis of plant oils.

[0013] Furthermore, the promoter, the recombinant vector, or the recombinant bacteria enhance lipid synthesis by regulating the expression of developmental regulatory factors.

[0014] Furthermore, the developmental regulatory factor is the GbLEC2 gene.

[0015] Furthermore, the plant includes sea island cotton.

[0016] The present invention discloses the following technical effects:

[0017] This invention clones four different GbLEC2 somatic embryo development-specific promoter fragments. The 946bp and 410bp GbLEC2 promoter fragments exhibit transcriptional activity during somatic embryogenesis and can be specifically expressed during somatic embryo development, demonstrating significant value in practical production. The GbLEC2 promoter holds promise as a candidate promoter for precisely regulating the expression of developmental regulatory factors, and may be applied in novel transgenic technology for island cotton mediated by a developmental regulatory factor-driven plant regeneration system. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Electrophoretic images showing PCR amplification and detection of different GbLEC2 promoter fragments using plant genome as template, where M: 2000bp Marker; 1: GbLEC2-1 promoter; 2: PCR negative control for GbLEC2-1 promoter; 3: GbLEC2-2 promoter; 4: PCR negative control for GbLEC2-2 promoter; 5: GbLEC2-3 promoter; 6: PCR negative control for GbLEC2-3 promoter; 7: GbLEC2-4 promoter; 8: PCR negative control for GbLEC2-4 promoter.

[0020] Figure 2 To transform plants with different promoters, the recombinant vector pCAMBIA1304::Pro was used. GbLEC2 ::GUS images of Escherichia coli culture PCR identification, where M: 2000bp Marker; 1: Culture PCR identification of Escherichia coli transformed with GbLEC2-1 promoter plant recombinant vector; 2: Culture PCR identification of Escherichia coli transformed with GbLEC2-2 promoter plant recombinant vector; 3: Culture PCR identification of Escherichia coli transformed with GbLEC2-3 promoter plant recombinant vector; 4: Culture PCR identification of Escherichia coli transformed with GbLEC2-4 promoter plant recombinant vector;

[0021] Figure 3 To transform plants with different promoters, the recombinant vector pCAMBIA1304::Pro was used. GbLEC2 ::GUS Agrobacterium tumefaciens culture PCR identification, where M: 2000bp Marker; 1: transformed with GbLEC2-1 promoter plant recombinant vector pCAMBIA1304::ProGbLEC2-1 ::GUS Agrobacterium tumefaciens culture PCR identification; 2: Transformed with GbLEC2-2 promoter plant recombinant vector pCAMBIA1304::Pro GbLEC2-2 ::GUS Agrobacterium tumefaciens culture PCR identification; 3: Transformed with GbLEC2-3 promoter plant recombinant vector pCAMBIA1304::Pro GbLEC2-3 ::GUS Agrobacterium tumefaciens culture PCR identification; 4: Transformed with GbLEC2-4 promoter plant recombinant vector pCAMBIA1304::Pro GbLEC2-4 :: PCR identification of Agrobacterium tumefaciens in GUS; 5: PCR negative control for GbLEC2-1 promoter; 6: PCR negative control for GbLEC2-2 promoter; 7: PCR negative control for GbLEC2-3 promoter; 8: PCR negative control for GbLEC2-4 promoter;

[0022] Figure 4 To detect GUS activity of different GbLEC2 promoter fragments in different cotton tissue cells;

[0023] Figure 5 The expression of the GbLEC2 gene in various tissues of sea island cotton is shown. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] The pCAMBIA1304 used in the following examples was purchased from Abcam.

[0030] Example 1: Cloning of the GbLEC2 promoter

[0031] The upstream nucleotide sequence of the start codon of the GbLEC2 gene (GB_A09G1025) was obtained from a cotton website. EcoRI and NcoI were selected as restriction enzyme sites. Seamless cloning primers with restriction enzyme sites were designed using the primer design website provided by Nanjing Novizan (https: / / crm.vazyme.com / cetool / simple.html). PCR amplification was performed using extracted sea island cotton DNA as a template. The promoter-specific primer sequences for different fragments of GbLEC2 are shown in Table 1.

[0032] Table 1 Primer sequences for PCR amplification of different GbLEC2 promoter fragments

[0033]

[0034] Table 2 PCR amplification systems for different promoter fragments

[0035]

[0036]

[0037] The amplification conditions were as follows: GbLEC2-1: 94℃ for 5 min; 94℃ for 30 s, 62.5℃ for 30 s, 72℃ for 120 s, 35 cycles; 72℃ for 10 min. GbLEC2-2: 94℃ for 5 min; 94℃ for 30 s, 62℃ for 30 s, 72℃ for 90 s, 35 cycles; 72℃ for 10 min. GbLEC2-3: 94℃ for 5 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 60 s, 35 cycles; 72℃ for 10 min. GbLEC2-4: 94℃ for 5 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 60 s, 35 cycles; 72℃ for 10 min. The amplification system is shown in Table 2. The obtained amplification products were detected by 1% agarose gel electrophoresis, and the results are as follows. Figure 1As shown, four different 1836bp GbLEC2 promoter fragments were cloned. 2-1 Promoter (SEQ ID NO.3), 1465bp GbLEC 2-2 Promoter (SEQ ID NO.4), 946bp GbLEC 2-3 Promoter (SEQ ID NO.5) and 410bp GbLEC 2-4 Promoter (SEQ ID NO.6).

[0038] GbLEC 2-1 The nucleotide sequence of the promoter (SEQ ID NO.3) is as follows:

[0039]

[0040] GbLEC 2-2 The nucleotide sequence of the promoter (SEQ ID NO.4) is as follows:

[0041]

[0042] GbLEC 2-3 The nucleotide sequence of the promoter (SEQ ID NO.5) is as follows:

[0043] 。

[0044] GbLEC 2-4The nucleotide sequence of the promoter (SEQ ID NO.6) is as follows:

[0045] TGCAATGAGTCCCAATTACTGCAATTTCCAATTTTTCACCTCAGTTTTTCGGACATCAAAGGCATATGGTTACTGATTAGTGTTTTGGACAATGTTTTCATGCATTAATCACCAATTTTTTCAGTTCATTTTGTCTTTTCCACAGGTTGAACCTGATGGCAGGTTTTGATCCTTCAAGTAGACGATAAAACAAGCCGGATATGGC AACCAAAAAGCCGCAGAGTGGAGTGGATGACAAACATTGTGAAGTTAAACACACATCTAAGACAACCCTCATGGAAACCGATATTCCTTATTCAATTTCAGCCACACCTTGATCCAAACCATTCCACCATCGTTTTACTCACAAATAGAGGCATTATCTAAAACCAATTTCAAAAACCAAGAAAACGAAAAAAAACTAGAAAAGC.

[0046] Example 2: Cis-Component Analysis of the GbLEC2 Promoter

[0047] The cis-regulatory elements in the promoter sequence upstream of the transcription start site of the GbLEC2 gene were predicted and analyzed using PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) and PLA CE (https: / / www.dna.affrc.go.jp / PLACE / ?action=newplace) online analysis software. The results are shown in Table 3. The GbLEC2 promoter sequence contains multiple cis-regulatory elements, including light-responsive, auxin-responsive, gibberellin-responsive, and salicylic acid-responsive elements, indicating that the GbLEC2 gene plays a certain role in the regulation of growth and development in sea island cotton. Notably, at 300 bp and 403 bp upstream of the GbLEC2 transcription start site, the Skn-1 element and RY REPEA element, which control seed-specific expression of the gene, are also present, which are closely related to the physiological role of GbLEC2 in seed development.

[0048] Table 3 Predictions of cis-elements in the GbLEC2 promoter sequence

[0049]

[0050]

[0051] Example 3: Construction of plant recombinant expression vector for promoter

[0052] Based on the GbLEC2 promoter nucleotide sequence disclosed in Example 1, GbLEC2-1 (1836bp, Pro) was amplified. GbLEC2-1 ), GbLEC2-2 (1465bp, Pro GbLEC2-2 ), GbLEC2-3 (946bp, Pro GbLEC2-3 ), GbLEC2-4 (410bp, Pro GbLEC2-4 The promoter was extracted, and the amplified product was recovered using a DNA fragment recovery kit (Novizan, Nanjing). The extracted product was then ligated into a T-vector and sent to Shanghai Sangon Biotech for sequencing. Subsequently, the correctly sequenced promoter fragment was constructed into the plant expression vector pCAMBIA1304 using a seamless cloning method. The specific method is as follows: EcoRI and NcoI were used to linearize the pCAMBIA1304 vector at 37℃ for 4 hours. The digestion products were recovered using a DNA fragment recovery kit (Novizan, Nanjing). The concentrations of the recovered promoter fragment PCR product and the linearized vector product were detected using NanoDrop (Coollab, Beijing). II. Recombination and transformation were performed using the formulas in the One Step Cloning Kit (Novizan, Nanjing) instructions. Positive clones were selected for colony PCR. Bacterial cultures with correct PCR bands were sent to Shanghai Sangon Biotech for sequencing, ultimately yielding the recombinant plant expression vector pCAMBIA1304::Pro. GbLEC2-1 ::GUS、pCAMBIA1304::Pro GbLEC2-2 ::GUS、pCAMBIA1304::Pro GbLEC2-3 ::GUS、pCAMBIA1304::Pro GbLEC2-4 ::GUS. (The GbLEC2 promoter was identified by E. coli culture PCR. See [link to relevant documentation]). Figure 2 ).

[0053] Example 4 Plant Recombinant Expression Vector Transformed into Agrobacterium

[0054] The constructed Escherichia coli recombinant plant expression vector was transformed into Agrobacterium GV3101 using the heat shock method, and then identified by PCR. (Identification results are shown below.) Figure 3 ).

[0055] Example 5: Agrobacterium-mediated transfection of different cotton tissues and cells

[0056] (1) Agrobacterium-mediated transformation of cotton leaves

[0057] Leaves of 'Xinhai 21' at the two-leaf-one-bud stage were selected. The needle of a 1mL plastic syringe was removed, and pressure was used to extract the OD... 600 Agrobacterium samples with values ​​of 1–1.5 (pCAMBIA1304::Pro) GbLEC2-1 ::GUS transforms Agrobacterium, pCAMBIA1304::Pro GbLEC2-2 ::GUS transforms Agrobacterium, pCAMBIA1304::Pro GbLEC2-3 ::GUS transforms Agrobacterium and pCAMBIA1304::Pro GbLEC2-4 Suspensions of Agrobacterium tumefaciens transformed with GUS and control group (positive: pCAMBIA1304 transformed with Agrobacterium tumefaciens; negative: empty Agrobacterium tumefaciens strain GV3101) were injected between the leaf veins (injected on the underside of the leaf). It is best to puncture the epidermis with a 1 mL plastic syringe needle beforehand and inject at the puncture site. After injection, the leaf was incubated under low light for 24 hours, followed by normal light exposure.

[0058] (2) Agrobacterium-mediated transformation of cotton stem, root, embryonic cells, globular embryos, torpedo embryos and cotyledonary cells

[0059] In a clean bench, the roots and stems of 5-day-old 'Xinhai 21' sterile seedlings were cut off, and the stems were cut into segments approximately 0.5–0.8 cm long. Embryogenic cells from different stages were selected and analyzed using OD... 600 Agrobacterium sample group with a value of 0.5 (pCAMBIA1304::Pro) GbLEC2-1 ::GUS transforms Agrobacterium, pCAMBIA1304::Pro GbLEC2-2 ::GUS transforms Agrobacterium, pCAMBIA1304::Pro GbLEC2-3 ::GUS transforms Agrobacterium and pCAMBIA1304::Pro GbLEC2-4 ::GUS-transformed Agrobacterium and control group (positive: pCAMBIA1304 transformed Agrobacterium; negative: Agrobacterium GV3101 empty strain) suspension were inoculated for 15 min, during which time the suspension was shaken continuously. The suspension was then discarded, rinsed 3 times with sterile distilled water for 30 s each time, and the water was blotted dry with filter paper and placed in co-culture medium for dark incubation for 48 h.

[0060] Example 6: GUS Activity Detection

[0061] The treated roots, stems, leaves, and four types of embryogenic cells were placed in freshly prepared GUS staining solution, vacuumed for 10 min, and stained at 37℃ for 12 h (until the positive control group showed results). The staining solution was discarded, and the cells were destained three times with 75% ethanol until the background was colorless. The GUS staining results are shown in Table 4, and photographs were taken and recorded as follows: Figure 4 .

[0062] GUS activity was observed in all four promoter fragments in embryogenic cells, globular embryos, torpedo-shaped embryos, and cotyledonary embryos, but not in leaves. This indicates that all GbLEC2 promoter fragments are transcriptionally active during somatic embryonic development, but not in leaves. In stems, only the GbLEC2-1 and GbLEC2-2 promoter fragments showed the same light blue GUS staining as the positive control group. In root tissues (including primary and lateral roots), only the GbLEC2-1 promoter fragment showed light blue GUS staining. These results indicate that different GbLEC2 promoters have promoter activity, but different transcriptional activities in different tissues and developmental stages. Specifically, the 946 bp and 410 bp GbLEC2 promoter fragments are transcriptionally active only during somatic embryogenesis, and have no transcriptional characteristics in other developmental stages, indicating the tissue-development specificity of their transcriptional regulation.

[0063] Table 4. GUS activity analysis of different GbLEC2 promoter fragments in cotton tissue cells.

[0064]

[0065]

[0066] Note: "–" indicates no activity, and "+" indicates activity.

[0067] Example 7 Semi-quantitative PCR

[0068] RNA was extracted from the roots, stems, leaves, embryogenic callus, globular embryos, torpedo embryos, and cotyledonary embryos of the Xinhai 21 cotton variety and converted into cDNA. A semi-quantitative analysis method was used, with the expression level of the housekeeping gene UBQ7 as a reference standard to observe the expression of GbLEC2 in the roots, stems, leaves, embryogenic callus, globular embryos, torpedo embryos, and cotyledonary embryos of Xinhai 21 cotton. cDNA from different samples was serially diluted to adjust the expression level of the housekeeping gene to a uniform level. The cDNA concentration at this point was then used as a template to detect the expression of the GbLEC2 gene in the roots, stems, leaves, embryogenic callus, globular embryos, torpedo embryos, and cotyledonary embryos of Xinhai 21. The amplification systems for GbLEC2 and the housekeeping gene UBQ7 are shown in Table 2. The amplification program was as follows: amplification conditions were: 94℃ for 5 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s (28 cycles for GbLEC2, 22 cycles for UBQ7); 72℃ for 10 min.

[0069] Semi-quantitative RT-PCR results by Figure 5As shown, the expression level of GbLEC2 differs significantly at each stage. GbLEC2 is not expressed or expressed at a very low level in the embryogenic callus tissue of roots, stems, and leaves. The expression order in globular embryos, torpedo embryos, and cotyledonary embryos is globular embryos > cotyledonary embryos > torpedo embryos, indicating that the GbLEC2 gene is specifically expressed during the embryonic development of *Cottonia spp.* Furthermore, the results also indicate that other regulatory elements in the *Cottonia spp.* genome sequence regulate the expression of GbLEC2 in root, stem, leaf, and embryogenic cells.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing a promoter, characterized in that, Using the whole genome of *Cotton Island* as a template, PCR amplification was performed using primer pairs to obtain the promoter; the primer pairs were Pro. GbLEC2-3 F and Pro GbLEC2-3 R, the Pro GbLEC2-3 The nucleotide sequence of F is shown in SEQ ID NO.11, and the Pro GbLEC2-3 The nucleotide sequence of R is shown in SEQ ID NO.

12.

2. The promoter constructed by the method of claim 1.

3. A recombinant vector, characterized in that, Includes the promoter described in claim 2.

4. A recombinant bacterium, characterized in that, Includes the recombinant vector as described in claim 3.

5. The application of the promoter as described in claim 2, the recombinant vector as described in claim 3, or the recombinant bacteria as described in claim 4 in the specific expression of embryogenic callus, globular embryo, torpedo embryo, and cotyledon embryo cells of sea island cotton.

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