A root-specific promoter derived from wheat and its applications

Abstract

This invention discloses a root-specific promoter derived from wheat and its application. The nucleotide sequence of the root-specific promoter is shown in SEQ ID NO:1. It can guide the target gene to be specifically highly expressed in the roots of plants, while not expressed or being expressed at low levels in other tissues of the plant. This promoter has great application potential in plant genetic engineering and genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of biological and genetic engineering technology, specifically relating to a root-specific promoter derived from the wheat TaRAX3 gene and its application in regulating the root-specific expression of exogenous genes. Background Technology

[0002] A promoter is a DNA sequence located upstream of the start codon (ATG) of a gene, directly determining the spatiotemporal expression characteristics and level of the gene. Based on their mode of action and function, promoters can be divided into three categories: constitutive promoters, tissue-specific promoters, and inducible promoters. In plant genetic engineering, promoters with different levels of expression regulation can be used to drive the expression of target genes. Although constitutive promoters can drive the non-specific high-level expression of target genes in various plant tissues, they often lead to the continuous synthesis and large accumulation of transgenic products, disrupting the original metabolic balance of transgenic plants, hindering normal plant growth, and sometimes even causing tissue toxicity, thus failing to obtain the desired phenotype. Tissue-specific promoters can restrict gene expression to specific developmental stages or tissue sites, not only allowing the expression product of the target gene to accumulate at the desired specific site but also avoiding unnecessary nutrient waste in the plant and reducing side effects on plant growth. Therefore, effectively utilizing tissue-specific promoters that can drive the timed and site-specific expression of target genes in plant tissues and organs is of great significance for gene function research and crop trait improvement.

[0003] Roots are the main organs for plants to absorb water and nutrients. By utilizing root-specific promoters, we can not only study the growth, development, and morphogenesis of plant roots, but also improve root growth and increase rhizosphere exudates, thereby enhancing the plant's ability to resist adversity and improve soil remediation. This is of great significance for research on basic crop functions and applications of genetic improvement.

[0004] Currently, the number of known, highly effective, and specific plant root-specific promoters available for use in the genetic improvement of crops using molecular biology and genetic engineering remains limited. Therefore, providing new plant root-specific promoters is of great significance for research on plant root development and the breeding of new varieties. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a novel plant root-specific promoter for guiding the specific high expression of a target gene in the plant root, while its expression is low or absent in other plant tissues.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In one aspect, the present invention provides a root-specific promoter derived from wheat, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0008] This invention discovered, isolated, and identified a 2020 bp DNA sequence (i.e., the sequence shown in SEQ ID NO:1) upstream of the wheat root-specifically highly expressed TaRAX3 gene, including the transcription start site, and determined that it can regulate the specific expression of exogenous genes in plant roots.

[0009] A second aspect of the present invention provides a primer set for amplifying the above-mentioned root-specific promoter, the nucleotide sequence of which is shown in SEQ ID NO:2-3.

[0010] A third aspect of the present invention provides a recombinant expression vector comprising a root-specific promoter having a nucleotide sequence as shown in SEQ ID NO:1.

[0011] A fourth aspect of the present invention further provides a method for constructing the above-mentioned recombinant expression vector, specifically comprising the following steps:

[0012] S1. Using wheat genomic DNA as a template, PCR amplification was performed using primer sets with sequences shown in SEQ ID NO:2-3;

[0013] S2. Using the PCR product obtained in S1 as a template, PCR amplification was performed using primer pairs with sequences as shown in SEQ ID NO:4-5. Then, the amplification product and plasmid vector were double-digested with Hind III and BamHI, respectively.

[0014] S3. Recover the PCR digestion products and vector and ligate them with DNA ligase to obtain the recombinant expression vector.

[0015] Furthermore, in the above technical solution, the reaction system for PCR amplification in step S1 includes 5 μL of 2×UltraHiFidelityPCRPreMix, 2 μL of 1 μM forward primer, 2 μL of 1 μM reverse primer, and 1 μL of genomic DNA, and its reaction program is 94℃ for 5 min, 94℃ for 15 sec, 60℃ for 15 sec, 72℃ for 30 sec, for 35 cycles.

[0016] Furthermore, in the above technical solution, the enzyme digestion reaction system in step S2 includes 5 μL 10×CutSmartBuffer, 1 μL Hind III-HF, 1 μL BamHI-HF, 1 μg PCR product or plasmid vector, and ddH2O added to 50 μL, and the reaction program is 37℃ for 1 h.

[0017] Furthermore, in the above technical solution, the reaction system for connection described in step S3 consists of 1 μL 10×T4 ligase buffer, 1 μL T4 ligase, 2 μL PCR digestion product, and 6 μL vector, and the reaction program is 16℃ for 12h.

[0018] The fifth aspect of the present invention provides a recombinant bacterium containing a root-specific promoter as shown in SEQ ID NO:1, or containing the above-mentioned recombinant expression vector. Specifically, the recombinant expression vector can be poured into the vector bacterium by electroporation. The vector bacterium can be Escherichia coli or Agrobacterium.

[0019] The sixth aspect of this invention provides the application of the above-mentioned root-specific promoter, recombinant expression vector, or recombinant bacteria in initiating the expression of a target gene in plants, specifically, the expression of the target gene is performed in plant roots.

[0020] In a specific embodiment of the present invention, a recombinant expression vector fused with the above-mentioned root-specific promoter and GUS gene is introduced into Agrobacterium tumefaciens and Arabidopsis thaliana is transformed using the Agrobacterium tumefaciens flower dipping method to obtain transgenic Arabidopsis thaliana. Detection of transgenic Arabidopsis thaliana plants reveals that the specific promoter provided by the present invention can regulate the specific expression of the target gene GUS in the roots of Arabidopsis thaliana.

[0021] The beneficial effects of this invention are as follows: It provides a new root-specific promoter that enables the target gene to be highly expressed specifically in plant roots but not in other tissues, thus enriching the selectivity of root-specific promoters and having important significance for plant root research and new variety breeding. Attached Figure Description

[0022] Figure 1 A graph comparing the expression levels of the TaRAX3 gene in different wheat tissues;

[0023] Figure 2 The results of PCR amplification of the promoter of the TaRAX3 gene;

[0024] Figure 3 This is a schematic diagram of the structure of the recombinant expression vector pBI101-TaRAX3 promoter-GUS;

[0025] Figure 4 The results are PCR detection results for transgenic plants. Among them, WT is a wild-type Arabidopsis thaliana plant, and L1, L2, and L3 are three different positive transgenic Arabidopsis thaliana lines.

[0026] Figure 5The results of GUS staining of positive transgenic Arabidopsis thaliana plants are shown. A represents 10-day-old seedlings, B represents roots at the flowering stage, C represents stems at the flowering stage, D represents rosette leaves at the flowering stage, E represents stem leaves at the flowering stage, F represents flowers, G represents immature pods, and H represents mature seeds. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments thereof. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0028] Unless otherwise specified, the methods described in the following examples are conventional; the reagents and materials described are commercially available unless otherwise specified.

[0029] Example 1

[0030] This embodiment analyzed the differential expression of the wheat TaRAX3 gene (GeneID: Traes CS5D02G415800) in different wheat tissues through the following process:

[0031] (1) Take the roots and leaves of wheat variety Chinese Spring 10-day seedlings, the roots, stems, leaves and ears of flowering wheat, and the seeds of mature wheat and preserve them in liquid nitrogen.

[0032] (2) After grinding the above wheat tissue samples into fine powder in liquid nitrogen, the total RNA of each sample was extracted using the TRNzol Universal (TIANGEN#DP424) method.

[0033] (3) Using the total RNA from the above wheat tissues as a template, III. Use the All-in-one RTSuperMix Perfect for qPCR kit (Vazyme#R333) for reverse transcription PCR to synthesize cDNA.

[0034] (4) Using the cDNA from the above wheat tissues as templates, real-time quantitative PCR was performed using the Talent qPCR PreMix kit (TIANGEN#FP209) to detect the relative expression level of the TaRAX3 gene in different wheat tissues.

[0035] The primers used to detect the TaRAX3 gene (target gene) are as follows:

[0036] The forward primer is 5'-GGAGGACGGACAATGACATC-3' (SEQ ID NO:6).

[0037] The reverse primer is 5'-TAAGCACCAGCAGCAAAGC-3' (SEQ ID NO:7);

[0038] The primers used to detect the β-Actin gene (internal reference gene) are as follows:

[0039] The forward primer is 5'-GGAATCCATGAGACCACCTAC-3' (SEQ ID NO:8).

[0040] The reverse primer is 5'-GACCCAGACAACTCGCAAC-3' (SEQ ID NO:9).

[0041] The reaction system for real-time quantitative PCR was as follows: 10 μL 2×Talent qPCR PreMix, 4 μL 1 μM forward primer, 4 μL 1 μM reverse primer, 1 μL cDNA template, and 1 μL ddH2O.

[0042] The reaction program for real-time quantitative PCR was as follows: 95℃ for 5 min, 95℃ for 15 sec, 60℃ for 15 s, 72℃ for 20 s, for 40 cycles.

[0043] The calculation method is: C = 2 -ΔCt ΔCt=Ct 目的基因 -Ct 内参基因 The average C-value of the three replicates was used as the relative expression level of the target gene. The results are as follows: Figure 1 As shown, the TaRAX3 gene is specifically highly expressed in wheat roots, but is not expressed in other tissues.

[0044] The promoter of the TaRAX3 gene was further cloned using the following method:

[0045] (1) Take the leaves of the wheat variety Chinese Spring, grind them into fine powder in liquid nitrogen, and extract genomic DNA using the CTAB (Coolaber#SL2071) method.

[0046] (2) Using the wheat leaf genomic DNA as a template, the promoter of the TaRAX3 gene was amplified by PCR using the Ultra HiFidelity PCR kit (TIANGEN#KP203); the primer set used to amplify the TaRAX3 gene promoter was as follows:

[0047] The forward primer is 5'-TCAGTCAGACCTCTTCAGCC-3' (SEQ ID NO:2).

[0048] The reverse primer is 5'-TGGTATCCTTCCTCCTCTTCTA-3' (SEQ ID NO:3).

[0049] The PCR reaction system consisted of: 5 μL 2×Ultra HiFidelityPCRPreMix, 2 μL 1 μM forward primer, 2 μL 1 μM reverse primer, and 1 μL genomic DNA.

[0050] The PCR reaction program was as follows: 94℃ for 5 min; 94℃ for 15 sec, 60℃ for 15 sec, 72℃ for 30 sec, for 35 cycles.

[0051] (3) The PCR products obtained in step (2) were detected and analyzed by 1% agarose gel electrophoresis, and sequencing analysis was performed by Qingke Biotechnology Co., Ltd.

[0052] Electrophoresis results (see) Figure 2 The sequencing results (SEQ ID NO:1) showed that the PCR product band was clear and single, and the DNA fragment size was 2020bp, which is the nucleotide sequence of the TaRAX3 gene promoter.

[0053] Example 2

[0054] This embodiment provides the construction process of the recombinant expression vector and recombinant bacteria, as detailed below:

[0055] (1) Using the PCR product obtained from the promoter cloning in Example 1 as a template, primers were used to amplify it by PCR.

[0056] The primer sequences used for amplification are as follows:

[0057] 5'-CCC AAGCTT TCAGTCAGACCTCTTCAGC-3' (SEQ ID NO:4, underlined is the recognition site of restriction endonuclease Hind III),

[0058] 5'-CGC GGATCC GGTGGTATCCTTCCTCCTCT-3' (SEQ ID NO:5, the underlined part is the recognition site of the restriction endonuclease BamHI).

[0059] The PCR amplification reaction system consisted of: 5 μL 2×Ultra HiFidelityPCRPreMix, 2 μL 1 μM forward primer, 2 μL 1 μM reverse primer, and 1 μL PCR product.

[0060] The PCR amplification reaction program was as follows: 94℃ for 5 min; 94℃ for 15 sec, 60℃ for 15 sec, 72℃ for 30 sec, for 35 cycles.

[0061] (2) The PCR product and pBI101 plasmid vector obtained from the above amplification were double-digested using Hind III (NEB#R3104) and BamHI (NEB#R3136). The digestion reaction system consisted of 5 μL 10×CutSmart Buffer, 1 μL Hind III-HF, 1 μL BamHI-HF, 1 μg PCR product / plasmid vector, and ddH2O added to a final volume of 50 μL. The digestion reaction program was 37℃ for 1 h.

[0062] (3) After the enzyme digestion products obtained in step (2) are separated by 1% agarose gel electrophoresis, the PCR enzyme digestion products and linearized vector backbones are recovered by using an agarose gel DNA recovery kit (TIANGEN#DP219).

[0063] (4) The recovered PCR digestion products and linearized vector backbone were ligated with T4 DNA ligase (NEB#M0202) to obtain the recombinant expression vector pBI101-TaRAX3 promoter-GUS, which fused the TaRAX3 gene promoter with the GUS gene.

[0064] The ligation reaction mixture consisted of: 1 μL 10×T4 ligase buffer, 1 μL T4 ligase, 2 μL PCR digestion product, and 6 μL linearized vector backbone. The ligation reaction program was 16℃ for 12 h.

[0065] (5) The above recombinant expression vector pBI101-TaRAX3 promoter-GUS was introduced into Escherichia coli DH5α by electroporation. Then, positive monoclonal strains were screened by bacterial PCR, and recombinant plasmids were extracted for sequencing analysis.

[0066] Based on the sequencing results, the structure of the recombinant expression vector pBI101-TaRAX3 promoter-GUS is described as follows: The small fragment between the Hind III and BamH I restriction endonuclease sites on the pBI101 vector is replaced with the DNA fragment shown in nucleotides 1 to 2020 from the 5' end of SEQ ID NO:1 in the sequence listing. A schematic diagram of the structure is shown below. Figure 3 .

[0067] Example 3

[0068] This embodiment uses Arabidopsis thaliana as an example to verify the specific expression of exogenous genes regulated by the root-specific promoter provided by the present invention, as follows:

[0069] (1) Preparation of transgenic Arabidopsis thaliana plants

[0070] First, the recombinant expression vector pBI101-TaRAX3 promoter-GUS constructed in Example 2 was introduced into Agrobacterium tumefaciens GV3103 using the freeze-thaw method. Then, the Agrobacterium tumefaciens flower immersion method was used to transform Colombian ecotype Arabidopsis thaliana (wild-type Arabidopsis thaliana, denoted as WT) to obtain seeds of T0 generation transgenic Arabidopsis thaliana.

[0071] After sterilization, T0 generation seeds were sown on MS solid medium containing 50 mg / L kanamycin. Transgenic seedlings with kanamycin resistance were screened and transplanted into nutrient soil for cultivation. The harvested seeds are the T1 generation seeds. (The process is repeated.)

[0072] The method for screening transgenic seedlings is as follows: leaves from T1 generation plants are collected, genomic DNA is extracted using the CTAB method, and positive transgenic Arabidopsis lines are identified by PCR amplification. The primer sequences used to detect the GUS gene (exogenous gene) are as follows:

[0073] The forward primer is 5'-GCATCAGCCGATTATCATCA-3' (SEQ ID NO:10).

[0074] The reverse primer is 5'-TCCCTTTCTTGTTACCGCC-3' (SEQ ID NO:11).

[0075] The reaction system used for PCR amplification and identification was: 5 μL 2×Ultra HiFidelityPCRPreMix, 2 μL 1 μM forward primer, 2 μL 1 μM reverse primer, and 1 μL genomic DNA.

[0076] The reaction program used for PCR amplification and identification was: 94℃, 5 min; 94℃, 15 sec; 60℃, 15 sec; 72℃, 10 sec, 35 cycles.

[0077] Results for some samples can be found Figure 4 The target band of the exogenous gene GUS was visible in the positive transgenic Arabidopsis lines (L1, L2, L3), but not in the wild-type Arabidopsis line (WT).

[0078] (2) Expression of GUS gene in various tissues of transgenic Arabidopsis thaliana

[0079] Ten-day-old seedlings of T1 generation positive transgenic Arabidopsis thaliana, roots, stems, rosette leaves, cauline leaves, fully opened flowers, immature pods, and mature seeds were collected. Each tissue sample was immersed in GUS staining solution (Coolaber#SL7160) and reacted overnight at 37°C. After destaining with 75% (v / v) ethanol, the GUS staining results were observed and photographed using a stereomicroscope.

[0080] The results are as follows Figure 5 As shown, in transgenic Arabidopsis plants, only the roots exhibit a distinct blue color, while the stems, leaves, flowers, and seeds show no coloration. This indicates that the TaRAX3 gene promoter is active and can only initiate the expression of the GUS gene in the roots of transgenic Arabidopsis. Therefore, the TaRAX3 gene promoter is a promoter capable of regulating the specific expression of the target gene in plant roots.

[0081] The above description is a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention. It should be noted that any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A root-specific promoter derived from wheat, characterized in that, The nucleotide sequence of the root-specific promoter is shown in SEQ ID NO:

1.

2. A primer set for amplifying the root-specific promoter of claim 1, characterized in that, The nucleotide sequences of the primer set are shown in SEQ ID NO:2-3.

3. A recombinant expression vector, characterized in that, Includes the root-specific promoter as described in claim 1.

4. A method for constructing the recombinant expression vector of claim 3, characterized in that, Includes the following steps: S1. Using wheat genomic DNA as a template, perform PCR amplification using the primer set described in claim 2; S2. Using the PCR product obtained in S1 as a template, PCR amplification was performed using primer pairs with sequences as shown in SEQ ID NO:4-5. The amplification product and plasmid vector were then digested with Hind III and BamHI, respectively. S3. Recover the PCR digestion products and vector and ligate them with DNA ligase to obtain the recombinant expression vector.

5. The method according to claim 4, characterized in that, The PCR amplification reaction system described in step S1 includes 5 μL of 2 × Ultra HiFidelity PCR Premix, 2 μL of 1 μM forward primer, 2 μL of 1 μM reverse primer, and 1 μL of genomic DNA. The reaction program is 94℃ for 5 min, 94℃ for 15 sec, 60℃ for 15 sec, and 72℃ for 30 sec, for 35 cycles.

6. The method according to claim 4, characterized in that, The enzyme digestion reaction system described in step S2 includes 5 μL of 10× CutSmart Buffer, 1 μL of Hind III-HF, 1 μL of BamH I-HF, 1 μg of PCR product or plasmid vector, and ddH2O added to 50 μL. The reaction program is 37°C for 1 h.

7. The method according to claim 4, characterized in that, The reaction system for the ligation in step S3 consists of 1 μL of 10 × T4 ligase buffer, 1 μL of T4 ligase, 2 μL of PCR digestion product, and 6 μL of vector, and the reaction program is 16 °C for 12 h.

8. A recombinant bacterium, characterized in that, It comprises the root-specific promoter of claim 1 or the recombinant expression vector of claim 3, wherein the recombinant bacteria is recombinant Escherichia coli or recombinant Agrobacterium.

9. The application of the root-specific promoter of claim 1, the recombinant expression vector of claim 3, or the recombinant bacteria of claim 8 in initiating the expression of the target gene in Arabidopsis thaliana.

10. The application according to claim 9, characterized in that, The target gene is specifically expressed in Arabidopsis roots.

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