A ce pro 722 promoter and uses thereof

Abstract

The present application relates to the field of agricultural biotechnology, and particularly relates to a CEPro722 promoter and application thereof.The CEPro722 promoter comprises a nucleotide sequence as shown in SEQ ID NO.1 or a nucleotide sequence as shown in SEQ ID NO.2.The present application is screened from a GRMZM2G069722 gene, and the CEPro722 promoter can drive gene expression in plants such as rice, wheat or corn; and particularly, the promoter can drive high and stable expression in various tissues such as plant callus, leaf in the vegetative growth period, young ear and seed, and can replace a non-plant source promoter.The CEPro722 promoter provided in the present application has important significance in the field of plant genetic engineering, and can effectively reduce potential safety risks of transgenic plants caused by exogenous DNA.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a CEPro722 promoter and its applications. Background Technology

[0002] Transgenic technology has become an indispensable technology in both basic and applied research on gene function. Promoters, as crucial cis-acting elements driving gene expression, occupy a vital position in transgenic technology. Promoters can be classified into three categories based on their expression patterns: constitutive promoters, inducible promoters, and spatiotemporally specific promoters. Constitutive promoters can initiate gene transcription in all or most tissues, giving gene expression spatiotemporal persistence and expression constancy. Inducible promoters can initiate or significantly enhance gene expression in response to certain physical or chemical signals; they possess enhancer, silencer, or similar functional sequence structures and exhibit significant specificity. Spatiotemporally specific promoters initiate gene expression only at specific growth stages or sites. In-depth research into promoter expression patterns is beneficial for understanding gene expression regulation mechanisms and biological functions, and also contributes to the effective regulation of exogenous gene expression.

[0003] Currently, the most commonly used promoters in plant transformation are the cauliflower floret virus promoter (CaMV35Spro) and the maize polyubiquitin protein gene promoter (ZmUbipro). CaMV35Spro is a plant DNA virus promoter, and its application in plant transgenics may raise unnecessary concerns. While ZmUbipro is derived from plants, frequent use of the same promoter during transformation can easily lead to transgene silencing. Therefore, discovering new, highly efficient constitutive promoters, especially plant-derived promoters with low biosafety risks, is particularly important. Summary of the Invention

[0004] To address the problems of existing technologies, this invention provides a CEPro722 promoter and its applications.

[0005] This invention provides a CEPro722 promoter, wherein the CEPro722 promoter comprises any of the following nucleotide sequences:

[0006] i) The nucleotide sequence shown in SEQ ID NO.1 or the nucleotide sequence shown in SEQ ID NO.2;

[0007] ii) A nucleotide sequence complementary to i);

[0008] iii) Nucleotide sequences with equivalent promoter function obtained by substituting, deleting, or adding one or more nucleotide sequences as shown in i).

[0009] The present invention further provides primer pairs for amplifying the CEPro722 promoter of claim 1, comprising: primer pairs as shown in SEQ ID NO. 3-4, and / or primer pairs as shown in SEQ ID NO. 5-6.

[0010] Furthermore, the primer pairs shown in SEQ ID NO.3-4 are used to amplify the nucleotide sequence shown in SEQ ID NO.1; and the primer pairs shown in SEQ ID NO.5-6 are used to amplify the nucleotide sequence shown in SEQ ID NO.2.

[0011] The present invention further provides biological materials containing the CEPro722 promoter, wherein the biological material is an expression cassette, a vector, or a transgenic cell.

[0012] Furthermore, when the biological material is an expression cassette, the expression cassette further includes a functional gene and a terminator.

[0013] Furthermore, the functional gene is a plant agronomic trait-related gene or marker gene.

[0014] Marker genes are genes that can play a specific marking role and are usually used to test whether gene transformation is successful. They are preferably one or more of the following: β-glucuronidase gene GUS, hygromycin phosphotransferase gene Hn, acetolactate synthase mutant gene ALS, Bar gene, resistance EPSPS gene, or NptII gene.

[0015] Plant agronomic traits are those characteristics of crop varieties, such as growth period, plant height, leaf area, fruit weight, quality, herbicide resistance, and pest and disease resistance. Correspondingly, the genes related to plant agronomic traits are the genes associated with these traits.

[0016] The present invention further provides a kit comprising the CEPro722 promoter, or the primer pair, or the biomaterial.

[0017] The present invention further provides the application of the CEPro722 promoter, or the primer pair, or the biological material, or the kit in the preparation of transgenic plants.

[0018] Furthermore, the application is to construct the CEPro722 promoter into a vector and then introduce it into a plant to prepare a transgenic plant; or, to introduce the biological material into a plant to prepare a transgenic plant.

[0019] Furthermore, after obtaining the transgenic plants, they are screened using marker genes.

[0020] As a preferred embodiment, the present invention provides a method for preparing transgenic rice, comprising:

[0021] The vector containing the CEPro722 promoter was transformed into rice callus using Agrobacterium-mediated transformation.

[0022] Rice seedlings were obtained by resistance screening and differentiation of the rice callus tissue;

[0023] Transgenic rice was obtained by rooting the rice seedlings.

[0024] Furthermore, the rice callus tissue was prepared by the following method:

[0025] After the rice seeds are dehulled and disinfected, the mature embryos are inoculated into an induction medium to induce embryogenic callus tissue, and cultured in the dark at 28-30℃ for 30-50 days.

[0026] Furthermore, after transforming the plant constitutive promoter CEPro432 into rice callus, co-culture is also included, wherein the co-culture is carried out in the dark at 22-24°C until bacterial cells appear on the surface of the callus.

[0027] Furthermore, the resistance screening involves inoculating co-cultured callus tissue into a screening medium supplemented with hygromycin, and incubating it in the dark at 28–30°C for 30–50 days to perform resistance screening.

[0028] Further, the differentiation involves adding the resistance-selected callus tissue to a differentiation medium supplemented with hygromycin and culturing it under light at 28–30°C for 25–40 days.

[0029] Furthermore, the rooting culture involves inoculating rice seedlings onto a rooting medium supplemented with hygromycin and cultivating them under light at 30–32°C for 5–20 days.

[0030] Furthermore, after rooting culture, including PCR testing, plants that test positive are selected for planting.

[0031] The present invention further provides the application of the CEPro722 promoter or the biological material in driving gene expression in plants, such as in one or more plant callus tissues, vegetative growth tissues or reproductive organs.

[0032] Furthermore, the genes include: functional genes, antisense genes of functional genes, or small RNA genes;

[0033] The functional genes preferably include plant agronomic trait-related genes or marker genes, and the small RNA genes are preferably small RNA genes capable of interfering with the expression of functional genes.

[0034] Furthermore, the plant is one or more of rice, corn, wheat, barley, soybean, cotton, rapeseed, sorghum, or millet.

[0035] The present invention has the following beneficial effects:

[0036] This invention screened and obtained a CEPro722 promoter, which is a constitutive promoter derived from maize. It can drive gene expression in callus tissue, major functional tissues (roots, leaves, flowers, seedlings, young ears, etc.) or reproductive organs of rice, maize or wheat, especially in seedlings at the 3-5 leaf stage.

[0037] The promoter CEPro722 provided by this invention can be combined with endogenous or exogenous plant selection marker genes to form a plant transgenic selection expression cassette or a plant genetic transformation selection vector, and other functional elements can be added for plant tissue culture or plant genetic transformation, providing an effective tool and method for screening plant genetic transformation.

[0038] The promoter CEPro722 provided by this invention can also drive the efficient expression of genes in the main functional tissues of the aboveground and underground parts of transformed seedlings during their vegetative growth period. Furthermore, since the promoter CEPro722 is an endogenous plant gene, no exogenous gene fragments such as bacteria are introduced during the transgenic process. This not only enriches the promoter resources for plant transgenics but also effectively reduces the potential safety risks of transgenic plants caused by exogenous genes and alleviates public concerns about the safety of transgenic plants. This is beneficial for the commercial application of transgenic plants and has good market value and social benefits. Attached Figure Description

[0039] Figure 1 The agarose gel electrophoresis results provided in Example 2 of the present invention are shown below; from left to right, they are the amplified fragments of promoter CEPro722-676, CEPro722-1542, and the 1300gusplus digestion fragment of the vector.

[0040] Figure 2 The vector spectrum of the 1300gusplus vector provided in Embodiment 2 of the present invention.

[0041] Figure 3Electrophoresis images of the 1300gusplus-722pro-676 and 1300gusplus-722pro-1542 vectors provided in Example 2 of this invention after digestion with BamHI and PstI; wherein, M is the Marker, CK- is the 1300gusplus backbone vector, 0 is the digested 1300gusplus backbone vector, ck1-ck4 are the undigested 1300gusplus-722Pro-676 recombinant plasmids, 1-4 are the digested 1300gusplus-722Pro-676 recombinant plasmids; ck5-ck8 are the undigested 1300gusplus-722Pro-1542 recombinant plasmids, and 5-8 are the digested 1300gusplus-722Pro-1542 recombinant plasmids.

[0042] Figure 4 The vector spectrum of the 1300gusplus-722Pro-676 vector provided in Embodiment 2 of the present invention.

[0043] Figure 5 The vector spectrum of the 1300gusplus-722Pro-1542 vector provided in Embodiment 2 of the present invention.

[0044] Figure 6 The above are the PCR detection electrophoresis results of Agrobacterium after transformation provided in Example 3 of the present invention; wherein, M is the Marker, ck+ is the positive control of 1300gusplus-722Pro-676 or 1300gusplus-722Pro-1542 recombinant plasmid, and 1-10 are Agrobacterium monoclonal bacterial culture samples transformed with 1300gusplus-722Pro-676 or 1300gusplus-722Pro-1542 recombinant plasmid.

[0045] Figure 7 This is a schematic diagram showing the results of screening callus tissue using hygromycin screening medium in Example 3 of the present invention.

[0046] Figure 8 This is an electrophoresis diagram of PCR detection of transgenic sample plants provided in Example 3 of the present invention; wherein, M is the marker, H2O is the blank control, ck- is the genomic DNA of ZH11 non-transgenic plants, ck+ is the positive control of 1300gusplus-722Pro-676 recombinant plasmid, and 1-13 are the genomic DNA of transgenic plants obtained by screening.

[0047] Figure 9The images show the GUS staining results of callus, differentiated seedlings, leaves, and seeds of the T0 generation transgenic lines of plasmids 1300gusplus-722Pro-676 and 1300gusplus-722Pro-1542 provided in Experimental Example 1 of this invention. Among them, Ck- represents the staining results of the negative control (ZH11) at each developmental stage, Ck+ represents the staining results of the positive control (pC1301) at each developmental stage, Pro-676 represents the staining results of the 1300gusplus-722Pro-676 transgenic line, and Pro-1542 represents the staining results of the 1300gusplus-722Pro-1542 transgenic line. Detailed Implementation

[0048] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0049] Unless otherwise specified, the experimental methods described in the following examples are conventional methods. Unless otherwise specified, the materials and reagents described in the following examples are commercially available.

[0050] Example 1

[0051] In this embodiment, bioinformatics analysis of the upstream sequence of the GRMZM2G069722 gene using promoter function prediction software such as PlantCARE and PlantPAN revealed that the sequence is rich in various promoter-related cis-acting elements, such as TATA-box and CAAT-box, indicating that the sequence possesses structural characteristics of plant cell promoters. Furthermore, PlantPAN analysis showed that the 1bp to 618bp sequence of this gene is rich in CpG islands, and CpG islands are also a sequence feature of eukaryotic promoters; therefore, this invention hypothesizes that this sequence should possess promoter activity.

[0052] In this embodiment, upstream sequences of the GRMZM2G069722 gene of different lengths were extracted for promoter activity identification. After continuous screening and comparison, the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 were finally determined as promoter sequences and named CEPro722. The CEPro722 promoter can drive the gene to be expressed efficiently in the callus tissue and the main tissues during the vegetative growth stage of rice.

[0053] The promoter CEPro722 can be amplified using the following primers:

[0054] The amplification primer sequences for the promoter shown in SEQ ID NO.1 are as follows:

[0055] SEQ ID NO.3: 5'-AGATCTACCATGGTACCGTGGATCCGGTCGTGGTCGTGGAGCAG-3';

[0056] SEQ ID NO. 4: 5'-CAAGCTTGCATGCCTGCAgACAATTTTTTTTACTCCCGTCGCA-3'.

[0057] The amplification primer sequences for the promoter shown in SEQ ID NO.2 are as follows:

[0058] SEQ ID NO.5: 5'-AGATCTACCATGGTACCGTGGATCCGGTCGTGGTCGTGGAGCAG-3';

[0059] SEQ ID NO. 6: 5'-CAAGCTTGCATGCCTGCAGTCACGGGGTGTAGATATGTAAGTATG-3'.

[0060] Example 2

[0061] In this embodiment, the promoter CEPro722-676 is constructed into the expression cassette and vector. The specific process is as follows:

[0062] 1. Preparation of plant transgenic expression cassettes containing promoter CEPro722-676

[0063] The method for constructing the plant transgenic expression cassette CEPro722-676-GUS-nosT (sequence as shown in SEQ ID NO.7) of the present invention is as follows:

[0064] Primers 1300-722Pro-F2 / 1300-722Pro-Rv2 were designed to amplify the promoter fragment CEPro722-676 from the maize B73 genome. Primer 1300-722Pro-F2 has a 25-nucleotide repeat at its 5' end with the corresponding ligation site in the vector; primer 1300-722Pro-Rv2 has a 19-nucleotide repeat at its 5' end with the corresponding ligation site in the vector, for subsequent recombination using Gibson Assembly.

[0065] The primer sequences are as follows:

[0066] 1300-722Pro-F2: 5'-AGATCTACCATGGTACCGTGGATCCGGTCGTGGTCGTGGAGCAG-3' (SEQ ID NO. 3);

[0067] 1300-722Pro-Rv2:5'-CAAGCTTGCATGCCTGCAGACAATTTTTTTTACTCCCGTCGCA-3' (SEQ ID NO. 4).

[0068] The PCR amplification reaction system is as follows:

[0069] Table 1 PCR amplification reaction system

[0070]

[0071] The PCR amplification program is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55-65℃ annealing for 30 s, 68℃ extension for 3 min, 35 cycles; 68℃ extension for 10 min, 16℃ to finish.

[0072] The PCR product amplified by primers 1300-722Pro-F2 and 1300-722Pro-RV2 was the CEPro722-676 fragment, which was recovered as a 676 bp product by 1.2% agarose gel electrophoresis (results are shown below). Figure 1 (As shown in the left figure).

[0073] 2. Preparation of plant transgenic expression cassettes containing promoter CEPro722-1542

[0074] The method for constructing the plant transgenic expression cassette CEPro722-1542-GUS-nosT (sequence as shown in SEQ ID NO.8) of the present invention is as follows:

[0075] Primers 1300-722Pro-F1 / 1300-722Pro-Rv1 were designed to amplify the promoter fragment CEPro722-1542 from the maize B73 genome. Primer 1300-722Pro-F1 has a 25-nucleotide repeat at its 5' end with the corresponding ligation site in the vector; primer 1300-722Pro-Rv1 has a 19-nucleotide repeat at its 5' end with the corresponding ligation site in the vector, for subsequent recombination using Gibson Assembly.

[0076] The primer sequences are as follows:

[0077] 1300-722Pro-F1:5'-AGATCTACCATGGTACCGTGGATCCGGTCGTGGTCGTGGAGCAG-3'(SEQID NO.5);

[0078] 1300-722Pro-Rv1:5'-CAAGCTTGCATGCCTGCAgTCACGGGGTGTAGATATGTAAGTATG-3' (SEQ ID NO.6);

[0079] The PCR amplification reaction system is as follows:

[0080] Table 2 PCR amplification reaction system

[0081]

[0082] The PCR amplification program is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55-65℃ annealing for 30 s, 68℃ extension for 3 min, 35 cycles; 68℃ extension for 10 min, 16℃ to finish.

[0083] The PCR product amplified by primers 1300-722Pro-F1 and 1300-722Pro-RV1 was the CEPro722-1542 fragment, which was recovered as a 1542bp product by 1.2% agarose gel electrophoresis (results are shown below). Figure 1 (As shown in the middle figure).

[0084] 3. Construction of plant genetic transformation vectors

[0085] Using the Gibson Assembly method, the amplification product from step 1 above was inserted into the 1300 gusplus vector (vector pattern shown). Figure 2 The specific method for the double restriction sites of BamHI and PstI is as follows:

[0086] (1) The vector plasmid 1300 gusplus was double-digested with BamHI+PstI, and after agarose gel electrophoresis, it was analyzed using EZNA. The extraction kit (Omega) recovers strips of about 10kb in size, yielding linear fragments of 1300 gus plus.

[0087] The BamHI+PstI double enzyme digestion reaction system is as follows:

[0088] Table 3 Enzyme digestion reaction system

[0089]

[0090] Enzyme digestion results as follows Figure 1 As shown in the right figure.

[0091] (2) 2×Lightening Cloning Kit Connector Ligate the CEPro722-676 or CEPro722-1542 fragments to the 1300 GUS Plus vector using the following ligation system:

[0092] Table 4 Connection System

[0093]

[0094] Connection procedure: 50℃, 30min.

[0095] (3) Transformation: Take 3 μl of the ligation product from step (2) and add it to E. coli competent cells. Mix gently and incubate on ice for half an hour. Transform E. coli competent cells using an electroporator at 1.8 KV. Add 1 ml of SOC medium and incubate at 37℃ and 220 rpm for 1 h with shaking. Centrifuge at 5000 rpm for 30 s, discard 800 μl of supernatant, mix the remaining cells with the medium, and spread on LB plates containing kanamycin. Incubate at 37℃ for about 16 h, pick single colonies, and perform colony PCR verification using specific primers (1300-722Pro-test-F and 1300-722Pro-test-R). Select positive colonies, incubate overnight at 37℃ and 220 rpm, and extract plasmids using a high-purity plasmid mini-prep kit (Zhongke Ruitai). After enzyme digestion and detection, if the results are correct (see results below). Figure 3 As shown, M represents the marker, CK- represents the 1300gusplus backbone vector, 0 represents the enzyme-digested 1300gusplus backbone vector, ck1-ck4 represent the undigested 1300gusplus-722Pro-676 recombinant plasmid, 1-4 represent the enzyme-digested 1300gusplus-722Pro-676 recombinant plasmid (each yielding a fragment of approximately 676 bp); ck5-ck8 represent the undigested 1300gusplus-722Pro-1542 recombinant plasmid, 5-8 represent the enzyme-digested 1300gusplus-722Pro-1542 recombinant plasmid (each yielding a fragment of approximately 1542 bp). The strain was preserved and sequenced. The resulting vectors were named 1300gusplus-722pro-676 and 1300gusplus-722pro-1542, and the vector diagrams are shown below. Figure 4 and Figure 5 .

[0096] Primer sequences:

[0097] 1300-722pro-test-F: 5'-CCACGGGTCTCGGTGTTGA-3' (SEQ ID NO.9);

[0098] 1300-722pro-test-R:5'-GCGAGCCCAACAAGAAATGC-3' (SEQ ID NO. 10).

[0099] Example 3

[0100] In this embodiment, the CEPro722 promoter is transformed into plants to prepare the corresponding transgenic plants. The specific process is as follows:

[0101] 1. Agrobacterium transformation and identification

[0102] Take *Agrobacterium* EHA105 competent cells stored at -80℃, add 1 μl of the correctly sequenced plasmids 1300gusplus-722Pro-676 and 1300gusplus-722Pro-1542 obtained in Example 2, and transform by electroporation at 2.5 kV. Spread on YEP culture plates containing kanamycin, rifampin, and streptomycin, and incubate at 28℃ for about 48 hours. Pick single colonies and shake them overnight. Verify the bacterial culture using PCR with specific primers (1300-722Pro-test-F and 1300-722Pro-test-R) (results are shown in Figure 1). Figure 6 As shown, M is the marker, ck+ is the positive control of 1300gusplus-722Pro-676 or 1300gusplus-722Pro-1542 recombinant plasmid, and 1-10 are Agrobacterium monoclonal bacterial culture samples transformed with 1300gusplus-722Pro-676 or 1300gusplus-722Pro-1542 recombinant plasmid (the amplified band size is 612bp, which is correct). The target fragment of about 612bp can be amplified. Positive clones (engineered Agrobacterium) are selected, and the culture is shaken for 36-48 hours. The bacterial culture is then preserved for infection.

[0103] 2. Agrobacterium-mediated genetic transformation

[0104] (1) Induction: After disinfecting with sodium hypochlorite, the seeds of Zhonghua 11 (ZH11) were placed on induction medium (N6 + 2.4-D 3 mg / L + CH 0.6 g / L + Pro 0.5 g / L + sucrose 30 g / L + Phytagel 3 g / L) and cultured in the dark at room temperature at 28℃ for 30-40 days. The induced callus was then subcultured for 30-40 days.

[0105] (2) Screening: The engineered Agrobacterium obtained in step 1 was used to transform the callus obtained in (1) using Agrobacterium-mediated genetic transformation. After co-culturing for 3 days, the callus was washed 5-6 times and transferred to a selection medium containing 50 mg / L hygromycin. The callus was then incubated in the dark at 30°C for 30-50 days. The results are as follows: Figure 7As shown, the callus selected after infection with Agrobacterium 1300gusplus-722pro-676 and 1300gusplus-722pro-1542 can be screened to obtain resistant callus;

[0106] (3) Differentiation: The resistant callus obtained by screening was transferred to a differentiation medium containing 50 mg / L hygromycin and positive seedlings were obtained after 25-30 days of differentiation;

[0107] (4) Rooting: Positive seedlings obtained after differentiation are transferred to a rooting medium containing 50 mg / L hygromycin. After 7-15 days of rooting, positive transgenic plants are finally obtained.

[0108] (5) Hardening off and transplanting: Open the bottle cap of the transformed strain with vigorous root growth, add sterile water to cover the culture medium 1-2cm thick, place it at room temperature to contact with air for hardening off for 2-3 days, and then transplant it to the greenhouse for cultivation.

[0109] 3. Identification of transgenic lines

[0110] To identify whether the lines obtained in step 2 are transgenic lines, this embodiment performs PCR verification on some positive transgenic plants obtained through screening culture, differentiation culture and rooting culture.

[0111] First, extract DNA from the sample. The DNA extraction steps are as follows: Take a rice leaf about 2 cm long and place it in a 2 ml centrifuge tube; add 800 μl of 1.5×CTAB to a mortar and grind the leaf into a homogenate and pour it back into the centrifuge tube; incubate in a 65℃ water bath for 20-30 min, inverting and mixing once every 5 min; centrifuge at 12000 rpm for 10 min; transfer 400 μl of supernatant to a new centrifuge tube, add 2 volumes of ice-cold anhydrous ethanol, and incubate at -20℃ for 20 min; centrifuge at 12000 rpm for 10 min; discard the supernatant, add 500 μl of 75% ethanol, invert and rinse, centrifuge at 8000 rpm for 5 min; discard the supernatant, place in a clean bench to air dry or air dry naturally, and add 100 μl of ddH2O to dissolve the DNA.

[0112] The genomic DNA samples of the transgenic line were amplified by PCR using hygromycin primers (Hn-F / Hn-R). This primer pair could not amplify the fragment using the endogenous rice genome as a template, but the fragment size obtained by amplification using transgenic seedlings was 561 bp.

[0113] The primer sequences are as follows:

[0114] Hn-F:5'-CTTAGCCAGACGAGCGGGTTC-3' (SEQ ID NO.11);

[0115] Hn-R: 5'-GCTTCTGCGGGCGATTTGT-3' (SEQ ID NO. 12).

[0116] ZH11 genomic DNA was used as a negative control, and water was used as a blank control. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 45 s, 55-65℃ annealing for 45 s; 72℃ extension for 1.5 min; 30-35 cycles; 72℃ further extension for 10 min; 16℃ final temperature.

[0117] The PCR reaction system is as follows:

[0118] Table 5 PCR reaction system

[0119]

[0120] The PCR products were subjected to agarose gel electrophoresis, and the results are as follows: Figure 8 As shown, the results indicate that most transgenic samples contained a 561bp transgenic band, the same size as the vector control; while the blank control and negative control ZH11 could not amplify the band.

[0121] Experimental Example 1

[0122] This experimental example further analyzes the transgenic lines obtained in Example 3, as follows:

[0123] 1. GUS staining analysis of plant tissues

[0124] GUS staining analysis was performed using a GUS staining kit (Zhongke Ruitai, catalog number: RTU4032). Positive callus tissue showed significant staining, as did the leaves and roots of the differentiated seedlings. Subsequent GUS staining of seedling leaves and seeds also yielded good results. (See attached image). Figure 9 As shown, Figure 9 The results showed that the expression levels of the GUS gene driven by the CEPro722-676 promoter and the CEPro722-1542 promoter were high. Among them, the leaf tissues of the negative control (ZH11) were not stained at any stage, while the positive control (positive line transgenic with pC1301 vector) were all stained.

[0125] 2. Tissue expression analysis in maize

[0126] Using a method similar to that used for rice in Example 3, transgenic maize plants were obtained. GUS staining of various tissues revealed that the selected positive callus tissue showed significant staining, and the leaves, roots, seedling leaves, and seeds of the differentiated seedlings all stained well with GUS. This demonstrates that the CEPro722-676 and CEPro722-1542 promoters can also drive stable expression of the GUS gene at the callus level, roots, seedling stage, mature leaves, and seeds in maize, making them highly efficient constitutive promoters.

[0127] 3. Tissue expression analysis in wheat

[0128] Using a method similar to that used for rice in Example 3, transgenic wheat plants were obtained. GUS staining of various tissues revealed that the selected positive callus tissue showed significant staining, and the leaves, roots, seedling leaves, and seeds of the differentiated seedlings all stained well with GUS. This demonstrates that the CEPro722-676 and CEPro722-1542 promoters can also drive stable expression of the GUS gene at the level of wheat callus tissue, roots, seedlings, mature leaves, and seeds, making them highly efficient constitutive promoters.

[0129] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Hainan Bolian Rice Gene Technology Co., Ltd. <120> A CEPro722 promoter and its application <130> KHP211119691.1 <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 676 <212> DNA <213> Artificial Sequence <400> 1 ggtcgtggtc gtggagcagg agatgttctg gcccacgagc tttgattgat cgatcgatcg 60 ctgctttggt gtttgtatgt ggcgctggca ccatcctgca tggctgcaca tatatataca 120 cacatacata cagcacccag ctgactggag gcgatcaacg acgccacgcc ttgaaggaga 180 agattgtcat acaaatattg cagacgcaat ggctgacgtt tggcgaggag gatggttcgt 240 gtagggtcag gatctcggcg atcatgttag ggagagaatg catgggagag ggcacgggcg 300 tctcccagga tgccgccctc cagcggccgg catttcttgt tgggctcgcg aaggccaact 360 tgaactgcga gatctgtgcg tgactttctt tggaagcgga actccattcg cgagtattat 420 acttcctggg gttttacttc tgatgaccaa tactggaacg ggtcctgatc agccgattct 480 tctgcaagta aagtcagcaa ggtcacggta acttactaac ttaaatgtaa acaatgagca 540 ttttcactcc accctgcgta ttggctgggc acccttgcgg ccttgtttgg atatttaata 600 aactaattta catctcaatt tatcttaata cattagatga gtgcctgtac gttgcgacgg 660 gagtaaaaaa aattgt 676 <210> 2 <211> 1542 <212> DNA <213> Artificial Sequence <400> 2 ggtcgtggtc gtggagcagg agatgttctg gcccacgagc tttgattgat cgatcgatcg 60 ctgctttggt gtttgtatgt ggcgctggca ccatcctgca tggctgcaca tatatataca 120 cacatacata cagcacccag ctgactggag gcgatcaacg acgccacgcc ttgaaggaga 180 agattgtcat acaaatattg cagacgcaat ggctgacgtt tggcgaggag gatggttcgt 240 gtagggtcag gatctcggcg atcatgttag ggagagaatg catgggagag ggcacgggcg 300 tctcccagga tgccgccctc cagcggccgg catttcttgt tgggctcgcg aaggccaact 360 tgaactgcga gatctgtgcg tgactttctt tggaagcgga actccattcg cgagtattat 420 acttcctgg gttttacttc tgatgaccaa tactggaacg ggtcctgatc agccgattct 480 tctgcaagta aagtcagcaa ggtcacggta acttactaac ttaaatgtaa acaatgagca 540 ttttcactcc accctgcgta ttggctgggc acccttgcgg ccttgtttgg attttaata 600 aactaattta catctcaatt tatcttaata cattagatga gtgcctgtac gttgcgacgg 660 gagtaaaaaa aattgtatga aacatagaaa cgtaacaaga aacatcacta tgatatccaa 720 aattcgtgtg gtaaatatta tcaatatcac acaaattatt tataagagtc aatttagaat 780 tttgtaatga cagacaaatg tgtcgacaac tatacactaa tctaatcctt tttagcgata 840 ccgataagtc attgttgtat gtttgcaggg atgatacatc gacacaaatt gtcctcataa 900 cttaacagat caatcacaaa gttcattcga agaatctttg ctttctacat acaaacacat 960 aagagaacaa aacgtcaaat ttgtattatg taaccagaat gtatgtttga aaatgaaaca 1020 aatgtactat actcacccta ataaattcta ttcgtctatt attcccccac atggtcatag 1080 cttgtagcat gaggtagctg gtattaagcc tatagaataa tgtttggcca actatgttat 1140 atacaatgat tatcatagaa aaatttaaac tgctgagaag gtgatacgaa gaaaatacac 1200 tcttaagtca attggaacac cagtcctaat tatttgttcc cacttaaaaa tatcctctgc 1260 ccatcctgaa tgtttcttgt tgtaggcaat cttatatttc tgaggctatg ataatcgctt 1320 ccgcgaacct cctgtatgac atgtgcttat accaatcttg agtcggtgta aagtcgataa 1380 aagtcacgtg ctttttaaca tgatctatta caaaaagggt gtgacattca tgaatttcca 1440 tggcataaga acctgcaaag tattagtcat caggattgca aagaagtgcc ctttataaat 1500 acatttaaaa tgaacacata cttacatatc tacaccccgt ga 1542 <210> 3 <211> 44 <212> DNA <213> Artificial Sequence <400> 3 agatctacca tggtaccgtg gatccggtcg tggtcgtgga gcag 44 <210> 4 <211> 43 <212> DNA <213> Artificial Sequence <400> 4 caagcttgca tgcctgcaga caattttttt tactcccgtc gca 43 <210> 5 <211> 44 <212> DNA <213> Artificial Sequence <400> 5 agatctacca tggtaccgtg gatccggtcg tggtcgtgga gcag 44 <210> 6 <211> 45 <212> DNA <213> Artificial Sequence <400> 6 caagcttgca tgcctgcagt cacggggtgt agatatgtaa gtatg 45 <210> 7 <211> 2987 <212> DNA <213> Artificial Sequence <400> 7 aatgtataat tgcgggactc taatcataaa aacccatctc ataaataacg tcatgcatta 60 catgttaatt attacatgct taacgtaatt caacagaaat tatatgataa tcatcgcaag 120 accggcaaca ggattcaatc ttaagaaact ttattgccaa atgtttgaac gatcggggaa 180 attcgagctc ggtagcaatt cccgaggctg tagccgacga tggtgcgcca ggagagttgt 240 tgattcacac gtgatggtga tggtgatggc tagcgttctt gtagccgaaa tctggaatgt 300 tggtccagcg ctcgcgaaag acgtgcgcgg cgagcttcgg cttgcggtca cgagtgaaca 360 cgcccttctt gtttccttgg acgcgcatca cgccctgaga ggtcgcgaag tccgcgaagt 420 tccacgcttg ctcacccacg aagttctcaa actcatcgaa cacgacgtgg ttcgcctggt 480 agtactcgac ttgatattcc tcggtgaaca tcactggatc aatgtcgtga aagcccgcaa 540 cggtgtctgc gccgtactca gtgatcatga tcggctttcc tgggcaacgc ttgttccacg 600 cgtgaaattc ctggcggaga tggactttgg ccgcttcgag atcaccgcca tcgaagtacc 660 atccgttata gcgattgagc gcgatgacgt caatcagttc ggcgactttg tccgtctccg 720 gggtagccat cacaaacagc acgatcgtga ccggacgctt ctgtgggtcg agttccttgg 780 tcagctccac caacggcttg aagtactcgt acgcgccctc ttcctcagtc gccgcctcgt 840 tggcgatgct ccacatcacg acgcttggat ggttcttgtc acgagacacc agttcacgga 900 gaacgtcttg atggtgctca aacgtccgaa tcttctccca ggtactgacg cgctcgctgc 960 cttcgccgag tcccgtggtg gccatgaagt tgaggtgcac gccaactgcc ggagtctcgt 1020 cgatcacgac cagaccctcg cgatccgcaa gacgcatcaa ctcttcagag tacggatagt 1080 gtgcggtccg gaagctgttg gcgccgatcc atttgaggat attgaaatcc atcacattgc 1140 tcgcttcgtt aaagccacgg ccgttgatag gagtgtcctc atgtttgcca aagcccttga 1200 agtagaacgg tttgttgttg atgaggaact tgccgtcgtt gacttccacg gtccgcacgc 1260 cgaacggctc ttcatagaca tcgatggtca gtccgtcgtt caccagttcc actttgatct 1320 ggtagagata cgtgttcagt ggttcccaga ggatgacatt cggaatctcc acgttaccgc 1380 tcaggccctc ggtgcttgcg accactttgc cttcctcatc cacgaccgac actttcacgg 1440 tctcggcttt gccttgaaag tccaccgtat aggtcacagt cccggttggg ccattgaagt 1500 cggtcacaac cgagatgtcc tcgacgtacg taaacggggt cgtgtagatt ttcaccggac 1560 ggtgcaggcc tgcatagttg aagaagtcga agttcggctt gttacgaatg acttttccga 1620 ggccctcttc gtggcgctcg ctgtacagcc ccaccgggag ggtgctatcg tcgaggatgt 1680 tgtccacggc gacggtgacg cgattcatgc catcacgcag cgagttgttg atttccgctt 1740 cgaatggcag gaatccgccc ttgtgctcca cgaccagctc accattgaca tagacaattg 1800 ctttgtgagt tgcagagccg aagcggagca cgatacgctg atccttcaga taggccggca 1860 ccgtgaactc acgttcgtac cagacatatc cgatatggtt gcggatttcc ttggtcacgc 1920 caatgtcatt gtaactgctt gggacggcca tactaatagt gtcggtcagc ttgctttcgt 1980 accacttctc ttccagtcct ttcccgtagt ccagcttgaa gttccagacg ccattgaggt 2040 cgaagacgcc acgggtctcg gtgttgatcg ggtacagact agttcgtcgg ttctgtaact 2100 atcatcatca tcatagacac acgaaataaa gtaatcagat tatcagttaa agctatgtaa 2160 tatttacacc ataaccaatc attaaaaaa tagatcagtt taaagaaaga tcaaagctca 2220 aaaaaataaa aagagaaaag ggtcctaacc aagaaaatga aggagaaaaa ctagaaattt 2280 accctcagat ctaccatggt accgtggatc cggtcgtggt cgtggagcag gagatgttct 2340 ggcccacgag ctttgattga tcgatcgatc gctgctttgg tgtttgtatg tggcgctggc 2400 accatcctgc atggctgcac atatatatac acacatacat acagcaccca gctgactgga 2460 ggcgatcaac gacgccacgc cttgaaggag aagattgtca tacaaatatt gcagacgcaa 2520 tggctgacgt ttggcgagga ggatggttcg tgtagggtca ggatctcggc gatcatgtta 2580 gggagagaat gcatgggaga gggcacgggc gtctcccagg atgccgccct ccagcggccg 2640 gcatttcttg ttgggctcgc gaaggccaac ttgaactgcg agatctgtgc gtgactttct 2700 ttggaagcgg aactccattc gcgagtatta tacttcctgg ggttttactt ctgatgacca 2760 atactggaac gggtcctgat cagccgattc ttctgcaagt aaagtcagca aggtcacggt 2820 aacttactaa cttaaatgta aacaatgagc attttcactc caccctgcgt attggctggg 2880 cacccttgcg gccttgtttg gatatttaat aaactaattt acatctcaat ttatcttaat 2940 acattagatg agtgcctgta cgttgcgacg ggagtaaaaa aaattgt 2987 <210> 8 <211> 3853 <212> DNA <213> Artificial Sequence <400> 8 aatgtataat tgcgggactc taatcataaa aacccatctc ataaataacg tcatgcatta 60 catgttaatt attacatgct taacgtaatt caacagaaat tatatgataa tcatcgcaag 120 accggcaaca ggattcaatc ttaagaaact ttattgccaa atgtttgaac gatcggggaa 180 attcgagctc ggtagcaatt cccgaggctg tagccgacga tggtgcgcca ggagagttgt 240 tgattcacac gtgatggtga tggtgatggc tagcgttctt gtagccgaaa tctggaatgt 300 tggtccagcg ctcgcgaaag acgtgcgcgg cgagcttcgg cttgcggtca cgagtgaaca 360 cgcccttctt gtttccttgg acgcgcatca cgccctgaga ggtcgcgaag tccgcgaagt 420 tccacgcttg ctcacccacg aagttctcaa actcatcgaa cacgacgtgg ttcgcctggt 480 agtactcgac ttgatattcc tcggtgaaca tcactggatc aatgtcgtga aagcccgcaa 540 cggtgtctgc gccgtactca gtgatcatga tcggctttcc tgggcaacgc ttgttccacg 600 cgtgaaattc ctggcggaga tggactttgg ccgcttcgag atcaccgcca tcgaagtacc 660 atccgttata gcgattgagc gcgatgacgt caatcagttc ggcgactttg tccgtctccg 720 gggtagccat cacaaacagc acgatcgtga ccggacgctt ctgtgggtcg agttccttgg 780 tcagctccac caacggcttg aagtactcgt acgcgccctc ttcctcagtc gccgcctcgt 840 tggcgatgct ccacatcacg acgcttggat ggttcttgtc acgagacacc agttcacgga 900 gaacgtcttg atggtgctca aacgtccgaa tcttctccca ggtactgacg cgctcgctgc cttcgccgag tcccgtggtg gccatgaagt tgaggtgcac gccaactgcc ggagtctcgt cgatcacgac cagaccctcg cgatccgcaa gacgcatcaa ctcttcagag tacggatagt gtgcggtccg gaagctgttg gcgccgatcc atttgaggat attgaaatcc atcacattgc tcgcttcgtt aaagccacgg ccgttgatag gagtgtcctc atgtttgcca aagcccttga agtagaacgg tttgttgttg atgaggaact tgccgtcgtt gacttccacg gtccgcacgc 1260 cgaacggctc ttcatagaca tcgatggtca gtccgtcgtt caccagttcc actttgatct ggtagata cgtgttcagt ggttcccaga ggatgacatt cggaatctcc acgttaccgc 1380 tcaggccctc ggtgcttgcg accactttgc cttcctcatc cacgaccgac actttcacgg 1440 tctcggcttt gccttgaaag tccaccgtat aggtcacagt cccggttggg ccattgaagt 1500 cggtcacaac cgagatgtcc tcgacgtacg taaacggggt cgtgtagatt ttcaccggac 1560 ggtgcaggcc tgcatagttg aagaagtcga agttcggctt gttacgaatg acttttccga 1620 ggccctcttc gtggcgctcg ctgtacagcc ccaccgggag ggtgctatcg tcgaggatgt 1680 tgtccacggc gacggtgacg cgattcatgc catcacgcag cgagttgttg attccgctt 1740 cgaatggcag gaatccgcccc ttgtgctcca cgaccagctc accattgaca tagacaattg 1800 ctttgtgagt tgcagagccg aagcggagca cgatacgctg atccttcaga taggccggca 1860 ccgtgaactc acgttcgtac cagacatatc cgatatggtt gcggatttcc ttggtcacgc 1920 caatgtcatt gtaactgctt gggacggcca tactaatagt gtcggtcagc ttgctttcgt 1980 accacttctc ttccagtcct ttcccgtagt ccagcttgaa gttccagacg ccattgaggt 2040 cgaagacgcc acgggtctcg gtgttgatcg ggtacagact agttcgtcgg ttctgtaact 2100 atcatcatca tcatagacac acgaaataaa gtaatcagat tatcagttaa agctatgtaa 2160 tatttacacc ataaccaatc attaaaaaa tagatcagtt taaagaaaga tcaaagctca 2220 aaaaaataaa aagagaaaag ggtcctaacc aagaaaatga aggagaaaaa ctagaaattt 2280 accctcagat ctaccatggt accgtggatc cggtcgtggt cgtggagcag gagatgttct 2340 ggcccacgag ctttgattga tcgatcgatc gctgctttgg tgtttgtatg tggcgctggc 2400 accatcctgc atggctgcac atatatatac acacatacat acagcaccca gctgactgga 2460 ggcgatcaac gacgccacgc cttgaaggag aagattgtca tacaaatatt gcagacgcaa 2520 tggctgacgt ttggcgagga ggatggttcg tgtagggtca ggatctcggc gatcatgtta 2580 gggagagaat gcatgggaga gggcacgggc gtctcccagg atgccgccct ccagcggccg 2640 gcatttcttg ttgggctcgc gaaggccaac ttgaactgcg agatctgtgc gtgactttct 2700 ttggaagcgg aactccattc gcgagtatta tacttcctgg ggttttactt ctgatgacca 2760 atactggaac gggtcctgat cagccgattc ttctgcaagt aaagtcagca aggtcacggt 2820 aacttactaa cttaaatgta aacaatgagc attttcactc caccctgcgt attggctgggg 2880 cacccttgcg gccttgtttg gatatttaat aaactaattt acatctcaat ttatcttaat 2940 acattagatg agtgcctgta cgttgcgacg ggagtaaaaa aaattgtatg aaacatagaa 3000 acgtaacaag aaacatcact atgatatcca aaattcgtgt ggtaaatatt atcaatatca 3060 cacaaattat ttataagagt caatttagaa ttttgtaatg acagacaaat gtgtcgacaa 3120 ctatacacta atctaatcct ttttagcgat accgataagt cattgttgta tgtttgcagg 3180 gatgatacat cgacacaaat tgtcctcata acttaacaga tcaatcacaa agttcattcg 3240 aagaatcttt gctttctaca tacaaacaca taagagaaca aaacgtcaaa tttgtattat 3300 gtaaccagaa tgtatgtttg aaaatgaaac aaatgtacta tactcaccct aataaattct 3360 attcgtctat tattccccca catggtcata gcttgtagca tgaggtagct ggtattaagc 3420 ctatagaata atgtttggcc aactatgtta tatacaatga ttatcataga aaaattttaaa 3480 ctgctgagaa ggtgatacga agaaaataca ctcttaagtc aattggaaca ccagtcctaa 3540 ttatttgttc ccacttaaaa atatcctctg cccatcctga atgtttcttg ttgtaggcaa 3600 tcttatattt ctgaggctat gataatcgct tccgcgaacc tcctgtatga catgtgctta 3660 taccaatctt gagtcggtgt aaagtcgata aaagtcacgt gctttttaac atgatctatt 3720 acaaaaaggg tgtgacattc atgaatttcc atggcataag aacctgcaaa gtattagtca 3780 tcaggattgc aaagaagtgc cctttataaa tacatttaaa atgaacacat acttacatat 3840 ctacaccccg tga 3853 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <400> 9 ccacgggtct cggtgttga 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 gcgagcccaa caagaaatgc 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <400> 11 cttagccaga cgagcgggtt c 21 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <400> 12 gcttctgcgg gcgatttgt 19

Claims

1. A CEPro722 promoter, characterized in that, The nucleotide sequence of the CEPro722 promoter is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A primer pair, characterized in that, The primer pair is used to amplify the CEPro722 promoter according to claim 1, and the primer pair is as shown in SEQ ID NO. 3-4 or as shown in SEQ ID NO. 5-6.

3. A biomaterial, characterized in that, The biological material is an expression cassette, vector, or transgenic cell containing the CEPro722 promoter as described in claim 1.

4. The biomaterial according to claim 3, characterized in that, When the biological material is an expression cassette, the expression cassette further includes a functional gene and a terminator; The functional genes are marker genes or genes related to plant agronomic traits.

5. A reagent kit, characterized in that, The kit contains one or more of the CEPro722 promoter of claim 1, the primer pair of claim 2, or the biological material of claim 3.

6. The use of the CEPro722 promoter of claim 1, or the primer pair of claim 2, or the biological material of claim 3 or 4, or the kit of claim 5 in the preparation of transgenic plants; wherein the plant is rice, corn or wheat.

7. The application according to claim 6, characterized in that, The application involves constructing the CEPro722 promoter of claim 1 onto a vector and then transforming it into a plant; the plant is rice, corn, or wheat.

8. The application of the CEPro722 promoter of claim 1, or the biomaterial of claim 3 or 4, in driving gene expression in plants; wherein the plant is rice, maize, or wheat.

9. The application according to claim 8, characterized in that, The gene is a functional gene, an antisense gene of a functional gene, or a small RNA gene; The functional genes are plant agronomic trait-related genes or marker genes.

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