CN115927302A - OsEPSPSpro promoter and application thereof - Google Patents

Abstract

The invention relates to the technical field of agricultural biology, in particular to an OsEPSPSpro promoter and application thereof. The OsEPSPSpro promoter comprises a nucleotide sequence shown as SEQ ID NO.1 or a nucleotide sequence shown as SEQ ID NO. 2. According to the invention, through research, an OsEPSPSpro promoter is screened and found in an OsEPSPS gene, and the promoter can drive the gene to express in a plant; especially in plant callus, leaf, young ear, seed and other tissues in vegetative growth period, and may be used in replacing non-plant promoter. The OsEPSPSpro promoter provided by the invention has important significance in the field of plant genetic engineering, and can effectively reduce potential safety risk of transgenic plants caused by exogenous DNA.

Description

OsEPSPSpro promoter and application thereof

Technical Field

The invention relates to the technical field of agricultural biology, in particular to an OsEPSPSpro promoter and application thereof.

Background

A promoter is a DNA sequence recognized, bound and initiated by RNA polymerase and contains conserved sequences required for RNA polymerase specific binding and transcription initiation, most of which are located upstream of the transcription initiation site of a structural gene, and is not transcribed per se. Promoters can be classified into constitutive promoters, inducible promoters and spatiotemporal specific promoters according to their expression modes. Constitutive promoters are capable of initiating gene transcription in all or most tissues, resulting in spatiotemporal persistence and expression constancy of gene expression.

At present, the promoters mainly applied in the plant transformation process are a cauliflower mosaic virus promoter (CaMV 35 Spro) and a maize polyubiquitin gene promoter (ZmUbipro). CaMV35Spro is a promoter of plant DNA virus, and can cause unnecessary concerns when applied to plant transgenes; zmUbipro is derived from plants, but frequently using the same promoter during transformation is prone to cause transgene silencing. Therefore, it is especially important to explore new efficient constitutive promoters, especially plant-derived promoters with low biosafety risk.

Disclosure of Invention

The invention provides an OsEPSPSpro promoter and application thereof, which are used for overcoming the defects that a plant-derived promoter with low biological safety risk is lacked in the prior art.

The invention provides an OsEPSPSpro promoter, which comprises any one of the following nucleotide sequences:

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

ii) a nucleotide sequence complementary to i);

iii) The nucleotide sequence with the same promoter function is obtained by substituting, deleting and adding one or more nucleotide sequences in the nucleotide sequence shown in i).

The invention also provides a primer pair for amplifying the OsEPSPSpro promoter.

Further, comprising: a primer pair shown as SEQ ID NO.3-4, and/or a primer pair shown as SEQ ID NO. 5-6.

Further, the primer pair shown as SEQ ID NO.3-4 is used for amplifying the nucleotide sequence shown as SEQ ID NO. 1; the primer pairs shown as SEQ ID NO.5-6 are used for amplifying the nucleotide sequence shown as SEQ ID NO. 2.

The invention also provides a biological material containing the OsEPSPSpro promoter, and the biological material is an expression cassette, a vector or a transgenic cell.

Further, when the biological material is an expression cassette, the expression cassette further comprises a functional gene and a terminator.

Further, the functional gene is a plant agronomic trait related gene or a marker gene.

The marker gene, i.e., a gene that can function as a specific marker, is usually used to check whether the gene transformation has succeeded or not, and is preferably one or more of β -glucuronidase gene GUS, hygromycin phosphotransferase gene Hn, acetolactate synthase mutant gene ALS, bar gene, resistant EPSPS gene, or NptII gene.

The agronomic characters of the plants, namely, the characters related to the growth period, the plant height, the leaf area, the fruit weight, the quality, the herbicide resistance, the pest resistance and the like of crops which can represent the characteristics of crop varieties. Accordingly, genes associated with agronomic traits in plants are genes associated with such traits.

The invention also provides a kit comprising the OsEPSPSpro promoter, the primer pair, or the biological material.

The invention also provides the application of the OsEPSPSpro promoter, the primer pair, the biological material or the kit in any one of the following steps:

1) Preparing a transgenic plant;

2) Driving expression of a gene in a plant;

3) Genetic breeding or germplasm improvement of plants;

4) And (4) carrying out plant hybridization seed production.

Further, the application is that the OsEPSPSpro promoter is constructed into a vector and then is introduced into a plant to prepare a transgenic plant; alternatively, the biological material is introduced into a plant to produce a transgenic plant.

Further, the application is that the target DNA operably linked to the OsEPSPSpro promoter is introduced into a plant.

Further, after the transgenic plant is prepared, screening is performed by a marker gene.

As a preferred embodiment, the present invention provides a method for producing transgenic rice, comprising:

transforming the vector comprising the OsEPSPSpro promoter into rice callus by an agrobacterium transformation method;

carrying out resistance screening and differentiation on the rice callus to obtain rice seedlings;

and carrying out rooting culture on the rice seedlings to obtain transgenic rice.

Further, the rice callus is prepared by the following method:

after rice seeds are shelled and disinfected, mature embryos are inoculated in an induction culture medium to induce embryonic callus, and dark culture is carried out for 30-50 days at the temperature of 28-30 ℃;

further, after the plant constitutive promoter OsEPSPSpro is transformed into the rice callus, co-culture is also included, and the co-culture is carried out at 22-24 ℃ in a dark culture mode until thalli appear on the surface of the callus;

further, the resistance screening is to inoculate the co-cultured callus to a screening culture medium added with hygromycin, and carry out dark culture for 30-50 days at 28-30 ℃ to carry out resistance screening;

further, the differentiation is that the callus which is selected by resistance is added to a differentiation medium which is added with hygromycin and is cultivated for 25 to 40 days by illumination at the temperature of between 28 and 30 ℃.

Further, the rooting culture is to inoculate the rice seedlings to a rooting culture medium added with hygromycin for rooting, and to culture the rice seedlings for 5 to 20 days at 30 to 32 ℃ under illumination.

Further, after rooting culture, PCR detection is included, and plants which are detected to be positive are selected for planting.

According to the application of the driving gene provided by the invention in expression in plants, the expression of the driving gene in the plants is specifically as follows: is the application of expression in one or more of plant callus, tissue in vegetative growth phase or reproductive organ.

Preferably, the expression is high in the pre-emergence/post-emergence inflorescence period of the seeds.

Further, the genes include: a functional gene, an antisense gene to a functional gene, or a small RNA gene;

the functional gene preferably comprises a plant agronomic trait related gene or a marker gene, and the small RNA gene is preferably a small RNA gene capable of interfering with the expression of the functional gene.

Further, the plant is one or more of rice, corn, wheat, barley, soybean, cotton, rape, sorghum or millet.

The invention has the following beneficial effects:

(1) The OsEPSPSpro promoter is obtained by screening, is a constitutive promoter, is derived from rice, and can drive genes to be efficiently expressed in callus, main functional tissues (roots, leaves, flowers, seedlings, scions and the like) in vegetative growth period or reproductive organs of the rice, particularly in the pre-bud/post-bud inflorescence period of the rice.

The promoter OsEPSPSpro provided by the invention can form a plant transgenic screening expression box with a plant endogenous or exogenous screening marker gene or a plant genetic transformation screening vector, and other functional elements are added for plant tissue culture or plant genetic transformation, so that an effective tool and method are provided for screening of plant genetic transformation.

(2) The primer pair provided by the invention can be used for efficiently amplifying the OsEPSPSpro promoter of the target gene.

(3) The biological material provided by the invention can improve the construction efficiency and genetic transformation efficiency of the vector.

(4) The promoter OsEPSPSpro provided by the invention can also drive the gene to be efficiently expressed in main functional tissues of the overground and underground parts of the vegetative growth period of the transformed seedling. In addition, the promoter OsEPSPSpro is a plant endogenous gene, and exogenous gene segments such as a bacterial source and the like are not introduced in the transgenic process, so that the transgenic promoter resource of the plant is enriched, the potential safety risk of the transgenic plant caused by the exogenous gene and the public worry about the safety of the transgenic plant can be effectively reduced, the commercial application of the transgenic plant is facilitated, and the market value and the social benefit are good.

Drawings

In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings needed for the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.

FIG. 1 shows the result of agarose gel electrophoresis provided in example 2 of the present invention;

wherein a, b and c are respectively an amplified fragment of a promoter OsEPSPSpro-770, an amplified fragment of OsEPSPSpro-2048 and a restriction enzyme fragment of a vector 1300 gusplus.

FIG. 2 is a vector map of 1300gusplus vector provided in example 2 of the present invention.

FIG. 3 is an electrophoretogram of 1300gusplus-OsEPSPSpro-770 and 1300gusplus-OsEPSPSpro-2048 vectors digested with BamHI and HindIII, which are provided in example 2 of the present invention;

wherein M is Marker, ck1-ck3 is 1300gusplus-OsEPSPSpro-770 recombinant plasmid which is not digested, and 1-3 is 1300 gusplus-OsEPSPro-770 recombinant plasmid which is digested; ck4-ck5 is 1300gusplus-OsEPSPSpro-2048 recombinant plasmid which is not cut by enzyme, and 4-5 is 1300gusplus-OsEPSPSpro-2048 recombinant plasmid which is cut by enzyme.

FIG. 4 is a vector map of 1300gusplus-OsEPSPSpro-770 vector provided in example 2 of the present invention.

FIG. 5 is a vector map of 1300gusplus-OsEPSPSpro-2048 vector provided in example 2 of the present invention.

FIG. 6 shows the result of electrophoresis of PCR detection of transformed Agrobacterium according to example 3 of the present invention; wherein, M is Marker, ck + is 1300gusplus-OsEPSPSpro-770 or 1300gusplus-OsEPSPSpro-2048 recombinant plasmid positive control, and 1-6 are 1300 gusplus-OsEPSPro-770 or 1300 gusplus-OsEPSPro-2048 recombinant plasmid agrobacterium monoclonal liquid samples.

FIG. 7 is a schematic diagram showing the results of screening callus with hygromycin screening medium according to example 3 of the present invention;

wherein WT is a schematic diagram of hygromycin for screening hot japonica 237 calluses, 1300gusplus-OsEPSPSpro-770 is a schematic diagram of hygromycin for screening 1300gusplus-OsEPSPSpro-770 trans-hot japonica 237 calluses, and 1300gusplus-OsEPSPSpro-2408 is a schematic diagram of hygromycin for screening 1300gusplus-OsEPSPSpro-2408 trans-hot japonica 237 calluses;

FIG. 8 is the electrophoresis diagram of the PCR detection of transgenic plants provided in example 3 of the present invention; wherein M is Marker, H ₂ O is blank control, ck-is hot round-grained non-transgenic plant genome DNA, ck + is 1300gusplus-OsEPSPSpro-770 recombinant plasmid positive control, and 1-13 is transgenic plant genome DNA obtained by screening.

FIG. 9 shows GUS staining results of transgenic T0 generation lines of callus, seedling leaves, heading stage (roots, stems, leaves, anthers) and grain filling for 30 days provided by 1300gusplus-OsEPSPSpro-770 plasmid and 1300 gusplus-OsEPSPro-2048 plasmid in Experimental example 1 of the present invention;

wherein Ck & lt- & gt is a negative control (hot japonica 237) staining result at each development stage, ck & lt + & gt is a positive control (pC 1301 vector strain) staining result at each development stage, pro & lt 770 & gt is a 1300gusplus-OsEPSPSpro & lt-770 transgenic strain staining result, and Pro & lt-2048 & gt is a 1300gusplus-OsEPSPSpro & lt-2048 & gt transgenic strain staining result.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The test methods described in the following examples are conventional unless otherwise specified. Materials and reagents described in the following examples are commercially available unless otherwise specified.

Example 1

In this example, bioinformatics analysis of the sequence upstream of the OsEPSPS gene by promoter function prediction software PlantCARE, plantPAN and the like revealed that the sequence is rich in various promoter-associated cis-acting elements, such as TATA-box, CAAT-box and the like, indicating that the sequence has structural characteristics of a plant cell promoter. Meanwhile, the result of the analysis of the plantan PAN shows that the gene sequence from 1bp to 770bp is rich in CpG islands which are also one of the sequence characteristics of eukaryotic promoters, so the invention speculates that the sequence has promoter activity.

In the embodiment, the upstream sequences of OsEPSPS genes with different lengths are further intercepted for carrying out promoter activity identification, and the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are finally determined to be used as promoter sequences through continuous screening and comparison, and are named as OsEPSpro, and the OsEPSpro promoter can drive the genes to be efficiently expressed in the callus tissues and the main tissues in the vegetative growth period of rice.

The promoter OsEPSPSpro can be obtained by amplifying the following primers:

the amplification primer sequence of the promoter shown as SEQ ID NO.1 is as follows:

SEQ ID NO.3：5’-tcagatctaccatggtaccgtggatcctgccggcggcgtggaggtc-3’；

SEQ ID NO.4：5’-taaaacgacggccagtgccaagcttccagctggtgtggacaaaatgt-3’。

the amplification primer sequence of the promoter shown as SEQ ID NO.2 is as follows:

SEQ ID NO.5：5’-tcagatctaccatggtaccgtggatcctgccggcggcgtggaggtc-3’；

SEQ ID NO.6：5’-taaaacgacggccagtgccaagcttagtagtcatgtgaccatccctggggt-3’。

example 2

In the embodiment, the promoter OsEPSPSpro is constructed into an expression cassette and a vector, and the specific process is as follows:

1. preparation of plant transgenic expression cassette containing promoter OsEPSPSpro

The construction method of the plant transgenic expression box OsEPSPSpro-GUS-nosT (the sequence is shown as SEQ ID NO. 7) of the invention is as follows:

designing a primer 1300-OsEPSPSpro-F1/1300-OsEPSPSpro-Rv1 to amplify a promoter OsEPSPSpro-770 fragment from a rice genome. Wherein, the 5' end of the primer 1300-OsEPSPSpro-F1 has 19 nucleotide sequences which are repeated with the corresponding connection positions of the vector; the 5' end of the primer 1300-OsEPSpro-Rv 1 has 25 nucleotide sequences which are repeated with the corresponding connection positions of the vector; so as to use the Clonexpress II One Step Cloning Kit to recombine and connect.

The primer sequences are as follows:

1300-EPSPSpro-F1：5’-tcagatctaccatggtaccgtggatcctgccggcggcgtggaggtc-3’(SEQ ID NO.3)；

1300-EPSPSpro-Rv1:5’-taaaacgacggccagtgccaagcttccagctggtgtggacaaaatgt-3’(SEQ ID NO.4)。

the PCR amplification reaction system is as follows:

TABLE 1 PCR amplification reaction System

The PCR amplification procedure was as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 55-65 ℃ for 30s, extension at 72 ℃ for 5min,35 cycles; extension was carried out at 68 ℃ for 10min and at 16 ℃ was complete.

PCR products amplified by the primers 1300-EPSPSpro-F1 and 1300-OsEPSPSpro-Rv1 are EPSPro-770 fragments, and products with the size of 770bp are recovered by 1.0% agarose gel electrophoresis (the result is shown in a picture in figure 1).

2. Preparation of plant transgenic expression cassette containing promoter OsEPSPro-2048

The construction method of the plant transgenic expression cassette OsEPSPSpro-2048-GUS-nosT (the sequence is shown as SEQ ID NO. 8) comprises the following steps:

the primer OsEPSPSpro-2048-F2/EPSPSpro-2048-Rv2 is designed to amplify the promoter OsEPSPSpro-2048 fragment from the rice genome. Wherein, the 5' end of the primer 1300-OsEPSPSpro-F2 has 19 nucleotide sequences which are repeated with the corresponding connecting positions of the vector; the 5' end of the primer 1300-OsEPSpro-Rv 2 has 25 nucleotide sequences which are repeated with the corresponding connection positions of the vector; so that the connection can be recombined by using the Clonexpress II One Step Cloning Kit.

The primer sequences are as follows:

1300-OsEPSPSpro-F2:

5’-TCAGATCTACCATGGTACCGTGGATCCTGCCGGCGGCGTGGAGGTC-3’(SEQ ID NO.5)；

1300-OsEPSPSpro-Rv2:5’

-TAAAACGACGGCCAGTGCCAAGCTTAGTAGTCATGTGACCATCCCTGGGGT-3’(SEQ ID NO.6)；

the PCR amplification reaction system is as follows:

TABLE 2 PCR amplification reaction System

The PCR amplification procedure was as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 55-65 ℃ for 30s, extension at 72 ℃ for 5min,35 cycles; extension was 10min at 68 ℃ and was completed at 16 ℃.

PCR products amplified by the primers 1300-OsEPSPSpro-F2 and 1300-OsEPSPSpro-Rv2 are OsEPSPSpro-2048 fragments, and products with the size of 2048bp are recovered by 1.0% agarose gel electrophoresis (the result is shown in a b picture in figure 1).

3. Construction of plant genetic transformation vectors

The amplified product obtained in Step 1 was inserted into 1300gusplus vector (vector map shown in FIG. 2), bamHI and HindIII double cleavage sites using Clonexpress II One Step Cloning Kit method as follows:

(1) The vector plasmid 1300gusplus was digested with BamHI + HindIII, electrophoresed on agarose gel and used

A band of about 10kb in size was recovered from the Gel Extraction kit (Omega, the same applies hereinafter), yielding a 1300gusplus linear fragment.

The BamHI + HindIII double digestion reaction system is as follows:

TABLE 3 digestion system

The cleavage result is shown in FIG. 1, panel c.

(2) The Clonexpress II One Step Cloning Kit ligation Kit was used to ligate the OsEPSPSpro-770 or OsEPSPSpro-2048 fragment to 1300gusplus vector as follows:

TABLE 4 connection System

And (3) connecting procedures: 30min at 37 ℃.

(3) And (3) transformation: adding 10 mu l of the ligation product obtained in the step (2) into 100 mu l of escherichia coli competent cells, slightly mixing uniformly, and carrying out ice bath for 30min; heat shock at 42 ℃ for 90s; incubating in ice water bath for 2min; adding 900. Mu.l SOC medium, shaking at 37 deg.C and 220rpm for 1h, centrifuging at 5000rpm for 30s, discarding 900. Mu.l supernatant, mixing the rest of the cells with the medium, and spreading on LB plate containing kanamycin. Culturing at 37 ℃ for about 16h, picking single colonies, carrying out colony PCR verification by using specific primers (1300-OsEPSPSpro-test-F and 1300 OsEPSPSpro-test-R), selecting positive colonies, shaking the colonies at 37 ℃ and 220rpm overnight, extracting plasmids by using a high-purity plasmid miniprep kit (Zhongkoitai), and carrying out enzyme digestion detection to obtain correct plasmids (the result is shown in figure 3, wherein M is Marker, ck1-ck3 is 1300 gusplus-OsEPSPro-770 recombinant plasmids which are not digested, 1-3 are 1300 gusplus-OsEPSPro-770 recombinant plasmids which are digested, fragments with sizes of about 770bp can be cut out, ck4-ck5 is 1300 guspsplus-OsEPSpro-2048 recombinant plasmids which are not digested, and 4-5 are 1300 guspsplus-OsEPSpro-2048 recombinant plasmids which are digested, fragments with sizes of about 2048bp can be cut out), storing strains and carrying out sequencing. The resulting vectors were designated 1300gusplus-OsEPSPSpro-770 and 1300 gusplus-OsEPSPro-2048, and the vector maps are shown in FIGS. 4 and 5.

The primer sequence is as follows:

1300-OsEPSPSpro-test-F：5’-TCTTCCAGTCCTTTCCCGTAGT-3’(SEQ ID NO.9)；

1300-OsEPSPSpro-test-R:5’-TGTTCACCCGGTACAACGCAC-3’(SEQ ID NO.10)。

example 3

In this example, the ospsps pro promoter is transformed into a plant to prepare a corresponding transgenic plant, and the specific process is as follows:

1. agrobacterium transformation and identification

Agrobacterium EHA105 competent cells preserved at-80 ℃ were added with 1. Mu.l of 1300gusplus-OsEPSPSpro-770 and 1300 gusplus-OsEPSPro-2048 plasmids obtained in example 2, which were correctly sequenced, and transformed by 2.5KV electroporation. Spread on YEP culture plate containing kanamycin, rifampicin and streptomycin, cultured at 28 ℃ for about 48h, picked single colony shake bacteria overnight, verified by PCR with specific primers (1300-OsEPSPSpro-test-F/1300-OsEPSPSpro-test-R and) bacterial liquid (the result is shown in FIG. 6, wherein M is Marker, ck + is 1300gusplus-OsEPSPSpro-770 or 1300gusplus-OsEPSPSpro-2048 recombinant plasmid positive control, 1-6 is 1300 gusplus-OsEPSpro-770 or 1300 guspsplus-OsEPSpro-2048 recombinant plasmid Agrobacterium monoclonal bacterial liquid sample, the amplified band size of which is 737bp or correct size), amplified to obtain target fragment of about 737bp, picked positive engineered bacterium (Agrobacterium), shaken for 36-48h, and the bacterial liquid is preserved for infection.

2. Agrobacterium mediated genetic transformation

(1) Induction: disinfecting seeds of the hot japonica 237 by sodium hypochlorite, placing the seeds on an induction culture medium (N6 +2.4-D3mg/L + CH 0.6g/L + Pro 0.5g/L + sucrose 30g/L + Phytagel 3 g/L), and carrying out dark culture at the normal temperature of 28 ℃ for 30-40D to obtain induced callus and then carrying out subculture for 30-40D;

(2) Screening: transforming the engineering agrobacterium obtained in the step 1 into the callus obtained in the step (1) by an agrobacterium-mediated genetic transformation method, co-culturing for 3 days, washing for 5-6 times, transferring to a screening culture medium containing 50mg/L hygromycin, and performing dark culture at 30 ℃ for 30-50 days, wherein the result is shown in figure 7, and the screened callus is infected by agrobacterium of 1300gusplus-722pro-676 and 1300gusplus-722pro-1542, so that resistant callus can be screened and obtained;

(3) Differentiation: transferring the resistance callus obtained by screening to a differentiation culture medium containing 50mg/L hygromycin, and differentiating for 25-30d to obtain a positive seedling;

(4) Rooting: transferring the positive seedling obtained by differentiation to a rooting culture medium containing 50mg/L hygromycin, and rooting for 7-15 days to finally obtain a positive transgenic plant;

(5) Hardening and transplanting seedlings: opening a bottleneck sealing film of a transformation strain with vigorous root system growth, adding sterile water to cover the thickness of the culture medium by 1-2cm, placing the transformation strain at room temperature to contact with air for hardening seedlings for 2-3d, and transplanting the transformation strain to a greenhouse for cultivation.

3. Identification of transgenic lines

In order to identify whether the line obtained in step 2 is a transgenic line, this example performed PCR verification of a part of positive transgenic plants obtained by screening culture, differentiation culture and rooting culture.

Firstly, extracting sample DNA, wherein the DNA extraction steps are as follows: taking rice leaves about 2cm long and placing the rice leaves in a 2ml centrifuge tube; add 800. Mu.l 1.5 × CTAB into mortar, grind the blade to homogenate and pour back into the centrifuge tube; water bath at 65 deg.C for 20-30min, and mixing by reversing every 5min for 1 time; centrifuging at 12000rpm for 10min; sucking 400 μ l of supernatant into a new centrifuge tube, adding 2 times volume of ice-precooled absolute ethyl alcohol, and standing at-20 deg.C for 20min; centrifuging at 12000rpm for 10min; discarding the supernatant, adding 500 μ l 75% ethanol, rinsing by inversion, and centrifuging at 8000rpm for 5min; is abandonedCleaning, drying in a clean room or naturally drying in the air, adding 100 μ l ddH ₂ O dissolves the DNA.

Hygromycin primers (Hn-F/Hn-R) are used for carrying out PCR amplification detection on a genome DNA sample of a transgenic strain, the primers cannot amplify fragments by taking endogenous rice genomes as templates, and the size of the fragments obtained by amplifying transgenic seedlings is 561bp.

The primer sequences are as follows:

Hn-F:5’-CTTAGCCAGACGAGCGGGTTC-3’(SEQ ID NO.11)；

Hn-R:5’-GCTTCTGCGGGCGATTTGT-3’(SEQ ID NO.12)。

hot round-grained rice 237 genomic DNA was used as a negative control, and water was used as a blank control. The PCR reaction procedure was as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, and annealing at 60 ℃ for 30s; extending for 5min at 72 ℃;30-35 cycles; further extension for 10min at 72 ℃; and finishing at 16 ℃.

The PCR reaction system is as follows:

TABLE 5 PCR reaction System

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The PCR product is subjected to agarose gel electrophoresis, and the result is shown in FIG. 8, and the result shows that most of transgenic samples contain a transgenic band of 561bp, which is the same as the vector control in size; while the blank control and the negative control ZH11 failed to produce a band.

Experimental example 1

The transgenic lines obtained in example 3 were further analyzed in this example, as follows:

1. GUS staining analysis of plant tissue

A GUS staining kit (Zhongke Ruitai, goods number: RTU 4032) is used for staining analysis, positive callus is screened and stained obviously, GUS staining is carried out on seedling-stage leaves, heading stages (roots, stems, leaves and anthers) and grains after 30 days of filling, the result is shown in figure 9, and figure 9 shows that the expression quantity of GUS genes driven by OsEPSPSpro-770 promoter and OsEPSPSpro-2048 promoter is high. Wherein, the tissues of the negative control leaves at all stages are not dyed, and the tissues of the positive control leaves are dyed.

2. Analysis of tissue expression in maize

A corn transgenic plant is obtained by a similar method of rice in the embodiment 3, and the dyeing of GUS of each tissue discovers that the dyeing of the screened positive callus of the corn is obvious, and the GUS dyeing of the obtained seedling stage leaves, heading stage (roots, stems, leaves and anthers) and grains after 30 days of filling is uniformly dyed and colored. It can be seen that the OsEPSPSpro-770 and OsEPSPSpro-2048 promoters can also drive GUS genes to be stably expressed in the maize callus level, the leaf at seedling stage and the heading stage (roots, stems, leaves, anthers and seeds 30 days after filling), and the promoter is a high-efficiency constitutive promoter.

3. Analysis of tissue expression in wheat

A wheat transgenic plant is obtained by a rice similar method in the example 3, the GUS staining of each tissue shows that the staining of the screened positive callus of the wheat is obvious, and the GUS staining is carried out on the obtained seedling stage leaves, heading stage (roots, stems, leaves and anthers) and grains 30 days after filling. It can be seen that the OsEPSPSpro-770 and OsEPSPSpro-2048 promoters can also drive GUS genes to be stably expressed in wheat callus level, seedling stage, leaf at mature stage, heading stage (root, stem, leaf and anther) and grain after 30 days of filling, and the promoter is a high-efficiency constitutive promoter.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Claims (10)

1. An OsEPSPSpro promoter, wherein the OsEPSPSpro promoter comprises any one of the following nucleotide sequences:

i) The nucleotide sequence shown in SEQ ID NO.1 and/or the nucleotide sequence shown in SEQ ID NO. 2;

ii) a nucleotide sequence complementary to i);

iii) The nucleotide sequence with the same promoter function is obtained by substituting, deleting and adding one or more nucleotide sequences in the nucleotide sequence shown in i).

2. A primer pair for amplifying the osepsspro promoter of claim 1;

preferably, the primer pair comprises: a primer pair shown as SEQ ID NO.3-4, and/or a primer pair shown as SEQ ID NO. 5-6.

3. A biomaterial comprising the osesps Spro promoter of claim 1, wherein the biomaterial is an expression cassette, a vector, or a transgenic cell.

4. The biomaterial according to claim 3, wherein when the biomaterial is an expression cassette, the expression cassette further comprises a functional gene and a terminator;

the functional gene is preferably a marker gene or a gene related to the agronomic traits of the plants.

5. A kit comprising one or more of the OsEPSPSpro promoter of claim 1, the primer pair of claim 2, or the biological material of claim 3 or 4.

6. Use of the ospepspspro promoter of claim 1, the primer pair of claim 2, the biological material of claim 3 or 4, or the kit of claim 5 in any one of:

1) Preparing a transgenic plant;

2) Driving expression of a gene in a plant;

3) Genetic breeding or germplasm improvement of plants;

4) And (4) plant hybridization seed production.

7. Use according to claim 6, wherein the expression of the driver gene in a plant is in particular: the driving gene is efficiently expressed in the callus, the functional tissue in the vegetative growth period or the reproductive organ of the plant;

preferably, the expression is high in the pre-and post-emergence inflorescence period of the seeds.

8. The use of claim 6, wherein said use is the introduction of a DNA of interest operably linked to the OsEPSPSpro promoter of claim 1 into a plant.

9. The use of claim 6, wherein the gene is a functional gene, an antisense gene to a functional gene, or a small RNA gene;

the functional gene is preferably a plant agronomic trait related gene or a marker gene.

10. Use according to any one of claims 6 to 9, wherein the plant comprises one or more of rice, maize, wheat, barley, soybean, cotton, oilseed rape, sorghum or millet.

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