CN112080501B - Recombinant promoter, gene expression cassette and application of recombinant promoter and gene expression cassette in plant breeding - Google Patents

Abstract

The invention provides a recombinant promoter, which consists of an FMV promoter and a corn heat shock 70kDa protein intron (iZmHSP70) which are sequentially connected in series. The invention also provides a gene expression cassette and application thereof in breeding. The specificity of the FMV promoter is utilized, the promoter is used for driving the herbicide gene, so that the herbicide is sprayed in a specific period, the effect of killing pollen can be achieved, and the male sterility is realized. The recombinant promoter solves the problem that the yield of female parent seed production is influenced in the seed production process of a commercial transformation event by using a 35S + iZmHsp70 promoter.

Description

Recombinant promoter, gene expression cassette and application of recombinant promoter and gene expression cassette in plant breeding

Technical Field

The invention belongs to the technical field of molecular biology and plant breeding, and particularly relates to a recombinant promoter, a gene expression cassette and application of the recombinant promoter and the gene expression cassette in plant breeding.

Background

The utilization of hybrid vigor is one of the important measures to improve crop yield and product quality. When the hybrid seeds are used in production, a large amount of hybrid seeds are prepared, and the male parent is removed for seed production. Detasseling is a very burdensome task. The manual castration is time-consuming and labor-consuming, and the production cost of the seeds is increased; if the castration is not timely or thorough, the quality of hybrid seeds can be reduced, and the yield increasing effect is influenced. The production of hybrid seeds by using plant male sterile lines is one of the most effective methods for reducing the production cost of hybrid seeds, ensuring the purity of hybrids and protecting intellectual property rights, and along with the development of the technology, the development of the technology is internationally provided with the character expression controlled by chemical reagents through gene recombination, for example, in the Chinese patent application with the application number of 201280032386.3, Monsanto applies for the technical scheme of regulating and controlling the expression of male pollen by herbicides, but domestic reports of related technologies are few, so that similar breeding technologies are monopolized by foreign enterprises and are not beneficial to the development of the breeding technology in China.

On the other hand, the current commercial promoter for breeding is the 35S + iZmHsp70 promoter, which significantly affects the yield of female parent seed production during seed production, so that modification and optimization are needed.

Disclosure of Invention

Based on the current situation, the invention aims to provide a recombinant gene promoter which can regulate the formation and expression of plant pollen by applying glyphosate and generate a male sterile line, is expected to break monopoly of foreign enterprises and promote the development of breeding technology in China. The specificity of the FMV promoter is utilized, the promoter is used for driving the herbicide gene, so that the effect of killing pollen can be achieved by spraying the herbicide in a specific period, and the male sterility is realized.

The invention provides a recombinant promoter, which consists of an FMV promoter and a corn heat shock 70kDa protein intron (iZmHSP70) which are sequentially connected in series.

In this latter embodiment according to the invention, the nucleotide sequence of the recombinant promoter is SEQ ID NO: 1.

preferably, the 5 'end and the 3' end of the recombinant promoter are respectively connected with the enzyme cutting site sequence of the restriction enzyme. More preferably, the recombinant promoter is connected with a Hindlll cleavage site sequence at the 5 'end and an Avrll cleavage site sequence at the 3' end. The above-mentioned cleavage sites are added to facilitate ligation of the corresponding cloning vectors.

The invention further provides a gene expression cassette for plant breeding, which comprises the recombinant promoter and a chemical resistance gene sequence under the control of the recombinant promoter.

In one embodiment according to the invention, the chemoresistance gene is selected from herbicide resistance genes, preferably the glyphosate resistance gene EPSPS.

The invention also provides a recombinant vector, a recombinant microorganism, a transgenic cell line, a callus or a plant containing the recombinant promoter.

The invention further provides application of the recombinant promoter in plant breeding.

Yet another aspect of the present invention provides a method of inducing male sterility in a transgenic plant, comprising:

a transgenic plant comprising the gene expression cassette as described above is grown and an effective amount of herbicide is applied during development of male reproductive tissue of the transgenic plant, thereby inducing male sterility in the transgenic plant.

Preferably, the transgenic plant is maize.

More preferably, wherein said developmental stage of said male reproductive tissue is one or more stages selected from the group consisting of V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, V13 and V14 stages of maize development. Preferably one or both of V8 and V10.

In this latter embodiment according to the invention, wherein the herbicide is selected from one or more of acetyl-coa carboxylase inhibitors, acetolactate synthase inhibitors, photosystem II inhibitors, protoporphyrinogen oxidase inhibitors, 4-hydroxyphenylpyruvate dioxygenase inhibitors, 5-enolpyruvylshikimate-3-phosphate synthase inhibitors, glutamine synthetase inhibitors or synthetic auxins.

Preferably, the herbicide is glyphosate, and further preferably, the application amount of the glyphosate is 150-.

The present invention still further provides a method of producing hybrid seed comprising: 1) applying an effective amount of a herbicide during development of male reproductive tissue of a transgenic plant containing the gene expression cassette described above, thereby inducing male sterility in the transgenic plant; 2) fertilizing the transgenic plant with pollen from a plant of another line; 3) harvesting hybrid seed from said transgenic plant. Preferably, the transgenic plant is maize.

More preferably, wherein said developmental stage of said male reproductive tissue is one or more stages selected from the group consisting of V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, V13 and V14 stages of maize development. Preferably one or both of V8 and V10.

In one embodiment according to the present invention, wherein the herbicide is selected from one or more of acetyl-coa carboxylase inhibitors, acetolactate synthase inhibitors, photosystem II inhibitors, protoporphyrinogen oxidase inhibitors, 4-hydroxyphenylpyruvate dioxygenase inhibitors, 5-enolpyruvylshikimate-3-phosphate synthase inhibitors, glutamine synthetase inhibitors or synthetic auxins.

Preferably, the herbicide is glyphosate, and further preferably, the application amount of the glyphosate is 150-.

The technical scheme provided by the invention has the following beneficial effects:

1) the recombinant promoter provided by the invention utilizes the specificity of the FMV promoter, and the promoter is used for driving herbicide genes, so that the effect of killing pollen can be achieved by spraying the herbicide in a specific period, and the male sterility is realized.

2) The recombinant promoter technology provided by the invention solves the problem that the yield of female parent seed production is influenced in the seed production process by using the 35S + iZmHsp70 promoter in a commercial transformation event.

3) The recombinant promoter provided by the invention can realize male sterility seed production in plant breeding, and compared with 35S + iZmHSP70-EPSPS (Mon87427), the yield of seed production is not influenced.

Drawings

FIG. 1 is a flow chart of construction of a recombinant cloning vector LP06-T containing pFMV + iZmHSP70-EPSPS nucleotide sequence according to the invention;

FIG. 2 is a method for preparing a male sterile vector of the invention: a flow chart for constructing a recombinant expression vector LP-PT06 containing a pFMV + iZmHSP70-EPSPS nucleotide sequence;

FIG. 3 is a PCR detection chart of the male sterile vector transformant of the present invention, wherein WT is a wild type plant, PC is a plasmid control, NC is a water control, and 1 to 15 positive transformants are selected, wherein 1 to 5 are OsAct-EPSPS transformants, 6 to 10 are 35S + iZmHSP70-EPSPS transformants and FMV + iZmHSP70-EPSPS transformants.

FIG. 4 is a graph showing the results of the determination of the yield of hybrid seeds of different transformants F1.

Detailed Description

The following examples are intended to illustrate the present application but are not intended to limit the scope of the present application.

Specific embodiments of the present application will be described in more detail below. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

Unless otherwise specified, the reagents used in the present invention are commercially available.

Example 1 obtaining and Synthesis of nucleotide sequence of promoter such as pFMMV + iZmHSP70

1. Obtaining the nucleotide sequence of pFMMV + iZmHSP70

The nucleotide sequence (1406 nucleotides) of the pFMV + iZmHSP70 combined promoter is shown as SEQ ID NO:1 is shown.

2. Synthesis of the above-mentioned pFMMV + iZmHSP70 nucleotide sequence

The nucleotide sequence of the pFMV + iZmHSP70 (shown as SEQ ID NO:1 in the sequence table) is synthesized by Nanjing Kinsley Biotech company; the 5 'end of the synthesized pFMV + iZmHSP70 nucleotide sequence (SEQ ID NO: 1) is connected with a Hindlll cleavage site, and the 3' end is connected with an Avrll cleavage site.

The synthetic nucleotide sequence of pFMV + iZmHSP70 was ligated to a cloning vector pEASY-T5(Transgen, Beijing, China, CAT: CT501-01) and the procedures were carried out according to the instruction of pEASY-T5 vector manufactured by Transgen corporation to obtain a recombinant cloning vector LP06-T, the construction procedure of which is shown in FIG. 1 (wherein Kan represents a kanamycin resistance gene; Amp represents an ampicillin resistance gene; pUC origin represents a replication region sequence of plasmid pUC which can direct a double-stranded DNA replication process; LacZ is a LacZ initiation codon; pFMV + iZmHSP70 is a nucleotide sequence of pFMV + iZHSP 70 (SEQ ID NO: 1).

The recombinant cloning vector LP06-T was then transformed into E.coli T1 competent cells (Transgen, Beijing, China; Cat. No: CD501) by a heat shock method under the following heat shock conditions: 50. mu.l of E.coli T1 competent cells, 10. mu.l of plasmid DNA (recombinant cloning vector LP01-T), water bath at 42 ℃ for 30 seconds; the ampicillin (100 mg/L) coated LB plates (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L, pH adjusted to 7.5 with NaOH) were grown overnight in a water bath at 37 ℃ for 45 minutes (shaking table at 200 rpm). White colonies were picked and cultured overnight in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, ampicillin 100mg/L, pH 7.5 adjusted with NaOH) at 37 ℃. Extracting the plasmid by an alkaline method: centrifuging the bacterial solution at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial solution with 100 μ l ice-precooled solution I (25mM Tris-HCl, 10mM EDTA (ethylene diamine tetraacetic acid), 50mM glucose, pH 8.0); add 150. mu.l of freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)), invert thetube 4 times, mix, and place on ice for 3-5 min; adding 150 μ l ice-cold solution III (4M potassium acetate, 2M acetic acid), mixing well immediately, and standing on ice for 5-10 min; centrifuging at 4 deg.C and 12000rpm for 5min, adding 2 times volume of anhydrous ethanol into the supernatant, mixing, and standing at room temperature for 5 min; centrifuging at 4 deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol, and air drying; the precipitate was dissolved by adding 30. mu.l of RNase (20. mu.g/ml) in TE (10mM Tris-HCl, 1mM EDTA, pH 8.0); bathing in water at 37 deg.C for 30min to digest RNA; storing at-20 deg.C for use.

After the extracted plasmid is subjected to enzyme digestion identification by Hindlll and Avrll, sequencing verification is carried out on positive clones, and the result shows that the nucleotide sequence of the pFMV + iZmHSP70 inserted into the recombinant cloning vector LP01-T is SEQ ID NO:1, i.e. the nucleotide sequence of pFMV + iZmHSP70 is correctly inserted.

According to the above method for constructing recombinant cloning vector LP06-T, the synthesized pOsAct1 nucleotide sequence is linked to cloning vector pEASY-T5 to obtain recombinant cloning vector LP06CK1-T, the synthesized p35s + iZmHSP70 nucleotide sequence is linked to cloning vector pEASY-T5 to obtain recombinant cloning vector LP06CK2-T, wherein pOsAct1 is pOsAct1 nucleotide sequence (SEQ ID NO: 2), and p35s + iZmHSP70 is p35s + iZmHSP70 nucleotide sequence (SEQ ID NO: 3). The nucleotide sequences of pOsAct1 and p35s + iZmHSP70 in the recombinant cloning vectors LP01CK1-T and LP01CK2-T are verified to be correctly inserted by enzyme digestion and sequencing.

2. Construction of recombinant expression vectors containing promoter sequences

The recombinant cloning vector LP06-T and the expression vector LP-BB2 (vector backbone: pCAMBIA3301 (available from CAMBIA organization)) were digested with restriction enzymes Hindll and Avrll, respectively, and the excised nucleotide sequence fragment of pFMV + iZmHSP70 was inserted between Hindll and Avrll sites of the expression vector LP-BB2, and the vectors constructed by conventional digestion methods were well known to those skilled in the art to construct a recombinant expression vector LP-PT06, whose construction flow is shown in FIG. 2 (Kan: kanamycin gene; RB: right border; pFMV + iZmHSP 70: pFMV + iZmHSP70 nucleotide sequence (SEQ ID NO: 1), AtCTP: Arabidopsis thaliana chloroplast signal transit peptide (SEQ ID NO: 4), EPSPS: glyphosate resistance gene (SEQ ID NO: 5), Nos: terminator of nopaline synthase (SEQ ID NO: 6), LB: left border).

Transforming the recombinant expression vector LP-PT06 into an escherichia coli T1 competent cell by a heat shock method, wherein the heat shock condition is as follows: 50 ul of Escherichia coli T1 competent cells, 10 ul of plasmid DNA (recombinant expression vector LP-PT06), water bath at 42 ℃ for 30 seconds; water bath at 37 ℃ for 1 hour (shaking table at 100 rpm); then, the cells were cultured on LB solid plates (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L, pH adjusted to 7.5 with NaOH) containing 50mg/L Kanamycin (Kanamycin) at 37 ℃ for 12 hours, and white colonies were picked up and cultured overnight at 37 ℃ in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Kanamycin 50mg/L, pH adjusted to 7.5 with NaOH). The plasmid is extracted by an alkaline method. The extracted plasmid is cut by restriction enzymes Hindll and Avrll and then identified, and the positive clone is sequenced and identified, and the result shows that the nucleotide sequence of the recombinant expression vector LP-PT06 between Hindll and Avrll sites is SEQ ID NO:1, i.e. the nucleotide sequence of pFMV + iZmHSP 70.

According to the above method for constructing recombinant expression vector LP-PT06, the nucleotide sequences cut by Hindll and Avrll enzyme digestion recombinant cloning vector LP06CK1-T and LP06CK2-T are inserted into expression vector LP-BB2, and recombinant expression vectors LP-PT06CK1 and LP-PT06CK2 are obtained. Enzyme digestion and sequencing verification shows that the nucleotide sequences of the recombinant expression vectors LP-PT06CK1 and LP-PT06CK2 between Hindlll sites and Avrll sites are the nucleotide sequences of the pOsAct1 and p35s + iZmHSP 70.

3. Recombinant expression vector transformation agrobacterium tumefaciens

The correctly constructed recombinant expression vectors LP-PT06, LP-PT06CK1 and LP-PT06CK2 are transformed into Agrobacterium LBA4404 (Invitron, Chicago, USA; Cat. No: 18313-015) by a liquid nitrogen method under the following transformation conditions: 100. mu.L Agrobacterium LBA4404, 3. mu.L plasmid DNA (recombinant expression vector); placing in liquid nitrogen for 10 minutes, and carrying out warm water bath at 37 ℃ for 10 minutes; inoculating the transformed Agrobacterium LBA4404 in LB test tube, culturing at 28 deg.C and 200rpm for 2 hours, spreading on LB plate containing 50mg/L Rifampicin (Rifamicin) and 50mg/L Kanamycin (Kanamycin) until positive monoclone grows out, picking monoclone to culture and extracting its plasmid, digesting the recombinant expression vectors LP-PT06, LP-PT06CK1 and LP-PT06CK2 with restriction enzymes Hindlll and Avrll, and verifying the digestion, the result shows that the recombinant expression vectors LP-PT06, LP-PT06CK1 and LP-PT06CK2 are completely correct.

Example 2 plant transformation experiments:

1. preparation of young maize embryos

The maize inbred line AX808 in a company is planted in a field or a greenhouse, and maize 8-10 days (summer) to 10-13 days (autumn) after artificial pollination is taken as a source of immature embryos.

2. Preparation of Agrobacterium

(1) Marking the transformed and identified agrobacterium tumefaciens glycerol on a YEP solid culture medium added with 100mg/L kan and 12mg/L tet, and culturing in dark at 28 ℃ for 2-3 days;

(2) adding 1ml of infection culture medium into a sterilized 2ml centrifugal tube, putting the agrobacterium of thestep 1 into the infection culture medium, and fully scattering and uniformly mixing the agrobacterium with a pipette gun;

(3) and (3) taking 1 sterilized 2ml centrifuge tube, and adjusting the concentration of the bacterial liquid by using an infection culture medium to ensure that the OD660 is 0.5-0.7.

3. Co-culture of young maize embryos and agrobacterium

(1) Removing the infection culture medium in the immature embryo centrifuge tube, and adding 1.5ml of fresh infection culture medium to clean the embryo once;

(2) removing the infection culture medium, and adding the adjusted agrobacterium liquid;

(3) oscillating at the maximum rotating speed for 30s, and standing at room temperature for 5 min;

(4) pouring the embryos onto a co-culture medium, and blotting the liquid;

(5) placing the embryo with the plane upward and the shield surface upward;

(6) the embryos are cultured in the dark at 22 ℃ for 2-3 days.

4. Induction and selection of calli

(1) Transferring the co-cultured embryo to an induced callus culture medium, and performing dark culture in an incubator at 28 ℃ for 7-10 days;

(2) transferring the induced callus to a screening culture medium for screening culture, wherein the screening pressure is 5.0mM glyphosate, and dark culture is carried out for 2-3 weeks at 28 ℃;

(3) taking the callus survived in the first screening to carry out the second screening, wherein the screening pressure is 5.0 mM;

5. regeneration and culture of transformed strains

(1) Placing the screened embryogenic callus on a pre-differentiation culture medium, and performing dark culture at 28 deg.C for 10-14 days;

(2) taking embryo healing wound on a differentiation culture medium, and performing light culture at 28 ℃ for 10-14 days until the seedling is differentiated;

(3) transferring the differentiated seedling to a rooting culture medium, and performing light culture at 28 ℃ until the root is completely developed;

(4) transplanting the well-grown seedlings into a greenhouse matrix.

And harvesting the transgenic plants after the transgenic plants blossom and fruit. The harvested seeds are sowed in a greenhouse, and when the plants grow to 4-6 leaf stages, expression analysis and detection are carried out by adopting a PCR technology.

2. General PCR verification of EPSPS Gene-transferred maize plants Using 2 × EasyTaq PCR Supermix (China, Beijing, Cat: AS111-11) from all-open gold

The primers for PCR were:

FMV-F：TTGGGTTCAATCAACAAGGT(SEQ ID NO：7)

FMV-R：CTTCAAATGGGAATGAATGC(SEQ ID NO：8)

fragment size 541bp

EPSPS-F：CTACGATTTCGACAGCACCT(SEQ ID NO：9)

EPSPS-R：AATCGGATATTCGTCGATCA(SEQ ID NO：10)

Fragment size 655bp

The conditions for the PCR reaction were: 30 cycles, each cycle being 95 ℃ 30 ' 58 ℃ 30 ' 72 ℃ 40 '.

Example 3 verification of copy number of maize plants transformed with FMV + iZmHSP70-EPSPS Gene by qRT-PCR

About 100mg of each leaf of a maize Plant into which an EPSPS nucleotide sequence was transferred was sampled, and the DNA of the maize Plant Genomic Kit (RNase A contained) by Transgen (Transgen, Beijing, China, Cat:

EE111-01) and detecting the copy number of the EPSPS gene by a TransStart Green fluorescent quantitative PCR method. Meanwhile, wild corn plants are used as a control, and detection and analysis are carried out according to the method. The experiment was repeated 3 times and the average was taken.

The specific method for detecting the copy number of the EPSPS gene comprises the following steps:

step 11, respectively taking 100mg of leaves of the maize plant with the transferred EPSPS nucleotide sequence and the wild maize plant, respectively grinding the leaves into homogenate in a mortar by using liquid nitrogen, and taking 3 samples for repetition;

step 12, extracting the Genomic DNA of the sample by using an easy pure Plant Genomic DNA Kit (containing RNase A) of Transgen (Transgen, Beijing, China, Cat: EE111-01), and referring to the product specification of the specific method;

step 13, measuring the genomic DNA concentration of the sample by using NanoDrop 2000(Thermo Scientific);

step 14, adjusting the genomic DNA concentration of the sample to the same concentration value, wherein the concentration value range is 80-100 ng/mu l;

step 15, identifying the copy number of the sample by adopting a TransStart Green fluorescent quantitative PCR method, taking the sample with known copy number after identification as a standard substance, taking the sample of a wild corn plant as a control, repeating each sample for 3 times, and taking the average value; the fluorescent quantitative PCR primer and the probe sequence are respectively as follows:

the following primers were used to detect the EPSPS nucleotide sequence:

EPSPS-qF：GTGTCGGAAAACCCTGTCAC(SEQ ID NO：11)

EPSPS-qR：CCTTCGTATCGGAGAGTTCG(SEQ ID NO：12)

the following primers were used to detect the 18s nucleotide sequence for internal reference leveling

18srRNA-F：CCATCCCTCCGTAGTTAGCTTCT(SEQ ID NO：13)

18srRNA-R：CCTGTCGGCCAAGGCTATATAC(SEQ ID NO：14)

The PCR reaction system is as follows:

the PCR reaction conditions are as follows:

repeating the steps for 2-3 and 40 times in a circulating way

Data were analyzed using SDS2.3 software (Applied Biosystems).

The experimental result shows that the EPSPS nucleotide sequence is integrated into the chromosome group of the detected corn plant, and the corn plants transferred with the EPSPS nucleotide sequence all obtain transgenic corn plants containing single copy.

Example 4 herbicide protein detection of transgenic maize plants

1. Detection of the content of herbicide proteins (EPSPS proteins) in transgenic maize plants

The solutions involved in this experiment were as follows:

extracting a buffer solution: 8g/L NaCl, 0.2g/L KH2PO4, 2.9g/L Na2HPO 4.12H 2O, 0.2g/L KCl, 5.5ml/L Tween 20(Tween-20), pH 7.4;

wash buffer PBST: 8g/L NaCl, 0.2g/L KH2PO4, 2.9g/L Na2HPO 4.12H 2O, 0.2g/L KCl, 0.5ml/L Tween 20(Tween-20), pH 7.4;

stopping liquid: 1M HCl.

Respectively taking 3mg of fresh leaves and pollen of a single-copy corn plant of the single-copy corn plant transferred into the nucleotide sequence of OsAct1-EPSPS and FMV + iZmHSP70-EPSPS as samples, grinding by using liquid nitrogen, adding 800 mu l of the extraction buffer solution, centrifuging for 10min at the rotating speed of 4000rpm, taking supernatant, diluting by 40 times by using the extraction buffer solution, and taking 80 mu l of diluted supernatant for ELISA detection. The proportion of the herbicide resistant protein (EPSPS protein) in the sample accounting for the fresh weight of the leaves is detected and analyzed by an ELISA (enzyme-linked immunosorbent assay) kit (ENVIRLOGIX, EPSPS kit), and the specific method refers to the product specification thereof.

Meanwhile, wild corn plants and corn plants which are identified as non-transgenic through fluorescent quantitative PCR are used as controls, and detection and analysis are carried out according to the method. Transferring 3 strains (O1, O2 and O3) of OsAct1-EPSPS nucleotide sequence, transferring 3 strains (S1, S2 and S3) of 35S + iZmHSP70-EPSPS nucleotide sequence, transferring 3 strains (F1, F2 and F3) of FMV + iZmHSP70-EPSPS nucleotide sequence, identifying 1 non-transgenic (NGM) strain and 1 wild type (CK) strain by fluorescence quantitative PCR; 3 strains from each line were selected for testing, each repeated 6 times.

The results of the experiment on the herbicide resistant protein (EPSPS protein) content in transgenic maize leaves are shown in table 1. The results respectively show that the average expression quantity (ng/g) of herbicide resistant protein (EPSPS protein) in the maize plant transferred with the EPSPS nucleotide sequence accounts for 4642.20, 4753.30 and 4555.42 respectively, and the results show that the EPSPS protein obtains higher expression quantity and stability in the maize leaf.

TABLE 1 average results of EPSPS protein expression measurements in leaves of transgenic maize plants

The results of the experiment on the herbicide resistant protein (EPSPS protein) content in transgenic maize pollen are shown in table 2. The average expression levels of herbicide resistant proteins (EPSPS proteins) in the corn plants transferred with the EPSPS nucleotide sequences are respectively determined to be 7331.08, 98.06 and 78.76, and the results show that the OsAct1-EPSPS protein has high expression level in pollen, the expression levels in FMV + iZmHSP70-EPSPS, 35S + iZmHSP70-EPSPS pollen are low, and the male sterility can be realized by spraying glyphosate herbicide in the pollen development period.

TABLE 2 average results of EPSPS protein expression measurements in transgenic maize plant pollen

Example 5 efficacy experiment of herbicide glyphosate spray on corn fertility:

three confirmed single copy T1 transgenic plant lines each resulting from transformation of OsAct-EPSPS, 35S + iZmHSP70-EPSPS and FMV + iZmHSP70-EPSPS were tested for efficacy of the expression cassettes in field trials. Data for a one year efficacy field trial for which the glyphosate spray treatment protocol was as follows for the three following fields are presented in table 3.

Treatment 1: 157 ml/acre glyphosate (weed control) at V3 stage;

thetreatment 2 comprises the following steps: 157 ml/acre glyphosate (weed control) at V3 stage, followed by 157 ml/acre at V8 stage, followed by 157 ml/acre at V10 stage;

and (3) treatment: consists of 157 ml/acre glyphosate (weed control) at V3 stage, followed by 262 ml/acre at V8 stage), followed by 262 ml/acre at V10 stage. The last two sprays (i.e., V8 and V10) are referred to as sterile sprays.

These results indicate that all plants, OsAct1-EPSPS, 35S + iZmHSP70-EPSPS, FMV + iZmHSP70-EPSPS, were male fertile with only the weed controlglyphosate spray treatment 1. 2, 3, OsAct1-EPSPS plants were fertile, 35S + iZmHSP70-EPSPS was sterile, FMV + iZmHSP70-EPSPS was sterile when sprayed with glyphosate sterility.

TABLE 3 influence of Glyphosate application at different periods on fertility of maize of different transformants

Example 6: f1 hybrid yield determination

This example illustrates the plant tolerance of transgenic plants field tested for yield to glyphosate spray and male sterility under glyphosate spray. One transformation event is selected from each construct of OsAct-EPSPS, 35S + iZmHSP70-EPSPS and FMV + iZmHSP70-EPSPS, 100 plants/plot land are planted in a standard plot land, and corresponding non-transgenic materials (glyphosate spraying is not carried out, isolation is carried out) are planted beside the transformation event for open pollination to prepare F1 hybrid seeds. For these transformant field trials, the spray treatments were:

treatment 1: consists of 314 ml/acre glyphosate (weed control) in the V3 stage;

and (3) treatment 2: consists of 314 ml/acre glyphosate at V3 stage, then 157 ml/acre at V8 stage, and then 157 ml/acre at V11 stage;

and (3) treatment: consists of 314 ml/acre glyphosate at stage V3, followed by 262 ml/acre at stage V8, and then 262 ml/acre at stage V10. Under all three glyphosate treatment protocols, each of 3 lines of OsAct-EPSPS, 35S + iZmHSP70-EPSPS and FMV + iZmHSP70-EPSPS showed good vegetative tolerance as measured by plant height. These three transformed lines were fully male-fertile when treated with the weed control glyphosate alone treatment protocol (treatment 1), but when treated withglyphosate treatment protocol 2 or 3. The results are shown in FIG. 4, the results of measuring the grain yield of 3 lines of OsAct-EPSPS, 35S + iZmHSP70-EPSPS and FMV + iZmHSP70-EPSPS sprayed with glyphosate show no obvious difference in the yield of OsAct-EPSPS and FMV-EPSPS, and the yield of 35S + iZmHSP70(Mon87427) is lower than that of OsAct-EPSPS and FMV + iZmHSP 70-EPSPS. The yield data shows that FMV + iZmHSP70-EPSPS can realize male sterile seed production, and compared with 35S + iZmHSP70-EPSPS (Mon87427), the yield data does not influence the seed production yield.

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

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Sequence listing

<110> Longping Biotechnology (Hainan) Co., Ltd

<120> recombinant promoter, gene expression cassette and application thereof in plant breeding

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atcgtgttct gcctgtgctg attacttgcc gtcctttgta gcagcaaaat atagggacat 780

ggtagtacga aacgaagata gaacctacac agcaatacga gaaatgtgta atttggtgct 840

tagcggtatt tatttaagca catgttggtg ttatagggca cttggattca gaagtttgct 900

gttaatttag gcacaggctt catactacat gggtcaatag tatagggatt catattatag 960

gcgatactat aataatttgt tcgtctgcag agcttattat ttgccaaaat tagatattcc 1020

tattctgttt ttgtttgtgt gctgttaaat tgttaacgcc tgaaggaata aatataaatg 1080

acgaaatttt gatgtttatc tctgctcctt tattgtgacc ataagtcaag atcagatgca 1140

cttgttttaa atattgttgt ctgaagaaat aagtactgac agtattttga tgcattgatc 1200

tgcttgtttg ttgtaacaaa atttaaaaat aaagagtttc ctttttgttg ctctccttac 1260

ctcctgatgg tatctagtat ctaccaactg acactatatt gcttctcttt acatacgtat 1320

cttgctcgat gccttctccc tagtgttgac cagtgttact cacatagtct ttgctcattt 1380

cattgtaatg cagataccaa gcggcc 1406

<210> 2

<211> 1411

<212> DNA

<213> OsAct1

<400> 2

tactcgaggt cattcatatg cttgagaaga gagtcgggat agtccaaaat aaaacaaagg 60

taagattacc tggtcaaaag tgaaaacatc agttaaaagg tggtataaag taaaatatcg 120

gtaataaaag gtggcccaaa gtgaaattta ctcttttcta ctattataaa aattgaggat 180

gtttttgtcg gtactttgat acgtcatttt tgtatgaatt ggtttttaag tttattcgct 240

tttggaaatg catatctgta tttgagtcgg gttttaagtt cgtttgcttt tgtaaataca 300

gagggatttg tataagaaat atctttagaa aaacccatat gctaatttga cataattttt 360

gagaaaaata tatattcagg cgaattctca caatgaacaa taataagatt aaaatagctt 420

tcccccgttg cagcgcatgg gtattttttc tagtaaaaat aaaagataaa cttagactca 480

aaacatttac aaaaacaacc cctaaagttc ctaaagccca aagtgctatc cacgatccat 540

agcaagccca gcccaaccca acccaaccca acccacccca gtccagccaa ctggacaata 600

gtctccacac ccccccacta tcaccgtgag ttgtccgcac gcaccgcacg tctcgcagcc 660

aaaaaaaaaa agaaagaaaa aaaagaaaaa gaaaaaacag caggtgggtc cgggtcgtgg 720

gggccggaaa cgcgaggagg atcgcgagcc agcgacgagg ccggccctcc ctccgcttcc 780

aaagaaacgc cccccatcgc cactatatac ataccccccc ctctcctccc atccccccaa 840

ccctaccacc accaccacca ccacctccac ctcctccccc ctcgctgccg gacgacgagc 900

tcctcccccc tccccctccg ccgccgccgc gccggtaacc accccgcccc tctcctcttt 960

ctttctccgt ttttttttcc gtctcggtct cgatctttgg ccttggtagt ttgggtgggc 1020

gagaggcggc ttcgtgcgcg cccagatcgg tgcgcgggag gggcgggatc tcgcggctgg 1080

ggctctcgcc ggcgtggatc cggcccggat ctcgcgggga atggggctct cggatgtaga 1140

tctgcgatcc gccgttgttg ggggagatga tggggggttt aaaatttccg ccgtgctaaa 1200

caagatcagg aagaggggaa aagggcacta tggtttatat ttttatatat ttctgctgct 1260

tcgtcaggct tagatgtgct agatctttct ttcttctttt tgtgggtaga atttgaatcc 1320

ctcagcattg ttcatcggta gtttttcttt tcatgatttg tgacaaatgc agcctcgtgc 1380

ggagcttttt tgtaggtaga agtgatcaac c 1411

<210> 3

<211> 1439

<212> DNA

<213> p35s

<400> 3

tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat 60

ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg 120

cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc 180

cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt 240

ggattgatgt gatggtccga ttgagacttt tcaacaaagg gtaatatccg gaaacctcct 300

cggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa aggaaggtgg 360

ctcctacaaa tgccatcatt gcgataaagg aaaggccatc gttgaagatg cctctgccga 420

cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc 480

aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc 540

acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga 600

gaggacacgc tgacaagctg actctagcag atctaccgtc ttcggtacgc gctcactccg 660

ccctctgcct ttgttactgc cacgtttctc tgaatgctct cttgtgtggt gattgctgag 720

agtggtttag ctggatctag aattacactc tgaaatcgtg ttctgcctgt gctgattact 780

tgccgtcctt tgtagcagca aaatataggg acatggtagt acgaaacgaa gatagaacct 840

acacagcaat acgagaaatg tgtaatttgg tgcttagcgg tatttattta agcacatgtt 900

ggtgttatag ggcacttgga ttcagaagtt tgctgttaat ttaggcacag gcttcatact 960

acatgggtca atagtatagg gattcatatt ataggcgata ctataataat ttgttcgtct 1020

gcagagctta ttatttgcca aaattagata ttcctattct gtttttgttt gtgtgctgtt 1080

aaattgttaa cgcctgaagg aataaatata aatgacgaaa ttttgatgtt tatctctgct 1140

cctttattgt gaccataagt caagatcaga tgcacttgtt ttaaatattg ttgtctgaag 1200

aaataagtac tgacagtatt ttgatgcatt gatctgcttg tttgttgtaa caaaatttaa 1260

aaataaagag tttccttttt gttgctctcc ttacctcctg atggtatcta gtatctacca 1320

actgacacta tattgcttct ctttacatac gtatcttgct cgatgccttc tccctagtgt 1380

tgaccagtgt tactcacata gtctttgctc atttcattgt aatgcagata ccaagcggc 1439

<210> 4

<211> 228

<212> DNA

<213> AtCTP

<400> 4

atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60

tcgaaatcca gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca 120

cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac gttaattggc 180

tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcgtgc 228

<210> 5

<211> 1368

<212> DNA

<213> EPSPS

<400> 5

atgcttcacg gtgcaagcag ccggcccgca accgcccgca aatcctctgg cctttccgga 60

accgtccgca ttcccggcga caagtcgatc tcccaccggt ccttcatgtt cggcggtctc 120

gcgagcggtg aaacgcgcat caccggcctt ctggaaggcg aggacgtcat caatacgggc 180

aaggccatgc aggcgatggg cgcccgcatc cgtaaggaag gcgacacctg gatcatcgat 240

ggcgtcggca atggcggcct cctggcgcct gaggcgccgc tcgatttcgg caatgccgcc 300

acgggctgcc gcctgacgat gggcctcgtc ggggtctacg atttcgacag caccttcatc 360

ggcgacgcct cgctcacaaa gcgcccgatg ggccgcgtgt tgaacccgct gcgcgaaatg 420

ggcgtgcagg tgaaatcgga agacggtgac cgtcttcccg ttaccttgcg cgggccgaag 480

acgccgacgc cgatcaccta ccgcgtgccg atggcctccg cacaggtgaa gtccgccgtg 540

ctgctcgccg gcctcaacac gcccggcatc acgacggtca tcgagccgat catgacgcgc 600

gatcatacgg aaaagatgct gcagggcttt ggcgccaacc ttaccgtcga gacggatgcg 660

gacggcgtgc gcaccatccg cctggaaggc cgcggcaagc tcaccggcca agtcatcgac 720

gtgccgggcg acccgtcctc gacggccttc ccgctggttg cggccctgct tgttccgggc 780

tccgacgtca ccatcctcaa cgtgctgatg aaccccaccc gcaccggcct catcctgacg 840

ctgcaggaaa tgggcgccga catcgaagtc atcaacccgc gccttgccgg cggcgaagac 900

gtggcggacc tgcgcgttcg ctcctccacg ctgaagggcg tcacggtgcc ggaagaccgc 960

gcgccttcga tgatcgacga atatccgatt ctcgctgtcg ccgccgcctt cgcggaaggg 1020

gcgaccgtga tgaacggtct ggaagaactc cgcgtcaagg aaagcgaccg cctctcggcc 1080

gtcgccaatg gcctcaagct caatggcgtg gattgcgatg agggcgagac gtcgctcgtc 1140

gtgcgtggcc gccctgacgg caaggggctc ggcaacgcct cgggcgccgc cgtcgccacc 1200

catctcgatc accgcatcgc catgagcttc ctcgtcatgg gcctcgtgtc ggaaaaccct 1260

gtcacggtgg acgatgccac gatgatcgcc acgagcttcc cggagttcat ggacctgatg 1320

gccgggctgg gcgcgaagat cgaactctcc gatacgaagg ctgcctga 1368

<210> 6

<211> 253

<212> DNA

<213> Nos

<400> 6

gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60

atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120

atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180

gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240

atgttactag atc 253

<210> 7

<211> 20

<212> DNA

<213> FMV-F

<400> 7

ttgggttcaa tcaacaaggt 20

<210> 8

<211> 20

<212> DNA

<213> FMV-R

<400> 8

cttcaaatgg gaatgaatgc 20

<210> 9

<211> 20

<212> DNA

<213> EPSPS-F

<400> 9

ctacgatttc gacagcacct 20

<210> 10

<211> 20

<212> DNA

<213> EPSPS-R

<400> 10

aatcggatat tcgtcgatca 20

<210> 11

<211> 20

<212> DNA

<213> EPSPS-qF

<400> 11

gtgtcggaaa accctgtcac 20

<210> 12

<211> 20

<212> DNA

<213> EPSPS-QR

<400> 12

ccttcgtatc ggagagttcg 20

<210> 13

<211> 23

<212> DNA

<213> 18srRNA-F

<400> 13

ccatccctcc gtagttagct tct 23

<210> 14

<211> 22

<212> DNA

<213> 18srRNA-R

<400> 14

cctgtcggcc aaggctatat ac 22

Claims (8)

1. A recombinant promoter is characterized in that the recombinant promoter consists of an FMV promoter and a corn heat shock 70kDa protein intron (iZmHSP70) which are connected in series in sequence; the nucleotide sequence of the recombinant promoter is SEQ ID NO. 1.

2. A gene expression cassette for use in plant breeding, comprising the recombinant promoter of claim 1, and a chemoresistance gene sequence whose expression is regulated by the recombinant promoter; the chemoresistance gene is selected from herbicide resistance genes; the plant is corn.

3. A recombinant vector or a recombinant microorganism comprising the gene expression cassette according to claim 2.

4. A method of inducing male sterility in a transgenic plant comprising:

cultivating a transgenic plant comprising the gene expression cassette of claim 2 and applying an effective amount of herbicide during development of male reproductive tissue of the transgenic plant, thereby inducing male sterility in the transgenic plant.

5. The method of claim 4, wherein said development of said male reproductive tissue is one or more stages selected from the group consisting of V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, V13, and V14 stages of maize development.

6. The method of claim 5, wherein said development of said male reproductive tissue is one or both selected from the group consisting of V8 and V10 of maize development.

7. The method of claim 4, wherein the herbicide is selected from one or more of acetyl-CoA carboxylase inhibitors, acetolactate synthase inhibitors, photosystem II inhibitors, protoporphyrinogen oxidase inhibitors, 4-hydroxyphenylpyruvate dioxygenase inhibitors, 5-enolpyruvylshikimate-3-phosphate synthase inhibitors, glutamine synthetase inhibitors or synthetic auxins.

8. A method of producing hybrid seed comprising:

1) applying an effective amount of a herbicide to a transgenic plant containing the gene expression cassette of claim 2 at a stage of male reproductive tissue development, thereby inducing male sterility in the transgenic plant;

2) fertilizing the transgenic plant with pollen from a plant of another line;

3) harvesting hybrid seed from said transgenic plant.

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