Promoter specifically expressed by rice endosperm and application thereof

Abstract

The invention relates to the field of plant genetic engineering and plant genetic breeding, in particular to a rice endosperm specific expression promoter and application thereof. The promoter is composed of a nucleotide sequence as shown in SEQ ID No.1 or a nucleotide sequence which is derived from the nucleotide as shown in SEQ ID No.1 through substitution, deletion and addition of one or more basic groups and can specifically induce specific expression of an exogenous gene in a rice endosperm tissue. When the nucleotide sequence is applied to plant genetic engineering, the specific expression of an exogenous gene in a rice endosperm tissue can be induced.

Description

Technical Field

[0001] The invention relates to the field of plant gene engineering and plant genetic breeding, in particular to a rice endosperm-specific expression promoter and application thereof. Background Art

[0002] Plants are the material basis for human survival and development. Plant production is primary production, and plant varieties are the material basis of plant production. The process of improving plant varieties is called plant breeding. The combination of plant genetic engineering and plant genetic breeding is the development direction of modern agriculture. Obtaining plant strains with high quality, high yield, high disease resistance and other special traits is the goal and driving force of agricultural development. The application of transgenic technology to plant genetic breeding is a new driving force for the development of modern plant breeding technology. Transgenic technology is to introduce artificially isolated and modified exogenous genes into the genome of the target organism, so as to achieve the purpose of transforming biological traits.

[0003] The key link in this technology is to place the foreign gene in a suitable promoter before the gene can be guided to be expressed in the target organism genome. A promoter refers to a deoxyribonucleic acid (DNA) sequence located upstream of a functional gene that enables the gene to be transcribed. The promoter can be recognized by RNA polymerase and initiates transcription, and the promoter plays a major role in determining the spatiotemporal pattern of gene expression. Currently, commonly used transgenic promoters are mainly constitutive expression promoters such as ubiquitin promoters and tobacco mosaic virus 35s promoters. For rice transgenics, this type of promoter can effectively induce foreign genes to be expressed in rice, but this type of promoter induces the expression of foreign genes without organ and tissue specificity. This promoter can induce foreign gene expression in various organs and tissues of rice, which limits the application value of this promoter. When we carry out transgenic breeding, we usually need to express the foreign genes we introduce at specific developmental stages and organs of rice, which is impossible for this type of promoter. Summary of the invention

[0004] In view of the problems in the prior art, the present invention provides a promoter for rice endosperm-specific expression and its application. In the present invention, we separated the promoter sequence of the rice endosperm-specific expression gene Os GASR6 from the genome of wild-type rice Nipponbare. The rice Os GASR6 gene belongs to the plant oligopeptide transport gene family. The gene is specifically expressed in rice endosperm. The promoter is applied to transgenic rice to induce the specific expression of functional genes in rice endosperm. By utilizing the function of this promoter, we can provide a method for specifically expressing foreign genes in transgenic rice endosperm, providing new impetus for the development of modern genetic breeding technology.

[0005] The technical solution adopted by the present invention to solve its technical problem is: in the first aspect of the present invention, the present invention provides a promoter for rice endosperm-specific expression, wherein the promoter is composed of the nucleotide sequence shown in SEQ ID No.1 or is a nucleotide sequence derived from the nucleotide shown in SEQ ID No.1 by replacing, deleting or adding one or more bases, which can specifically induce the specific expression of exogenous genes in rice endosperm.

[0006] Preferably, the nucleotide sequence of the promoter is SEQ ID No.1.

[0007] In a second aspect of the present invention, the present invention provides an expression cassette comprising the nucleotide sequence shown in SEQ ID No. 1 as a promoter, wherein the expression cassette is capable of specifically expressing a target gene connected to the expression cassette in rice endosperm.

[0008] Preferably, the present invention provides a recombinant vector comprising the above expression cassette, wherein the recombinant vector is capable of specifically expressing a target gene connected to the recombinant vector in rice endosperm.

[0009] Preferably, the vector is a vector capable of being expressed in rice, such as a pCAMBIA1301 vector.

[0010] This vector can be an Agrobacterium binary expression vector or a plant expression vector transferred into the recipient plant genome by other means. The coding nucleotide sequence of the exogenous gene that needs to be specifically expressed in rice endosperm is added after the promoter to induce the specific expression of the gene in rice endosperm. It is also possible to express purified protein by introducing various expression tags downstream of the coding sequence. In addition, different resistance genes can be added to the vector to cope with different screening methods according to specific experimental materials and methods.

[0011] In the third aspect of the present invention, the present invention provides the application of the nucleotide sequence shown in SEQ ID No.1 or the expression cassette or recombinant vector containing the nucleotide sequence shown in SEQ ID No.1 as a promoter in rice breeding. In the application, the exogenous functional gene for improving rice traits is connected to the expression cassette or recombinant vector so that it is expressed under the control of the promoter, and then the expression cassette or the recombinant vector connected with the exogenous gene is transfected into the rice plant, so that the exogenous functional gene is specifically expressed in the rice endosperm, and transgenic rice having the function of the exogenous functional gene is screened, thereby playing a role in improving rice traits. Specifically, the utilization rate of rice nitrogen can be improved. For example, the specific expression of efficient nitrogen transport genes in the endosperm can improve the nitrogen transport efficiency of rice, thereby increasing rice yield, but the present invention is not limited to this application.

[0012] In the fourth aspect of the present invention, the present invention provides a method for breeding rice varieties, the method comprising connecting an exogenous functional gene for improving rice traits to the expression cassette or recombinant vector described in the second aspect of the present invention, so that the exogenous functional gene is expressed under the control of the promoter described in the first aspect of the present invention, and then transfecting the expression cassette or the recombinant vector connected with the exogenous gene into a rice plant, so that the exogenous functional gene is specifically expressed in the rice endosperm, and then screening transgenic rice having the function of the exogenous functional gene, thereby playing a role in improving rice traits.

[0013] Those skilled in the art should understand that if the exogenous functional gene is not derived from rice, in order to fully express it in rice, codon optimization can be performed according to the rice codon usage preference, and the nucleotide sequence can be optimized to enable it to be fully expressed in rice without changing the amino acid sequence encoded by the exogenous functional gene. The technology for codon optimization is well known to those skilled in the art. In addition, the method for transfecting the expression cassette or recombinant vector into rice plants is also a conventional method in the art, for example, Agrobacterium-mediated transfection technology can be used.

[0014] In the fifth aspect of the present invention, the present invention also provides a recombinant cell comprising the expression cassette or recombinant vector described in the second aspect of the present invention, wherein the cell is selected from microbial cells or fungal cells, for example, but not limited to, Escherichia coli cells, Agrobacterium cells, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0016] Figure 1 :Schematic diagram of the construction of Agrobacterium transformation vector OsGASR6Promoter-GUS for inducing GUS gene expression by rice OsGASR6 gene promoter.

[0017] Figure 2 : PCR identification results of T0 generation transgenic rice; Lane 1 is the positive control with OsGASR6Promoter-GUS as template; Lanes 2, 3, 4, 5, and 6 are transgenic positive plants; Lane 7 is transgenic negative plants; Lane 8 is negative plant control.

[0018] Figure 3 : GUS staining results of transgenic rice Os GASR6Promoter-GUS endosperm; a: embryo piece of wild-type Nipponbare; b: Os GASR6Promoter-GUS endosperm. DETAILED DESCRIPTION

[0019] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0020] The experimental methods in the following examples are all conventional experimental methods unless otherwise specified. The kits and experimental instruments used in the experiments can be purchased from biological instrument and reagent companies unless otherwise specified.

[0021] Rice OsGASR6 gene promoter induces GUS gene to express specifically in rice endosperm:

[0022] 1. Based on, specific primers were designed to isolate and clone the promoter sequence (1539bp) of rice oligopeptide transport gene Os GASR6.

[0023] 2. Construction of the rice OsGASR6 gene promoter-induced GUS gene Agrobacterium recombinant expression vector OsGASR6Promoter-GUS;

[0024] The Os GASR6 promoter was amplified using an upstream primer with a PstⅠ restriction site and a downstream primer with a NcoⅠ restriction site. After the PCR product was recovered, it was connected to the pMDtm19-T vector. After correct sequencing, the pGEM-Os GASR6P vector was obtained. The Os GASR6 promoter was then inserted into the upstream of the GUS gene of the Agrobacterium transformation vector 1301 through restriction digestion, connection and transformation to obtain the Os GASR6Promoter-GUS Agrobacterium recombinant expression vector.

[0025] 3. The recombinant expression vector Os GASR6 Promoter-GUS was transferred into Agrobacterium EHA105 by freeze-thaw method; 3.1. A single colony of Agrobacterium EHA105 was picked and activated in 2 ml YEP medium (1 liter of YEP medium composition: yeast extract 1g, peptone 10g, sucrose 5g, MgSO4.7H2O1.027g, adjusted to pH 7.0, containing Rif 20mg / ml,) at 28°C overnight;

[0026] 3.2. Take 2 ml of overnight bacterial solution and inoculate it into 50 ml of YEP medium and grow it at 28°C until OD600 is about 0.5 (4 hr);

[0027] 3.3, centrifuge at 5krpm for 5 minutes;

[0028] 3.4. Suspend cells in 10 ml of 0.15 M NaCl;

[0029] 3.5. Centrifuge at 5krpm for 5 minutes and suspend the cells in 20ml ice-cold CaCl2 to obtain Agrobacterium competent cells.

[0030] 3.6. Add 1ug DNA to 200ul Agrobacterium competent cells and place on ice for 30 minutes;

[0031] 3.7. Freeze in liquid nitrogen for 1 minute;

[0032] 3.8. Thaw the Agrobacterium cells in a 37°C water bath;

[0033] 3.9. Add 1 ml of YEP medium and culture in a shaking incubator at 28°C for 2-4 hours at a low speed.

[0034] 3.10. Centrifuge for 1 minute and resuspend the Agrobacterium cells in 100ul YEP medium;

[0035] 3.11. Take out the bacterial solution and spread it on the YEP plate containing the corresponding antibiotics (AMP+Rif), and culture it at 28℃ for 2-3 days. The colonies that grow are Agrobacterium containing the OsGASR6Promoter-GUS recombinant expression vector.

[0036] 4. Agrobacterium-mediated rice transformation

[0037] Preparation of rice transformation recipients:

[0038] Take mature rice seeds, shell them, select plump, smooth and free of sterile spots, and disinfect them according to the following steps: Pre-disinfect the surface of the seeds with 70% ethanol for 30s-1min in a clean bench, pour off the ethanol, and rinse with sterile water 2-3 times. Add 5.0% sodium hypochlorite solution, shake and disinfect for 50min, pour off the sodium hypochlorite solution, rinse with sterile water 3-4 times, and soak for 30min for the last time.

[0039] Induction and subculture: Place the seeds on sterilized filter paper to dry, place the seeds in the induction medium, press gently, seal the culture dish with sealing film, culture in the dark at 25℃ for one month, open the culture dish on the clean bench, pick up the naturally divided embryonic callus with tweezers, place it in the subculture medium, and subculture at 25℃ for 6-8 days.

[0040] Cultivation of Agrobacterium:

[0041] Agrobacterium EHA105 containing the Os GASR6 Promoter-GUS recombinant expression vector was streaked on a YEP plate containing 100 mg / L Kan and 50 mg / L Str, and cultured in the dark at 28°C for 3 days. A single clone was picked and cultured in liquid YEP with the same antibiotics for 12-24 h, and then expanded in the same liquid YEP (50 ml) at a ratio of 1:50 (v / v) for 4-5 hours. The bacterial liquid was collected by centrifugation at 5000 rpm for 5 min, resuspended in liquid AAM medium (20 ml), and cultured in a shaker at 28 degrees with slight shaking for 30 min-3 h. The Agrobacterium suspension can be used to transform rice callus.

[0042] Co-cultivation of rice callus and Agrobacterium:

[0043] Select callus tissues in good condition (5-7 days of subculture, light yellow in color) and place them in a 50ml sterile Erlenmeyer flask, add the prepared Agrobacterium suspension, and place it in a 28℃ shaker for 20 minutes. Pour out the bacterial solution, place the callus tissue on sterile filter paper to absorb the excess bacterial solution, and then transfer it to a solid co-culture medium covered with a layer of sterile filter paper and culture it in the dark at 25℃ for 2-3 days.

[0044] Screening of resistant callus:

[0045] The co-cultured callus was placed on a screening medium containing 50 mg / L hygromycin and 500 mg / L cephalosporin, cultured in the dark at 26°C for 15 days, and then transferred to a freshly prepared screening medium for another 15 days. Most of the callus turned brown about 10 days after screening, and then milky white resistant callus regrew at the edge of the browned tissue.

[0046] Differentiation of resistant callus:

[0047] From the resistant callus grown after two rounds of screening, select the creamy yellow and dense resistant callus and first transfer it to the pre-differentiation medium containing 10mg / L PPT and 250mg / L cef for dark culture for 7-15 days, then transfer it to the differentiation medium for culture under 15h / d light conditions. Generally, green dots will appear after 6-10 days. After 20-30 days, it will further differentiate into seedlings.

[0048] Rooting, seedling strengthening and transplanting:

[0049] When the buds differentiated from the resistant callus grow to about 2 cm, the seedlings are moved to the rooting medium and cultured for about two weeks. Select seedlings about 10 cm tall with well-developed root systems, wash off the medium, and transplant them into the soil in the greenhouse.

[0050] The following media were used in tissue culture and transformation:

[0051] Table 1. MS basic medium

[0052]

[0053] Co-culture medium: MS basic medium + GLn (146 mg / l) + hydrolyzed chelating agent (200 mg / l) + 2 mg / l 2,4-D + sucrose (30 g / l) + 0.7% agar + 200 mM AS;

[0054] Callus induction and subculture medium: MS basic medium + GLn (146 mg / l) + hydrolyzed lysine (200 mg / l) + 2 mg / l 2,4-D + sucrose (30 g / l) + 0.7% agar + 200 mM AS;

[0055] Screening medium: MS basic medium + GLn (146 mg / l) + hydrolyzed chelating agent (200 mg / l) + 2 mg / l 2,4-D + sucrose (30 g / l) + 0.7% agar + 200 mM AS + 3-5 mg / l Bialaphos + 150 mg / l Timentin;

[0056] Predifferentiation medium: MS basic medium + 0.5 mg / l IAA + 0.5 mg / l 6-BA + sucrose (25 g / l) + 0.7% agar + 150 g / l Timentin;

[0057] Differentiation medium: MS basic medium + 0.5 mg / l IAA + 1.0 mg / l 6-BA + sucrose (25 g / l) + 0.7% agar + 150 g / l Timentin;

[0058] Seedling medium: 1 / 2MS basic medium + 0.5mg / l IAA + 0.2mg / l 6-BA + sucrose (20g / l) + 0.7% agar + 1mg / l paclobutrazol;

[0059] Rooting medium: 1 / 2MS basic medium + 0.2 mg / l IAA + sucrose (20 g / l) + 0.7% agar.

[0060] 5. PCR detection of transgenic plants

[0061] DNA extraction of transgenic plants (CTAB mini-extraction method):

[0062] (1) Cut an appropriate amount of leaves from the regenerated plants and place them in a 1.5 ml centrifuge tube, add liquid nitrogen and grind them into powder;

[0063] (2) Add 600 μl of lysis buffer {1.3% CTAB (Hexadecyl TrimethylAmmonium Bromide), 133 mmol / L Tris-HCl pH 8.0, 13 mmol / L EDTA, 0.93 mol / L NaCl, 0.66% PVP3600, 0.18 mol / L β-mercaptoethanol} preheated at 65°C and incubate in a 65°C water bath for 40 min, mixing by inverting 2-3 times;

[0064] (3) Add 800 μl of chloroform: isoamyl alcohol (24:1 v / v) mixture, invert more than 25 times, let stand at room temperature for 5 min, centrifuge at 12,000 rpm for 15 min (25°C), transfer the supernatant to a 1.5 ml centrifuge tube, add 0.6 volume of pre-cooled isopropanol, slowly invert 30 times, mix, let stand for 10 min, centrifuge at 12,000 rpm for 10 min (RT), discard the supernatant, add 1 ml of 75% ethanol to the precipitate for washing, centrifuge at 12,000 rpm for 1 min. Pour off the supernatant and dry under ventilation for 20 min;

[0065] (4) Add 100 μl TE buffer (containing 20 μg / ml RNase A) to dissolve the DNA (4°C, more than 30 min) and store at -20°C.

[0066] PCR detection of transgenic plants:

[0067] In the PCR reaction system, primer 1 and primer 2 are a pair of primers specific to the GUS gene.

[0068] GUS-QC-F: 5'-ATGTTACGTCCTGTAGAAACCCCAA-3'

[0069] GUS-QC-R: 5'-TCATTGTTTGCCTCCCTGCTGC-3'

[0070] The PCR reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 sec, Tm value annealing for 45 sec, extension at 72°C for 2 min, 30 cycles; and extension at 72°C for 10 min.

[0071] Table 2. PCR reaction system for identifying transgenic plants (20 μl)

[0072]

[0073] The results are as follows Figure 2As shown, lane 1 is a positive control using OsGASR6Promoter-GUS as a template; lanes 2, 3, 5, and 6 are transgenic positive plants; lane 7 is a transgenic negative plant; and lane 8 is a negative plant control.

[0074] 6. GUS staining of transgenic rice:

[0075] The roots, stems, leaves and other tissues and organs of the isolated positive transgenic rice were placed in a 1.5 ml centrifuge tube, and an appropriate amount of GUS staining solution was added. The tubes were incubated at 37°C for 16-24 hours (the specific time was based on sufficient staining), and then decolorized and stored with 70% ethanol.

[0076] The results are as follows Figure 3 As shown, GUS gene expression was detected strongly in endosperm tissue, but no GUS gene expression was observed in Nipponbare endosperm.

[0077] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of the present invention is defined by the attached claims and their equivalents.

Claims

1. A rice endosperm-specific expression promoter, characterized in that: The promoter is composed of the nucleotide sequence shown in SEQ ID No. 1 or is a nucleotide sequence derived from the nucleotide shown in SEQ ID No. 1 by replacing, deleting or adding one or more bases, and is capable of specifically inducing the specific expression of foreign genes in rice endosperm.

2. A rice endosperm-specific expression promoter according to claim 1, characterized in that: An expression cassette comprising the promoter, wherein the expression cassette is capable of causing a target gene connected to the expression cassette to be specifically expressed in rice endosperm; The recombinant vector comprising the promoter can make the target gene connected in the vector specifically expressed in rice endosperm.

3. A rice endosperm-specific expression promoter according to claim 2, characterized in that: A recombinant cell comprising the expression cassette or the recombinant vector, wherein the recombinant cell is selected from a microbial cell or a fungal cell.

4. A rice endosperm-specific expression promoter according to claim 3, characterized in that: The recombinant cell is an Escherichia coli cell or an Agrobacterium cell.

5. A method for breeding rice varieties, the method comprising connecting an exogenous functional gene for improving rice traits to the expression cassette and the recombinant vector of claim 2, so that the exogenous functional gene is expressed under the control of the promoter of claim 1, and then transfecting the expression cassette or the recombinant vector connected with the exogenous gene into a rice plant, so that the exogenous functional gene is specifically expressed in the rice endosperm, and then screening transgenic rice having the function of the exogenous functional gene, thereby improving the rice traits.

6. Application of the rice endosperm-specific expression promoter shown in SEQ ID No. 1 in breeding rice varieties, wherein: In the application, the exogenous functional gene for improving rice traits is connected to an expression cassette containing the promoter shown in SEQ ID No.1 or a recombinant vector containing the promoter shown in SEQ ID No.1, so that the exogenous functional gene is expressed under the control of the promoter shown in SEQ ID No.1, and then the expression cassette or the recombinant vector connected with the exogenous gene is transfected into rice plants, so that the exogenous functional gene is specifically expressed in rice endosperm, and then transgenic rice having the function of the exogenous functional gene is screened, thereby playing a role in improving rice traits.

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