Rice Os04g0650800 gene promoter and application thereof

Abstract

The invention discloses a rice Os04g0650800 gene promoter and application thereof, belongs to the technical field of plant biology, and successfully constructs a yellow fluorescent protein gene recombinant expression vector pPGDH-YFP driven by the promoter of the gene by cloning the promoter sequence of the Os04g0650800 gene. Agrobacterium tumefaciens infects Nipponbare rice, and positive rice plants are screened. Xanthomonas oryzae pv.oryzae, Xoo is inoculated to the positive rice seedlings of the generation T0, a confocal microscope is used for observation, and it is found that YFP expression can be induced by infection of the positive rice plants with Xoo. Genetic engineering modification is carried out on rice through the promoter, for example, high-efficiency expression of a target gene is regulated and controlled through the promoter, and a rice plant responding to Xoo infection will be obtained; if the promoter is used for driving the expression of disease-resistant related genes in rice, rice plants resistant to bacterial blight can be obtained.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and in particular relates to a rice Os04g0650800 gene promoter and application thereof. Background Art

[0002] Rice is one of the most important food crops for human beings and has a long history of cultivation and consumption in my country. According to statistics, the rice planting area in my country accounts for about 35% of the planting area of ​​food crops, and the output accounts for 30% of the total domestic grain output. Ricebacterial blight, caused by Xanthomonas oryzae pv.oryzae (Xoo), is a common and important disease in rice cultivation worldwide. Due to the use of disease-resistant varieties, rice blight disappeared in my country for a period of time. However, in recent years, the disease has shown a trend of worsening year by year, especially in some local areas. The most economical and effective means of preventing and controlling rice bacterial blight is to use the disease resistance of rice itself.

[0003] In the early stage, the laboratory analyzed a large number of gene chip data of rice infected by Xanthomonas oryzae (Xoo) and Magnaporthe oryzae (Mor), and found that the rice Os04g0650800 gene strongly responded to Xoo and Mor infection and up-regulated its expression. Based on this result, the laboratory further found that the rice Os04g0650800 gene responded strongly to Xoo induction and up-regulated its expression. Studies have reported that the protein encoded by its homologous gene is the first key enzyme in plant serine synthesis, and serine is an important intermediate product in cell metabolism and is closely related to plant stress resistance.

[0004] However, there are currently no reports on the relationship between the serine biosynthesis pathway and rice disease resistance. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a rice Os04g0650800 gene promoter.

[0008] In order to solve the above technical problems, the present invention provides the following technical solution: a rice Os04g0650800 gene promoter, the nucleotide sequence of the rice Os04g0650800 gene promoter is shown in SEQ ID No.1.

[0009] As a preferred embodiment of the promoter of the present invention, the nucleotide sequences of primers for amplifying the promoter of rice Os04g0650800 gene are shown in SEQ ID No.2 and SEQ ID No.3.

[0010] Another object of the present invention is to overcome the deficiencies in the prior art and provide a recombinant vector containing the rice Os04g0650800 gene promoter.

[0011] As a preferred embodiment of the recombinant vector of the present invention, the recombinant vector is obtained by connecting the rice Os04g0650800 gene promoter shown in SEQ ID No. 1 with the pRHEcYFP plasmid digested with restriction endonucleases BamH I and Hind III through homologous recombinase.

[0012] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of a rice Os04g0650800 gene promoter in inducing the expression of a target gene in transgenic rice in response to bacterial blight pathogen.

[0013] Beneficial effects of the present invention:

[0014] (1) By cloning the Os04g0650800 promoter sequence, the recombinant vector pPGDH-YFP driven by the promoter was successfully constructed. The positive plants were screened by infecting Nipponbare rice with Agrobacterium tumefaciens, and the leaves of the T0 generation positive plants were inoculated with Xoo at the seedling stage. The leaves of the plants were observed under a confocal microscope, and it was found that Xoo infection of the positive plants could induce YFP expression. YFP expression refers to the successful translation of the YFP-related tag sequence in the recombinant vector into yellow fluorescent protein, and yellow fluorescent signals can be observed in rice leaf tissues, indicating that the promoter sequence is highly induced by Xoo, proving that the promoter has the expression activity and characteristics of a Xoo-induced promoter.

[0015] (2) The promoter of the present invention has the expression activity and characteristics of a pathogen (Xoo)-induced promoter, and can be used as a pathogen-induced expression promoter in rice genetic engineering research.

[0016] (3) The promoter of the present invention is strongly induced by rice bacterial blight pathogen. Adding a relevant disease-resistant gene to the promoter to construct a vector and forming a transgenic plant mediated by Agrobacterium can achieve the characterization of rice resistance to rice bacterial blight pathogen, thereby increasing the quality and yield of rice grains. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0018] Figure 1 The electrophoresis diagram of the Os04g0650800 gene promoter in the embodiment of the present invention; wherein, M: DL5000maker, 1-2: Os04g0650800 gene promoter.

[0019] Figure 2 The electrophoresis diagram of the Os04g0650800 recombinant gene promoter (including the homology arms of the restriction site) in the embodiment of the present invention; wherein, M: DL15000 maker; 1-4: Os04g0650800 recombinant gene promoter.

[0020] Figure 3 This is the electrophoresis diagram for double restriction enzyme digestion verification of pPGDH-YFP in the embodiment of the present invention; wherein, M: DL15000bp Maker; 1-3: pPGDH-YFP.

[0021] Figure 4 It is a structural diagram of the plant expression vector pPGDH-YFP in the embodiment of the present invention.

[0022] Figure 5 This is a PCR verification diagram of Agrobacterium colonies containing pPGDH-YFP in the embodiment of the present invention; wherein, M: DL15000bpMaker; 1-2: PCR product of Agrobacterium colonies containing pPGDH-YFP.

[0023] Figure 6 This is the electrophoresis diagram of positive plant detection in the embodiment of the present invention, wherein M: DL2000bp Maker; 1-24: transgenic plant DNA.

[0024] Figure 7 It is the relative expression level of YFP at 0, 12, 24 and 48h of Xoo infection in the embodiment of the present invention; wherein, Mock is a transgenic plant containing the pPGDH-YFP vector but not infected by Xoo, and the marked Xoo is a transgenic plant containing the pPGDH-YFP vector and infected by Xoo.

[0025] Figure 8 This is an observation diagram of the yellow fluorescence signal under a confocal microscope in an embodiment of the present invention, wherein the labeled WT is Nipponbare rice. Since the wavelength of the yellow fluorescence signal is similar to the spontaneous green fluorescence signal of the chloroplasts in the rice leaves, WT presents a weak fluorescence signal in the YFP fluorescence field. The labeled Mock is a transgenic plant containing the pPGDH-YFP vector that is not infected by Xoo, and the labeled Xoo is a transgenic plant containing the pPGDH-YFP vector that is infected by Xoo. DETAILED DESCRIPTION

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0027] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0028] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0029] Example 1

[0030] Cloning of Rice Os04g0650800 Gene Promoter

[0031] The promoter sequence 3000 bp upstream of the translation start site (ATG) of rice Os04g0650800 gene was searched on NCBI, and promoter primers pPGDH.F / R were designed. The designed primer sequences are shown in Table 1.

[0032] Table 1. Primer sequences for amplification of the promoter of Os04g0650800 gene

[0033]

[0034] Using the genomic DNA of rice Nipponbare as a template, primers pPGDH.F and pPGDH.R and high-fidelity DNA polymerase were used to amplify the promoter sequence of 3000 bp upstream of the Os04g0650800 gene. The PCR reaction system and PCR reaction conditions used for amplification are shown in Tables 2 and 3.

[0035] Table 2. Reaction system for amplifying the promoter sequence of Os04g0650800 gene

[0036]

[0037] Total Volume 50.0μL

[0038] Table 3. PCR reaction procedure for amplifying the promoter of Os04g0650800 gene

[0039]

[0040] After the PCR reaction is completed, take 5 μL of PCR product and use 1% agarose gel electrophoresis to check whether the cloned band meets the expected size. The electrophoresis results are as follows: Figure 1 As shown, the size of the cloned fragment is about 2700 bp (SEQ ID No. 1), which is consistent with the target fragment. The target fragment was recovered from agarose gel and sequenced and confirmed.

[0041] Example 2

[0042] Construction of plant expression vector driven by promoter YFP:

[0043] The rice Os04g0650800 gene promoter and the pRHEcYFP plasmid were connected by homologous recombination method. Homologous recombination primers containing BamH I and HindIII restriction sites were designed. The designed primer sequences are shown in Table 4.

[0044] Table 4. Primer sequences for promoter homologous recombination amplification

[0045]

[0046] Note: Underline indicates restriction site.

[0047] The cloned Os04g0650800 gene promoter was used as a template, a high-fidelity DNA polymerase was used, and primers BamHI-if-21 and HindIII-if-21 were used to add restriction sites and homology arms to the Os04g0650800 gene promoter. The PCR reaction system and PCR reaction conditions for amplifying the promoter are shown in Tables 5 and 6.

[0048] Table 5. Reaction system for amplifying the promoter of Os04g0650800 gene

[0049]

[0050] Table 6. PCR reaction procedure for amplifying the promoter of Os04g0650800 gene

[0051]

[0052] After the PCR reaction is completed, take 5 μL of PCR product and use 1% agarose gel electrophoresis to check whether the cloned band meets the expected size. The electrophoresis results are as follows: Figure 2 As shown, the size of the cloned fragment is about 2700 bp, which is consistent with the target fragment. The target fragment was recovered from the agarose gel.

[0053] The vector plasmid pRHEcYFP was double-digested with restriction endonucleases BamHI and Hind III provided by Novazonics (not limited to), and the double-digestion system is shown in Table 7.

[0054] Table 7. Double enzyme digestion system

[0055]

[0056] After reacting at 37°C for 1 h, 1% agarose gel electrophoresis was used to detect whether the enzyme digestion was complete, and the linearized vector backbone after digestion was recovered by gel electrophoresis.

[0057] The linear vector pRHEcYFP (vector enzyme cutting backbone) after restriction digestion and the promoter fragment (including restriction sites and homology arms) prepared in Example 2 were recombined using 2×Clone Express mixtiure produced by Novozymes (not limited to), and the following recombinase reaction system (as shown in Table 8) was configured in a 1.5 mL centrifuge tube.

[0058] Table 8. Recombination reaction system

[0059]

[0060] After reacting at 16℃ for 1h, all E. coli DH5α were transformed. Then single clone plaques were selected for liquid culture. After the bacterial solution became turbid, the recombinant plasmid was extracted and double enzyme digestion was performed using restriction endonucleases BamHI and HindIII. The electrophoresis results were as follows: Figure 3 As shown, the enzyme digestion products are all correct bands. The correct recombinant plasmid was sequenced and aligned using MegAlign software. The similarity between the recombinant plasmid sequence and the target sequence reached 100%, indicating that the plant expression vector pPGDH-YFP ( Figure 4 ).

[0061] Example 3

[0062] Plant recombinant expression vector transformed into Agrobacterium EHA105

[0063] The promoter vector pPGDH-YFP was transformed into Agrobacterium EHA105. The competent Agrobacterium was placed on ice, 1 μg of plasmid DNA was added and mixed thoroughly. After standing on ice for 30 minutes, it was placed in liquid nitrogen for cooling for 1 minute, and then quickly transferred to 37°C to be melted. 1 mL of antibiotic-free YM liquid medium was added and cultured at 28°C 230 r / m for 2-4 hours. After that, the bacteria were collected by centrifugation at 3000 r / min for 2 minutes, resuspended with 100 μL of antibiotic-free YM liquid medium, and then spread on YM medium containing rifampicin and cultured at 28°C for 48 hours. After that, a single colony was picked and inoculated into YM liquid medium containing rifampicin and cultured at 28°C 230 r / m for 48 hours. After plasmid extraction, electrophoresis and sequencing detection, the results are shown in Figure 5 , it can be confirmed that the promoter vector pPGDH-YFP has been successfully transformed into Agrobacterium EHA105.

[0064] Example 4

[0065] Agrobacterium infection of rice callus:

[0066] The callus tissue was transformed into Nipponbare rice by Agrobacterium infection. The rice callus prepared from hulled rice seeds was inoculated with Agrobacterium suspension for 20 min, dried with sterile filter paper, and then transferred to N 6 D 2 C medium at 25°C for 3 days, wash the callus 4-5 times with sterile water containing 300 mg / L cephalosporin, dry it with sterile filter paper and transfer it to N 6 D 2 S 1 Two weeks later, the cells were transferred to N 6 D 2 S 2 The second generation was screened on the culture medium. The resistant callus that had grown vigorously after three generations of screening was taken out and transferred to the pre-differentiation culture medium, and cultured in a differentiation incubator (12-hour photoperiod, 28°C during the day and 25°C at night) for 7 days; then transferred to the differentiation culture medium and cultured in the differentiation incubator until regenerated seedlings were produced, thereby obtaining transgenic plants with Os04g06508()() gene promoter driving YFP expression. DNA was extracted from the leaves of the transgenic plants and tested by PCR. The appearance of the target size band was a positive plant.

[0067] like Figure 6 As shown, a total of 23 transgenic plants expressing YFP driven by the Os04g0650800 gene promoter were obtained. The transgenic plants were cultured to the tillering stage and used for subsequent XoO infection rice leaf experiments.

[0068] In order to clarify whether the Os04g0650800 gene promoter is induced by Xoo infection, T 0The Xoo infection experiment was conducted on the leaves of positive rice plants at the seedling stage. The Xoo grown in NA medium was rinsed with 10mM magnesium chloride solution to prepare a bacterial solution. The concentration of the Xoo bacterial solution was measured using a spectrophotometer, and the concentration of the bacterial solution was adjusted to OD600nm=0.6 with 10mM magnesium chloride solution. Sterilized scissors were dipped into the prepared bacterial solution and the tip of the rice leaf was cut off at an angle. The inoculated rice plants were observed daily and samples were taken at regular intervals.

[0069] The expression of YFP gene was detected by qRT-PCR experiment and the intensity of yellow fluorescence signal was observed by confocal microscope. Figure 7 , Figure 8 As shown, the promoter of the Os04g0650800 gene was found to be induced by Xoo infection and drive YFP expression.

[0070] Therefore, the rice Os04g0650800 promoter of the present invention has the expression activity and characteristics of an inducible promoter, and can be used as an inducible promoter that strongly responds to pathogen infection and is applied in rice genetic engineering research.

[0071] SEQ ID No.1

[0072] >AP014960.1:33163525-33167059Oryza sativa Japonica Group DNA,chromosome 4,cultivar:Nipponbare

[0073]

[0074] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A rice Os04g0650800 gene promoter, characterized in that: The nucleotide sequence of the rice Os04g0650800 gene promoter is shown in SEQ ID No.

1.

2. The promoter according to claim 1, characterized in that: The nucleotide sequences of the primers for amplifying the promoter of rice Os04g0650800 gene are shown in SEQ ID No.2 and SEQ ID No.

3.

3. A recombinant vector containing the rice Os04g0650800 gene promoter according to claim 1.

4. The recombinant vector according to claim 3, characterized in that: The recombinant vector is obtained by connecting the rice Os04g0650800 gene promoter shown in SEQ ID No. 1 with the pRHEcYFP plasmid cut with restriction endonucleases BamH I and Hind III through homologous recombinase.

5. Use of the rice Os04g0650800 gene promoter according to claim 1 or 2 in inducing the expression of a target gene in transgenic rice in response to bacterial blight pathogen.

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