Rice os04g0650800 gene promoter and application thereof

Abstract

This invention discloses the promoter of the rice Os04g0650800 gene and its applications, belonging to the field of plant biotechnology. By cloning the promoter sequence of the Os04g0650800 gene, a recombinant expression vector pPGDH-YFP driving the yellow fluorescent protein gene was successfully constructed. Positive rice plants were screened by infecting Nipponbare rice with Agrobacterium. The T0 generation of positive rice seedlings were inoculated with Xanthomonas oryzae pv. oryzae. Xoo Using a confocal microscope, it was discovered that... Xoo Infection of positive rice plants can induce YFP expression. Genetic engineering of rice using this promoter, such as regulating the efficient expression of target genes through this promoter, will yield positive results. Xoo Infected rice plants; if this promoter is used to drive the expression of disease resistance-related genes in rice, rice plants resistant to bacterial blight will be obtained.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to the rice Os04g0650800 gene promoter and its applications. Background Technology

[0002] Rice is one of the most important food crops for humankind, with a long history of cultivation and consumption in my country. Statistics show that rice cultivation area accounts for approximately 35% of the total grain crop cultivation area in my country, and its yield accounts for 30% of the country's total grain output. Rice bacterial blight, caused by the pathogenic strain *Xanthomonas oryzae* pv. oryzae (Xoo), is a common and important disease in rice cultivation worldwide. Due to the use of resistant varieties, rice bacterial blight had disappeared in my country for a period of time. However, in recent years, the disease has shown a trend of increasing severity year after year, especially in some localized areas. The most economical and effective means of controlling rice bacterial blight is to utilize the rice's own disease resistance.

[0003] Previous analysis of extensive gene chip data from rice infected with Xoo (bacterial blight) and Mor (blast fungus) revealed that the rice Os04g0650800 gene showed a strong upregulation in response to Xoo and Mor infection. Based on this result, further research showed that the rice Os04g0650800 gene also exhibits a dramatic upregulation in response to Xoo induction. Previous studies have reported that the protein encoded by its homologous gene is the first key enzyme in serine synthesis in plants, and serine is an important intermediate product in cellular metabolism, closely related to plant stress resistance.

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

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

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

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

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

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

[0010] Another objective of this invention is to overcome the shortcomings of the prior art and provide a recombinant vector containing the promoter of the rice Os04g0650800 gene.

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

[0012] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of the rice Os04g0650800 gene promoter in transgenic rice in response to bacterial blight-induced expression of the target gene.

[0013] Beneficial effects of this invention:

[0014] (1) By cloning the Os04g0650800 promoter sequence, the recombinant vector pPGDH-YFP driven by the promoter was successfully constructed. Positive plants were screened by infecting Nipponbare rice with Agrobacterium tumefaciens. The leaves of the T0 generation positive plants were inoculated with Xoo. The plant leaves were observed by confocal microscopy. It was found that Xoo infection of positive plants could induce YFP expression. YFP expression means that the YFP-related tag sequence in the recombinant vector can be successfully translated into yellow fluorescent protein, and a yellow fluorescent signal can be observed in the rice leaf tissue. This indicates that the promoter sequence is highly induced by Xoo, which proves that the promoter has the expression activity and characteristics of Xoo-induced promoter.

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

[0016] (3) The promoter of this invention is strongly induced by rice bacterial blight. By adding relevant disease resistance genes after the promoter to construct a vector and forming transgenic plants mediated by Agrobacterium, the resistance of rice to rice bacterial blight can be characterized, thereby increasing the quality and yield of rice grains. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0018] Figure 1 This is an electrophoresis image of the Os04g0650800 gene promoter in an embodiment of the present invention; wherein, M: DL5000 maker, 1-2: Os04g0650800 gene promoter.

[0019] Figure 2 This is an electrophoresis image of the Os04g0650800 recombinant gene promoter (containing homologous arms of restriction enzyme sites) in an embodiment of the present invention; wherein, M: DL15000 maker; 1-4: Os04g0650800 recombinant gene promoter.

[0020] Figure 3 This is an electrophoresis image of pPGDH-YFP double enzyme digestion verification in an embodiment of the present invention; where M: DL15000bp Maker; 1-3: pPGDH-YFP.

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

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

[0023] Figure 6 This is an electrophoresis image of a positive plant detected in an embodiment of the present invention, where M: DL2000bp Maker; 1-24: DNA of transgenic plants.

[0024] Figure 7 The values ​​represent the relative expression levels of YFP at 0, 12, 24, and 48 hours after Xoo infection in this embodiment of the invention; where Mock represents a transgenic plant containing the pPGDH-YFP vector that was not infected by Xoo, and Xoo represents a transgenic plant containing the pPGDH-YFP vector that was infected by Xoo.

[0025] Figure 8 This is an image of yellow fluorescence signal observed under a confocal microscope in an embodiment of the present invention. WT is labeled as Nipponbare rice. Because the wavelength of the yellow fluorescence signal is similar to the spontaneous green fluorescence signal of chloroplasts in rice leaves, WT exhibits a weak fluorescence signal in the YFP fluorescence field. Mock is labeled as a transgenic plant containing the pPGDH-YFP vector that has not been infected by Xoo, and Xoo is labeled as a transgenic plant containing the pPGDH-YFP vector that has been infected by Xoo. Detailed Implementation

[0026] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0027] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the 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 phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0029] Example 1

[0030] Rice Os04g0650800 gene promoter cloning

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

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

[0033]

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

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

[0036]

[0037] Total Volume 50.0μL

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

[0039]

[0040] After the PCR reaction was completed, 5 μL of the PCR product was used for 1% agarose gel electrophoresis to check whether the clone bands met the expected size. The electrophoresis results are as follows: Figure 1 As shown, the cloned fragment is approximately 2700 bp in size (SEQ ID No. 1), consistent with the target fragment. The target fragment was recovered from the agarose gel and its sequence was confirmed by sequencing.

[0041] Example 2

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

[0043] Homologous recombination was used to ligate the promoter of the rice Os04g0650800 gene and the pRHEcYFP plasmid. Homologous recombination primers containing BamHI and HindIII restriction sites were designed, and the primer sequences are shown in Table 4.

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

[0045]

[0046] Note: Underlined areas indicate enzyme cleavage sites.

[0047] Using the cloned Os04g0650800 gene promoter as a template, high-fidelity DNA polymerase was used, and primers BamHI-if-21 and HindIII-if-21 were used to add restriction sites and homologous 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 Os04g0650800 gene promoter

[0049]

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

[0051]

[0052] After the PCR reaction was completed, 5 μL of the PCR product was used for 1% agarose gel electrophoresis to check whether the clone bands met the expected size. The electrophoresis results are as follows: Figure 2 As shown, the cloned fragment is approximately 2700 bp in size, consistent with the target fragment. The target fragment was recovered from the agarose gel.

[0053] The vector plasmid pRHEcYFP was double-digested using restriction endonucleases BamHI and Hind III provided by Novizan (not limited to). 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 hour, 1% agarose gel electrophoresis was used to check whether the enzyme digestion was complete, and the linearized vector backbone after enzyme digestion was recovered by gel extraction.

[0057] The linear vector pRHEcYFP (vector digestion backbone) and the promoter fragment (containing restriction sites and homologous arms) prepared in Example 2 were recombined using Novizan (not limited to) 2×Clone Express mixtiure. The recombinase reaction system was prepared in a 1.5 mL centrifuge tube as shown in Table 8.

[0058] Table 8. Recombination Reaction System

[0059]

[0060] After reacting at 16℃ for 1 hour, all cells were transformed into *E. coli* DH5α. Single colonies were then selected for liquid culture. Once the culture became turbid, the recombinant plasmid was extracted and double-digested with restriction endonucleases BamHI and HindIII. Electrophoresis results are shown below. Figure 3 As shown, all enzyme digestion products were correct bands. The correctly identified recombinant plasmid was sequenced and aligned using MegAlign software. The recombinant plasmid sequence showed 100% similarity to the target sequence, indicating successful construction of the plant expression vector pPGDH-YFP (driven by the rice Os04g0650800 gene promoter) for YFP. Figure 4 ).

[0061] Example 3

[0062] Plant recombinant expression vector transformed Agrobacterium EHA105

[0063] The promoter vector pPGDH-YFP was transformed into Agrobacterium EHA105. Competent Agrobacterium cells were placed on ice, and 1 μg of plasmid DNA was added and mixed thoroughly. After standing on ice for 30 min, the cells were cooled in liquid nitrogen for 1 min, and then rapidly transferred to 37°C to thaw. 1 mL of antibiotic-free YM liquid medium was added, and the cells were cultured at 28°C and 230 rpm for 2–4 h. The cells were then collected by centrifugation at 3000 rpm for 2 min, resuspended in 100 μL of antibiotic-free YM liquid medium, and plated onto YM medium containing rifampicin. The cells were cultured at 28°C for 48 h. Single colonies were then picked and inoculated into YM liquid medium containing rifampicin and cultured at 28°C and 230 rpm for 48 h. Results after plasmid extraction, electrophoresis, and sequencing are shown below. 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] Nipponbare rice was transformed using the Agrobacterium-mediated callus inoculation method. Rice callus prepared from dehulled rice seeds was inoculated with Agrobacterium suspension for 20 min, blotted dry with sterile filter paper, and transferred to N6D2C medium for 3 days of dark incubation at 25°C. The callus was then washed 4-5 times with sterile water containing 300 mg / L cephalosporin, blotted dry with sterile filter paper, and transferred to N6D2S1 medium for first-generation selection. Two weeks later, it was transferred to N6D2S2 medium for second-generation selection. Vigorous growth of resistant callus after three generations of selection was collected and transferred to pre-differentiation medium, cultured in a differentiation incubator (12-hour photoperiod, 28°C during the day, 25°C at night) for 7 days. Then, it was transferred to differentiation medium and cultured in a differentiation incubator until regenerated seedlings were produced, thus obtaining transgenic plants expressing YFP driven by the Os04g0650800 gene promoter. DNA was extracted from the leaves of the transgenic plants and analyzed by PCR. The presence of a band of the target size indicates 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. These transgenic plants were cultured to the tillering stage for subsequent XoO infection experiments on rice leaves.

[0068] To determine whether the Os04g0650800 gene promoter is induced by Xoo infection, Xoo infection experiments were conducted on leaves from T0 generation positive rice seedlings. Xoo grown on NA medium was rinsed with 10 mM magnesium chloride solution to prepare a bacterial suspension. The concentration of the Xoo bacterial suspension was measured using a spectrophotometer, and the concentration was adjusted to OD600nm = 0.6 using 10 mM magnesium chloride solution. Sterilized scissors were dipped into the prepared bacterial suspension, and the tips of the rice leaves were obliquely cut off. The inoculated rice plants were observed daily, and samples were taken at regular intervals.

[0069] The expression level of the YFP gene was detected by qRT-PCR and the intensity of the yellow fluorescence signal was observed by confocal microscopy. The results are as follows: Figure 7 , Figure 8 As shown, the Os04g0650800 gene promoter was found to drive YFP expression by Xoo infection.

[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 a strongly pathogen-responsive inducible promoter 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 and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

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

1.

2. The promoter as described in claim 1, characterized in that: The nucleotide sequences of the primers used to amplify the promoter of the 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 as described in claim 1.

4. The recombinant vector as described in claim 3, characterized in that: The recombinant vector was obtained by ligating the promoter of the rice Os04g0650800 gene shown in SEQ ID No.1 with the pRHEcYFP plasmid digested by restriction endonucleases BamHI and HindIII via homologous recombinase.

5. The application of the rice Os04g0650800 gene promoter as described in claim 1 or 2 in the expression of the target gene in transgenic rice in response to bacterial blight induction.

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