Coding gene of benzyl benzoate 2-hydroxylase participating in rice PAL-dependent salicylic acid synthesis route and application of coding gene

Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses a coding gene of benzyl benzoate 2-hydroxylase participating in a rice PAL-dependent salicylic acid synthesis pathway and application of the coding gene, and the nucleotide sequence of OSD3 is SEQ ID NO: 1; the nucleotide sequence of a coding protein region is SEQ ID NO: 2; the amino acid sequence of the coded protein is SEQ ID NO: 3; according to the invention, an OSD3 gene is knocked out through CRISPR / Cas9 under the background of Zhonghua 11, a mutant osd3 of which the SA of a rice leaf is reduced to 20% of that of a wild type is obtained, and an OSD3 overexpression plant is created by constructing an OSD3-pMDC43 overexpression vector.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of plant genetic engineering technology, and particularly relates to a gene encoding benzyl benzoate 2-hydroxylase that participates in PAL-dependent salicylic acid synthesis in rice, regulates the content of endogenous salicylic acid and disease resistance in rice, and its application. Background Technology

[0002] Salicylic acid (SA) is an important plant hormone that participates directly or through crosstalk with other hormones in plant growth and development, as well as in resistance to external stresses. SA is one of many phenolic compounds synthesized by plants, containing hydroxyl groups or their derivatives, and participates in multiple life processes, including seed germination, cellular respiration, cell growth, stomatal opening and closing, flowering, and senescence. Furthermore, SA is a key signal for regulating heat production and disease resistance.

[0003] Plant ammonia synthesis primarily occurs through two secondary metabolic pathways originating from shikimic acid: the isochorismate synthase (ICS) pathway and the phenylalanine ammonia-lyase (PAL) pathway. SA signaling pathways play a crucial regulatory role in plant disease resistance and defense responses. In rice, ICS1 mainly participates in chloroquinone biosynthesis but not in SA biosynthesis. Conversely, the biochemical pathway of PAL in rice SA synthesis still requires further elucidation. However, the loss of function of the gene AIM1 (ABNORMALINFLORESCENCE MERISTEM 1), which is involved in the PAL-dependent SA synthesis pathway, reduces the endogenous SA content in rice to approximately 10% of the wild-type level, indicating that the PAL pathway is the main pathway for SA biosynthesis in rice. Furthermore, it has been reported that benzoic acid 2-hydroxylase (BA2H) in tobacco can catalyze the production of salicylic acid (SA) from benzoic acid (BA), a cytochrome P450 monooxygenase with a size of 160 kDa. In barley inoculated with *Pseudomonas syringae* pv. *syringae*, endogenous SA levels increased during pathogen infection. However, in rice, SA levels did not increase during infection with *Pseudomonas syringae*, *Magnaporthe grisea*, or *Rhizoctonia solani*. Salicylate hydroxylasase (NahG) can block the accumulation of SA in vivo. In NahG transgenic Arabidopsis, SA levels increased significantly after invasion by the Pto DC3000 pathogen, indicating that SA plays an important role in Arabidopsis disease resistance. Rice expressing NahG showed a significant increase in ROS content after invasion by *Bacillus oryzae*, indicating that decreased SA leads to increased susceptibility to *Bacillus oryzae*. Mutations in OsS5h increased SA content and resistance to *Bacterium oryzae*, while overexpression of OsS5h reduced endogenous SA and increased susceptibility to *Bacterium oryzae*, demonstrating a positive correlation between SA content and resistance to *Bacterium oryzae*. Recent studies suggest that SA may also mediate defense signaling in wheat. In summary, these results indicate that, at least in some monocotyledonous plants, SA is a signal of the defense response, and endogenous SA content is positively correlated with plant disease resistance.

[0004] Rice is the staple crop for more than half the world's population and a monocotyledonous model plant with a high basal salicylic acid (SA) level. Rice production is frequently affected by various field diseases, the most destructive of which is bacterial bacterial blight caused by Xanthomonas oryzae. Bacterial blight can affect rice growth, development, and reproduction, causing severe yield losses of up to 50%. It is estimated that with population growth, global food production will need to increase by at least 50% by 2050 to meet global demand. Therefore, developing high-yielding and stable-yielding new crop varieties is of great significance for ensuring food security.

[0005] Rice cannot accumulate large amounts of saprophytic acid (SA) after pathogen invasion, which prevents the plant from effectively utilizing SA to resist external pathogen invasion. In plant disease resistance improvement, any increase in resistance is often accompanied by changes in growth and development and a decrease in yield. Therefore, it is crucial to improve rice's resistance to pathogens while ensuring stable yields.

[0006] Current technologies still have significant limitations in elucidating the PAL-dependent SA biosynthesis pathway in rice. Currently, only a few key enzymes, such as AIM1, have been clearly located in this pathway, while the remaining functional members involved have not been systematically identified or functionally resolved. Although research as early as thirty years ago reported the existence of a class of cytochrome P450 monooxygenases, BA2H, in tobacco that catalyzes the hydroxylation of benzoic acid to SA, the specific gene encoding this enzyme has not yet been successfully cloned. Therefore, the enzymatic composition of the PAL-dependent SA synthesis pathway remains incomplete, limiting a deeper understanding of this metabolic network. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a gene involved in rice PAL-dependent salicylic acid synthesis, OSD3, that regulates the endogenous salicylic acid content and disease resistance in rice, and its application.

[0008] This invention is achieved by providing a gene involved in PAL-dependent salicylic acid synthesis in rice, the gene being OSD3, and the nucleotide sequence of the gene being SEQ ID NO: 1.

[0009] Furthermore, the gene involved in rice PAL-dependent salicylic acid synthesis further includes a DNA sequence with more than 90% homology to the DNA sequence shown in SEQ ID NO: 1;

[0010] The salicylic acid synthesis gene involved in rice PAL-dependent synthesis further includes one or more alleles or gene derivatives with base alterations produced by base substitution, deletion, or addition.

[0011] The gene involved in rice PAL-dependent salicylic acid synthesis further includes a DNA molecule capable of hybridizing with the DNA sequence shown in SEQ ID NO: 1 under stringent conditions.

[0012] Furthermore, the protein encoded by the gene involved in rice PAL-dependent salicylic acid synthesis has the amino acid sequence of SEQ ID NO: 3.

[0013] Furthermore, the protein encoded by the gene involved in the synthesis of SA in rice leaves and the regulation of rice tolerance to bacterial blight is characterized in that the encoded protein nucleotide sequence is SEQ ID NO: 2.

[0014] Furthermore, the protein encoded by the gene involved in rice PAL-dependent salicylic acid synthesis is characterized by:

[0015] The encoded protein further includes an amino acid sequence that has more than 90% homology with the amino acid sequence shown in SEQ ID NO: 3;

[0016] The encoded protein further includes proteins and protein analogs that have undergone one or more amino acid alterations based on the amino acid sequence shown in SEQ ID NO: 3 through amino acid substitution, deletion, or addition.

[0017] The encoded protein further includes a fusion protein formed by linking the protein shown in SEQ ID NO: 3 with other tagged proteins.

[0018] Another objective of this invention is to provide a method for catalyzing the production of benzyl salicylate (BS) using the aforementioned gene involved in rice PAL-dependent salicylate synthesis with benzyl benzoate (BB) as a substrate.

[0019] Another objective of this invention is to provide a plant genetic transformation vector constructed using the aforementioned gene involved in rice PAL-dependent SA synthesis, wherein the plant genetic transformation vector includes an upregulation expression vector; the upregulation expression vector comprises a fusion expression vector constructed using a recombinant promoter or an organ-specific promoter.

[0020] The plant genetic transformation vector further comprises: a DNA sequence containing the DNA sequence shown in SEQ ID NO: 1, or a DNA sequence having more than 90% homology with the DNA sequence shown in SEQ ID NO: 1, or an allele or gene derivative with one or more base changes generated by base substitution, deletion, or addition of the nucleotide sequence shown in SEQ ID NO: 1, or a DNA molecule that can hybridize with the DNA sequence shown in SEQ ID NO: 1 under strict conditions.

[0021] Furthermore, the plant genetic transformation vector further includes a gene expression downregulation vector, which downregulates the expression of the gene shown in SEQ ID NO: 3 through CRISPR / Cas9 technology, T-DNA insertion technology, EMS mutagenesis, RNA interference technology, and gene silencing technology.

[0022] The plant genetic transformation vector upregulates or downregulates the expression level or activity of the protein shown in SEQ ID NO: 3 using relevant protein regulators.

[0023] Another objective of this invention is to provide a recombinant bacterium, plant callus, and cell line expressed by the aforementioned plant genetic transformation vector.

[0024] Another objective of this invention is to provide a method for regulating the resistance of rice leaves to bacterial blight. Knocking out the OSD3 gene leads to a decrease in the salinity (SA) of rice leaves, making them more susceptible to bacterial blight. Overexpression of the OSD3 gene increases the resistance of rice to bacterial blight.

[0025] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0026] This invention provides the rice SA synthesis gene OSD3 and its application, illustrating that rice OSD3 can regulate rice SA synthesis. Figures 1-3 It catalyzes the formation of BS using BB as a substrate. Figure 4 ); Create overexpression materials to increase rice disease resistance ( Figure 5 ). Attached Figure Description

[0027] Figure 1 To identify the gene OSD3 involved in the SA biosynthesis pathway in rice: A, Location of the Cas9-induced mutagenesis target motif (red arrow) within the OSD3 gene coding region (orange box); B, Sanger sequencing peak diagram of the osd3 mutant; C, Encoding amino acid sequence information of wild-type and osd3 mutant; D, Phenotype of wild-type and osd3 mutant plants at the heading stage. Scale bar: 10 cm; E, SA and SAG content in leaves of 14-day-old wild-type and osd3 mutant plants; F, Phenotype and lesion length statistics of wild-type and osd3 mutant plants 14 days after Xoo inoculation. Scale bar: 3 cm. Statistical analysis was performed using a two-tailed student's t-test. **p < 0.01, ***p < 0.001. Data are expressed as mean ± standard deviation, n = 4 (E) and n = 20 (F) biologically independent samples.

[0028] Figure 2The expression patterns of OSD3 are as follows: A. OSD3 gene expression in the roots, stems, leaves, and panicles of 70-day-old Zhonghua 11 plants. The gene expression levels were normalized to the internal reference gene OsUBQ5. B. OSD3 gene expression in the leaves of Zhonghua 11 plants inoculated with Xoo at 0, 12, 24, 48, and 72 hours. The OSD3 gene expression levels after Xoo inoculation were normalized to the corresponding control groups. The MOCK group was rice wild-type ZH11 infected with water, and the P6 treated group was infected with OD. 600 P6 (Xoo) bacterial suspension with a concentration of 1.0 was used to infect wild-type rice ZH11.

[0029] Statistical analysis was performed using the two-tailed student's t-test. *p < 0.05, **p < 0.01. Data are expressed as mean ± standard deviation, with n = 3 (A and B) biologically independent samples.

[0030] Figure 3 Subcellular localization of OSD3: Subcellular localization of OSD3 in rice protoplasts. mCherry-HDEL was used as an endoplasmic reticulum localization marker. Scale bar: 10 μm.

[0031] Figure 4 Identification of OSD3 enzyme activity products: (A) Purification of recombinant protein Flag-OSD3 from yeast strain WAT11. Lane 1, molecular weight standard label; lanes 2 and 3, yeast microsomes with empty vector (EV) or Flag-OSD3, respectively. Expression of recombinant protein was detected by immunofluorescence; (B) In vitro MRM (multiple reaction monitoring) spectra of BA and SA in OSD3 products with BA as substrate. No SA was formed in the OSD3 product, indicating that the Flag-OSD3 recombinant protein cannot catalyze the formation of SA with BA as substrate; (C) Biochemical reaction catalyzed by OSD3 in vitro; (D) Comparison of spectra of OSD3 in vitro enzyme activity reaction products and BS standard; (E) DAD chromatograms of OSD3-catalyzed BB formation products and BS standard. The retention time of the enzyme product was the same as that of the BS standard; (F) Structure and putative fragments of the OSD3 product; (G) MS fragmentation pattern of the OSD3-catalyzed BB product was the same as that of the BS standard; (H) Effect of pH on Flag-OSD3 enzyme activity; (I) Effect of temperature on Flag-OSD3 enzyme activity; (J) Reaction of Flag-OSD3 with different substrates including methyl benzoate, ethyl benzoate, phenyl benzoate, phenylethyl benzoate, benzyl benzoate, and benzoic acid. Data are presented as mean ± standard deviation, n = 3 (H–J) biologically independent samples.

[0032] Figure 5 To demonstrate that OSD3 gene overexpression enhances disease resistance in rice: A, OSD3-pMDC43 / ZH11 overexpression material expression level at the tillering stage in the field. B, Xoo phenotype in field-grown plants of Zhonghua 11 and OSD3-pMDC43 / ZH11 after inoculation at the tillering stage. Scale bar: 3 cm; C, Statistical analysis of lesion length on leaves of Zhonghua 11 and three OSD3 overexpression lines 14 days after inoculation with Xoo pathogen; Statistical analysis was performed using a two-tailed Student's t-test. ***p < 0.001, NS, no statistical significance. Data are expressed as mean ± standard deviation, n = 3 (A), and n = 20 (C) independent biological replicates. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] This invention provides a gene involved in PAL-dependent salicylic acid synthesis in rice, which is OSD3 and has the nucleotide sequence SEQ ID NO: 1.

[0035] The gene involved in rice PAL-dependent salicylic acid synthesis further includes a DNA sequence with more than 90% homology to the DNA sequence shown in SEQ ID NO: 1;

[0036] The salicylic acid synthesis gene involved in rice PAL-dependent synthesis further includes one or more alleles or gene derivatives with base alterations produced by base substitution, deletion, or addition.

[0037] The gene involved in rice PAL-dependent salicylic acid synthesis further includes a DNA molecule capable of hybridizing with the DNA sequence shown in SEQ ID NO: 1 under stringent conditions.

[0038] The protein encoded by the gene involved in rice PAL-dependent salicylic acid synthesis has the amino acid sequence SEQ ID NO: 3.

[0039] The protein encoded by the gene involved in the synthesis of SA in rice leaves and the regulation of rice tolerance to bacterial blight is characterized in that the encoded protein nucleotide sequence is SEQ ID NO: 2.

[0040] The protein encoded by the gene involved in rice PAL-dependent salicylic acid synthesis is characterized by:

[0041] The encoded protein further includes an amino acid sequence that has more than 90% homology with the amino acid sequence shown in SEQ ID NO: 3;

[0042] The encoded protein further includes proteins and protein analogs that have undergone one or more amino acid alterations based on the amino acid sequence shown in SEQ ID NO: 3 through amino acid substitution, deletion, or addition.

[0043] The encoded protein further includes a fusion protein formed by linking the protein shown in SEQ ID NO: 3 with other tagged proteins.

[0044] This invention provides a method for using the rice PAL-dependent salicylic acid synthesis gene to catalyze the production of benzyl salicylate using benzyl benzoate as a substrate.

[0045] This invention provides a plant genetic transformation vector constructed using the aforementioned gene involved in rice PAL-dependent salicylic acid synthesis. The plant genetic transformation vector includes an upregulation expression vector; the upregulation expression vector comprises a fusion expression vector constructed using a recombinant promoter or an organ-specific promoter.

[0046] The plant genetic transformation vector further comprises: a DNA sequence containing the DNA sequence shown in SEQ ID NO: 1, or a DNA sequence having more than 90% homology with the DNA sequence shown in SEQ ID NO: 1, or an allele or gene derivative with one or more base changes generated by base substitution, deletion, or addition of the nucleotide sequence shown in SEQ ID NO: 1, or a DNA molecule that can hybridize with the DNA sequence shown in SEQ ID NO: 1 under strict conditions.

[0047] The plant genetic transformation vector further includes a gene expression downregulation vector, which downregulates the expression of the gene shown in SEQ ID NO: 3 through CRISPR / Cas9 technology, T-DNA insertion technology, EMS mutagenesis, RNA interference technology, and gene silencing technology.

[0048] The plant genetic transformation vector upregulates or downregulates the expression level or activity of the protein shown in SEQ ID NO: 3 using relevant protein regulators.

[0049] This invention provides a recombinant bacterium, plant callus, and cell line expressed using the plant genetic transformation vector described above.

[0050] This invention provides a method for regulating the resistance of rice leaves to bacterial blight. Knocking out the OSD3 gene leads to a decrease in the salinity (SA) of rice leaves, making them more susceptible to bacterial blight. Overexpression of the OSD3 gene increases the resistance of rice to bacterial blight.

[0051] Inoculation experiments with *Bacillus cereus* showed that the osd3 mutant was more susceptible to *Bacillus cereus*, and the OSD3 overexpression material exhibited stronger resistance. The OSD3-pESC-URA vector was constructed, and its in vitro enzyme activity was verified. In vitro enzyme activity results indicated that OSD3 can catalyze the direct conversion of BB to BS. Based on the fact that OSD3 loss of function leads to a significant decrease in SA content in rice, resulting in greater sensitivity to *Bacillus cereus*, and that OSD3 overexpression provides strong resistance to *Bacillus cereus*, this study aims to elucidate the function of OSD3 in the rice SA synthesis pathway and explore the application of this gene in crop disease resistance improvement.

[0052] Example 1: Identification of OSD3 gene involved in the rice SA biosynthesis pathway

[0053] To elucidate the PAL pathway in SA biosynthesis, we used CRISPR / Cas9 technology to knock out the Os09g0441400 gene and validate its function. Os09g0441400 was predicted to be cytochrome P450 71A1 and was named OSD3 (Oryza Sativa SA-Deficient gene 3). This gene is 2477 bp in length, contains an intron, and encodes a protein composed of 514 amino acids. The protein was knocked out using CRISPR / Cas9 technology, yielding three homozygous lines. The osd3-1 line had a T base inserted at position 75 of the ATG, resulting in a frameshift mutation at amino acid position 25 and premature termination at amino acid position 29. The osd3-2 line had a 30-base deletion at position 50 of the ATG, resulting in a 10-amino acid deletion in its encoded protein. The osd3-3 line had an A base inserted at position 75 of the ATG, resulting in a frameshift mutation at amino acid position 25 and premature termination at amino acid position 29. Figure 1 A–C). The osd3 mutant showed no significant difference from WT during growth. Figure 1 D), the determination of endogenous SA and SAG content in the leaves of 14-day-old plants showed that both SA and SAG content decreased to less than 20% of that in wild type. Figure 1 E), indicating that the OSD3 gene plays a crucial role in the synthesis of SA in rice. Subsequently, Xoo inoculation experiments were conducted in the field at the tillering stage on wild-type and osd3 mutants. Disease incidence was statistically analyzed 14 days after inoculation. The results showed that the lesion length of the osd3 mutant was significantly increased compared to the wild type, with increases of 59.4%, 44.3%, and 30.5% in three independent lines compared to the wild type, respectively. Figure 1 F). The above results indicate that OSD3 is involved in the synthesis of SA in rice, and the deletion of the OSD3 gene leads to a significant decrease in the SA content of rice, making it more susceptible to pathogens.

[0054] Example 2: OSD3 gene expression and inducible expression

[0055] The transcriptional expression pattern and regulation of genes are crucial to their biological functions. To investigate the transcriptional expression pattern of the OSD3 gene, qRT-PCR was used to detect the transcriptional levels in the roots, stems, leaves, and panicles of wild-type ZH11 rice at the heading stage. The results showed that the OSD3 gene was expressed in all tissues of rice. Figure 2 A) Because rice is more sensitive to Xoo after SA deficiency ( Figure 1 Therefore, samples were taken from ZH11 within 72 hours after inoculation to detect the induction of the OSD3 gene by Xoo. The results showed that OSD3 was induced by Xoo 24 hours after inoculation, with the expression level increasing by 13.9 times. Figure 2 B). The above results indicate that SA synthesized with the participation of OSD3 plays an important role in the disease resistance of rice.

[0056] Example 3: Subcellular localization of OSD3 protein

[0057] Subcellular localization of genes is often closely related to their biological functions. To study the subcellular localization of the OSD3 protein, an OSD3 protein vector fused to the N-terminus of the 35S promoter with GFP protein was constructed. Transformation was performed using rice protoplasts, and the subcellular localization was observed. The GFP-OSD3 fusion protein co-localized with the endoplasmic reticulum marker protein mCHerry-HDEL, indicating that OSD3 is located on the endoplasmic reticulum (Figure 3).

[0058] Example 4: OSD3 catalyzes the production of BS from BB substrate instead of SA from BA substrate.

[0059] To verify the biological function of OSD3, the OSD3-pESC-URA vector was constructed, and the Flag-OSD3 fusion protein was purified using yeast strain WAT11. The purified Flag-OSD3 fusion protein was approximately 58.1 kDa in size. Figure 4 A). It has been reported that a BA2H enzyme exists in tobacco that catalyzes the production of SA from BA; it is a 160 kDa cytochrome P450 monooxygenase. To verify whether OSD3 is BA2H from rice, an enzymatic reaction was performed using BA as a substrate, and the product was detected by LC-MS. The results showed that OSD3 had no retention peak at the retention time of the SA standard. Figure 4 B), which indicates that OSD3 cannot generate SA using BA as a substrate. Therefore, we hypothesize that OSD3 can generate BS using BB as a substrate. Figure 4 C). The product was analyzed by LC-MS, and its spectrum and retention time were consistent with those of BB. Figure 4Based on their molecular formulas (D, E), possible fragmentation modes were inferred, and MS fragmentation was compared with BB's standard. The results showed that the MS fragments of the two were identical. Figure 4 F, G), indicating that OSD3 catalyzes the formation of BS using BB as a substrate. Based on its biochemical function, we named it benzyl benzoate 2-hydroxylase (BB2H). pH and temperature experiments showed that the optimal pH for OSD3 catalysis is 7.0 (F, G). Figure 4 H), the optimal temperature is 30℃ ( Figure 4 I). The substrate specificity of OSD3 was determined under optimal conditions using BB, methyl benzoate, ethyl benzoate, phenyl benzoate, phenyl benzoate, phenylethyl benzoate, and BA as substrates. The results showed that OSD3 could only catalyze BB, and had no catalytic activity for other substrates, indicating that OSD3 has strong substrate specificity. Figure 4 J). The above results indicate that OSD3 can react with BB as a substrate to generate BS, with an optimal pH of 7.0, an optimal temperature of 30℃, and BB as the optimal substrate, demonstrating strong substrate specificity.

[0060] Example 5: OSD3 overexpression plants increased rice resistance to Xoo.

[0061] Overexpression of the OSD3 gene was performed using the 35S promoter in the background of Zhonghua 11. Three overexpressing lines were screened and named OSD3-pMDC42 / ZH11-2, OSD3-pMDC42 / ZH11-8, and OSD3-pMDC42 / ZH11-10, respectively. Their expression levels were 10.8, 3.6, and 5.3 times that of Zhonghua 11, respectively. Figure 5 A). Xoo inoculation experiments were conducted on the tillering stage, and the disease incidence was observed and statistically analyzed 14 days later. The results showed that, compared with the wild type, the lesion length of the three overexpression lines was reduced by 28.0%, 33.6%, and 28.4%, respectively. Figure 5 (B, C) indicates that overexpression of OSD3 can increase the resistance of rice to Xoo.

[0062] Based on Examples 1-5 above, it is demonstrated that OSD3 is a gene involved in the biosynthesis of PAL-dependent SA. OSD3 catalyzes the formation of BS from BB but not from BA. The OSD3 gene is expressed in all plant tissues, its expression is induced by Xoo, and it is located in the endoplasmic reticulum. Overexpression of OSD3 in rice increases the rice's resistance to Xoo.

[0063] SEQ ID NO:1:

[0064]

[0065] SEQ ID NO:2:

[0066]

[0067] SEQ ID NO: 3:

[0068] MELMLPPWASFVGVVLATVLFLKAVLGRSRRVYNLPPGPKPWPVIGNLNLVGTLPHRSIHNLSKKYGPLMYLRFGSFPVVVGSSVEMAKFFLKTHDVVFTDRPKTAAGKHTTYNYSDITWSPYGAYWR QARKMCLTELFSAKRLESYEYIRGEEVRALLRDLHGAAGGVVVLKDYLSTVSLNVITRMVLGKKYLDKDAGGSVTTPEEFKWMLDELFLLLNGVLNIGDSIPWLDWLDLQGYIKRMKKLGKMFDRFLEHV VDEHNERRRREGESFVAKDMVDVLLQFADNPNLEVKLKREGVKAFTQDLIAGGTESSAVTVEWALSELLKKPEVFAKATEELDRVVGRGRWVTEKDVPSLTYVDAIVKETMRLHPVAPMLVPRLSREDT SVDGYDIPAGTRVLVSVWTIGRDPKLWDAPEEFMPERFIGNKIDVKGQDFELLPFGSGRRMCPGYSLGLKVIQLSLANLLHGFAWRLPDGVTREQLSMEEIFGLSTPRKFPLEAVVEPKLPAHLYAAA*

[0069] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A gene involved in PAL-dependent salicylic acid synthesis in rice, characterized in that: The gene is OSD3, and its nucleotide sequence is SEQ ID NO:

1.

2. The protein encoded by the gene involved in rice PAL-dependent salicylic acid synthesis as described in claim 1, characterized in that: The amino acid sequence of the protein is SEQ ID NO:

3.

3. The protein encoded by the gene involved in rice PAL-dependent salicylic acid synthesis according to claim 2, characterized in that: The encoded protein nucleotide sequence is SEQ ID NO:

2.

4. The gene as described in claim 1, characterized in that, The gene encodes a protein with BB2H or related metabolic enzyme activity, which can catalyze a key intermediate reaction step in the PAL-dependent salicylic acid biosynthesis pathway—catalyzing the conversion of BB to BS—and promote the enhancement of PAL-dependent salicylic acid biosynthesis metabolic flux in rice.

5. A plant genetic transformation vector constructed using the salicylic acid synthesis gene involved in rice PAL dependence as described in claim 1, characterized in that, The plant genetic transformation vector includes an up-regulation expression vector; the up-regulation expression vector includes a fusion expression vector constructed from a recombinant promoter or an organ-specific promoter.

6. The plant genetic transformation vector as described in claim 5, characterized in that, It also contains a CRISPR / Cas9 editing element for loss-of-function gene expression of SEQ ID NO:

1.

7. A method for regulating the resistance of rice leaves to bacterial blight bacteria, characterized in that: Knocking out the OSD3 gene leads to a decrease in the salinity (SA) of rice leaves, making them more susceptible to bacterial blight. Overexpression of the OSD3 gene increases the resistance of rice to bacterial blight.

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