Gene participating in rice PAL-dependent salicylic acid synthesis route and application thereof

Abstract

The invention belongs to the field of plant genetic engineering, and discloses a gene participating in a rice PAL-dependent SA synthesis pathway and application of the gene, and the nucleotide sequence of OSD2 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; the OSD2 gene is knocked out through CRISPR / Cas9 under the background of Zhonghua 11, a mutant OSD2 with the SA of rice leaves reduced to 15% of the wild type level is obtained, phenotypic analysis and hormone determination are carried out, a pUbi-OSD2-pMDC32 overexpression vector is constructed, and an OSD2 overexpression plant is created. Based on the fact that OSD2 function deficiency leads to the fact that the SA content of rice is remarkably reduced, the rice is more sensitive to xanthomonas oryzae pv. Oryzae, overexpression of OSD2 has high resistance to xanthomonas oryzae pv. Oryzae, therefore, the function of OSD2 in a rice SA synthesis pathway is analyzed, application of the gene in crop disease resistance improvement is explored, and a new thought can be provided for preventing rice diseases in production.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the technical field of plant genetic engineering, and in particular relates to a gene involved in a rice PAL-dependent salicylic acid synthesis pathway and its application. Background Art

[0002] Salicylic acid (SA) is an important plant hormone that participates in plant growth, development, and stress responses, both directly and indirectly through crosstalk with other hormones. SA is one of the many phenolic compounds synthesized by plants and is involved in multiple life processes, including seed germination, cellular respiration, cell growth, stomatal opening and closing, flowering, and aging. Furthermore, SA is a key signal regulating heat production and disease resistance.

[0003] SA (Salt-Acid) synthesis in plants primarily occurs through two secondary metabolic pathways, originating from shikimic acid: the isochorismate synthase (ICS) pathway and the phenylalanine ammonia lyase (PAL) pathway. Plants synthesize SA through both the ICS and PAL pathways, and SA signaling plays a crucial role in plant disease resistance. In rice, ICS1 is primarily involved in chlorophyllin quinone biosynthesis but not SA biosynthesis. The PAL pathway remains largely uncharacterized in rice. Studies have shown that functional deletion of AIM1 (ABNORMALINFLORESCENCE MERISTEM 1), a gene involved in the PAL-dependent SA biosynthesis pathway, reduces endogenous SA content in rice to approximately 10% of that in wild-type plants, indicating that the PAL pathway is the primary pathway for SA synthesis in rice. Barley inoculated with Pseudomonas syringae pv. syringae exhibited an increase in endogenous SA during infection, whereas rice did not experience an increase in SA levels following infection with P. syringae, the rice blast pathogen (Magnaporthe grisea), or Rhizoctonia solani. Salicylate hydroxylases (NahG), which block SA accumulation in vivo, significantly increased in NahG-transgenic Arabidopsis plants following infection with the pathogen Pto DC3000, suggesting a key role for SA in disease resistance. Rice expressing NahG exhibited a significant increase in ROS levels following infection with P. oryzae, indicating that reduced SA contributes to increased susceptibility to the pathogen. Mutation of OsS5h leads to increased SA content in rice and enhanced resistance to bacterial blight. Overexpression of OsS5h reduces endogenous SA in rice and makes it susceptible to bacterial blight, indicating that SA content is positively correlated with resistance to bacterial blight. Recent studies suggest that SA may also mediate defense signaling in wheat. Taken together, these results indicate that, at least in some monocotyledons, SA serves as a signal for defense responses, and that endogenous SA content is positively correlated with plant disease resistance.

[0004] Rice, a staple food crop for more than half the world's population, is a model monocot plant with high basal SA levels. Rice production is frequently impacted by various field diseases, the most devastating of which is bacterial leaf blight caused by Xanthomonas oryzae. Bacterial leaf blight can affect rice growth, development, or reproduction, resulting in 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 new high-yield and stable crop varieties is crucial for ensuring food security.

[0005] In view of the above analysis, the technical problems that need to be solved urgently in the existing technology are:

[0006] Rice SA cannot accumulate in large quantities after pathogen invasion, resulting in the plant being unable to effectively utilize SA to resist the invasion of external pathogens. In plant disease resistance improvement, any improvement in disease resistance is often accompanied by changes in growth and development and a decrease in yield. Therefore, it is crucial to improve rice resistance to pathogens while ensuring stable rice yields. Summary of the Invention

[0007] In response to the problems existing in the prior art, the present invention provides a gene involved in the rice PAL-dependent salicylic acid synthesis pathway and its application.

[0008] The present invention is achieved by providing a gene involved in the rice PAL-dependent salicylic acid synthesis pathway, characterized in that the gene involved in the rice PAL-dependent salicylic acid synthesis pathway is OSD2 (Oryza Sativa SA-Deficient gene 2), and the nucleotide sequence is SEQ ID NO: 1.

[0009] Furthermore, the salicylic acid synthesis gene further comprises a DNA sequence having a homology of more than 90% with the DNA sequence shown in SEQ ID NO: 1.

[0010] Furthermore, the salicylic acid synthesis gene further includes alleles or gene derivatives with one or more base changes produced by base substitution, deletion, or addition.

[0011] Furthermore, the salicylic acid synthesis gene further comprises: a DNA molecule that can hybridize with the DNA sequence shown in SEQ ID NO: 1 under stringent conditions.

[0012] Another object of the present invention is to use the protein encoded by the rice salicylic acid synthesis gene, wherein the nucleotide sequence of the encoded protein is SEQ ID NO: 2.

[0013] Furthermore, the amino acid sequence of the encoded protein is SEQ ID NO: 3;

[0014] The encoded protein further comprises an amino acid sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 3;

[0015] The encoded protein further includes proteins and protein analogs having one or more amino acid changes produced by amino acid substitution, deletion, or addition based on the amino acid sequence shown in SEQ ID NO: 3;

[0016] The encoded protein further includes a fusion protein formed by linking the protein shown in SEQ ID NO: 3 with other tag proteins.

[0017] Another object of the present invention is a plant genetic transformation vector constructed using the SA synthetic gene, wherein the plant genetic transformation vector includes an expression vector for up-regulating OSD2; the expression vector for up-regulating OSD2 includes a fusion expression vector constructed with a recombinant promoter or a tissue-specific promoter;

[0018] The plant genetic transformation vector further comprises: a DNA sequence as shown in SEQ ID NO: 1, or a DNA sequence having a homology of more than 90% with the DNA sequence as shown in SEQ ID NO: 1, or an allele or gene derivative with one or more base changes produced by base substitution, deletion, or addition of the nucleotide sequence as shown in SEQ ID NO: 1, or a DNA molecule capable of hybridizing with the DNA sequence as shown in SEQ ID NO: 1 under stringent conditions.

[0019] Furthermore, the plant genetic transformation vector further includes an up-regulation or down-regulation OSD2 expression vector, which down-regulates the expression of the gene shown in SEQ ID NO: 3 by CRISPR / Cas9 technology, T-DNA insertion technology, EMS mutagenesis, RNA interference technology, or gene silencing technology;

[0020] The plant genetic transformation vector up-regulates or down-regulates the expression level or activity of the protein shown in SEQ ID NO: 3 through a related protein regulator.

[0021] Another object of the present invention is a recombinant bacterium, plant callus and cell line using the plant genetic transformation vector for expression.

[0022] Another object of the present invention is to provide a method for regulating the resistance of rice leaves to bacterial blight bacteria, which specifically includes: knocking out the OSD2 gene, resulting in a decrease in SA of rice leaves and sensitivity to bacterial blight bacteria, and overexpressing the OSD2 gene, thereby increasing the resistance of rice to bacterial blight bacteria.

[0023] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0024] The present invention provides a rice SA synthesis gene OSD2 and its application, and explains that rice OSD2 can regulate rice SA synthesis, create its over-expression material, and increase rice disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Identification of the gene OSD2 involved in the rice SA biosynthesis pathway provided by the embodiments of the present invention: A, location of the Cas9-induced mutagenesis target motif (red arrow) within the coding region of the OSD2 gene (orange frame), B, Sanger sequencing peak plot of the osd2 mutant, C, amino acid sequence information encoded by the wild type and osd2 mutant; D, phenotype of wild type and osd2 mutant plants at heading stage, scale bar, 10 cm, E, SA and SAG content in leaves of wild type and osd2 mutant 14-day-old plants, F, lesion phenotype and lesion length statistics of wild type and osd2 mutant plants 14 days after Xoo inoculation, scale bar, 3 cm, statistical analysis was performed using a two-tailed student's t-test, ***p < 0.001, data are expressed as mean ± SD, n = 4 (E) and n = 20 (F) biologically independent samples;

[0026] Figure 2 The expression pattern of OSD2 provided in the embodiments of the present invention is as follows: A. Expression of the OSD2 gene 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. Expression of the OSD2 gene in the leaves of Zhonghua 11 plants inoculated with Xoo at 0, 12, 24, 48, and 72 hours. The OSD2 gene expression levels after Xoo inoculation were normalized to the corresponding control group. Statistical analysis was performed using a two-tailed student's t-test. *p < 0.05, ***p < 0.001. Data are expressed as mean ± SD. n = 3 biologically independent samples (A and B).

[0027] Figure 3 The subcellular localization of OSD2 provided in the embodiments of the present invention is as follows: Subcellular localization of OSD2 in rice protoplasts, mCherry-AIM1 is used as a peroxisome localization marker, scale bar is 10 μm;

[0028] Figure 4 Overexpression of the OSD2 gene provided in the embodiments of the present invention can enhance rice disease resistance: A. Expression level of the pUbi-OSD2-pMDC32 / ZH11 overexpression material at the tillering stage in the field; B. Field-grown plants of Zhonghua 11 and pUbi-OSD2-pMDC32 / ZH11 inoculated with the Xoo phenotype at the tillering stage; scale bar, 3 cm; C. Statistics of lesion length on leaves of Zhonghua 11 and three OSD2-overexpressing lines 14 days after inoculation with the Xoo strain. Statistical analysis was performed using a two-tailed Student's t-test, ***p < 0.001. Data are expressed as mean ± SD, n = 3 (A) and n = 20 (C) independent biological replicates. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] An embodiment of the present invention provides a gene involved in the PAL-dependent salicylic acid synthesis pathway in rice. The gene is OSD2, and the nucleotide sequence is SEQ ID NO: 1.

[0031] The salicylic acid synthesis gene further includes a DNA sequence having a homology of more than 90% with the DNA sequence shown in SEQ ID NO: 1.

[0032] The salicylic acid synthesis gene further includes alleles or gene derivatives with one or more base changes produced by base substitution, deletion, or addition.

[0033] The salicylic acid synthesis gene further comprises: a DNA molecule that can hybridize with the DNA sequence shown in SEQ ID NO: 1 under stringent conditions.

[0034] An embodiment of the present invention provides a protein encoded by the rice salicylic acid synthesis gene, wherein the nucleotide sequence of the encoded protein is SEQ ID NO: 2.

[0035] The amino acid sequence of the encoded protein is SEQ ID NO: 3;

[0036] The encoded protein further comprises an amino acid sequence having a homology of more than 90% with the amino acid sequence shown in SEQ ID NO: 3;

[0037] The encoded protein further includes proteins and protein analogs having one or more amino acid changes produced by amino acid substitution, deletion, or addition based on the amino acid sequence shown in SEQ ID NO: 3;

[0038] The encoded protein further includes a fusion protein formed by linking the protein shown in SEQ ID NO: 3 with other tag proteins.

[0039] The embodiment of the present invention provides a plant genetic transformation vector constructed using the SA synthetic gene, wherein the plant genetic transformation vector includes an expression vector for up-regulating OSD2; the expression vector for up-regulating OSD2 includes a fusion expression vector constructed with a recombinant promoter or a tissue-specific promoter;

[0040] The plant genetic transformation vector further comprises: a DNA sequence as shown in SEQ ID NO: 1, or a DNA sequence having a homology of more than 90% with the DNA sequence as shown in SEQ ID NO: 1, or an allele or gene derivative with one or more base changes produced by base substitution, deletion, or addition of the nucleotide sequence as shown in SEQ ID NO: 1, or a DNA molecule capable of hybridizing with the DNA sequence as shown in SEQ ID NO: 1 under stringent conditions.

[0041] The plant genetic transformation vector further includes an up-regulation or down-regulation OSD2 expression vector, which down-regulates the expression of the gene shown in SEQ ID NO: 3 by CRISPR / Cas9 technology, T-DNA insertion technology, EMS mutagenesis, RNA interference technology, or gene silencing technology;

[0042] The plant genetic transformation vector up-regulates or down-regulates the expression level or activity of the protein shown in SEQ ID NO: 3 through a related protein regulator.

[0043] The embodiments of the present invention provide a recombinant bacterium, plant callus tissue and cell line using the plant genetic transformation vector for expression.

[0044] An embodiment of the present invention provides a method for regulating the resistance of rice leaves to bacterial blight bacteria, which specifically includes: knocking out the OSD2 gene, resulting in a decrease in SA of rice leaves and sensitivity to bacterial blight bacteria, and overexpressing the OSD2 gene, thereby increasing the resistance of rice to bacterial blight bacteria.

[0045] The present invention uses CRISPR / Cas9 technology to knock out the Os10g0503300 gene and verify its function. By studying its basic biological functions, it is found that it can participate in the PAL-dependent SA synthesis pathway and participate in rice disease resistance.

[0046] Example 1: Identification of the gene OSD2 involved in the rice SA biosynthesis pathway

[0047] To elucidate the PAL pathway of SA biosynthesis, we used CRISPR / Cas9 technology to knock out the Os10g0503300 gene and verify its function. Os10g0503300 is a predicted benzyl alcohol O-benzoyltransferase, named OSD2. The gene is 1428 bp long, has no introns, and encodes a protein composed of 475 amino acids. Using CRISPR / Cas9 technology to knock out the gene, three independent homozygous strains were obtained. The osd2-1 strain had an A insertion at position 334 from the ATG, resulting in a frameshift mutation in its protein starting at position 112. The osd2-2 strain had a T insertion at position 334 from the ATG, resulting in a frameshift mutation in its protein starting at position 112. The osd2-3 strain had a C insertion at position 335 from the ATG, resulting in a frameshift mutation in its protein starting at position 112 ( Figure 1 A–C). There is no significant difference between the osd2 mutant and WT during growth ( Figure 1 D), the determination of endogenous SA and SAG contents in its 14-day-old leaves showed that both SA and SAG contents decreased to less than 15% of the wild type ( Figure 1 E), indicating that the OSD2 gene plays a crucial role in the synthesis of rice SA. Subsequently, wild-type and osd2 mutants were inoculated with Xoo in the field during the tillering stage. Statistical analysis of disease incidence 14 days after inoculation revealed that the lesion length of the osd2 mutant was significantly increased compared to the wild-type, with increases of 60.3%, 43.7%, and 41.7% in three independent lines, respectively, compared to the wild-type. Figure 1 F). The above results indicate that OSD2 is involved in the synthesis of rice SA. The deletion of OSD2 gene leads to a significant decrease in rice SA content and makes rice more sensitive to pathogens.

[0048] Example 2: OSD2 gene expression and induced expression

[0049] The transcriptional expression pattern and expression regulation of genes are crucial to their biological functions. To study the transcriptional expression pattern of the OSD2 gene, qRT-PCR was used to detect the transcriptional levels in the roots, stems, leaves, and panicles of wild-type ZH11 at the heading stage. The results showed that the OSD2 gene was expressed in all tissues of rice ( Figure 2 A) Rice is more sensitive to Xoo after SA deficiency ( Figure 1 F), therefore, ZH11 was sampled within 72 hours after inoculation to detect the induction of OSD2 gene by Xoo. The results showed that 24 hours after inoculation, OSD2 was induced by Xoo and its expression increased by 10.1 times ( Figure 1 B). The above results indicate that SA plays an important role in rice disease resistance.

[0050] Example 3: Subcellular localization of OSD2 protein

[0051] The subcellular localization of a gene is often closely related to its biological function. To study the subcellular localization of OSD2 protein, an OSD2 protein vector containing an N-terminal GFP protein fused to the 35S promoter was constructed. Rice protoplasts were used for transformation and its subcellular localization was observed. The results showed that the GFP-OSD2 fusion protein colocalized with the peroxisomal marker protein mCHerry-AIM1, indicating that OSD2 is localized in peroxisomes ( Figure 3 ).

[0052] Example 4: OSD2 overexpression in rice plants increases resistance to Xoo

[0053] The OSD2 gene was overexpressed in the Zhonghua 11 background using the ubiquitin promoter (Ubi). Three overexpression lines were screened, and their expression levels were 2.4, 5.9, and 11.6 times that of Zhonghua 11, respectively. Figure 4 A). Xoo inoculation experiments were conducted during the tillering stage. 14 days later, the disease progression was observed and statistically analyzed. The results showed that compared with the wild type, the lesion lengths of the three overexpression lines were reduced by 32.1%, 34.8%, and 31.2%, respectively. Figure 4 B, C), indicating that overexpression of OSD2 can increase rice resistance to Xoo.

[0054] According to the above Examples 1-4, OSD2 is a gene involved in the biosynthesis of PAL-dependent SA, expressed in all tissues of the plant, its expression level is induced by Xoo, and is located in the peroxisome. Overexpression of the OSD2 gene can increase rice resistance to Xoo.

[0055] SEQ ID NO: 1:

[0056]

[0057] SEQ ID NO:2:

[0058]

[0059] SEQ ID NO: 3:

[0060] MAGSTAAALKFTVRRKPAELVAPAGPTPRELKKLSDIDDQDGLRFHIPVIQFYRRSAAMGGRDPAPVIRAAVARALVSYYPFAGRLRELEGRKLAVDCTGEGVLFIEADADVRLEHFGG ALQPPFPCLEELVFDVPGSSEVLGSPLLLFQVTRLACGGFILAVRLHHTMADAQGLVQFLGAVAEMARGGAAAAPSVAPVWGREMLEARSPPRPAFAHREYDEVPDTKGTIIPLDDMAH RSFFFGAREVAAVRSHLAPGIRERATTFEVLTGCLWRCRTAALAPDDDEVMRMICIVNARGGGKSGGGAGMIPEGYYGNAFAFPVAVATAGELRARPLGYAVELVRAAKGEVSVEYMRS VADLMVQRGRPHFTVVRAYLVSDVTKAGFGDLDFGWGKPAYGGPAKGGVGAIPGVASFLIPFKNAKGEDGIVVPMCLPGPAMDKFVEEMGKLMKPAAAATAATRQQPADMFAMIKSAL*

[0061] The above description is only 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 any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A gene involved in the PAL-dependent salicylic acid biosynthesis pathway in rice, characterized in that The gene is OSD2, and the nucleotide sequence of the gene is SEQ ID NO:

1.

2. The gene according to claim 1, characterized in that The gene also includes a nucleotide sequence having a homology of not less than 90% with the sequence of SEQ ID NO: 1, or an allele having one or more base changes produced by base substitution, deletion or addition.

3. A protein encoded by the gene according to claim 1 or 2, characterized in that The amino acid sequence of the protein is SEQ ID NO:

3.

4. The protein according to claim 3, characterized in that The protein also includes an amino acid sequence having no less than 90% homology to the sequence of SEQ ID NO: 3, or a protein homolog having one or more amino acids changed by amino acid substitution, deletion or addition.

5. The protein according to claim 3, characterized in that The protein is fused with the tag protein to form a fusion protein.

6. A plant genetic transformation vector constructed using the gene according to claim 1, characterized in that The vector comprises a promoter and a terminator for driving the expression of the sequence of SEQ ID NO:

1.

7. The carrier according to claim 6, characterized in that It also contains a CRISPR / Cas9 editing element or an RNA interference element for downregulating the expression of the gene of SEQ ID NO:

1.

8. A recombinant microbial strain, characterized in that The strain carries the plant genetic transformation vector according to claim 6 or 7 and expresses the gene product of SEQ ID NO: 1 in vitro.

9. A transgenic rice plant, characterized in that The plant genetic transformation vector according to claim 6 or 7 is integrated into the plant genome.

10. A method for regulating resistance of rice leaves to bacterial blight bacteria, characterized by: Knocking out the OSD2 gene leads to a decrease in SA in rice leaves and sensitivity to bacterial blight pathogen. Overexpression of the OSD2 gene increases the resistance of rice to Xoo pathogen.

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