A benzyl alcohol synthesis gene and application thereof

Abstract

This invention discloses a benzyl alcohol synthesis gene and its application, relating to the field of plant genetic engineering. Specifically, the benzyl alcohol synthesis gene is disclosed as OsCAD6, with the nucleotide sequence SEQ ID NO: 1; the nucleotide sequence encoding the protein region is SEQ ID NO: 2; and the amino acid sequence encoding the protein is SEQ ID NO: 3. By knocking out the OsCAD6 gene in the background of Zhonghua 11 rice using CRISPR / Cas9, a mutant oscad6 was obtained, reducing benzyl alcohol content in rice leaves to less than 30% of the wild type. Phenotypic analysis and metabolite determination were conducted, and the Ubipro-OsCAD6-pMDC32 overexpression vector was constructed to create OsCAD6 overexpression plants. This invention is based on the fact that OsCAD6 loss of function leads to a significant decrease in benzyl alcohol content in rice, while OsCAD6 overexpression leads to a highly significant increase in benzyl alcohol and SA content in rice.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically to a benzyl alcohol synthesis gene and its application. Background Technology

[0002] Benzyl alcohol (BAlc) is a simple aromatic alcohol commonly found in the flowers, fruits, and leaves of seed plants. While its content in volatile compounds is generally low in most seed plants, it plays a crucial role in plant-environment interactions, participating extensively in various life activities such as insect pollination, pathogen defense, interplant communication, and cell growth.

[0003] The biosynthesis of benzyl alcohol in plants originates from phenylalanine (Phe). In petunias, isotope labeling experiments confirmed the following biosynthetic pathway for benzyl alcohol: phenylalanine → cinnamic acid → cinnamoyl-CoA → benzoylacetyl-CoA → benzoyl-CoA → benzaldehyde → benzyl alcohol. This pathway highly overlaps with the salicylic acid (SA) biosynthetic pathway dependent on phenylalanine ammonialyase (PAL). Recent studies have isolated and characterized the catalytic enzymes responsible for the conversion of benzaldehyde from phenylalanine. However, the key enzyme responsible for catalyzing the production of benzyl alcohol from benzaldehyde remains unclear.

[0004] Benzyl alcohol, a high-value-added industrial chemical, has wide applications in the soap, perfume, cosmetic, condiment, and pharmaceutical industries. Due to its volatility and strong polarity, it is also a highly efficient solvent in the polymer and coating industries, widely used in the production of inks, paints, adhesives, and automobile tires.

[0005] Traditional benzyl alcohol chemical synthesis faces the dual challenges of "difficult chlorine removal" and "high pollution and consumption," limiting its application in fields such as pharmaceuticals, food additives, high-end fragrances, and cosmetics where high purity and efficiency are paramount. Green biosynthesis of benzyl alcohol using microorganisms (such as E. coli or yeast) is considered an ideal pathway to overcome these bottlenecks. However, the key to its success lies in obtaining enzymes capable of efficiently and specifically catalyzing the conversion of benzaldehyde to benzyl alcohol. Therefore, the discovery and identification of novel, highly efficient benzyl alcohol synthases are crucial for achieving the green biosynthesis of benzyl alcohol.

[0006] Rice, as a monocotyledonous model plant, feeds more than half of the world's population. The PAL-dependent synthesis pathway is the main route for amino acid (SA) synthesis in rice. Benzyl alcohol is a key intermediate in the PAL-dependent SA synthesis pathway, but its synthesis route is not well understood. Therefore, elucidating the benzyl alcohol synthesis pathway is crucial for a complete understanding of the PAL-dependent SA synthesis pathway.

[0007] Currently, the key enzymes involved in the biosynthesis of benzyl alcohol in plants have not been characterized, which not only limits the development of efficient benzyl alcohol synthesis systems using biological methods but also hinders its green manufacturing. Therefore, identifying benzyl alcohol synthesis genes has become a crucial breakthrough for achieving the green synthesis of benzyl alcohol. Summary of the Invention

[0008] In view of this, the present invention provides a benzyl alcohol synthesis gene and its application.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A benzyl alcohol synthesis gene, wherein the benzyl alcohol synthesis gene is OsCAD6, and the DNA sequence is shown in SEQ ID NO: 1.

[0011] Furthermore, the benzyl alcohol synthesis gene also includes a DNA sequence with more than 90% homology to the DNA sequence shown in SEQ ID NO: 1; The benzyl alcohol synthesis gene also includes one or more alleles or gene derivatives with base alterations generated by base substitution, deletion or addition based on the DNA sequence shown in SEQ ID NO: 1. The benzyl alcohol synthesis gene also includes a DNA molecule that can hybridize with the DNA sequence shown in SEQ ID NO: 1 under stringent conditions.

[0012] The protein expressed by the benzyl alcohol synthesis gene, the amino acid sequence of which is shown in SEQ ID NO: 3; The DNA sequence encoding the protein is shown in SEQ ID NO: 2.

[0013] Furthermore, the encoded protein also includes an amino acid sequence that has more than 90% homology with the amino acid sequence shown in SEQ ID NO: 3; The encoded protein also 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. The encoded protein also includes fusion proteins formed by linking the protein shown in SEQ ID NO:3 with other tagged proteins.

[0014] A benzyl alcohol synthesis gene, which encodes a protease with BAD or related metabolic enzyme activity, which catalyzes intermediate reaction steps in the PAL-dependent benzyl alcohol biosynthesis pathway, thereby enhancing the PAL-dependent benzyl alcohol biosynthesis metabolic flux in plants. The intermediate reaction step is to catalyze the production of benzaldehyde from benzyl alcohol.

[0015] A plant genetic transformation vector constructed using a benzyl alcohol synthesis gene, the plant genetic transformation vector comprising an upregulation expression vector; the upregulation expression vector comprising a fusion expression vector constructed using a recombinant promoter or an organ-specific promoter; The plant genetic transformation vector also includes the DNA sequence of the benzyl alcohol synthesis gene as described in claim 1 or 2.

[0016] Furthermore, the plant genetic transformation vector also includes a gene expression downregulation vector, which downregulates the expression of the gene shown in SEQ ID NO: 2 through CRISPR / Cas9 technology, T-DNA insertion technology, EMS mutagenesis, RNA interference technology, and gene silencing technology. 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.

[0017] A recombinant microbial strain carrying the plant genetic transformation vector as described in claim 6 or 7, and expressing the benzyl alcohol synthesis gene product as described in claim 1 or 2 in vitro.

[0018] Furthermore, the gene product expressed by the strain has the activity of catalyzing the production of benzaldehyde from benzyl alcohol.

[0019] A transgenic rice plant, wherein the plant's genome integrates the plant's genetic transformation vector.

[0020] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides the rice benzyl alcohol synthesis gene OsCAD6 and its applications, elucidating that rice OsCAD6 can catalyze the synthesis of benzyl alcohol. Based on the fact that OsCAD6 loss of function leads to a significant decrease in rice benzyl alcohol content, while OsCAD6 overexpression leads to a highly significant increase in both benzyl alcohol and SA content, this invention aims to elucidate the core role of OsCAD6 in the rice benzyl alcohol synthesis pathway and explore its application as a catalytic element in microbial synthesis systems. This opens up new pathways for the green industrial biosynthesis of benzyl alcohol, and also demonstrates the feasibility of using it to enhance plant SA content and increase plant stress resistance. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 This shows the location of the Cas9-induced mutagenesis target motif (red arrow) within the coding region of the OsCAD6 gene (blue box); Figure 2 This is a Sanger sequencing peak diagram of the oscad6 mutant; Figure 3 The information includes the amino acid sequence information encoded by the wild type and the oscad6 mutant; Figure 4 The benzyl alcohol content in the leaves of 60-day-old wild-type and oscad6 mutant plants was measured; statistical analysis was performed using the two-tailed Student's-St. test. p<0.05, p<0.01, p<0.001, data are expressed as mean ± standard deviation, n=3 biologically independent samples; Figure 5 The total SA (SA and SAG) content in the leaves of 14-day-old wild-type and oscad6 mutant plants was measured; statistical analysis was performed using a two-tailed Student's t-test. p<0.05, p<0.01, p<0.001, data are expressed as mean ± standard deviation, n = 3 biologically independent samples; Figure 6 The expression pattern of OsCAD6 provided in this embodiment of the invention: the expression of the OsCAD6 gene in the roots, internodes, leaves, leaf veins, spikes and leaf sheaths of 70-day-old Zhonghua 11 plants. The gene expression level was normalized with the internal reference gene OsUBQ5. The data are expressed as mean ± standard deviation, n = 3 biologically independent samples. Figure 7 The subcellular localization of OsCAD6 provided in this embodiment of the invention: subcellular localization of OsCAD6 in rice protoplasts, with mCherry-OsAIM1 used as a peroxisome localization marker, and a scale bar of 10 μm. Figure 8 It is a biochemical reaction catalyzed by OsCAD6 in vitro; Figure 9 This is a comparison of the total ion chromatograms of the OsCAD6 in vitro enzyme activity reaction product and the benzyl alcohol standard; Figure 10 This is a comparison of the quality spectra of the OsCAD6 in vitro enzyme activity reaction product and the benzyl alcohol standard; Figure 11 The optimal pH for OsCAD6 catalysis; Figure 12 This is a comparison of the suitable catalytic temperatures for OsCAD6; Figure 13 This is a substrate-specific comparison of OsCAD6; Figure 14 The expression level of Ubipro-OsCAD6-pMDC32 / ZH11 overexpression material at the field tillering stage was measured; statistical analysis was performed using a two-tailed Student's t-test. p<0.05, p<0.01, data are expressed as mean ± standard deviation, n=3 biologically independent samples; Figure 15 The benzyl alcohol content in the leaves of 60-day-old plants of wild-type and OsCAD6-overexpressing lines was measured; statistical analysis was performed using a two-tailed Student's t-test. p<0.05, p<0.01, data are expressed as mean ± standard deviation, n=3 biologically independent samples; Figure 16 The total SA (SA and SAG) content in the leaves of 14-day-old plants from wild-type and OsCAD6-overexpressing lines was measured; statistical analysis was performed using a two-tailed Student's t-test. p<0.05, p<0.01, data are expressed as mean ± standard deviation, n = 3 biological independent samples. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1: Identification of OsCAD6 gene involved in the rice benzyl alcohol biosynthesis pathway To elucidate the biosynthetic pathway of benzyl alcohol, this invention used CRISPR / Cas9 technology to knock out the Os04g0229100 gene and verify its function. Os04g0229100 was predicted to be a cinnamyl alcohol dehydrogenase, named OsCAD6. This gene is 1083 bp in length and encodes a protein composed of 360 amino acids. Knockout using CRISPR / Cas9 technology yielded two independent homozygous lines. The oscad6-1 line had a G base inserted at position 439 of the ATG, resulting in a frameshift mutation starting at position 115 in its protein. The oscad6-2 line had a C base inserted at position 439 of the ATG, also resulting in a frameshift mutation starting at position 115 in its protein. Figure 1 , Figure 2 , Figure 3 The determination of benzyl alcohol content in 14-day-old leaves of the oscad6 mutant and WT showed that the benzyl alcohol content decreased to below 44.0% and 26.1% of that of the wild type, respectively. Figure 4 This indicates that the OsCAD6 gene plays a crucial role in the synthesis of benzyl alcohol in rice. The total SA content in 14-day-old leaves of the oscad6 mutant and WT showed that the total SA content decreased to below 13.8% and 10.5% of the wild type, respectively. Figure 5 This indicates that the OsCAD6 gene is further involved in SA synthesis. These results demonstrate that OsCAD6 participates in the synthesis of benzyl alcohol in rice and further influences SA synthesis.

[0025] Example 2: OsCAD6 gene tissue expression The transcriptional expression pattern and regulation of genes are crucial to their biological functions. To investigate the transcriptional expression pattern of the OsCAD6 gene, RT-qPCR was used to detect the transcriptional levels in roots, stems, internodes, leaves, leaf veins, panicles, and leaf sheaths of wild-type ZH11 rice at the heading stage. The results showed that the OsCAD6 gene was expressed in all tissues of rice. Figure 6 ).

[0026] Example 3: Subcellular localization of OsCAD6 protein Subcellular localization of genes is often closely related to their biological functions. To study the subcellular localization of the OsCAD6 protein, an OsCAD6 protein vector fused with GFP protein at the N-terminus of the 35S promoter was constructed. Transformation was performed using rice protoplasts, and the subcellular localization was observed. The results showed that the GFP-OsCAD6 fusion protein co-localized with the peroxisome marker protein mCherry-OsAIM1, indicating that OsCAD6 is localized in the peroxisome (…). Figure 7 ).

[0027] Example 4: OsCAD6 catalyzes the production of benzyl alcohol (BAlc) from benzaldehyde (BAld) as a substrate. To verify the biological function of OsCAD6, the OsCAD6-pMAL vector was constructed, and the protein was purified using *E. coli* BL21 to obtain MBP-OsCAD6. To confirm whether it is a benzaldehyde reductase, an enzymatic activity reaction was performed using benzaldehyde as a substrate and NADPH as a coenzyme. Figure 8 The product was detected by HRGC-MS, and the results showed that the enzyme activity product was consistent with the benzyl alcohol standard, both showing the same chromatographic peak at 5.88 min. Figure 9 Meanwhile, MS results showed that the enzyme activity product had the same fragmentation as the benzyl alcohol standard (77.0385 m / z, 79.0541 m / z, 107.0491 m / z and 108.0568 m / z). Figure 10 This indicates that the enzyme's active product is benzyl alcohol, and OsCAD6 catalyzes the production of benzyl alcohol from benzaldehyde. Based on its biochemical function, this invention also names it benzyl alcohol dehydrogenase (BAD). pH and temperature experiments have shown that the optimal pH for OsCAD6 catalysis is 7.0. Figure 11 The optimal temperature is 40℃. Figure 12 Using coniferylaldehyde and sinapaldehyde as substrates, the substrate specificity of OsCAD6 was tested under optimal conditions. The results showed that OsCAD6 also exhibited reducing activity towards coniferylaldehyde and sinapaldehyde, but its catalytic activity towards benzaldehyde was stronger. Figure 13 The above results indicate that OsCAD6 can react with benzaldehyde as a substrate to produce benzyl alcohol, with an optimal pH of 7.0, an optimal temperature of 40℃, and benzaldehyde as the optimal substrate, demonstrating strong substrate specificity.

[0028] Example 5: OsCAD6 overexpression increased the endogenous benzyl alcohol content in rice. OsCAD6 gene was overexpressed using the maize ubiquitin promoter (Ubi) in the background of Zhonghua 11. Two overexpressing lines were screened, with expression levels 348 and 624 times higher than those of Zhonghua 11, respectively. Figure 14 The total SA content of the two overexpression lines and Zhonghua 11 was measured and found to be increased to 202.8% and 162.6% of the wild type, respectively. Figure 15The total SA content of the two overexpression lines and Zhonghua 11 was measured and found to be increased to 192.1% and 195.4% of the wild type, respectively. Figure 16 ).

[0029] Based on Examples 1-5 above, it is shown that OsCAD6 is a gene involved in PAL-dependent benzyl alcohol biosynthesis. It can catalyze the production of benzaldehyde to benzyl alcohol. It is expressed in various plant tissues and located in peroxisomes. Overexpression of the OsCAD6 gene can increase the endogenous benzyl alcohol content in rice and, at the same time, increase the synthesis of SA as a substrate.

[0030] The benzyl alcohol biosynthesis gene (1739 bp) is shown in SEQ ID NO: 1: The nucleotide sequence encoding benzyl alcohol synthase (1083 bp) is shown in SEQ ID NO: 2: The benzyl alcohol biosynthesis gene encodes a benzyl alcohol synthase (360 amino acid residues), as shown in SEQ ID NO: 3: MEVTPNHTQTVSGWAAMDESGKIVPFVFKRRENGVDDVTIKVKYCGMCHTDLHFIHNDWGITMYPVVPGHEITGVVTKVGTNVAGFKVGDRVGVGCIAASCLDCEHCRRSEENYCDKVALTYNGIFWDGSITYGGYSGMLVAHKRFVVRIPDTLPLDAAAPLLCAGITVYSPMKQHGMLQ ADAAGRRLGVVGLGGLGHVAVKFGKAFGLHVTVISTSPAKEREARENLKADNFVVSTDQKQMQAMTRSLDYIIDTVAATHSLGPILELLKVNGKLVLVGAPEKPVELPSFPLIFGKRTVSGSMTGGMKETQEMMDICGEHNITCDIEIVSTDRINDALARLARNDVRYRFVINVGGDSKL .

[0031] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A benzyl alcohol synthesis gene, characterized in that, The benzyl alcohol synthesis gene is OsCAD6, and its DNA sequence is shown in SEQ ID NO:

1.

2. The benzyl alcohol synthesis gene according to claim 1, characterized in that, The benzyl alcohol synthesis gene also includes a DNA sequence with more than 90% homology to the DNA sequence shown in SEQ ID NO: 1; The benzyl alcohol synthesis gene also includes one or more alleles or gene derivatives with base alterations generated by base substitution, deletion or addition based on the DNA sequence shown in SEQ ID NO:

1. The benzyl alcohol synthesis gene also includes a DNA molecule that can hybridize with the DNA sequence shown in SEQ ID NO: 1 under stringent conditions.

3. The protein expressed by the benzyl alcohol synthesis gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO: 3; The DNA sequence encoding the protein is shown in SEQ ID NO:

2.

4. The protein according to claim 3, characterized in that, The encoded protein also includes an amino acid sequence that has more than 90% homology with the amino acid sequence shown in SEQ ID NO: 3; The encoded protein also 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. The encoded protein also includes fusion proteins formed by linking the protein shown in SEQ ID NO:3 with other tagged proteins.

5. The benzyl alcohol synthesis gene according to any one of claims 1 or 2, characterized in that, The gene encodes a protease with BAD or related metabolic enzyme activity, which can catalyze intermediate reaction steps in the PAL-dependent benzyl alcohol biosynthesis pathway and promote enhanced PAL-dependent benzyl alcohol biosynthesis metabolic flux in plants. The intermediate reaction step is to catalyze the formation of benzaldehyde from benzyl alcohol.

6. A plant genetic transformation vector constructed using the benzyl alcohol synthesis gene as described in claim 1 or 2, 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. The plant genetic transformation vector also includes the DNA sequence of the benzyl alcohol synthesis gene as described in claim 1 or 2.

7. The plant genetic transformation vector according to claim 6, characterized in that, The plant genetic transformation vector also includes a gene expression downregulation vector, which downregulates the expression of the gene shown in SEQ ID NO: 2 through CRISPR / Cas9 technology, T-DNA insertion technology, EMS mutagenesis, RNA interference technology, and gene silencing technology. 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.

8. A recombinant microbial strain, characterized in that, The strain carries the plant genetic transformation vector as described in claim 6 or 7 and expresses the benzyl alcohol synthesis gene product as described in claim 1 or 2 in vitro.

9. The recombinant microbial strain according to claim 8, characterized in that, The gene product expressed by the strain has the activity of catalyzing the production of benzaldehyde from benzyl alcohol.

10. A transgenic rice plant, characterized in that, The plant genome integrates the plant genetic transformation vector as described in claim 6 or 7.

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