Creation method and application of 2-phenethyl alcohol biosynthetic rice with rose fragrance characteristic

Abstract

The invention discloses a creation method of 2-phenethyl alcohol biosynthetic rice with a rose fragrance type characteristic. The creation method comprises the following steps: taking an mDHS1 gene, a PAAS gene and a PAR gene to construct a pRHVc-mDHS1-PAAS-PAR recombinant plasmid, transforming the pRHVc-mDHS1-PAAS-PAR recombinant plasmid into a callus through an agrobacterium tumefaciens-mediated method, and carrying out screening and plant regeneration of resistant calluses to obtain rice with overexpressed mDHS1, PAAS and PAR genes. According to the rice, 2-PE can be effectively synthesized in rice endosperm. Based on a synthetic biological technology and metabolic engineering, the pRHVc vector is used for overexpressing mDHS1, PAAS and PAR genes in rice endosperm through a transgenic means, 2-PE is efficiently and directionally synthesized and stably enriched in rice grains, and the economic added value of rice crops is remarkably increased.

Description

A method for the creation and application of 2-phenylethanol biosynthetic rice with rose-scented characteristics. Technical Field

[0001] This invention relates to rice breeding, and more particularly to a method for creating 2-phenylethanol biosynthesized rice with rose-scented characteristics and its application. Background Technology

[0002] Rice (Oryza sativa L.), a major global food crop, possesses advantages such as large biomass, low planting costs, and a mature genetic transformation system, making it an ideal plant bioreactor. In recent years, metabolic engineering strategies have been successfully applied to the synthesis of high-value-added products in rice, such as vitamins and medicinal proteins. However, research on the targeted synthesis of products like 2-phenylethanol (2-PE) remains lacking. The technical bottlenecks lie in the complexity of plant secondary metabolic networks and the inability to accumulate large amounts of substrate feedback inhibition. This makes it difficult to achieve efficient conversion from shikimic acid to 2-phenylethanol throughout the entire process. Summary of the Invention

[0003] This invention provides a method for creating rice with rose-scented biosynthesis of 2-phenylethanol and its application. Targeting the metabolic characteristics of rice, this invention constructs a "metabolic modular enhancement" strategy by synergistically regulating key nodes in the shikimic acid pathway (mDHS1), phenylacetaldehyde synthase gene (PAAS), and phenylacetaldehyde reductase gene (PAR). This overcomes the limitations of precursor supply and the accumulation of intermediate products. Furthermore, by utilizing rice grains as a natural, closed storage unit, it achieves efficient, targeted synthesis and stable enrichment of 2-PE, providing an innovative solution for the cross-disciplinary integration of agriculture and industrial production.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for creating 2-phenylethanol biosynthetic rice with rose-scented characteristics, constructing the pRHVc-mDHS1-PAAS-PAR recombinant plasmid, transforming it into callus tissue through Agrobacterium tumefaciens-mediated transformation, and obtaining rice overexpressing the mDHS1, PAAS and PAR genes through screening of resistant callus tissue and plant regeneration.

[0005] The rice constructed in this invention that overexpresses the mDHS1, PAAS, and PAR genes can promote the accumulation of 2-PE in rice grains by overexpressing the mDHS1, PAAS, and PAR genes in rice plants through the endosperm promoter.

[0006] The mDHS1 gene originates from rice, the PAAS gene from roses, and the PAR gene from tomatoes. Of course, it can also originate from homologous genes from other different species.

[0007] The nucleotide sequence of the mDH1S gene is shown in SEQ ID NO.1, the nucleotide sequence of the PAAS gene is shown in SEQ ID NO.2, and the nucleotide sequence of the PAR gene is shown in SEQ ID NO.3.

[0008] In this invention, the pRHVc-mDHS1-PAAS-PAR recombinant plasmid is constructed as follows: Gene fragments of the promoter PGluB4, mDHS1, and terminator T35s are inserted into the pSAT6 vector to obtain the pSAT6-GluB4-mDHS-T35s recombinant intermediate vector; gene fragments of the promoter PGlb1 and PAAS are inserted into the pSAT6 vector to obtain the pSAT6-PGlb1-PAAS recombinant intermediate vector; gene fragments of the promoter PGluC, PAR, and terminator Tmas are inserted into the pSAT6 vector to obtain the PAST6-PGluC-PAR-Tmas recombinant intermediate vector. The inserted fragments on the three pSAT6 recombinant intermediate vectors are then amplified to obtain three fragments. Finally, the three fragments are inserted into the pRHVc vector to obtain the pRHVc-mDHS1-PAAS-PAR recombinant plasmid.

[0009] The specific construction method is as follows: (1) Using plasmid as template, PCR amplification was performed using primers SAT6-B4-F and DHS-B4-R3 to obtain the PGluB4 gene fragment; PCR amplification was performed using primers DHS-35-F3 and SAT6-T35s-R to obtain the T35s gene fragment; PCR amplification was performed using primers SAT6-Glb1-F and PAAS-Glb1-R to obtain the Glb1 gene fragment; PCR amplification was performed using primers SAT6-luC-F and PAR1-luC-R to obtain the PGluC gene fragment; PCR amplification was performed using primers PAR1-mas-F and SAT6-mas-R to obtain the Tmas gene fragment; ( 2) Using rice cDNA as a template, PCR amplification was performed using primer pairs B4-DHS-F3 and DHS2-R1; DHS2-F2 and 35-DHS-R3 to obtain the mDHS1 gene fragment; using rose cDNA as a template, PCR amplification was performed using primer pairs Glb1-PAAS-F and SAT6-PAAS-R to obtain the PAAS gene fragment; using tomato cDNA as a template, PCR amplification was performed using primer pairs luC-PAR1-F and mas-PAR1-R to obtain the PAR gene fragment; (3) The vector pSAT6 was selected as the backbone vector for vector construction, and PI-PspI was selected as the restriction enzyme site for vector construction. Homologous recombination was used to transfer the DNA from step (1) to (2). The obtained PGluB4, mDHS1, T35s gene fragments and PGlb1, PAAS, Tnos gene fragments and PGluC, PAR, Tmas gene fragments were respectively ligated with the backbone vector to obtain three recombinant intermediate vectors containing promoters, genes, and terminators: pSAT6-GluB4-mDHS1-T35s, pSAT6-PGlb1-PAAS, and PAST6-PGluC-PAR-Tmas; (4) Using pSAT6-GluB4-mDHS1-T35s as a template, primer pairs pB4DHS-F0 and p35DHS-R0 were used for PCR amplification to obtain the GluB4-mDHS1-T35s gene fragment; pSA Using T6-PGlb1-PAAS-Tnos as a template, primers pb1PAAS-F0 and pPAAS-R0 were used for PCR amplification to obtain the PGlb1-PAAS gene fragment; using PAST6-PGluC-PAR-Tmas as a template, primers pCPAR1-F0 and pCPAR1-R0 were used for PCR amplification to obtain the PGluC-PAR-Tmas gene fragment; (5) pRHVc was selected as the backbone vector for vector construction, and HpaI and BamHI were used as restriction enzyme sites for vector construction. The GluB4-mDHS1-T35s gene fragment was inserted using homologous recombination to obtain the pRHVc-mDHS1 recombinant plasmid;The pRHVc-mDHS1 vector was selected as the backbone vector for vector construction, and XbaI and BamHI were used as restriction enzyme sites. Homologous recombination was used to insert the PGlb1-PAAS gene fragment, resulting in the pRHVc-mDHS1-PAAS recombinant plasmid. Alternatively, the pRHVc-mDHS1-PAAS vector was selected as the backbone vector for vector construction, and HpaI and I-SceI were used as restriction enzyme sites. Homologous recombination was used to insert the PGluC-PAR-Tmas gene fragment, resulting in the pRHVc-mDHS1-PAAS-PAR recombinant plasmid.

[0010] For example, PCR sequencing data:SAT6-B4-F:5'-gtgccacccataatacccataatacagggttccttgcgtgaaga-3';DHS-B4-R3:5'-GGCTCTCGGTTTGATT tggaaact-3';DHS-35-F3:5'-CTCTGAGACGTCGTCCGCAAAAATCACCAGTCTCT-3';SAT6-T35s-R:5'-catgattactggcaaacagcttatGTCACTGGATTTTGGT TTTAGGAATTAGAAATTTTATTGA-3';SAT6-Glb1-F:5'-gtgccacccataatacccataaGgcgcctggagggga-3';PAAS-Glb1-R:5'-GAAAGAGCCCATtgatc aatcagacaatcactagaagtgt-3';SAT6-luC-F:5'-gtgccacccataatacccataaGTCCAAGATTTTTTTGGTATTTAATTTACTTGCTT-3';PAR1-luC-R:5'-TGT CACACTCATTCCCACTGCTCTTGGTGatg-3';PAR1-mas-F:5'-ATCTTCTATGTAACTTGGACTCCCATGTTGGCA-3';SAT6-mas-R:5'-gattactggcaaacagcttattat GATAATTTATTTGAAAATTCATAAGAAAAGCAAACG-3';B4-DHS-F3:5'-caaatagctGACGTCATGCCCCTCGCGCCATGC-3';DHS2-R1:5'-TGGCAGCATAAgaTCtAGTA GCAAAAGCCCGCAGC-3';DHS2-F2:5'-GCTACTaGAtcTTATGCTGCCATGCAGAGGGTAACACA-3';35-DHS-R3:5'-CGGACGACGTCTCAGAGCCCCAAAGATGGGATGTTG -3';Glb1-PAAS-F:5'-gattgatcatcaATGGGCTCTTTCCCGTTCC-3';SAT6-PAAS-R:5'-atgattactggcaaacagcttattatTCAATAGGTTGAGAGGATGGCCT-3';luC-PAR1-F:5'-CAGTGGGAATGAGTTGTGACAGCGAAAACag-3'; mas-PAR1-R:5 '-GGGAGTCCAAGTTACATAGAAGATGAACCTCCAAAAAACTTCT-3';pB4DHS-F0:5'-CCGAGCTCACCCGGGGATCCCGCTCTAGAtacagggttccttgcgtgaaga ag-3';p35DHS-R0:5'-ACGGCTTTATGCgttaaccagctattatGTCACTGGATTTTGGTTTTAGGA-3';pb1PAAS-F0:5'-cacgcaaggaaccctgtaTtaaGgc gcctggagggag-3';pPAAS-R0:5'-TACCGAGCTCACCCGGGTCAATAGGTTGAGAGGATGGCCTGG-3';pCPAR1-F0:5'-AGCGAGCTCTTAATTAATAGGGATAA GTTCAAGATTTATTTTTGGTATTTAATTTACTT-3';pCPAR1-R0:5'-ATCCAGTGACataagctggttGATAATTTATTTGAAAATTCATAAGAAAAGCAAACG-3'. ;

[0011] The Agrobacterium mentioned is Agrobacterium EHA105.

[0012] The rice variety is japonica rice. The preferred japonica rice variety is ZH11.

[0013] A second objective of this invention is to provide the application of rice with the ability to biosynthesize 2-phenylethanol, which has a rose-like fragrance, in rice breeding.

[0014] Specifically, the application of rice with the ability to biosynthesize 2-phenylethanol, a characteristic of rose-like fragrance, in rice breeding involves cultivating rice plants capable of synthesizing 2-phenylethanol, specifically rice plants overexpressing the mDHS1, PAAS, and PAR genes. Once mature, rice seeds are obtained. During the cultivation process, the mDHS1, PAAS, and PAR genes are overexpressed in the rice plants via endosperm promoters to promote the accumulation of 2-phenylethanol (2-PE) in the rice grains.

[0015] The beneficial effects of this invention compared with the prior art are as follows: Based on synthetic biology technology and metabolic engineering strategy, this invention uses the pRHVc vector to overexpress the mDHS1, PAAS and PAR genes in rice endosperm through transgenic means, thereby directionally synthesizing and stably enriching 2-PE in rice grains, and endowing rice with rose-scented characteristics, significantly improving the economic added value of rice crops, and providing technical support for the development of innovative rice varieties that have both food function and industrial raw material value. Attached Figure Description

[0016] Figure 1 shows the vector construction diagram; Figure 2 shows the 2-PE assay results; ZH11 represents wild-type ZH11, and PAR / ZH11 represents transgenic plants; T-test was used for significance analysis, with the error bar representing standard error (SD, n=3), and *** representing P<0.001. Detailed Implementation

[0017] 1. The pRHVc overexpression method was used to increase the expression levels of the mDHS1 (nucleotide sequence shown in SEQ ID NO.1), PAAS (nucleotide sequence shown in SEQ ID NO.2), and PAR (nucleotide sequence shown in SEQ ID NO.3) genes in rice.

[0018]

[0019] For example, PCR sequencing data:SAT6-B4-F:5'-gtgccacccataatacccataatacagggttccttgcgtgaaga-3';DHS-B4-R3:5'-GGCTCTCGGTTTGATT tggaaact-3';DHS-35-F3:5'-CTCTGAGACGTCGTCCGCAAAAATCACCAGTCTCT-3';SAT6-T35s-R:5'-catgattactggcaaacagcttatGTCACTGGATTTTGGT TTTAGGAATTAGAAATTTTATTGA-3';SAT6-Glb1-F:5'-gtgccacccataatacccataaGgcgcctggagggga-3';PAAS-Glb1-R:5'-GAAAGAGCCCATtgatc aatcagacaatcactagaagtgt-3';SAT6-luC-F:5'-gtgccacccataatacccataaGTCCAAGATTTTTTTGGTATTTAATTTACTTGCTT-3';PAR1-luC-R:5'-TGT CACACTCATTCCCACTGCTCTTGGTGatg-3';PAR1-mas-F:5'-ATCTTCTATGTAACTTGGACTCCCATGTTGGCA-3';SAT6-mas-R:5'-gattactggcaaacagcttattat GATAATTTATTTGAAAATTCATAAGAAAAGCAAACG-3';B4-DHS-F3:5'-caaatagctGACGTCATGCCCCTCGCGCCATGC-3';DHS2-R1:5'-TGGCAGCATAAgaTCtAGTA GCAAAAGCCCGCAGC-3';DHS2-F2:5'-GCTACTaGAtcTTATGCTGCCATGCAGAGGGTAACACA-3';35-DHS-R3:5'-CGGACGACGTCTCAGAGCCCCAAAGATGGGATGTTG -3';Glb1-PAAS-F:5'-gattgatcatcaATGGGCTCTTTCCCGTTCC-3';SAT6-PAAS-R:5'-atgattactggcaaacagcttattatTCAATAGGTTGAGAGGATGGCCT-3';luC-PAR1-F:5'-CAGTGGGAATGAGGTTGACAGCGAAAACag-3'; mas-PAR1-R:5'-GGGAGTCCAAGTTACATAGAAGATGAACCTCCAAAAAACTTCT-3'; pB4DHS-F0:5'-CCGAGC TCACCCGGGGATCCCGCTCTAGAtacagggttccttgcgtgaagaag-3';p35DHS-R0:5'-ACGGCTTTATGCgttaaccagctattatGTCACTGGATTTTGGTTTTAGGA-3';pb1PAAS-F 0:5'-cacgcaaggaaccctgtaTtaaGgcgcctggagggag-3';pPAAS-R0:5'-TACCGAGCTCACCCGGGTCAATAGGTTGAGAGGATGGCCTGG-3';pCPAR1-F0:5'-AGCGAGCTCTT AATTAATAGGGATAAGTTCAAGATTTATTTTTGGTATTTAATTTACTT-3';pCPAR1-R0:5'-ATCCAGTGACataatagctggttGATAATTTATTTGAAAATTCATAAGAAAAGCAAACG-3'. ;

[0020] The rice variety is ZH11.

[0021] When overexpressing the mDHS1, PAAS, and PAR genes, the nucleotide sequence of the PGluB4 promoter is shown in SEQ ID NO.4; the nucleotide sequence of the terminator T35s is shown in SEQ ID NO.5; the nucleotide sequence of the PGlb1 promoter is shown in SEQ ID NO.6; the nucleotide sequence of the PGluC promoter is shown in SEQ ID NO.7; and the nucleotide sequence of the terminator Tmas is shown in SEQ ID NO.8.

[0022] 1.1 Obtaining mDHS1, PAAS and PAR gene fragments (1) Using plasmid as template, PCR amplification was performed using primers SAT6-B4-F and DHS-B4-R3 to obtain the PGluB4 gene fragment; PCR amplification was performed using primers DHS-35-F3 and SAT6-T35s-R to obtain the T35s gene fragment; PCR amplification was performed using primers SAT6-Glb1-F and PAAS-Glb1-R to obtain the Glb1 gene fragment; PCR amplification was performed using primers SAT6-luC-F and PAR1-luC-R to obtain the PGluC gene fragment; PCR amplification was performed using primers PAR1-mas-F and SAT6-mas-R to obtain the Tmas gene fragment.

[0023] (2) Using rice cDNA as a template, PCR amplification was performed using primer pairs B4-DHS-F3, DHS2-R1; DHS2-F2, 35-DHS-R3 to obtain the mDHS1 gene fragment; using rose cDNA as a template, PCR amplification was performed using primer pairs Glb1-PAAS-F, SAT6-PAAS-R to obtain the PAAS gene fragment; using tomato cDNA as a template, PCR amplification was performed using primer pairs luC-PAR1-F, mas-PAR1-R to obtain the PAR gene fragment.

[0024] (3) Electrophoresis detection: After PCR amplification, 1% agarose gel electrophoresis was performed, and the results showed that there were target bands of the same size.

[0025] (3) Purification of target fragment: After electrophoresis, the PCR product is purified and recovered.

[0026] 1.2 Construction of recombinant vectors (1) Select vector pSAT6 as the backbone vector for vector construction, and PI-PspI as the restriction enzyme site for vector construction. Using homologous recombination, the PGluB4, mDHS1, T35s gene fragments and PGlb1, PAAS, Tnos gene fragments and PGluC, PAR, Tmas gene fragments obtained in steps (1) and (2) are respectively ligated to the backbone vector to obtain three intermediate recombinant vectors containing promoter, gene and terminator: pSAT6-GluB4-mDHS1-T35s, pSAT6-PGlb1-PAAS and PAST6-PGluC-PAR-Tmas.

[0027] (2) Using pSAT6-GluB4-mDHS1-T35s as a template, PCR amplification was performed using primer pairs pB4DHS-F0 and p35DHS-R0 to obtain the GluB4-mDHS1-T35s gene fragment; using pSAT6-PGlb1-PAAS-Tnos as a template, PCR amplification was performed using primer pairs pb1PAAS-F0 and pPAAS-R0 to obtain the PGlb1-PAAS gene fragment; using PAST6-PGluC-PAR-Tmas as a template, PCR amplification was performed using primer pairs pCPAR1-F0 and pCPAR1-R0 to obtain the PGluC-PAR-Tmas gene fragment.

[0028] (3) Selecting pRHVc as the backbone vector for vector construction, and HpaI and BamHI as restriction enzyme sites for vector construction, the GluB4-mDHS1-T35s gene fragment was inserted using homologous recombination to obtain the pRHVc-mDHS1 recombinant plasmid; selecting pRHVc-mDHS1 as the backbone vector for vector construction, and XbaI and BamHI as restriction enzyme sites for vector construction, the PGlb1-PAAS gene fragment was inserted using homologous recombination to obtain the pRHVc-mDHS1-PAAS recombinant plasmid; selecting pRHVc-mDHS1-PAAS as the backbone vector for vector construction, and HpaI and I-SceI as restriction enzyme sites for vector construction, the PGluC-PAR-Tmas gene fragment was inserted using homologous recombination to obtain the pRHVc-mDHS1-PAAS-PAR recombinant plasmid.

[0029] (4) The DH5α strain was transformed by heat shock and evenly spread on LB solid plates containing kanamycin for screening. An appropriate amount of colonies were selected for PCR verification. The positive plasmid with correct sequencing was selected, named pRHVc-mDHS1-PAAS-PAR, and stored at -20℃ for later use.

[0030] 1.3 Transformation of recombinant vector into EHA105 Agrobacterium (1) Add 2 μL of plasmid (about 500 ng) to EHA105 Agrobacterium competent cells, place on ice for 5 min, then transfer to liquid nitrogen for 5 min, then transfer to a 37℃ water bath for 5 min of heat shock, and finally ice bath for 5 min.

[0031] (2) Add 500 μL of anti-YEP liquid and activate it for 2 h at 200 rmp / min in a shaker at 28℃.

[0032] (3) The transformed bacterial culture was plated on YEP plates containing kanamycin and rifampicin antibiotics for screening to obtain positive strains.

[0033] 1.4 Transformation of callus (1) Obtaining rice callus: 1) Take mature ZH11 seeds that are fully developed and free of mold lesions, remove the hulls with a small rice huller, and keep the whole rice grains.

[0034] 2) In the ultra-clean workbench, add an appropriate amount of rice grains to a sterile Erlenmeyer flask, clean and disinfect the surface with 75% alcohol for 1 minute, and then rinse with sterile distilled water 3-5 times.

[0035] 3) Add 10% sodium hypochlorite solution, cover with sealing film, and sterilize in a shaker at room temperature (160 rpm) for 30 minutes.

[0036] 4) Discard the sodium hypochlorite solution, wash the seeds 3-5 times with sterile distilled water, transfer them to sterile filter paper and air dry until the surface turns white.

[0037] 5) Transfer the seeds to a plate of callus induction medium and incubate at 30°C under light for 4-6 days.

[0038] (2) Agrobacterium tumefaciens-mediated transformation of rice callus 1) Take EHA105 Agrobacterium tumefaciens containing pRHVc-mDHS-PAAS-PAR plasmid stored at -80℃, streak on a plate (containing 50 mg / L kanamycin and 50 mg / L rifampin), and incubate at 28℃ for two days.

[0039] 2) Pick a single colony and incubate it in 3 mL of YEP liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin) at 28°C and 200 rpm for 24 hours.

[0040] 3) Take 50 µL of the bacterial culture from the previous step and add it to 50 mL of YEP liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin). Incubate at 28°C and 200 rpm until the absorbance OD600 reaches 0.5.

[0041] 4) Centrifuge the expanded bacterial culture at 6000 rpm for 10 minutes at room temperature, discard the supernatant, and collect the precipitated bacterial cells. Resuspend the cells in AAM liquid medium (containing 150 uM acetylsuccine) and adjust the OD600 to 0.1.

[0042] 5) In the clean bench, select pale yellow, dense, healthy rice callus tissue with a size of 1-2 mm, immerse the callus tissue in the prepared mycelium suspension, and gently shake for 1 minute.

[0043] 6) Discard the culture medium, wash 2-3 times with sterile distilled water, and then place it on sterile filter paper to air dry.

[0044] 7) Place a filter paper soaked in liquid co-culture medium on the surface of the solid co-culture medium, spread the callus tissue on it, and incubate in a dark room at 25-26 ℃ for 2 days.

[0045] (3) Screening of resistant callus and plant regeneration 1) After dark culture, the callus was transferred to a sterile Erlenmeyer flask, rinsed 5 times with sterile distilled water, added sterile distilled water containing 500 mg / L carbenicillin, and shaken at 160 rpm for 20 min; the callus was then removed and air-dried on sterile filter paper.

[0046] 2) Transfer the callus tissue to selective medium and incubate in the dark at 30°C, replacing the selective medium every two weeks.

[0047] 3) After the callus tissue on the selective medium develops into bright yellow granular resistant callus, it is transferred to the selective differentiation medium and cultured at 30°C under light. The differentiation medium is replaced every two weeks.

[0048] 4) After the callus tissue grows resistant green seedlings, it is transferred to rooting medium and cultured at 30 ℃ with light for 16 h / d.

[0049] 5) After the resistant seedlings have rooted in 3-4 weeks, select plants with well-developed root systems, open the top cover, add distilled water, and carry out hardening-off cultivation.

[0050] 2. Experimental Materials: Transgenic plants were cultivated in the transgenic experimental field of South China Agricultural University, following conventional water and fertilizer management methods. After the rice grains filled, they were harvested, flash-frozen in liquid nitrogen, stored at -80℃, and then sent to the company for 2-PE content testing.

[0051] 3. GC-MS was used to detect 2-PE. The T-test was used to analyze the significant difference between the wild-type and the transgenic plants PAR / ZH11 of the present invention. As shown in Figure 1, the 2-PE content of the transgenic plants of the present invention was significantly higher than that of the wild-type.

Claims

1. A method for creating rice with rose-scented characteristics through the biosynthesis of 2-phenylethanol, characterized in that, The mDHS1, PAAS, and PAR genes were used to construct the pRHVc-mDHS1-PAAS-PAR recombinant plasmid. This plasmid was then transformed into callus tissue using Agrobacterium tumefaciens-mediated transformation. After screening of resistant callus tissue and plant regeneration, rice overexpressing the mDHS1, PAAS, and PAR genes was obtained.

2. The method for creating 2-phenylethanol biosynthetic rice with rose-scented characteristics according to claim 1, characterized in that, The mDHS1 gene is derived from a homologous gene of rice or other different species, the PAAS gene is derived from a homologous gene of rose or other different species, and the PAR gene is derived from a homologous gene of tomato or other different species.

3. The method for creating 2-phenylethanol biosynthetic rice with rose-scented characteristics according to claim 1, characterized in that, The pRHVc-mDHS1-PAAS-PAR recombinant plasmid was constructed as follows: Gene fragments of the promoter PGluB4, mDHS1, and terminator T35s were inserted into the pSAT6 vector to obtain the pSAT6-GluB4-mDHS1-T35s recombinant intermediate vector; gene fragments of the promoter PGlb1 and PAAS were inserted into the pSAT6 vector to obtain the pSAT6-PGlb1-PAAS recombinant intermediate vector; gene fragments of the promoter PGluC, PAR, and terminator Tmas were inserted into the pSAT6 vector to obtain the PAST6-PGluC-PAR-Tmas recombinant intermediate vector. The inserted fragments on the three pSAT6 recombinant intermediate vectors were then amplified to obtain three fragments. Finally, the three fragments were inserted into the pRHVc vector to obtain the pRHVc-mDHS1-PAAS-PAR recombinant plasmid.

4. The method for creating 2-phenylethanol biosynthetic rice with rose-scented characteristics according to claim 1, characterized in that, The construction method of the pRHVc-mDHS1-PAAS-PAR recombinant plasmid is as follows: (1) Using the plasmid as a template, PCR amplification was performed using primers SAT6-B4-F and DHS-B4-R3 to obtain the PGluB4 gene fragment; PCR amplification was performed using primers DHS-35-F3 and SAT6-T35s-R to obtain the T35s gene fragment; PCR amplification was performed using primers SAT6-Glb1-F and PAAS-Glb1-R to obtain the Glb1 gene fragment; PCR amplification was performed using primers SAT6-luC-F and PAR1-luC-R to obtain the PGluC gene fragment; PCR amplification was performed using primers PAR1-mas-F and SAT6-mas-R to obtain the Tmas gene fragment; (2) Using rice cDNA as a template, PCR amplification was performed using primers B4-DHS-F3 and DHS2-R1; DHS2-F2 and 35-DHS-R3 to obtain the PGluB4 gene fragment; The mDHS1 gene fragment was obtained; using rose cDNA as a template, PCR amplification was performed using primer pairs Glb1-PAAS-F and SAT6-PAAS-R to obtain the PAAS gene fragment; using tomato cDNA as a template, PCR amplification was performed using primer pairs luC-PAR1-F and mas-PAR1-R to obtain the PAR gene fragment; (3) pSAT6 was selected as the backbone vector for vector construction, and PI-PspI was used as the restriction enzyme site for vector construction. Homologous recombination was used to connect the PGluB4, mDHS1, T35s gene fragments and PGlb1, PAAS, Tnos gene fragments and PGluC, PAR, Tmas gene fragments obtained in steps (1)-(2) to the backbone vector respectively to obtain pSAT6-GluB4-mDHS1-T35s, pSAT6-PGlb1-PAAS and PAST6-PGluC-PAR-Tmas containing promoter, gene and terminator. Three recombinant intermediate vectors; (4) Using pSAT6-GluB4-mDHS1-T35s as a template, PCR amplification was performed using primers pB4DHS-F0 and p35DHS-R0 to obtain the GluB4-mDHS1-T35s gene fragment. Using pSAT6-PGlb1-PAAS-Tnos as a template, primer pairs pb1PAAS-F0 and pPAAS-R0 were employed. PCR amplification was performed to obtain the PGlb1-PAAS gene fragment; using PAST6-PGluC-PAR-Tmas as a template, primer pairs pCPAR1-F0 and pCPAR1-R0 were used to perform PCR amplification to obtain the PGluC-PAR-Tmas gene fragment; (5) the vector pRHVc was selected as the backbone vector for vector construction, and HpaI and BamHI were selected as the restriction enzyme sites for vector construction. The GluB4-mDHS1-T35s gene fragment was inserted using homologous recombination to obtain the pRHVc-mDHS1 recombinant plasmid; the vector pR was selected as the backbone vector for vector construction. Using HVc-mDHS1 as the backbone vector and XbaI and BamHI as restriction enzyme sites, the PGlb1-PAAS gene fragment was inserted using homologous recombination to obtain the pRHVc-mDHS1-PAAS recombinant plasmid. Alternatively, using pRHVc-mDHS1-PAAS as the backbone vector and HpaI and I-SceI as restriction enzyme sites, the PGluC-PAR-Tmas gene fragment was inserted using homologous recombination to obtain the pRHVc-mDHS1-PAAS-PAR recombinant plasmid. The primer pairs used for PCR amplification have the following sequences: SAT6-B4-F: 5'-gtgccacccataatacccataatacagggttccttgcgtgaaga-3'; DHS-B4-R3: 5'-GGCATGACGTCagctatttgaggatgttattggaaact-3'; DHS-35-F3: 5'-CTCTGAGACGTCGTCCGCAAAAATCACCAGTCTCT-3'; SAT6-T35s-R: 5'-catgattactggcaaacagctattatGTCACTGGATTTTGGTTTTAGGAATTAGAAATTTTATTGA-3'; SAT6-Glb1-F: 5' -gtgccacccataatacccataaGgcgcctggagggagga-3';PAAS-Glb1-R:5'-GAAAGAGCCCATtgatgatcaatcagacaatcactagaagtgt-3';SAT6-luC-F:5'-gtgccacccataatacccataa GTTCAAGATTTATTTTTGGTATTTAATTTACTTGCTT-3'; PAR1-luC-R:5'-TGTCACACTCATTCCCACTGCTCTTGGTGatg-3'; PAR1-mas-F:5'-ATCTTCTATGTAACTTGGACTCCCATGTTGGCA-3';SAT6-mas-R:5'-gattactggcaaacagcttatGATAATTTATTTGAAAATTCATAAGAAAAGCAAACG-3';B4-DHS-F3:5'-caaatagctGACGTCATGCCCCTC GCGCCATGC-3';DHS2-R1:5'-TGGCAGCATAAgaTCtAGTAGCAAAAGCCCGCAGC-3';DHS2-F2:5'-GCTACTaGAtcTTATGCTGCCATGCAGAGGGTAACACA-3 ';35-DHS-R3:5'-CGGACGACGTCTCAGAGCCCCAAAGATGGGATGTTG-3';Glb1-PAAS-F:5'-gattgatcatcaATGGGCTCTTTCCCGTTCC-3';SAT6-PAAS -R:5'-atgattactggcaaacagctattatTCAATAGGTTGAGAGGATGGCCT-3';luC-PAR1-F:5'-CAGTGGGAATGAGTGTGACAGCGAAAACag-3';mas-PAR1 -R:5'-GGGAGTCCAAGTTACATAGAAGATGAACCTCCAAAAACTTCT-3';pB4DHS-F0:5'-CCGAGCTCACCCGGGGATCCCGCTCTAGAtacagggttccttgcgtga agaag-3';p35DHS-R0:5'-ACGGCTTTATGCgttaaccagctattatGTCACTGGATTTTGGTTTTAGGA-3';pb1PAAS-F0:5'-cacgcaaggaaccctgtaTtaaG gcgcctggagggag-3';pPAAS-R0:5'-TACCGAGCTCACCCGGGTCAATAGGTTGAGAGGATGGCCTGG-3';pCPAR1-F0:5'-AGCGAGCTCTTAATTAATAGGGATA AGTTCAAGATTTTTTTGGTATTTAATTTACTT-3':pCPAR1-R0:5'-ATCCAGTGACataatagctggttGATAATTTTTGAAAATTCATAAGAAAAGCAAACG-3'。:

5. The method for creating 2-phenylethanol biosynthetic rice with rose-scented characteristics according to claim 1, characterized in that, The Agrobacterium mentioned is Agrobacterium EHA105.

6. The method for creating 2-phenylethanol biosynthetic rice with rose-scented characteristics according to claim 1, characterized in that, The rice variety mentioned is ZH11.

7. Rice with 2-phenylethanol biosynthesis capability having a rose-scented characteristic, created by the method according to any one of claims 1-6.

8. The application of the rice with 2-phenylethanol biosynthesis capability as described in claim 7 in rice breeding, characterized in that, The rice plants with the ability to biosynthesize 2-phenylethanol, which has a rose-like fragrance, are cultivated into plants, and rice seeds are obtained after they mature.

Images