Application of transcription factor OsMPS in increasing 2-acetyl-1-pyrroline content in rice

Abstract

The present invention relates to the technical field of plant genetic engineering, and in particular to the application of transcription factor OsMPS in increasing the content of 2-acetyl-1-pyrroline in rice. The amino acid sequence of the transcription factor OsMPS is shown in SEQ ID NO.27. The present invention significantly increases the content of 2-acetyl-1-pyrroline in rice by overexpressing the OsMPS gene driven by a constitutive promoter in rice; while no 2-acetyl-1-pyrroline is detected in the knockout mutant. The advantage is that OsMPS, as a positive regulatory factor, promotes the synthesis of 2-acetyl-1-pyrroline in rice, and plays an important role in improving the aroma quality of rice.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to application of transcription factor OsMPS in improving the content of 2-acetyl-1-pyrroline in rice. Background Art

[0002] Rice is the most important staple food crop in the world and has made a significant contribution to human health. With the increase in rice yield, people have higher requirements for rice quality, changing from "eating enough" to "eating well". Fragrance is one of the most important traits of rice quality. Fragrant rice is called "the treasure of rice". It has the advantages of excellent quality, rich fragrance, mellow taste and rich nutrition. It has been loved by people for a long time. The fragrance of rice can effectively improve the quality and value of rice. Many consumers are attracted by the unique aroma of fragrant rice. Fragrant rice broadens the genetic diversity of rice varieties, adds new high-quality gene resources to the rice gene bank, improves the quality traits of rice, and lays a solid foundation for future breeding work. Therefore, the molecular mechanism of rice fragrance traits and its application in breeding have attracted the attention of many scholars.

[0003] Studies have shown that the key aromatic substance of aromatic rice is 2-acetyl-1-pyrroline (2-AP), which has a strong aroma similar to that of popcorn, is highly volatile, has a low olfactory threshold, and is mainly found in rice leaves and grains. It has the effects of promoting appetite and improving metabolism. The biosynthesis of 2-acetyl-1-pyrroline can be carried out through two pathways: non-enzymatic pathway and enzymatic pathway. The non-enzymatic pathway starts from precursors, including proline, ornithine, and glutamate, while the enzymatic pathway starts from ornithine. The two form common intermediates 1-pyrroline-5-carboxylic acid (P5C) and γ-aminobutyraldehyde (GABald), respectively. The former is converted into 2-acetyl-1-pyrroline, while the latter forms 2-acetyl-1-pyrroline or γ-aminobutyric acid (GABA). Studies have shown that the main contributor to aroma, 2-acetyl-1-pyrroline, is produced by the enzyme betaine aldehyde dehydrogenase 2 (Betaine Aldehyde Dehydrogenase Homologue 2, located on chromosome 8 of rice. OsBADH2 ) gene control. Under normal circumstances Badh2 Encodes betaine aldehyde dehydrogenase, which catalyzes the conversion of γ-aminobutyraldehyde to γ-aminobutyric acid. However, after the gene mutation leads to loss of function, γ-aminobutyraldehyde cannot be converted into γ-aminobutyric acid, which is converted into 1-pyrroline and further generates 2-acetyl-1-pyrroline ( Figure 1 ).

[0004] Genome-wide association study (GWAS) was first used as a powerful tool to study the genetic structure of human diseases. With the continuous advancement of sequencing technology and the gradual completion of high-quality reference genome sequencing of many species, more and more genotype data have been generated, making the application of genome-wide association study in plants more and more extensive, and becoming a powerful tool for identifying genetic loci for complex traits in plants. Volatile metabolites are used as phenotypic traits, and the volatile-based genome-wide association study (vGWAS) combined with volatileomics is used to analyze the genetic mechanisms behind volatile diversity and its association with complex plant traits.

[0005] MYB transcription factors are one of the most important transcription factor (TFs) families, which are widely distributed in eukaryotes. Studies have found that R2R3-MYB family members not only play an important regulatory role in plant secondary metabolite synthesis, biotic stress and abiotic stress response, growth and development and other life processes, but also play an important role in fragrance regulation. SlMYB75-OE The content of volatile metabolites in tomatoes is significantly higher than that in wild type, especially aldehydes and terpenes. Petunia is often used as a model plant for studying fragrance because of its large flowers that can produce and release a large amount of phenylpropanoid volatiles. Studies have shown that the R2R3-MYB transcription factor EVER controls the release of volatile metabolites in petunia flowers by regulating the biosynthesis of epidermal wax in petals. In lilies, LiMYB1 、 LiMYB305 and LiMYB330 interact with each other and play an important role in terpenoid biosynthesis in Lilium plants, providing new insights into the transcriptional regulation of volatile terpenoids in flowers.

[0006] At present, the research reports on the regulation of 2-acetyl-1-pyrroline synthesis mainly focus on OsBADH2, while there are few reports on the transcriptional regulation mechanism. Summary of the invention

[0007] To solve the above problems, one of the objects of the present invention is to provide an application of the transcription factor OsMPS in increasing the 2-acetyl-1-pyrroline content in rice; a second object of the present invention is to provide a method for increasing the 2-acetyl-1-pyrroline content in rice.

[0008] The technical solution adopted by the present invention to achieve the technical purpose is:

[0009] The present invention provides application of transcription factor OsMPS in increasing 2-acetyl-1-pyrroline content in rice. The amino acid sequence of the transcription factor OsMPS is shown in SEQ ID NO.27.

[0010] Preferably, the nucleotide sequence of the transcription factor OsMPS is shown as SEQ ID NO.26.

[0011] Preferably, the transcription factor OsMPS inhibits the expression of 2-acetyl-1-pyrroline in rice. OsBADH2 Gene expression, promoting the synthesis of 2-acetyl-1-pyrroline.

[0012] The present invention also provides a method for increasing the 2-acetyl-1-pyrroline content of rice, comprising: overexpressing the transcription factor OsMPS in rice, wherein the amino acid sequence of the transcription factor OsMPS is shown in SEQ ID NO.27.

[0013] Preferably, the nucleotide sequence of the transcription factor OsMPS is shown as SEQ ID NO.26.

[0014] Preferably, the method for overexpressing the transcription factor OsMPS in rice comprises: inserting the transcription factor OsMPS into a plant transformation plasmid containing the overexpression gene function to obtain a plant overexpression vector of the transcription factor OsMPS.

[0015] More preferably, the plant overexpression vector is constructed using the Gateway system, and the desired gene fragment is amplified using rice cDNA as a template, and the primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0016] More preferably, pH2GW7 is used as the starting vector of the plant overexpression vector.

[0017] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0018] The present invention discloses the application of transcription factor OsMPS in increasing the content of 2-acetyl-1-pyrroline in rice. OsBADH2 Gene expression, thereby promoting the synthesis of 2-acetyl-1-pyrroline. Overexpression of this gene can significantly increase the content of 2-acetyl-1-pyrroline, indicating that the transcription factor OsMPS, as a positive regulatory factor, promotes the synthesis of 2-acetyl-1-pyrroline in rice, which has important guiding significance and broad market prospects for improving the aroma quality of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The synthetic route of 2-acetyl-1-pyrroline putatively described in the prior art is shown.

[0020] Figure 2The expression of OsMPS gene in rice leaves of OsMPS overexpressing strains detected by real-time fluorescence quantitative PCR provided in the embodiments of the present invention (A), and the expression of OsMPS gene in rice leaves of OsMPS overexpressing strains and knockout mutant strains OsBADH2 Gene expression (B); OsMPS-OE15 and OsMPS-OE27: different transgenic rice lines overexpressing OE - OsMPS; osmps-2, osmps-4: indicate different transgenic rice lines of knockout mutants CR-OsMPS; wild type: wild type-ZH11.

[0021] Figure 3 A bar graph (A) and a standard curve (B) of 2-acetyl-1-pyrroline in leaves of overexpressed and mutant OsMPS strains detected by GC-MS for the embodiment of the present invention; OsMPS-OE15, OsMPS-OE27: different transgenic rice strains OE-OsMPS overexpressed; osmps - 2.osmps - 4: Different transgenic rice lines representing knockout mutants: CR-OsMPS; wild type: wild type-ZH11.

[0022] Figure 4 The domain (A: OsBADH2 Promotor region motif analysis of genes; B: OsMPS yeast one-hybrid assay results); proOsBADH2: OsBADH2 Promoter OsBADH2 .

[0023] Figure 5 The present invention provides a dual-luciferase reporter assay (Dual-LUC) to verify that OsMPS binds to proOsBADH2, thereby inhibiting transcription. OsBADH2 Expression of OsMPS (A: vector diagram; B: OsBADH2 promoter-based dual-luciferase reporter gene activity); proOsBADH2 :express OsBADH2 Promoter OsBADH2 promoter. DETAILED DESCRIPTION

[0024] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the same modules are represented by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, the detailed description thereof will not be repeated.

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0026] Example 1 Rice OsMPS and OsBADH2 Promoter gene cloning

[0027] 1. Extraction of Total RNA from Rice Leaves

[0028] Fresh rice leaves were taken, quickly frozen in liquid nitrogen, and fully ground; 0.1 g of powder was added to 1 ml of Trizol, and total RNA was extracted according to the kit method of Quanshijin Company. The RNA quality was tested by agarose gel electrophoresis, and the RNA concentration was tested by NanoDrop2000 spectrophotometer.

[0029] 2. Cloning of Rice Genes

[0030] The extracted total RNA was used as a template to synthesize cDNA using a one-step reverse transcription kit from Beijing Quanshijin Company; PCR amplification primers were designed based on the OsMPS gene sequence, and the primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, see Table 1.

[0031] Table 1 Cloning primers for rice OsMPS

[0032]

[0033] The obtained cDNA was used as a template for PCR amplification, and the program was: 94°C pre-denaturation for 5 min, 98°C denaturation for 10 s, 68°C annealing for 5 s, 72°C extension for 30 s, repeated 28 cycles; 72°C final extension for 5 min; stored at 25°C. PCR amplification was performed with the OsMPS gene primers in Table 1, and the product was purified and sequenced to obtain the rice OsMPS sequence. The sequencing results showed that the obtained OsMPS gene was 870 bp in length, and its nucleotide sequence was shown in SEQ ID NO.26; the obtained OsMPS gene encodes 289 amino acids, and its amino acid sequence is shown in SEQ ID NO.27.

[0034] 3. Cloning of Rice Promoter

[0035] Refer to the CTAB extraction method of plant genome to extract rice leaf DNA. proOsBADH2 PCR amplification primers were designed based on the 1646 bp sequence of the promoter. The primer sequences are shown in Table 2 as SEQ ID NO.3 and SEQ ID NO.4.

[0036] Table 2 Rice proOsBADH2 Amplification primers

[0037]

[0038] The obtained DNA was used as a template for PCR amplification, and the program was 94℃ pre-denaturation for 5min, 98℃ denaturation for 10s, 68℃ annealing for 5s, 72℃ extension for 50s, repeated 30 cycles; 72℃ final extension for 5min; stored at 25℃. The PCR amplification product was excised and recovered and sequenced to obtain rice proOsBADH2 The sequencing results showed that the obtained proOsBADH2 The promoter sequence is 1646 bp in length, and its sequence is shown in SEQ ID NO.28.

[0039] Example 2 Construction of plant overexpression vector

[0040] The construction of plant overexpression vectors was carried out using the Gateway system. First, the desired gene fragment was amplified using rice cDNA as a template. The primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, as shown in Table 3. The gel recovery product with the pDONR207 recombination linker was obtained by PCR, and BP (LR) enzyme, pH2GW7+pUbi (Smedley MA, Harwood WA. Gateway-compatible plant transformation vectors. Methods Mol Biol. 2015;1223:3-16.doi: 10.1007 / 978-1-4939-1695-5_1. PMID: 25300827.), and water were added to form a 2µL system (see Table 4), and the mixture was placed in a dry bath at 25°C for 4 hours, and then transformed into DH5α. The final vector pH2GW7_OE was obtained after sequencing verification.

[0041] Table 3 Amplification primers for rice overexpression vector

[0042]

[0043] Table 4 BP, LR reaction system

[0044]

[0045] Example 3 Construction of plant gene knockout vector

[0046] To construct a plant gene knockout vector, the gene editing target site was first designed using the online software CRISPR-P2.0 (http: / / crispr.hzau.edu.cn / CRISPR2), and a three-target site strategy was adopted. A 50 μL PCR amplification system was used, and transformation and colony PCR identification were performed after restriction digestion and ligation. The target site sequences of OsMPS are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9 (see Table 5). The OsMPS editing sequence was amplified with SEQ ID NO.10 and SEQ ID NO.11, and then pH-Ubi-cas9-7 (purchased from Addgene; catalog number: 106331) was connected to obtain a plant transformation plasmid containing the OsMPS gene editing function of CRISPR / Cas9. The recombinant plasmid was the knockout vector (the vector was constructed by Boyuan Biotechnology Co., Ltd.).

[0047] Table 5 Target site design information

[0048]

[0049] Table 6 Amplification primers

[0050]

[0051] Example 4 Stable genetic transformation of rice mediated by Agrobacterium tumefaciens

[0052] The plant overexpression vector and plant gene knockout vector of OsMPS were introduced into Agrobacterium tumefaciens EHA105 for stable genetic transformation of rice. The specific steps are as follows:

[0053] Agrobacterium positive single clone identification: Take 1 μL of plasmid and add it to 30 μL of EHA105 Agrobacterium tumefaciens competent cells, mix well, add 1 mL of YEB liquid medium after electroporation, shake and culture for 30 min at 28°C and 180 rpm on a shaker, inoculate it into YEB solid medium, and culture it in the dark at 28°C for 48 h. After PCR identification, positive Agrobacterium single clones were obtained, and the identification primer sequences are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, see Table 3, Table 6 and Table 7.

[0054] Rice genetic transformation: Select complete, plump and uniform rice grains, disinfect with 70% alcohol for 2 minutes, wash with sterile water, disinfect with 30% sodium hypochlorite for 10 minutes, wash with sterile water, place in 1 / 2MS medium, and culture under light for 7-8 days. The grown callus is infecting with positive Agrobacterium solution for 25 minutes, placed on sterile filter paper to dry in the shade, spread on co-culture medium, and co-cultured in the dark at 25℃ for 2.5-3 days. The callus is evenly placed on the screening medium, screened and cultured in the dark at 25℃ for 2-3 weeks, transferred to the pre-differentiation medium, cultured at room temperature with 14h / 10h light and dark alternation for two weeks, and transferred to the differentiation medium. The rice callus that has grown to 2 leaves or 2 leaves and one heart is transferred to the rooting medium. When the rice seedlings grow to about 10cm, they are placed in clean water for 3-4 days of seedling hardening and moved into soil pots (the transgenic material is created by Boyuan Biotechnology Co., Ltd.).

[0055] The positive plants of the T0 generation were identified by PCR, and the identification primer sequences were shown in SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17, as shown in Table 7. The positive plants of the T1 generation were obtained by self-pollination for subsequent experiments and analysis of the content of related metabolites.

[0056] Table 7 PCR primers for positive plant identification

[0057]

[0058] Quantitative PCR was used to determine the expression of OsMPS and OsBADH2 The gene expression status and the detection primer sequences are shown in SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21, see Table 8.

[0059] Table 8 qPCR identification primers

[0060]

[0061] Test results such as Figure 2 As shown in Figure 2, the expression level of OsMPS gene in rice plants of OE-OsMPS overexpression line increased significantly, while OsBADH2 The expression of the gene was significantly reduced, while the CR knockout material was the opposite, indicating that OsMPS can inhibit OsBADH2 Gene expression.

[0062] Example 5 Determination of the content of volatile metabolites in rice leaves

[0063] Headspace solid phase microextraction (HS-SPME) can meet the requirements for accurate qualitative and quantitative determination of volatile metabolites. Its operation process is briefly as follows:

[0064] Sample preparation: Mix 1 g of brown rice sample with 300 μL of ddHO. 2 O was transferred to a 22 mL glass headspace vial for GC-MS analysis.

[0065] Before extraction using headspace solid phase microextraction (HS-SPME), samples were preheated at 80°C for 10 min to simulate the cooking process. Volatile metabolites were detected using an Agilent 7890 A GC system coupled to an Agilent 7000 D triple quadrupole mass detector. Volatile metabolites were adsorbed onto a 1 cm long 50 / 30 divinylbenzene / carbon molecular sieve / polydimethylsiloxane stabilized elastic fiber at 80°C. The adsorption process lasted for 20 min with constant shaking, followed by desorption for 25 s in a gas chromatography-mass spectrometer (GC-MS).

[0066] Volatile metabolites were detected by headspace solid phase microextraction. HP-5 mass spectrometry capillary column (30 mm* 0.25 mmi.d., 0.25 mm film thickness; Agilent Technologies) was used to separate volatile metabolites. The initial column temperature was 50°C, held for 2 min, the temperature was increased by 5°C / min to 200°C, held for 2 min, and then the temperature was increased by 20°C / min to a final temperature of 250°C, held for 2 min. The ion source temperature was set to 230°C, the quadrupole temperature was set to 150°C, and the electron energy was set to 70 electron volts; the He flow rate was 1.0 ml / min (99.999%). The mass spectrometer was operated in full scan mode with a scan range of 35-500 m / z.

[0067] Standard curve establishment: 2-acetyl-1-pyrroline standard solution was prepared by dissolving 2-acetyl-1-pyrroline (purity 95%, CAS number: 85213-225, provided by Beijing J&K Technology Co., Ltd.) in toluene, with an initial concentration of 100 mg / ml. The stock solution was diluted with n-hexane to obtain standard solutions with concentrations of 10, 15, 20 and 25 mg / ml, respectively. 1 μl of each standard solution was injected, and each sample was repeated three times. Then, a standard calibration curve was constructed based on the 2-acetyl-1-pyrroline peak area and the corresponding injected 2-acetyl-1-pyrroline concentration.

[0068] The results are as follows Figure 3As shown, the content of 2-acetyl-1-pyrroline in the OsMPS overexpression strain was significantly increased compared with the wild type, but 2-acetyl-1-pyrroline was not detected in the mutant strain.

[0069] Example 6 OsMPS and proOsBADH2 Interaction status and action site verification

[0070] The OsMPS cloned in Example 1 was connected to the vector pB7WG2.0 (Dual-LUC) (Cat. No. VT13019, Qingdao Crespo Biotechnology Co., Ltd.) to obtain the recombinant vector OsMPS-pB7WG2.0 (Dual-LUC). proOsBADH2 The sequences were constructed into the vector pGEXT4-1 to obtain proOsBADH2-pGEXT4-1. The construction of the vector referred to the Gateway system of Example 2. The interaction was verified using mature Dual-LUC and yeast single hybrid. Dual-LUC was verified in tobacco leaves, and the binding of DNA and protein was determined by fluorescence signals. Yeast single hybrid is to connect the OsMPS obtained by cloning in Example 1 into the vector pGADT7 (Article No.: HG-VJC0483, Changsha Abiwei Biotechnology Co., Ltd.) to obtain the recombinant vector OsMPS-pGADT7. The upstream 1636bp proOsBADH2 sequence was constructed into the vector pHIS2 (Article No.: HG-VJC0481, Changsha Abiwei Biotechnology Co., Ltd.) to obtain proOsBADH2-pHIS2, and the Gateway system of Reference Example 2 was constructed. The Y1H system is a yeast competent system using Y187. The pGADT7 and pHIS2 containing the target gene can grow plaques normally on the triple-deficient plate with 3-AT, which can determine the interaction between DNA and protein. The vector and primer sequences are shown in SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO.25, see Table 9.

[0071] Table 9 Vector and primer information

[0072]

[0073] The results are as follows Figure 4 and Figure 5 As shown, OsMPS can bind to OsBADH2 The promoter region of the gene significantly inhibited its expression. Yeast one-hybrid experiments further confirmed that OsMPS binds to proOsBADH2 on the MYB domain.

[0074] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps described in the disclosure of the present invention can be performed in parallel, sequentially or in different orders, as long as the desired results of the technical solution disclosed in the present invention can be achieved, and this document does not limit this.

[0075] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of transcription factor OsMPS in increasing the content of 2-acetyl-1-pyrroline in rice, characterized in that: The amino acid sequence of the transcription factor OsMPS is shown in SEQ ID NO.

27.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the transcription factor OsMPS is shown as SEQ ID NO.

26.

3. The use according to claim 1 or 2, characterized in that: The transcription factor OsMPS inhibits the OsBADH2 Gene expression, promoting the synthesis of 2-acetyl-1-pyrroline.

4. A method for increasing the content of 2-acetyl-1-pyrroline in rice, characterized in that: The method comprises: over-expressing the transcription factor OsMPS in rice, wherein the amino acid sequence of the transcription factor OsMPS is shown as SEQ ID NO.

27.

5. The method according to claim 4, characterized in that The nucleotide sequence of the transcription factor OsMPS is shown as SEQ ID NO.

26.

6. The method according to claim 4, characterized in that The method for overexpressing the transcription factor OsMPS in rice comprises: inserting the transcription factor OsMPS into a plant transformation plasmid containing an overexpression gene function to obtain a plant overexpression vector of the transcription factor OsMPS.

7. The method according to claim 6, characterized in that The plant overexpression vector was constructed using the Gateway system, and the desired gene fragment was amplified using rice cDNA as a template. The primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.

6.

8. The method according to claim 7, characterized in that pH2GW7 was used as the starting vector for plant overexpression vector.

Images