BADH2 gene for regulating millet aroma, application and method

Abstract

The invention provides a method for regulating the aroma of millet BADH2 Gene, application and method, belonging to the field of biotechnology, the BADH2 Genes include BADH2‑1 Genes and BADH2‑2 gene; the application is by using BADH2‑1 Genes and BADH2‑2 The genes are simultaneously inactivated or reduced in activity to increase the content of the aromatic substance 2-AP in millet; the method is to plant BADH2‑1 Genes and BADH2‑2 The transgenic millet in which the genes are simultaneously inactive or have reduced activity is used to increase the content of the aromatic substance 2-AP in the millet. Compared with the wild-type millet, the transgenic millet obtained by the present invention BADH2 The 2-AP content in leaves of the transgenic millet lines with gene mutations was significantly increased, indicating that BADH2 Losing or reducing the activity of the gene can significantly increase the content of 2-AP in millet leaves.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to plant transgenic biotechnology breeding, and specifically relates to a method for regulating the aroma of millet. BADH2 Genes, applications and methods. Background Art

[0002] Aroma is one of the important quality traits of grains, an important indicator of the cooking and taste quality of grains, and one of the key factors that determine the market price. The aroma of fragrant rice is overflowing and fragrant, which is deeply favored by consumers and has high economic value. According to the different aromas produced by mature rice, it can be divided into popcorn type, jasmine type, violet type, toast type, pecan type, etc. Among them, most of the fragrant rice is dominated by popcorn aroma. Current research shows that the main substance that makes rice produce popcorn aroma is 2-acetyl-1-pyrroline (2-acetyl-1-pyrroline, 2-AP). BADH2 The gene is the main effect gene that controls the popcorn aroma trait of rice and other crops. The gene encodes betaine aldehyde dehydrogenase. The loss of its function will lead to an increase in the content of 2-AP in rice, resulting in fragrant rice. BADH2 Genes have been reported to be used to obtain fragrant rice, millet, corn and sorghum.

[0003] Millet Panicum miliaceum L. ) belongs to the genus Millet of the Poaceae family. It is rich in nutrients and has high nutritional value. Millet was once an important cultivated crop in northern my country, but with the development of agricultural production, it gradually withdrew from the status of a major crop and became a regional specialty crop. The main reason is that there has been no major breakthrough in millet yield and no fundamental improvement in quality. If a new germplasm of fragrant millet can be improved and created, it will surely improve the taste quality of millet, thereby increasing its added value and market value, which is of great significance to the development of the millet industry.

[0004] So far, no BADH2 The fragrant millet materials produced by gene mutations have the disadvantages of unpredictability, high cost, low efficiency and large screening workload when screening natural variations and traditional mutagenesis methods. The development of modern genetic engineering technology can provide a fast and effective breeding method for millet, but there is no research on knocking out millet using gene editing technology. BADH2 Report on genetic creation of fragrant millet. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for regulating the aroma of millet BADH2 (Betaine aldehyde dehydrogenase 2) genes, applications and methods.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] A method for regulating the aroma of millet BADH2 Gene, BADH2 Genes include BADH2-1 Genes and BADH2-2 Gene;

[0008] BADH2-1 The sequence of the gene is shown in SEQ ID NO: 1, and the gene accession number is PM13G12800;

[0009] BADH2-1 The gene is 5530 bp in length, contains 15 exons, and the CDS coding region is 1518 bp in length, encoding 505 amino acids. The amino acid sequence is shown in SEQ ID NO: 4;

[0010] BADH2-2 The sequence of the gene is shown in SEQ ID NO: 2, and the gene accession number is PM14G14600;

[0011] BADH2-2 The gene is 5540 bp in length, contains 15 exons, and the CDS coding region is 1518 bp in length, encoding 505 amino acids. The amino acid sequence is shown in SEQ ID NO: 5;

[0012] By mutating millet BADH2-1 Genes and BADH2-2 The gene can increase the content of the aromatic substance 2-AP in millet, thereby obtaining aromatic millet.

[0013] A method for regulating the aroma of millet BADH2 Application of genes, wherein the application is to use gene editing technology to make the above BADH2-1 Genes and BADH2-2 The genes lose their activity or reduce their activity at the same time to increase the content of the aromatic substance 2-AP in millet.

[0014] Furthermore, the application is to simultaneously knock out by gene editing BADH2-1 Genes and BADH2-2 Gene to increase the content of 2-AP, a fragrant substance in millet;

[0015] BADH2-1 Genes and BADH2-2 The gene editing sites in the gene are the same, and the sequence is shown in SEQ ID NO: 3.

[0016] Further, the application is millet BADH2 Gene editing recombinant vector or containing BADH2 The recombinant strain of gene editing recombinant vector was transformed into millet, making millet BADH2-1Genes and BADH2-2 Genes simultaneously become inactive or less active.

[0017] Furthermore, the application is to construct a method for making millet by using CRISPR / Cas9 gene editing. BADH2-1 Genes and BADH2-2 Millet with genes simultaneously inactivated or reduced in activity BADH2 Gene editing recombinant vector, reuse of millet BADH2 Gene editing recombinant vector was used to transform millet to make millet BADH2-1 Genes and BADH2-2 Genes simultaneously become inactive or less active.

[0018] Furthermore, millet BADH2 The gene editing recombinant vector selected three intermediate vectors pJG310, pJG338 and pJG471 of the sgRNA multiplex vector system, and a backbone vector pRLG103, and finally constructed a millet containing one editing site BADH2 Gene editing recombinant vector.

[0019] Furthermore, the application comprises the following steps:

[0020] Building millet BADH2 Gene editing recombinant vector;

[0021] Using millet BADH2 Gene editing recombinant vector construction includes BADH2 A recombinant strain of a gene editing recombinant vector; the recombinant strain is Agrobacterium, preferably Agrobacterium AGL1;

[0022] Utilize the included BADH2 The recombinant strain of gene editing recombinant vector was transformed into millet to make millet BADH2-1 Genes and BADH2-2 Genes simultaneously become inactive or less active.

[0023] Furthermore, the transformation of millet is carried out by using mature seeds of millet Jishu No. 5 as explants to induce embryonic callus, and then using BADH2 The recombinant strain containing the gene editing recombinant vector is transformed to BADH2-1 Genes and BADH2-2 Transgenic millet strains in which the genes are simultaneously inactive or have reduced activity;

[0024] Furthermore, the specific steps of the conversion are as follows:

[0025] 1) Induction of embryonic callus of millet

[0026] The callus induction medium contained 4.4 g / L MS dry powder, 30.0 g / L sucrose, and ZnSO4 7H 2 O 0.035 g / L, CuSO 4 ·5H 2 O 0.0006 g / L, kinetin 0.0005 g / L, 2,4-dichlorophenoxyacetic acid 0.002 g / L, and phytogels 9.0 g / L, pH 5.8;

[0027] The dry mature seeds of millet treated with dormancy are selected, the seed coats are removed, the seeds are disinfected and sterilized with a sodium hypochlorite solution and a Tween 20 solution, and then the seeds are washed with sterile water, and the seeds are inoculated on a callus induction medium, and the seed buds are directed upward during inoculation, and the buds do not directly contact the medium. During the culture process, light germination is first performed, and dark culture is performed after germination. After the dark culture is completed, induced embryonic callus tissue is picked from the buds, transferred to a new callus induction medium, and dark culture is continued to obtain pre-cultured millet embryonic callus tissue;

[0028] 2) Preparation of Agrobacterium culture solution

[0029] LB medium contained 10.0 g / L peptone, 5.0 g / L yeast powder, and 10.0 g / L NaCl;

[0030] The millet infection solution contained 2.2 g / L MS dry powder, 30.0 g / L sucrose, 1.0 g / L morpholineethanesulfonic acid, 0.04 g / L acetosyringone, and 0.1% of the surfactant poloxamer in a mass volume ratio, with a pH value of 5.4;

[0031] Take millet BADH2 The recombinant strain of the gene editing recombinant vector was plated on an LB plate containing carbenicillin and kanamycin, cultured in the dark, and a single colony was isolated;

[0032] Pick a single colony and place it in LB culture medium containing carbenicillin and kanamycin at the same time, and culture it with shaking. BADH2 The recombinant bacterial culture of the gene editing recombinant vector was centrifuged, the culture medium was poured off, the bacteria were collected, and then the millet infection solution was added, shaken, and the OD was adjusted. 600 value, and then gently shake the culture at a low speed for activation to obtain the activated Agrobacterium liquid;

[0033] 3) Infection and co-culture

[0034] The co-culture medium contained 2.2 g / L MS dry powder, 30.0 g / L sucrose, 1.0 g / L morpholineethanesulfonic acid, 0.002 g / L 2,4-dichlorophenoxyacetic acid, 0.0005 g / L kinetin, 0.04 g / L acetosyringone, and 4.0 g / L phytagel, with a pH of 5.4;

[0035] Add the embryonic callus of millet to the activated Agrobacterium bacterial solution, shake gently to infect, pour out the bacterial solution after the infection is completed, pour the infected millet embryonic callus on sterile filter paper, absorb the excess bacterial solution, blow dry properly on the clean bench for about half an hour, and then transfer to the co-cultivation medium with sterile filter paper, culture in the dark, and obtain the millet embryonic callus after co-cultivation;

[0036] 4) Screening of resistant callus

[0037] The callus screening medium contained 4.4 g / L MS dry powder, 30.0 g / L sucrose, and ZnSO 4 7H 2 O 0.035 g / L, CuSO 4 ·5H 2 O 0.0006 g / L, kinetin 0.0005 g / L, 2,4-dichlorophenoxyacetic acid 0.002 g / L, phytagel 9.0 g / L, antibiotic timentin 0.5 g / L to inhibit Agrobacterium, and selection agent hygromycin 0.025 g / L, pH 5.8;

[0038] The co-cultivated millet embryonic callus was transferred to a callus screening medium, cultured in the dark to complete one subculture, and then transferred to light culture to complete a second subculture to obtain resistant callus.

[0039] 5) Differentiation and rooting

[0040] The first stage differentiation medium contained 4.4 g / L MS dry powder, 30.0 g / L sucrose, 1.0 mL / L MS trace element stock solution (1000x), 1.0 mL / L MS vitamin (1000x), 0.003 g / L 6-benzylaminopurine, 0.003 g / L kinetin, 0.001 g / L 2,4-dichlorophenoxyacetic acid, 5% (v / v) coconut water, 0.5 g / L timentin, 0.015 g / L hygromycin, 1.0 mL / L chondroitin diluted 5000 times, and 9.0 g / L phytogel, with a pH of 5.8;

[0041] The second stage differentiation medium contained 4.4 g / L MS dry powder, 20.0 g / L sucrose, 1.0 mL / L MS trace element stock solution (1000x), 1.0 mL / L MS vitamin (1000x), 0.001 g / L 6-benzylaminopurine, 0.001 g / L kinetin, 0.0005 g / L 2,4-dichlorophenoxyacetic acid, 5% (v / v) coconut water, 0.5 g / L timentin, 0.015 g / L hygromycin, 1.0 mL / L chondroitin diluted 5000 times, and 9.0 g / L phytogel, with a pH of 5.8;

[0042] The rooting medium contained 2.2 g / L of MS dry powder, 15.0 g / L of sucrose, 1.0 mL / L of MS trace element stock solution (1000x), 1.0 mL / L of MS vitamins (1000x), 0.0005 g / L of 3-indolebutyric acid, 0.5 g / L of timentin, 0.015 g / L of hygromycin, and 9.0 g / L of phytagel, with a pH of 5.8;

[0043] First, the resistant callus is transferred to the first stage differentiation medium and cultured in light using the light / dark culture method, and then transferred to the second stage differentiation medium and cultured in light using the light / dark culture method to differentiate seedlings;

[0044] The differentiated seedlings are inoculated into a rooting medium and cultured in a light / dark manner to obtain resistant regenerated plants of millet after the rooting is completed.

[0045] 6) PCR identification of resistant regenerated plants

[0046] The culture medium was washed off the resistant regenerated plants of millet with water, and the plants were transplanted into a plant culture box and cultivated with nutrient soil. When the transgenic plants grew to about 20.0 cm in height, leaves were cut to extract DNA for PCR detection to obtain transgenic millet lines.

[0047] A method for regulating the aroma of millet, the method comprising planting BADH2-1 Genes and BADH2-2 A transgenic millet strain in which the genes are simultaneously inactive or have reduced activity, so as to increase the content of the aromatic substance 2-AP in millet;

[0048] BADH2-1 The sequence of the gene is shown in SEQ ID NO: 1;

[0049] BADH2-2 The sequence of the gene is shown in SEQ ID NO:2.

[0050] Furthermore, the BADH2-1 Genes and BADH2-2 Genetically modified millet with genes that have lost or reduced activity is produced by gene editing. BADH2-1 Genes and BADH2-2 Transgenic millet strains in which the genes are simultaneously inactive or have reduced activity;

[0051] BADH2-1 Genes and BADH2-2 The gene editing sites in the gene are identical, and the sequence is shown in SEQ ID NO: 3;

[0052] Specifically, first construct the BADH2-1 Genes andBADH2-2 Millet with genes simultaneously inactivated or reduced in activity BADH2 The gene editing recombinant vector was then introduced into millet through genetic transformation, making the millet BADH2-1 Genes and BADH2-2 The genes simultaneously lose their activity or reduce their activity due to base mutations, resulting in a transgenic millet strain.

[0053] A method for regulating the aroma of millet of the present invention BADH2 The beneficial effects of the gene, application and method are:

[0054] The present invention finds that by mutating millet BADH2 Genes that make millet's two BADH2 The simultaneous inactivation of genes can increase the content of the aromatic substance 2-AP in millet leaves, providing a research basis for the study of the synthesis and metabolic pathway of 2-AP in millet and providing new gene resources for the development of aromatic millet.

[0055] The present invention constructs millet BADH2 Gene editing vector, obtained after genetic transformation BADH2 The content of 2-AP, a flavor substance in the leaves of the transgenic millet strains with gene mutation, increased significantly, indicating that the directional mutation of this gene can affect the content of 2-AP in millet leaves and has important application value in improving the flavor of millet.

[0056] The invention discloses a method for making millet by using gene editing technology BADH2 The gene inactivation method provides a new approach for creating new varieties of millet with rich flavor.

[0057] Compared with wild-type millet, the BADH2 The 2-AP content in the leaves of the transgenic millet lines with gene mutations increased significantly, indicating that BADH2 Losing or reducing the activity of the gene can significantly increase the content of 2-AP in millet leaves;

[0058] Millet disclosed in the present invention BAHD2 The gene has important application value in improving the flavor of millet. The invention also provides a new method for creating new varieties of fragrant millet, provides certain theoretical guidance for the breeding process of millet flavor, and has certain practical significance and socio-economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is a structural diagram of the plant gene editing vector in Example 1 of the present invention;

[0060] Figure 2 Inventive Example 2 BADH2PCR identification results of the resistant regenerated plants of gene-edited millet; where M is DL2000 Marker, the unit is bp, and the molecular weight from top to bottom is 2000, 1000, 750, 500, 250 and 100; numbers 1 to 15 represent the resistant regenerated plants of some millet; K represents sterile water as a blank control, K- represents wild-type millet Jishu No. 5 as a wild-type control, and K+ represents 103- PmBADH2 Plasmid served as a positive control;

[0061] Figure 3 is the positive transformation seedling of millet in Example 3 of the present invention BADH2-1 and BADH2-2 PCR identification results of editing sites; where M is DL2000 Marker, the unit is bp, and the molecular weight from top to bottom is 2000, 1000, 750, 500, 250 and 100; numbers 1 to 10, 12, 13 and 15 represent the positive transformation seedlings of some millet; K represents sterile water as a blank control, and the two K- represent two wild-type millet Jishu No. 5 as wild-type controls;

[0062] Figure 4 The gene-edited millet in Example 4 of the present invention BADH2 The relative expression of genes was measured;

[0063] Figure 5 This is the result of determining the content of the aromatic substance 2-AP in the gene-edited millet leaves in Example 5 of the present invention. DETAILED DESCRIPTION

[0064] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0065] In addition, if no specific techniques or conditions are specified in the specific examples disclosed below, the techniques or conditions described in the literature in this field (for example, refer to "Molecular Cloning Experiment Guide" written by Sambrook et al., translated by Huang Peitang et al., third edition, Science Press) or the product instructions are used. If the manufacturer of the reagents used is not specified, they are all conventional products that can be purchased.

[0066] Example 1: Millet BADH2 Construction of gene editing vector

[0067] The plant gene editing vector system used in the present invention is a sgRNA multiplex vector system specifically used for plant gene editing obtained from the Voytas Laboratory of the University of Minnesota. The present invention selected three intermediate vectors pJG310, pJG338 and pJG471 of the system, and a backbone vector pRLG103, and finally constructed a plant gene editing vector containing one editing site (see Figure 1 ).

[0068] The specific construction method of the plant gene editing vector is as follows:

[0069] S1. Select two of millet BADH2 Gene, BADH2 It is a betaine aldehyde dehydrogenase, with the gene accession number PM13G12800 ( BADH2-1 ) and PM14G14600 ( BADH2-2 ), the gene sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2. BADH2-1 The gene is 5530 bp in length, contains 15 exons, the CDS coding region is 1518 bp in length, encodes 505 amino acids, and the amino acid sequence is shown in SEQ ID NO:4. BADH2-2 The gene is 5540 bp in length, contains 15 exons, the CDS coding region is 1518 bp in length, encodes 505 amino acids, and the amino acid sequence is shown in SEQ ID NO:5. BADH2-1 and BADH2-2 The gene editing sites in the gene are the same, and the sequence is shown in SEQ ID NO: 3.

[0070] S2. Selection of gene editing targets and primer design. The nucleotide sequence of the gene was analyzed on the gene editing target online design website (http: / / crispor.tefor.net / ), and a suitable target was selected according to the specificity, editing efficiency, off-target rate and position of the target. The sequence is shown in SEQ ID NO: 3, and then annealing primers were designed according to the target sequence, as shown in Table 1.

[0071] Table 1 Primers for gene editing targets

[0072]

[0073] Note: The underlined sequence is the editing site recognition sequence.

[0074] S3, double-strand the annealed primer, and then digest the double-stranded fragment with endonuclease BsaI, and connect it into the intermediate vector pJG338 to obtain the connection product; transform the connection product into E. coli DH5α, and select a single clone for sequencing verification. Finally, the pJG338 vector, pJG310 and the pJG471 vector containing the cas9 enzyme that have been verified to be connected to the correct target sequence are digested with AarI, and then connected together into the backbone vector pRLG103. The resulting connection product is transformed into E. coli DH5α, and the single clone bacterial solution is selected for bacterial solution PCR identification and sequence determination to screen positive clones. Among them, the PCR primers of the bacterial solution are shown in Table 2, and the reaction system is: 9.5 μL of sterile water, 1.0 μL of primers (10 μM), 12.5 μL of Taq Mix, and 1.0 μL of bacterial solution template. The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 1 min for 35 cycles; and finally extension at 72°C for 7 min.

[0075] Table 2 Primers for PCR identification of bacterial liquid

[0076]

[0077] The amplified products of the monoclonal bacterial solution after PCR amplification were separated and detected by 2% agarose gel electrophoresis. According to the results of the bacterial solution PCR test, several positive clones were selected and sent to the sequencing company for sequence determination. The positive clones with correct sequencing were saved, and the plasmids were extracted and named 103- PmBADH2 Plasmid (i.e. BADH2 Gene editing recombinant vector), and then transformed into Agrobacterium AGL1 by liquid nitrogen freeze-thaw method to obtain a 103- PmBADH2 Plasmid AGL1 bacteria (i.e. containing millet BADH2 The recombinant strain containing the gene editing recombinant vector was stored in glycerol at -80°C for future use.

[0078] Example 2 Transformation of millet with recombinant gene editing vector

[0079] The embryonic callus induction tissue of millet was induced by using mature seeds of millet Jishu No. 5, and the recombinant gene editing vector strain was transformed, which specifically includes the following steps:

[0080] 1) Induction of embryonic callus of millet

[0081] The embryonic callus induction medium contained 4.4 g / L MS dry powder, 30.0 g / L sucrose, and ZnSO 4 7H 2 O 0.035g / L, CuSO 4 ·5H 2O 0.0006 g / L, kinetin 0.0005 g / L, 2,4-dichlorophenoxyacetic acid 0.002 g / L and phytogel 9.0 g / L, with a pH of 5.8.

[0082] The dried mature seeds of millet (Ji Shu No. 5) that had been dormant were selected, the seed coat was removed, and about 2.0 g of seeds were placed in a 50.0 mL sterile centrifuge tube. The seeds were sterilized for 20 min with 20.0 mL of sodium hypochlorite solution with an effective concentration of 3% and 50.0 μL of Tween 20 solution. Then, the seeds were washed with sterile water for several times and inoculated on the embryonic callus induction medium. During inoculation, the bud points of the seeds should face upward and the bud points should not directly contact the culture medium. About 20 seeds were inoculated per dish of culture medium. The culture temperature was constant temperature culture at 24 °C and the humidity was 55%. During the culture process, the light germination was first carried out in a light / dark culture mode of 12 h / 12 ​​h, and then dark culture was switched after germination. After 28 days of dark culture, the induced embryonic callus tissue was picked from the bud point. The embryonic callus tissue of millet was dense and not water-soaked. It was transferred to a new embryonic callus induction medium and continued to be transformed after 7 days of dark culture to obtain the embryonic callus induction tissue of millet.

[0083] 2) Preparation of Agrobacterium culture solution

[0084] LB medium contained 10.0 g / L peptone, 5.0 g / L yeast powder, and 10.0 g / L NaCl.

[0085] Take LB culture medium, add 50.0 mg of carbenicillin specific to AGL1 Agrobacterium and 50.0 mg of kanamycin corresponding to the vector per liter to obtain Agrobacterium culture medium.

[0086] The infection solution of millet contained 2.2 g / L MS dry powder, 30.0 g / L sucrose, 1.0 g / L morpholineethanesulfonic acid, 0.04 g / L acetosyringone and 0.1% poloxamer as a surfactant in a mass volume ratio, and the pH value was 5.4.

[0087] Take millet BADH2 The recombinant strain of the gene editing recombinant vector was streaked on an LB plate containing carbenicillin and kanamycin, cultured in the dark at 28°C for 3 days, and a single colony was isolated.

[0088] Pick 3 to 5 single colonies and place them in LB culture medium containing 50.0 μg / mL carbenicillin and 50.0 μg / mL kanamycin. Culture at 28°C with shaking until the OD value of the bacterial solution reaches 0. 600 is 0.6, and the obtained BADH2 The recombinant bacterial solution of the gene editing recombinant vector was centrifuged at 5000 rpm for 10 min, the culture medium was poured off, the bacteria were collected, and then the millet infection solution was added, shaken, and the OD was adjusted.600 The agarose gel was 0.6, and then the culture was activated by gentle shaking at 100 rpm at 20°C for 3 h to obtain the activated Agrobacterium solution.

[0089] 3) Infection and co-culture

[0090] The co-culture medium contained 2.2 g / L MS dry powder, 30.0 g / L sucrose, 1.0 g / L morpholineethanesulfonic acid, 0.002 g / L 2,4-dichlorophenoxyacetic acid, 0.0005 g / L kinetin, 0.04 g / L acetosyringone and 4.0 g / L phytagel, with a pH value of 5.4.

[0091] Add the embryonic callus-inducing tissue of millet to the activated Agrobacterium bacterial solution, at a ratio of 60 to 80 pieces of millet embryonic callus-inducing tissue with a diameter of about 0.5 cm per 30.0 mL of activated Agrobacterium bacterial solution, and infect with gentle shaking at 120 rpm at 20°C for 60 min (shaking infection uses dark culture). After the infection is completed, pour out the bacterial solution to obtain the infected millet embryonic callus tissue.

[0092] Pour the infected millet embryonic callus onto sterile filter paper, absorb the excess bacterial solution, and blow dry it properly on a clean bench for about half an hour, then transfer it to the co-cultivation medium with sterile filter paper and culture it in the dark at 20℃ for 6 days to obtain the millet embryonic callus after co-cultivation.

[0093] 4) Screening of resistant callus

[0094] The callus screening medium contained 4.4 g / L MS dry powder, 30.0 g / L sucrose, and ZnSO 4 7H 2 O 0.035 g / L, CuSO 4 ·5H 2 O 0.0006 g / L, kinetin 0.0005 g / L, 2,4-dichlorophenoxyacetic acid 0.002 g / L, phytagel 9.0 g / L, timentin 0.5 g / L and hygromycin 0.025 g / L, with a pH of 5.8.

[0095] The co-cultivated millet embryonic callus was transferred to the callus screening medium, with about 20 to 30 grains of co-cultivated millet embryonic callus per dish. It was cultured in the dark at 24°C for 2 weeks to complete one subculture, and then transferred to light culture for 2 weeks (light culture was cultured at 24°C with 12 h / 12 ​​h light / dark) to complete the second subculture. The entire co-cultivation screening process was subcultured twice, once every 2 weeks. The total time for co-cultivation screening was 1 month, and resistant callus was obtained.

[0096] 5) Differentiation and rooting

[0097] The first stage differentiation medium contained 4.4 g / L MS dry powder, 30.0 g / L sucrose, 1.0 mL / L MS trace element stock solution (1000x), 1.0 mL / L MS vitamin (1000x), 0.003 g / L 6-benzylaminopurine, 0.003 g / L kinetin, 0.001 g / L 2,4-dichlorophenoxyacetic acid, 5% (v / v) coconut water, 0.5 g / L timentin, 0.015 g / L hygromycin, 1.0 mL / L chondroitin diluted 5000 times, and 9.0 g / L plant gel, with a pH of 5.8.

[0098] The second stage differentiation medium contained 4.4 g / L MS dry powder, 20.0 g / L sucrose, 1.0 mL / L MS trace element stock solution (1000x), 1.0 mL / L MS vitamin (1000x), 0.001 g / L 6-benzylaminopurine, 0.001 g / L kinetin, 0.0005 g / L 2,4-dichlorophenoxyacetic acid, 5% (v / v) coconut water, 0.5 g / L timentin, 0.015 g / L hygromycin, 1.0 mL / L chondroitin diluted 5000 times, and 9.0 g / L plant gel, with a pH of 5.8.

[0099] The chondroitin in this embodiment is sodium chondroitin sulfate produced by Beijing Putang Biotechnology Co., Ltd.

[0100] The rooting medium contained 2.2 g / L MS dry powder, 15.0 g / L sucrose, 1.0 mL / L MS trace element stock solution (1000x), 1.0 mL / L MS vitamin (1000x), 0.0005 g / L 3-indolebutyric acid, 0.5 g / L timentin, 0.015 g / L hygromycin and 9.0 g / L plant gel, with a pH value of 5.8.

[0101] First, the resistant callus tissue was transferred from the callus screening medium to the first stage differentiation medium, and cultured in the light at 24°C with a light / dark culture method of 12 h / 12 ​​h for 2 weeks, and then transferred to the second stage differentiation medium, and cultured in the light at 24°C with a light / dark culture method of 12 h / 12 ​​h for 2 weeks to obtain differentiated seedlings with a height of about 2.0 cm.

[0102] The differentiated seedlings were inoculated into rooting medium and cultured for rooting at 24°C using a 12 h / 12 ​​h light / dark culture method for 1 to 2 weeks. The specific rooting culture time was determined according to the size of the seedlings. After the rooting was completed, the resistant regenerated plants of millet were obtained, which were the resistant seedlings. The entire transformation cycle was approximately 12 to 16 weeks.

[0103] 5) PCR identification of resistant regenerated plants

[0104] The culture medium was washed off the resistant regenerated plants of millet with water, and the plants were transplanted into a plant culture box and cultivated with nutrient soil. During the cultivation process, when the transgenic plants grew to about 20.0 cm in height, the leaves were cut and their genomic DNA was extracted using a plant genomic DNA extraction kit (Tiangen DP305). Using the above DNA as a template, PCR detection of vector-specific primers (Cas1 gene on the vector) was performed on the resistant regenerated plants, and Jishu No. 5 was used as the wild-type control K- (i.e., negative control), 103- PmBADH2 The plasmid was used as the positive control K+, and sterile water was used as the blank control K. The detection primers used are shown in Table 3. The enzyme used for PCR detection was ordinary Taq Mix, and the reaction system was: 9.5 μL of sterile water, 1.0 μL of each primer (10.0 μM), 12.5 μL of Taq Mix, and 1.0 μL of DNA template. The PCR amplification program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s for 35 cycles; and finally extension at 72°C for 7 min. The PCR amplification products were separated and detected by 2% agarose gel electrophoresis. The electrophoresis diagram of the PCR products of the Cas1 gene of some regenerated plants is shown in Figure 2 .from Figure 2 It can be seen that the resistant regenerated plants 1-10, 12, 13 and 15 can amplify a band of about 375 bp, which is consistent with the positive control, while the wild type control and water control do not have this band, indicating that the resistant regenerated plants 1-10, 12, 13 and 15 are all positive transformation seedlings. The positive transformation seedlings in the regenerated millet plants were screened according to the PCR test results of Cas1.

[0105] Table 3 Primer sequences for detecting regenerated plants with resistance to millet

[0106]

[0107] Example 3 Positive regeneration of plants BADH2 Mutation identification and analysis of editing sites

[0108] In millet BADH2-1 and BADH2-2 PCR primers were designed upstream and downstream of the editing site of the gene (sequences are shown in Table 4) and the 13 positive transformed seedlings were cloned. BADH2-1 and BADH2-2The editing site of the gene was identified by PCR and sequencing, and two wild-type millet strains Jishu No. 5 were used as wild-type controls K-, and sterile water was used as blank control K. The enzyme used for PCR was TaKaRa LA Taq with GC Buffer from Takara. The PCR reaction system was: 4.25 μL of sterile water, 1.0 μL of each primer (10.0 μM), 12.5 μL of Buffer I, 4.0 μL of dNTP buffer, 0.25 μL of LA Taq, and 2.0 μL of DNA template. The PCR amplification program was: pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, and 35 cycles; finally extension at 72°C for 5 min. The PCR amplification products were separated and detected by 2% agarose gel electrophoresis, and some positive transformed seedlings BADH2-1 and BADH2-2 The PCR electrophoresis diagram of the gene editing site is shown in Figure 3 .from Figure 3 It can be seen that both the positive transformed seedlings and the wild-type control can amplify a band of about 907 bp and a band of about 867 bp. The PCR product was sent to the company for sequencing, and the editing effect was judged based on the sequencing peak graph and the sequencing sequence to obtain the mutation of the editing site. If the sequencing peak graph is a single peak and the sequence is different from the wild-type sequence, it means a homozygous mutation; if double peaks or multiple peaks appear in the sequencing peak graph starting from the editing site or its upstream, it means a heterozygous mutation, but the specific mutation form cannot be determined. The specific mutation form can only be determined by continuing to plant the next generation to isolate homozygous mutant plants.

[0109] Table 4 Primers for editing site identification

[0110]

[0111] According to the positive transformation BADH2-1 and BADH2-2 The sequencing results of the PCR products of the gene showed that some positive transformed seedlings BADH2-1 and BADH2-2 The gene has a double peak starting from the editing site, indicating a heterozygous mutant strain, T 0 No homozygous mutant strains were found in the next generation. The seeds of the heterozygous mutant strains (selected in this example) Figure 3 The seeds corresponding to the positive transformed seedling numbered 11 in the figure) are used to continue planting T 1 Generation and T 2 Generation, filter out BADH2-1 and BADH2-2 Transgenic lines 1 and 2 with homozygous mutations at the editing site of the gene (both transgenic lines 1 and 2 are BADH2 Gene-edited millet with homozygous mutation of the gene).

[0112] Among them, transgenic line 1 BADH2-1 and BADH2-2 At the editing site of the gene (a base T is inserted (GGG T GCCGTCCGGGCCAAGTA), resulting in a base shift mutation, BADH2-1 and BADH2-2 The amino acid sequence of the gene changes and translation is terminated prematurely at the third exon.

[0113] Transgenic line 2 BADH2-1 There are two editing methods at the editing site of the gene. One is to insert a base G (GGG G GCCGTCCGGGCCAAGTA), and another is to insert a base T (GGG T GCCGTCCGGGCCAAGTA), but both lead to base shift mutations, making BADH2-1 The amino acid sequence of the gene changes, and the translation is terminated prematurely at the third exon. BADH2-2 A base T (GGG) is also inserted at the editing site of the gene T GCCGTCCGGGCCAAGTA), resulting in a base shift mutation, BADH2-2 The amino acid sequence of the gene changes and translation is terminated prematurely at the third exon.

[0114] Example 4 Transgenic Line 1 and Transgenic Line 2 BADH2 Determination of gene expression

[0115] Due to millet BADH2-1 and BADH2-2 The homology of the genes is over 97%, making it difficult to design qPCR primers to identify the expression levels of the two genes separately. Therefore, a pair of qPCR primers that can amplify the two genes simultaneously was designed. BADH2-F and BADH2-R The results of using this pair of primers to identify these two BADH2 The total expression of the gene. ACTIN1 The wild type millet Jishu 5 was used as the reference sample (expression level was 1), and the expression of transgenic lines 1 and 2 was calculated. BADH2 The relative expression levels of genes and primer sequences are shown in Table 5.

[0116] Leaf samples of transgenic strain 1, transgenic strain 2 and wild-type millet Jishu 5 were taken, and total RNA was extracted using the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen Biochemical Technology, Beijing), and the total RNA was reverse transcribed into cDNA using the PrimeScriptTM RT Reverse Transcription Kit (Takara, Dalian). The cDNA was used as a template for qPCR. BADH2 and ACTIN1 The primers were used to perform qPCR reaction in QuantStudioTM 1 Plus real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific, Shanghai). The enzyme used in qPCR was NOVA Taq SYBR ® Green qPCR Premix, reaction system: TaqSYBR ® Green qPCR Premix (universal) 10.0 μL, primers (10.0 μM) 0.5 μL each, cDNA template 1.0 μL, Nuclease-Free Water 8.0 μL. BADH2 and ACTIN1 The qPCR reaction program was: 95°C pre-denaturation for 30 s; 95°C denaturation for 10 s, 60°C annealing for 30 s, and 40 cycles. Each sample was biologically replicated three times, and the measured data were analyzed by one-way ANOVA using DPSV15.10 software. Duncan's new multiple range method was used to detect significant differences and annotated. The results are shown in the figure. Figure 4 As shown, transgenic lines 1 and 2 BADH2 The expression levels of the genes were significantly lower than those of the wild type millet Jishu 5, indicating BADH2 Insertional mutations in genes affect BADH2 Normal gene expression.

[0117] Table 5 qPCR primers

[0118]

[0119] Example 5 Determination of 2-AP content in leaves of transgenic line 1 and transgenic line 2

[0120] Leaves of transgenic strain 1 and transgenic strain 2 and wild-type millet Jishu No. 5 were collected, and 2-AP in the samples was quantitatively analyzed using GC-QQQ gas chromatography-triple quadrupole mass spectrometry (Agilent 7890A-7000). Each sample was biologically repeated 3 times, and the measured data were analyzed by one-way ANOVA using DPS V15.10 software, and Duncan's new multiple range method was used for significant difference detection and annotation. The quantitative analysis of 2-AP used the internal standard method, and the internal standard was 2,4,6-trimethylpyridine (TMP). The specific steps are as follows:

[0121] 1) Prepare standard solution: Accurately weigh 1.0 mg of 2-AP standard and place it in a 1.0 mL sample bottle. Dissolve it with chromatographic grade ethanol solvent and make up to volume to obtain a standard stock solution with a mass concentration of 1.0 mg / mL. Store at -18℃ away from light.

[0122] 2) Preparation of extract: Add TMP to a mixed solution of anhydrous ethanol and dichloromethane (volume ratio of 1:1) to obtain an extract containing 50.0 ng / mL TMP.

[0123] 3) Grind the sample into 200 mesh powder using a grinder, weigh 0.2 g of powder into a 5.0 mL crimp bottle, add 1.0 mL of extracting solution, seal the bottle tightly with a crimp, and extract in an 80°C water bath for 2 h; take out and cool to room temperature, centrifuge for 5 min, transfer the supernatant to an injection bottle with a 200.0 μL inner tube, let it stand for 0.5 h, and then detect it on the machine.

[0124] Gas chromatography parameters: The chromatographic column was HP-5MS (30.0 m×0.25 mm×0.25 μm), and the column temperature program was 50°C for 2 min, then increased to 120°C at 10°C / min, and maintained for 0 min; then increased to 250°C at 30°C / min, and maintained for 3 min. The injection port temperature was 170°C; the carrier gas was high-purity (purity>99.999%) helium; constant pressure splitless injection, the injection volume was 1.0 μL.

[0125] Mass spectrometry parameters: electron impact (EI) ion source, ion source temperature of 230 °C; ionization energy of 70 eV; connection line temperature of 280 °C; quadrupole temperature of 150 °C; MRM scanning mode, scanning range of m / z 35~500.

[0126] The results of 2-AP content determination are shown in Table 6 and Figure 5As shown in the figure, the 2-AP content in the leaves of wild-type millet Jishu 5 was 0.009 mg / kg, while the 2-AP content in the leaves of transgenic line 1 was 0.439 mg / kg, and the 2-AP content in the leaves of transgenic line 2 was 0.418 mg / kg. The 2-AP content in the leaves of transgenic line 1 and transgenic line 2 was significantly higher than that of wild-type millet Jishu 5. Therefore, using gene editing technology to make millet BADH2-1 and BADH2-2 The genes of the two genes mutate at the same time, and millet germplasm materials with improved aroma traits can be obtained.

[0127] Table 6 BADH2 2-AP content in gene-edited millet

[0128]

[0129] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, rather than all the embodiments. A person skilled in the art can also obtain other embodiments based on the embodiments without inventiveness, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A method for regulating the aroma of millet BADH2 The application of genes is characterized in that Said BADH2 Genes include BADH2-1 Genes and BADH2-2 Gene; BADH2-1 The sequence of the gene is shown in SEQ ID NO: 1; BADH2-2 The sequence of the gene is shown in SEQ ID NO: 2; The application is to simultaneously knock out BADH2-1 Genes and BADH2-2 Gene to increase the content of the aromatic substance 2-AP in millet.

2. The method for regulating and controlling millet fragrance according to claim 1 BADH2 The application of genes is characterized in that BADH2-1 Genes and BADH2-2 The gene editing sites in the genes are the same, and the sequence is shown in SEQ ID NO:

3.

3. The method for regulating and controlling millet fragrance according to claim 1 BADH2 The application of genes is characterized in that The application is millet BADH2 Gene editing recombinant vector or containing BADH2 The recombinant strain of gene editing recombinant vector was transformed into millet, making millet BADH2-1 Genes and BADH2-2 Gene knockout simultaneously.

4. The method for regulating and controlling millet flavor according to claim 1 BADH2 The application of genes is characterized in that The application uses CRISPR / Cas9 gene editing method to construct BADH2-1 Genes and BADH2-2 Broomcorn millet with gene knockout BADH2 Gene editing recombinant vector, reuse of millet BADH2 Gene editing recombinant vector was used to transform millet to make millet BADH2-1 Genes and BADH2-2 Gene knockout at the same time; The process of converting millet also includes BADH2-1 Genes and BADH2-2 Gene identification; BADH2-1 The PCR primer sequences for the genes are: BADH2-1-F: 5'-CGTTCTTTGCCGTAACTGCCA-3'; BADH2-1-R: 5'-CAAACTTGGCCAACTTTGACC-3'; Identification BADH2-2 The PCR primer sequences for the genes are: BADH2-2-F: 5'-CTTCCATGATTTTCCCCAGCC-3'; BADH2-2-R: 5'-GTGACAGCACAGCCACTAACC-3'.

5. according to claim 3 or 4, the method for regulating and controlling millet fragrance BADH2 The application of genes is characterized in that Millet BADH2 The gene editing recombinant vector selected three intermediate vectors pJG310, pJG338 and pJG471 of the sgRNA multiplex vector system, and a backbone vector pRLG103, and finally constructed a millet containing one editing site BADH2 Gene editing recombinant vector.

6. according to any one of claims 1-4, regulating millet flavor BADH2 The application of genes is characterized in that The application comprises the following steps: Building millet BADH2 Gene editing recombinant vector; Using millet BADH2 Gene editing recombinant vector construction includes BADH2 Recombinant strains of gene-editing recombinant vectors; Utilize the included BADH2 The recombinant strain of gene editing recombinant vector was transformed into millet to make millet BADH2-1 Genes and BADH2-2 Gene knockout simultaneously.

7. The method for regulating and controlling millet flavor according to claim 6 BADH2 The application of genes is characterized in that The method for transforming millet is to use mature seeds of millet as explants, induce embryonic callus, and then use BADH2 The recombinant strain containing the gene editing recombinant vector is transformed to BADH2-1 Genes and BADH2-2 Transgenic millet strains with simultaneous gene knockout.

8. A method for regulating the aroma of millet, characterized in that: The method for regulating the aroma of millet is to plant the millet using a gene editing method. BADH2-1 Genes and BADH2-2 Gene knockout of transgenic millet to increase the content of 2-AP, a flavor substance in millet; BADH2-1 The sequence of the gene is shown in SEQ ID NO: 1; BADH2-2 The sequence of the gene is shown in SEQ ID NO:

2.

9. The method for regulating the flavor of millet according to claim 8, characterized in that: BADH2-1 Genes and BADH2-2 The gene editing sites in the genes are the same, and the sequence is shown in SEQ ID NO: 3.

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