A method for increasing ethylene release in plants

Abstract

The present invention provides a method for increasing the ethylene release of plants, wherein specific guide RNA is designed according to the sequence of an acid reductone dioxygenase gene (MpARD), a CRISPR / Cas9 gene editing vector is constructed, and Agrobacterium is used to infect and transform liverwort, and the ethylene release of gene-edited mutant plants is significantly increased by more than 3 times. The acid reductone dioxygenase MpARD gene can be edited to enhance the ethylene synthesis level of plants.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular biotechnology and genetic engineering, and relates to a method for increasing the ethylene release of plants. Background Art

[0002] Ethylene is an important regulatory hormone for plant growth and development, and is particularly important for the ripening of respiratory climacteric fruits such as bananas, apples, and tomatoes. Exogenous ethylene treatment can promote fruit ripening and form the fruit's unique color, flavor, texture, aroma, and nutritional quality. In commercial production, the application of ethephon solution, a stable substitute for ethylene, is usually used to promote fruit ripening. During the operation, it not only consumes a lot of labor and economic investment, but also easily causes mechanical damage and pathogen corruption of the fruit, reducing the commercial value of the fruit. Therefore, the study found that genes that can promote ethylene release levels are of great significance for achieving fruit ripening control and quality maintenance.

[0003] Acid reductone dioxygenase ARD. The acid reductone dioxygenase (English name: acireductone dioxygenase) in the non-seed plant Marchantia polymorpha (Latin name: Marchantia polymorpha) was named MpARD. The acid reductone dioxygenase MpARD in the Marchantia polymorpha plant was discovered using biotechnology. After the gene was mutated using CRISPR / Cas9 gene editing technology, the level of ethylene release in the plant was promoted. Summary of the invention

[0004] The object of the present invention is to provide a method for increasing the ethylene release of plants, which is achieved by the following steps:

[0005] 1. Acid reductone dioxygenase MpARD, whose genomic sequence is shown in SEQ ID NO.1, and whose protein amino acid sequence is shown in SEQ ID NO.2; its coding region consists of 600 nucleotide base pairs, and the protein consists of 199 amino acid residues. According to the MpARD gene sequence, guide RNA is designed, with sequences SEQ ID NO.3 and SEQ ID NO.10, and the pMpGE_En03 vector is used as a template, and the MpU6-1 sequence is amplified with primers, and the gRNA backbone is amplified with primers, and the purified fragment is cloned into the GE010 vector linearized by MaubI and AflII restriction endonucleases through a one-step ligation reaction kit. The product obtained by one-step ligation is used to transform the Escherichia coli vector, obtain the MpARD gene CRISPR / Cas9 guide RNA gene knockout vector, and transform the correct vector into Agrobacterium. Agrobacterium monoclonal was inoculated into LB liquid medium and cultured for 2 days. After centrifugation, it was resuspended in 1 / 2 Gamborg's B5, 1% (w / v) sucrose and 100 μM acetosyringone liquid medium and activated in a shaking in the dark at 28°C for 6 hours to obtain Agrobacterium transformation liquid.

[0006] 2. The wild-type Marchantia polyphylla buds were planted on sterile culture medium for 14 days. After the tissues including the meristem were removed, the remaining thallus tissue was divided into four parts and continued to grow on a medium of 1 / 2 Gamborg's B5, 1% (w / v) sucrose and 1.3% agar for 3 days. The Agrobacterium transformation liquid prepared and activated above was added to the Gamborg's B5 liquid culture medium and regenerated thallus containing 2% sucrose and 100 μM acetosyringone, and co-cultured for 3 days. The co-cultured tissue was washed with sterile water and then soaked in sterile water containing 1g / L cefotaxime for 30 minutes. The thallus tissue was placed on a 1 / 2 Gamborg's B5 culture medium containing 1.3% agar, 10mg / L hygromycin and 100mg / L cefotaxime for screening. After the positive plants of the transformed generation were screened to produce buds, they were screened on hygromycin culture medium for one generation. Extract genomic DNA and sequence and compare to screen mutant plants with gene editing, and obtain different MpARD protein mutation types, sequences SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16;

[0007] 3. The wild-type and mutant plant materials were cultivated in a constant temperature tissue culture room at 23°C, with a light condition of 16 hours light and 8 hours dark. The 10-day-old wild-type plants and Mpard mutant plants were placed in a headspace bottle containing a liquid culture medium containing 1 / 2 Gamborg's B5 and 1% (w / v) sucrose. After being placed in the original growth environment for a period of time, the ethylene production was measured by gas chromatography. The ethylene release level per unit weight of the plant was calculated based on the ethylene release in the headspace measured by gas chromatography.

[0008] The present invention provides two CRISPR / Cas9 guide RNA gene editing targets and a gene editing vector, and the present invention provides four MpARD protein mutation types.

[0009] The present invention provides an acid reductone dioxygenase encoding gene MpARD gene and its sequence, CDS sequence, and protein sequence. The gene is derived from liverwort. By designing two guide RNA gene editing target primers, constructing CRISPR / Cas9 vectors, and genetically transforming to obtain transgenic plants, it is found that the ethylene release of plants of four different mutants of the MpARD gene increases from 0.395 μL / hr / g FW to between 1.131 μL / hr / g FW and 1.184 μL / hr / g FW, or from 0.237 μL / hr / g FW to between 0.690 μL / hr / g FW and 0.788 μL / hr / g FW, with an increase of about 3 times. The method of gene editing the acid reductone dioxygenase MpARD gene can be used to increase the ethylene release of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The ethylene release level in liverwort plants with MpARD gene mutation types I and II was created using SEQ ID NO.3 as a guide RNA.

[0011] Figure 2 The ethylene release level in liverwort plants with MpARD gene mutation types III and IV was created using SEQ ID NO.10 as a guide RNA. DETAILED DESCRIPTION

[0012] The present invention is further described in conjunction with the accompanying drawings and embodiments.

[0013] Example 1. Determination of ethylene release from Marchantia polyphylla mutants with MpARD gene knockout by CRISPR / Cas9 guide RNA1

[0014] Research Methodology:

[0015] 1. Construction of CRISPR / Cas9 guide RNA1 vector for Marchantia polymorpha MpARD gene

[0016] The online software CRISPR direct (https: / / crispr.dbcls.jp / ) was used to design CRISPR / Cas9 guide RNA1 (SEQ ID NO.3) for the MpARD gene sequence. According to the designed guide RNA1 sequence, the pMpGE_En03 vector was used as a template, and the MpU6-1 sequence was amplified with primers SEQ ID NO.4 and SEQ ID NO.5. The gRNA backbone was amplified with primers SEQ ID NO.6 and SEQ ID NO.7. The reaction system was 50 μL, and MAX Super Fidelity DNA polymerases (Vazyme, China) were used. 2.0 μL (10 μM) of primer pair SEQ ID NO.4 and SEQ ID NO.5 or SEQ ID NO.6 and SEQ ID NO.7, 1.0 μL of dNTP (10 mM), 1.0 μL of pMpGE_En03 vector (100 ng), 1.0 μL of DNA polymearase, 25 μL of 2×PhantaMax Buffer, 18 μL of ddH2O, and a total reaction system of 50 μL. After adding, stir the bottom of the tube to mix well, and then centrifuge it slightly to remove the liquid from the tube wall.

[0017] The PCR reaction program was 98°C for 1.5 min; 98°C for 20 s, 62°C for 20 s, 72°C for 30 s, and 38 cycles. 10x loading buffer was added to the PCR product, mixed, and centrifuged slightly to concentrate the liquid at the bottom of the tube. The desired PCR fragments were separated by agarose gel electrophoresis and then recovered (Easy Gel Extraction & Clean-up Kit, China).

[0018] The purified fragment was cloned into the GE010 vector linearized by MaubI and AflII restriction enzymes using a one-step ligation kit (Vazyme, China). n ) to the insert fragment y in a molecular ratio of 1:1 to 1:3, the maximum total volume of x and y is 7 μL, 5×CE II Buffer 2.0 μL, CE II Enzyme 1.0 μL, and ddH2O to make up the volume to a total of 10 μL.

[0019] The PCR reaction program was 37°C for 30 min.

[0020] The product obtained by one-step ligation was used to transform the vector of Escherichia coli (DH5α competent, Shanghai Weidi Biology), cultured in non-antibiotic LB liquid medium, incubated on a 37°C shaker for 45 minutes, and then the bacterial solution was spread on LB solid medium supplemented with spectinomycin, placed in a 37°C incubator for overnight culture, and several monoclonal colonies were picked for Sanger sequencing (Youkang Biology, China), and the correct vector was transformed into Agrobacterium (GV3101, Shanghai Weidi Biology), and glycerol bacteria were preserved. Agrobacterium monoclonal inoculation was added to 5mL LB liquid medium and cultured for 2 days. After centrifugation and resuspension, it was resuspended in 25mL of liquid medium containing 1 / 2Gamborg's B5, 1% (w / v) sucrose and 100μM acetosyringone, and activated for 6 hours in a shaker at 180rpm and 28°C in the dark.

[0021] 2. Genetic Transformation of Marchantia polymorpha Mpard Mutant Plants

[0022] The wild-type buds were planted on sterile culture medium for 14 days, and the tissue including the meristem was removed 2-3 mm from the edge of the thallus. The remaining thallus tissue was divided into four parts and continued to grow on a medium of 1 / 2 Gamborg's B5, 1% (w / v) sucrose and 1.3% agar for 3 days. In a 200 mL beaker, 50 mL of Gamborg's B5 liquid culture medium containing 2% sucrose and 100 μM acetosyringone and 20 regenerated thallus were added, and 1 mL of the Agrobacterium liquid prepared and activated above was added, and placed under continuous white light, 22°C, 130rpm shaking incubator for co-cultivation for 3 days. The co-cultured tissue was washed with sterile water for 5-6 times, and then soaked in sterile water containing 1 g / L cefotaxime for 30 minutes. The thallus tissue was placed on 1 / 2 Gamborg's B5 medium containing 1.3% agar, 10 mg / L hygromycin and 100 mg / L cefotaxime for screening. After the positive plants of the current generation were screened for budding, they were screened for one generation on 1 / 2 Gamborg's B5 medium containing 1.3% agar, 10 mg / L hygromycin and 100 mg / L cefotaxime. Genomic DNA was extracted, and the MpARD gene sequence was amplified using primer sequences SEQ ID NO.8 and SEQ ID NO.9. The PCR reaction program was 95℃5min; 95℃30s, 55℃30s, 72℃60s, 38 cycles. Sequencing comparison was performed to screen the mutant plants L1 (mutant protein type I sequence SEQ ID NO.13) and L2 (mutant protein type II sequence SEQ ID NO.14) that underwent gene editing.

[0023] 2. Ethylene content determination of WT and Mpard mutant

[0024] Marchantia polymorpha materials were cultivated in a tissue culture room at a constant temperature of 23°C at the Fruit Institute of Zijingang Campus of Zhejiang University. The lighting conditions were 16 hours of light and 8 hours of darkness. The selection criteria for experimental materials were uniform plant size and 10-day-old Marchantia plants.

[0025] Wild-type Marchantia polyphylla and Mpard mutant plants were placed in 5 mL headspace bottles containing 0.3 mL liquid culture medium and placed in the original growth environment for 48 hours. 1 mL of gas was extracted from the headspace and the ethylene production was determined by gas chromatography. Five biological replicates were set for each group.

[0026] According to the ethylene release in the headspace measured by gas chromatography, the ethylene release level of the plant per unit weight was calculated and expressed in nL / hr / g FW. The calculation formula is:

[0027] Where A s —Gas chromatography measured the ethylene peak area of ​​the sample headspace; A eth - Gas chromatography measured the peak area of ​​standard concentration of ethylene;

[0028] C eth —standard ethylene gas concentration, μL / L; t—incubation time, hr; V h —Headspace area of ​​sample bottle, mL; m s —Sample weight, g.

[0029] Results: Compared with the wild type (WT), the ethylene release of Mpard mutant plants (L1 and L2) was significantly increased (see Appendix Figure 1 ).

[0030] Example 2. Determination of ethylene release from Marchantia polyphylla mutants with MpARD gene knockout by CRISPR / cas9 guide RNA2

[0031] Research Methodology:

[0032] 1. Construction of CRISPR / cas9 guide RNA2 vector for Marchantia polymorpha MpARD gene

[0033] CRISPR / Cas9 guide RNA2 (SEQ ID NO.10) was designed according to the MpARD gene sequence, and the pMpGE_En03 vector was used as a template. The MpU6-1 sequence was amplified with primers SEQ ID NO.4 and SEQ ID NO.11, and the gRNA backbone was amplified with primers SEQ ID NO.12 and SEQ ID NO.7. The reaction system was 50 μL, MAX Super Fidelity DNA polymerases (Vazyme, China). Primer pairs SEQ ID NO.4 and SEQ ID NO.11 or SEQ ID NO.12 and SEQ ID NO.7 were 2.0 μL (10 μM) each, dNTP (10 mM) 1.0 μL, pMpGE_En03 vector 1.0 μL (100 ng), DNA polymearase 1.0 μL, 2×Phanta Max Buffer 25 μL, ddH2O 18 μL, and the total reaction system was 50 μL.

[0034] The PCR reaction program was 98°C for 1.5 min, 98°C for 20 s, 62°C for 20 s, 72°C for 30 s, and 38 cycles. The desired PCR fragments were separated by agarose gel electrophoresis and then recovered.

[0035] The purified fragment was cloned into the GE010 vector linearized by MaubI and AflII restriction enzymes through a one-step ligation reaction. n ) to the insert fragment y in a ratio of 1:1 to 1:3, the total volume of x and y is 7 μL at most, 5×CE II Buffer 2.0 μL, CE II Enzyme 1.0 μL, and ddH2O to make up the total volume to 10 μL.

[0036] The PCR reaction program was 37°C for 30 min.

[0037] The product obtained by one-step ligation was used to transform the vector of Escherichia coli (DH5α competent state), cultured in LB liquid medium without antibiotics, incubated in a shaking incubator at 37°C for 45 minutes, and then the bacterial solution was spread on LB solid medium supplemented with spectinomycin, placed in a 37°C incubator for overnight culture, and several monoclonal colonies were picked for Sanger sequencing (Youkang Bio, China), and the correct vector was transformed into Agrobacterium (GV3101, Shanghai Weidi Bio), and glycerol bacteria were preserved. Agrobacterium monoclonal inoculation was added to 5mL LB liquid medium and cultured for 2 days. After centrifugation and resuspension, it was resuspended in 25mL 1 / 2Gamborg's B5, 1% (w / v) sucrose and 100μM acetosyringone liquid medium, and activated in a shaking incubator at 180 rpm and 28°C for 6 hours in the dark.

[0038] 2. Genetic Transformation of Marchantia polymorpha Mpard Mutant Plants

[0039] The wild-type buds were planted on sterile culture medium for 14 days, and the tissue including the meristem was removed 2-3 mm from the edge of the thallus. The remaining thallus tissue was divided into four parts and continued to grow on a medium of 1 / 2 Gamborg's B5, 1% (w / v) sucrose and 1.3% agar for 3 days. In a 200 mL beaker, 50 mL of Gamborg's B5 liquid culture medium containing 2% sucrose and 100 μM acetosyringone and 20 regenerated thallus were added, and 1 mL of the Agrobacterium liquid prepared and activated as described above was added, and placed under continuous white light, 22°C, 130 shaking incubator for co-cultivation for 3 days. The co-cultured tissue was washed with sterile water for 5-6 times, and then soaked in sterile water containing 1 g / L cefotaxime for 30 minutes. The thallus tissue was placed on 1 / 2 Gamborg's B5 medium containing 1.3% agar, 10 mg / L hygromycin and 100 mg / L cefotaxime for screening. After the positive plants of the current generation were screened for budding, they were screened on hygromycin medium for another generation. Genomic DNA was extracted, and the MpARD gene sequence was amplified using primer sequences SEQ ID NO.8 and SEQ ID NO.9. The PCR reaction program was 95℃5min; 95℃30s, 55℃30s, 72℃60s, 38 cycles. Sequencing comparison was performed to screen the mutant plants L3 (mutant protein type III sequence SEQ IDNO.15) and L4 (mutant protein type IV sequence SEQ ID NO.16) that underwent gene editing.

[0040] 3. Ethylene content determination of WT and Mpard mutant

[0041] Marchantia polymorpha materials were cultivated in a tissue culture room at a constant temperature of 23°C at the Fruit Institute of Zijingang Campus of Zhejiang University. The lighting conditions were 16 hours of light and 8 hours of darkness. The selection criteria for experimental materials were uniform plant size and 10-day-old Marchantia plants.

[0042] Wild-type Marchantia polyphylla and Mpard mutant plants were placed in 5 mL headspace bottles containing 0.3 mL liquid culture medium and placed in the original growth environment for 48 hours. 1 mL of gas was extracted from the headspace and the ethylene production was determined by gas chromatography. Five biological replicates were set for each group.

[0043] According to the ethylene release in the headspace measured by gas chromatography, the ethylene release level of the plant per unit weight was calculated and expressed in nL / hr / g FW. The calculation formula is:

[0044] Where A s—Gas chromatography measured the ethylene peak area of ​​the sample headspace; A eth - Gas chromatography measured the peak area of ​​standard concentration of ethylene;

[0045] C eth —standard ethylene gas concentration, μL / L; t—incubation time, hr; V h —Headspace area of ​​sample bottle, mL; m s —Sample weight, g.

[0046] Results: Compared with the wild type (WT), the ethylene release of Mpard mutant plants (L3 and L4) was significantly increased (see Appendix Figure 2 ).

Claims

1. A method for increasing ethylene release in plants, characterized in that: This is accomplished by following these steps: (1) Designing a guide RNA according to the acid reductone dioxygenase MpARD gene sequence, wherein the MpARD gene sequence is shown in SEQ ID NO.1, and the protein amino acid sequence is shown in SEQ ID NO.

2. Using the pMpGE_En03 vector as a template, amplifying the MpU6-1 sequence with primers, amplifying the gRNA backbone with primers, and cloning the purified fragment into the GE010 vector linearized by MaubI and AflII restriction endonucleases using a one-step ligation reaction kit. The product obtained by the one-step ligation was used to transform the Escherichia coli vector to obtain Mp ARD Gene CRISPR / Cas9 guide RNA gene knockout vector, transforming the correct vector into Agrobacterium, inoculating the Agrobacterium monoclone into LB liquid culture medium and culturing for 2 days, centrifuging, resuspending in liquid culture medium containing 1 / 2 Gamborg's B5, 1% (w / v) sucrose and 100 μM acetosyringone, and activating in a shaker at 28°C in the dark for 6 hours to obtain Agrobacterium activated bacterial solution; the sequence of the guide RNA is shown in sequence SEQ ID NO.3 or SEQ ID NO.10; (2) The wild-type Marchantia polymorpha buds were grown on sterile culture medium for 14 days. After the tissues including the meristem were removed, the remaining thallus tissue was divided into four parts and continued to grow on a medium containing 1 / 2 Gamborg's B5, 1% (w / v) sucrose and 1.3% agar for 3 days. The Agrobacterium activated bacterial solution prepared above was added to the Gamborg's B5 liquid culture medium and regenerated thallus containing 2% sucrose and 100 μM acetosyringone and co-cultured for 3 days. The co-cultured tissue was washed with sterile water and then immersed in sterile water containing 1 g / L ceftriaxone for 30 min. The thallus tissue was screened on a 1 / 2 Gamborg's B5 medium containing 1.3% agar, 10 mg / L hygromycin and 100 mg / L ceftriaxone. After the positive plants were transformed and screened to produce buds, they were continued to be screened on a medium containing 1.3% agar, 10 mg / L hygromycin and 100 The first generation was screened on Gamborg's B5 medium containing 1 / 2 mg / L cefotaxime, and the genomic DNA was extracted and sequenced to screen the mutant plants with gene editing, and different MpARD protein mutation types were obtained; (3) The wild-type and mutant plant materials were cultured in a tissue culture room at a constant temperature of 23°C and a light condition of 16 hours of light and 8 hours of darkness. Mpard The mutant plants were placed in a headspace bottle containing 1 / 2 Gamborg's B5 and 1% (w / v) sucrose liquid culture medium. After being placed in the original growth environment for a period of time, the ethylene production was measured by gas chromatography. The ethylene release level of the plant per unit weight was calculated based on the ethylene release in the headspace measured by gas chromatography.

2. The method according to claim 1, characterized in that The primers for amplifying the MpU6-1 sequence are SEQ ID NO.4 and SEQ ID NO.5, and the primers for amplifying the gRNA backbone are SEQ ID NO.6 and SEQ ID NO.

7.

3. The method of claim 1 is used to increase the ethylene release of plants, characterized in that: The plant is liverwort.

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