Application of GhMRG gene in regulation and control of cottonseed oil synthesis

Abstract

The invention discloses an application of a GhMRG gene in regulation and control of cottonseed oil synthesis. According to the present invention, the editing target of the CRISPR / Cas9 system is designed according to the DNA sequence of the GhMRG gene CDS full-length fragment, and the GhMRG target is finally determined to be SG-RNA 1 and SG-RNA 2; and then constructing a gene editing vector CAS9-GhMRG, introducing agrobacterium, and transforming cotton by using an agrobacterium-mediated method to obtain a transgenic line. Finally, the editing condition of the transgenic strain KO-GhMRG is identified, and the result shows that compared with a ZM49 control material, the content of the cottonseed oil in the gene editing knockout strain KO-GhMRG material is remarkably increased, which indicates that the KO-GhMRG promotes the synthesis of the cottonseed oil and suggests that the GhMRG gene negatively regulates the synthesis of the cottonseed oil. The GhMRG gene is cloned from upland cotton, a new cotton variety is created through a gene editing technology and a genetic transformation technology, and the cottonseed oil content is remarkably increased. Theoretical basis and technical support are provided for cultivation of new varieties of high-oil cotton, and the method has important application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to application of a GhMRG gene in regulating synthesis of cottonseed oil content. BACKGROUND

[0002] Cotton is an important economic crop in China, which is not only an important raw material for the textile industry, but also an important oil crop, providing edible oil and protein feedstock for humans and animals. Cotton seeds are rich in oil, which is a potential source of edible oil and biodiesel. However, at present, cotton planting mainly targets fiber yield, and the cottonseed oil content is generally low, which limits its application in the oil industry. Traditional breeding methods have made slow progress in improving cottonseed oil content, which is difficult to meet market demand.

[0003] In recent years, genetic engineering technology has provided a new way for crop quality improvement. Oil crops are generally rich in vegetable oil, and the regulation network of their development and maturation process is a highly integrated system, involving multiple levels of coordinated regulation such as transcription factor interaction, hormone signal transduction, dynamic accumulation of nutrients, and metabolic reprogramming. Among them, transcription factors, by regulating fatty acid biosynthesis, triglyceride assembly, and oil body formation in the oil synthesis pathway, become an important target for genetic improvement strategies. Metabolite regulatory generator (GhMRG) is a homologous gene of Arabidopsis TT2 (TRANSPARENT TESTA 2), which belongs to one of the R2R3-MYB family members, mainly expressed in seed coat and endosperm, and regulates flavonoid metabolic pathways, especially the synthesis of proanthocyanidins. Studies have shown that the TT2 gene plays an important role in seed size, seed coat color, and cotton fiber color, but there are few reports on the function of GhMRG gene in cottonseed oil synthesis. SUMMARY

[0004] In view of the deficiencies in the prior art, the purpose of the present application is to provide the application of GhMRG gene in regulating cottonseed oil synthesis. By constructing a gene editing vector of GhMRG, the cotton is transformed by Agrobacterium-mediated method to obtain transgenic plants. Experiments have proved that knocking out the GhMRG gene can significantly improve the cottonseed oil content, providing a new way for cotton quality improvement.

[0005] To achieve the above purpose, the technical scheme of the present application provides an application of GhMRG gene in regulating cottonseed oil synthesis.

[0006] Further, the GhMRG gene negatively regulates the synthesis of cottonseed oil content.

[0007] Further, the coding nucleotide sequence of the GhMRG gene is shown in SEQ ID NO. 1.

[0008] Further, the GhMRG gene is amplified by using the primer shown in SEQ ID NO. 5 and SEQ ID NO. 6 as a template of the Gossypium hirsutum ZM49 genome.

[0009] Further, the amino acid sequence coded by the GhMRG gene is shown in SEQ ID NO. 2.

[0010] Further, the GhMRG gene is constructed on a plant editing vector CAS9 to obtain an editing vector CAS9-GhMRG, and the cotton is transformed by using the agrobacterium-mediated method to obtain a homozygous edited GhMRG strain, and the cottonseed oil content is improved.

[0011] Further, the core sequence of the editing vector CAS9-GhMRG is shown in SEQ ID NO. 8.

[0012] Further, the cottonseed variety is Gossypium hirsutum ZM49.

[0013] The technical scheme of the present application is further provided with a high-oil-content transgenic strain KO-GhMRG.

[0014] Further, the editing target points SG-RNA 1 and SG-RNA 2 of the CRISPR / Cas9 system are designed according to the DNA sequence of the CDS fragment of the GhMRG gene, a gene editing vector CAS9-GhMRG is constructed, and then the agrobacterium is introduced, and the cotton is transformed by using the agrobacterium-mediated method to obtain a transgenic strain KO-GhMRG.

[0015] The present application has the following beneficial effects: The application designs the editing target point of CRISPR / Cas9 system according to the DNA sequence of the full-length fragment of CDS of GhMRG (Gh_A07G014800) gene, and evaluates off-targeting in order to achieve the expected gene editing effect, and finally determines that the target point of GhMRG is SG-RNA 1: 5'-AGGCCTTGCTGTGCCAAAGAAGG-3', and SG-RNA2: 5'-CCATCCATGGTGAAGGCAAATGG-3'. Then the gene editing vector CAS9-GhMRG is constructed, introduced into Agrobacterium EHA105, and transformed into cotton by Agrobacterium-mediated method to obtain transgenic lines. The primers for specifically detecting the target point are designed, and the editing condition of the transgenic line KO-GhMRG is identified, and the results show that compared with the ZM49 control material, the cotton seed oil content in the gene editing knockout line KO-GhMRG material is significantly increased, which indicates that KO-GhMRG promotes the synthesis of cotton seed oil, and implies that GhMRG gene negatively regulates the synthesis of cotton seed oil. Meanwhile, the fatty acid content can be adjusted. The application clones GhMRG gene from Gossypium hirsutum, creates new cotton varieties by gene editing technology and genetic transformation technology, obtains new germplasm resource KO-GhMRG material with high oil content, and significantly improves the cotton seed oil content. This finding provides a theoretical basis and technical support for breeding new cotton varieties with high oil content, and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is an evolutionary tree, a conserved domain and a gene structure analysis of the GhTT2-like gene family of Gossypium hirsutum.

[0017] Figure 2 It is a GhTT2 amino acid sequence alignment.

[0018] Figure 3 It is the editing target point information of the GhMRG gene of the cotton transgenic plant.

[0019] Figure 4 It is a partial core map of At-26 plasmid.

[0020] Figure 5 It is a schematic diagram of the editing vector structure of CAS9-GhMRG.

[0021] Figure 6 It is the gene editing condition of KO-GhMRG knockout material.

[0022] Figure 7 It is the oil content in cotton seeds of KO-GhMRG knockout material.

[0023] Figure 8 It is the fatty acid content in cotton seeds of KO-GhMRG knockout material. DETAILED DESCRIPTION

[0024] The specific embodiments of the present application are further described in detail below with reference to the accompanying examples.

[0025] The technical solutions in the embodiments of the present application are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0026] The cotton in the following examples is Gossypium hirsutum L., and the variety is ZM49 / ZMS49, which is from the Germplasm Bank of the Cotton Institute of the Chinese Academy of Agricultural Sciences, and is referred to as ZM49 hereinafter. The hormones in the following examples are from Sigma Company; the culture medium and other products are from reagent companies.

[0027] MSB culture medium: solutes and their concentrations are as follows: NH4NO3 1650 mg / L, KNO3 1900 mg / L, KH2PO4 170 mg / L, MgSO4·7H2O 370 mg / L, CaCl2·2H2O 440 mg / L, FeSO4·7H2O 27.85 mg / L, Na2EDTA 37.25 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, myo-inositol 100 mg / L, VB1 10 mg / L, VB6 1.0 mg / L, nicotinic acid 1.0 mg / L, sucrose 28 g / L, calcium gluconate 1.29 g / L; pH 5.6.

[0028] Dipping liquid mother liquor (QR): solutes and their concentrations are as follows: MgSO4 10 g / L, (NH4)2SO4 5.36 g / L, CaCl2 6 g / L, Na3PO4 6 g / L, boric acid 30 mg / L, MnSO4 100 mg / L, KI 8 mg / L, CuSO4 250 mg / L, KNO3 100 g / L.

[0029] Immersion medium: QR 10 mL, glucose 30 g, 2-(N-morpholino) ethanesulfonic acid (MES) 4.2 g, liquid VB5 (Gamborg Vitamin Solution (1000x), G219) 0.1 mL, 6-benzyladenine 6-BA (1 mg / mL) and 1-naphthaleneacetic acid NAA (0.1 mg / mL) 1 mL, acetyl-syringone (AS) (100 mM) 2 mL, appropriate amount of surfactant Silwet-L77 (reference concentration: 0.02% (v / v)), constant volume to 1 L.

[0030] Co-culture medium: QR 10 mL, glucose 30 g, MES 4.2 g, liquid VB5 0.1 mL, 6-BA (1 mg / mL) and NAA (0.1 mg / mL) 1 mL, cysteine (50 mg / mL) 5 mL, AS (100 mM) 2 mL; Agar 6 g / L, pH 5.6.

[0031] Primary screening medium: add 6-BA (1 mg / mL) and NAA (0.1 mg / mL) 1 mL, carbenicillin (100 mg / mL) 1 mL, cephalosporin (100 mg / mL) 1 mL, spectinomycin (100 mg / mL) 1 mL to MSB medium (1 L), appropriate amount of plant tissue culture antibacterial agent (200 mg / mL of timentin stock solution: add 1 mL of timentin stock solution to 1 L of medium); Agar 6 g / L, pH 5.6.

[0032] Secondary screening medium: add 6-BA (1 mg / mL) and NAA (0.1 mg / mL) 1 mL, carbenicillin (100 mg / mL) 1 mL, cephalosporin (100 mg / mL) 1 mL, spectinomycin (100 mg / mL) 4 mL to MSB medium (1 L), appropriate amount of plant tissue culture antibacterial agent (200 mg / mL of timentin stock solution: add 1 mL of timentin stock solution to 1 L of medium); Agar 6 g / L, pH 5.6.

[0033] Rooting medium: add 6-BA to MSB medium to a content of 0.1 mol / L; Agar 6 g / L, pH 5.6.

[0034] Example 1: Obtaining of GhMRG gene edited strain 1. Identification of GhMRG gene: We analyzed the gene structure, evolutionary relationship and conserved domain of the GhTT2-like gene family in Gossypium hirsutum L. (GhTT2-like) (Wang et al., 2019) Figure 1To explore upland cotton in detail TT2 To investigate the phylogenetic relationships and structural characteristics of homologous genes, this study selected 14 representative upland cotton species. TT2 Gene family members were systematically aligned and phylogenetic trees were constructed using MEGA11 (a molecular evolutionary genetic analysis software) to their full-length protein sequences. Simultaneously, the online bioinformatics tool MEME was used to further analyze... TT2 Motif prediction and functional analysis were performed on the amino acid sequences encoded by the genes. All genes contain three exons and two endons, and the exon lengths of the gene coding sequences show little difference, indicating a degree of gene conservation. In summary, based on phylogenetic classification and the consistency analysis of gene sequence length, motif composition, and gene structure, we can confidently conclude that these 14 upland cotton genes... TT2 Homologous genes did indeed form four stable subclasses according to certain evolutionary rules, and maintained a high degree of sequence and structural consistency within their respective categories, thus strongly verifying the effectiveness and accuracy of the phylogenetic tree construction.

[0035] We selected three of them. TT2 Amino acid sequence alignment of genes (Gh_A06G085200, Gh_A07G014800 and Gh_A07G020100) was performed. Figure 2 The study found that these three TT2-like genes have the same conserved domain, but differ in amino acid sequence.

[0036] Based on a comprehensive evaluation, we selected Gh_A07G014800 as... TT2 The candidate gene was named GhMRG.

[0037] The CDS sequence of GhMRG is as follows: >Gh_A07G014800 ATGGGGAGGAGGCCTTGCTGTGCCAAAGAAGGGCTAAACAAAGGAGCATGGACTGCAAATGAAGACCAAATCCTCAAAAACTACATCACCATCCATGGTGAAGGCAAATGGAGAGACCTTCCTCAAAAAGCTGGGTTGAAGAGATGTGGGAAGAGTTGCCGGCTCCGGTGGCTGAATTACTTGAGGCCGGACATTAAGAGAGGGAACATCTCTATTGAAGAAGAAGAGCTCATTATTAGACTACACAAGCTCCTTGGTAACAGGTGGTCTCTGATTGCTGGAAGATTACCGGGGCGAACAGACAATGAAATAAAGAACTATTGGAACACAAATTTAAGCAAAAGGATGCAAGGCCAAAAAAGGAATATTTCTCCAAATACCCCCCAAGTTTCTTCTCACAATGTGATTAGGACCAAAGCAGTGAAGTGCACCAAGGCCGTCAATGTTGATACTTTAGACCCCAAATCCGAAACTCTAGTCAACAGTCCTAGTGAGACCCCTTCTTCTTCAGCCATCAATGAGGATAATACTAACAACAATTCCATGGATTTCTTGGTCGATTTCGATATCAATGAACTTCTCGCATTCGAATACAACGCGATCCCGGAGTTGTATCGAACCCAAAACGGCGGTGATAAAATGGGCAATAATGTGGCGGACAACGGGGAGTACAGTCATGGTACCGAGTTTTGTCAGGCAGGTGAACACTTGGATCTTCGAGGTCTAGAATCTTATTTGAATCTTGAAGATGAATGGATCAACTAA (SEQ ID NO. 1) The amino acid sequence encoded by GhMRG is: Gh_A07G014800 MGRRPCCAKEGLNKGAWTANEDQILKNYITIHGEGKWRDLPQKAGLKRCGKSCRLRWLNYLRPDIKRGNISIEEEELIIRLHKLLGNRWSLIAGRLPGRTDNEIKNYWNTNLSKRMQGQKRNISPNTPQVSSHNVIRTKAVKCTKAVNVDTLDPKSETLVNSPSETPSSSAINEDNTNNNSMDFLVDFDINELLAFEYNAIPELYRTQNGGDKMGNNVADNGEYSHGTEFCQAGEHLDLRGLESYLNLEDEWIN (SEQ ID NO. 2) 2. Construction of CAS9-GhMRG gene editing vector: Design of gene target: select high-score target on CRISPRscan website http: / / www.rgenome.net / cas-offinder / and evaluate off-target, finally select two editing targets SG-RNA1 and SG-RNA2 of GhMRG (Gh_A07G014800) Figure 3 ): SG-RNA1: 5'-AGGCCTTGCTGTGCCAAAGAAGG-3' (SEQ ID NO. 3) SG-RNA2: 5'-CCATCCATGGTGAAGGCAAATGG-3' (SEQ ID NO. 4) Design of gene editing primer: CAS9-GhMRG-F: 5'-agagtcgaagtagtgattgGGCCTTGCTGTGCCAAAGAgttttagagctagaaata-3' (SEQ ID NO. 5) CAS9-GhMRG-R: 5'-TATTTCTAGCTCTAAAACTTTGCCTTCACCATGGATGCAATCACTACTTCGACTC-3' (SEQ ID NO. 6) Using At-26 plasmid (core sequence as shown in SEQ ID NO. 7, part of core map see Figure 4) PCR amplification as a template, reaction system: 2 x Gflex PCR Buffer 25 μL, Tks Gflex DNA Polymerase 1 μL, CAS9-GhMRG-F and CAS9-GhMRG-R 1 μL (10 μM) each, At-26 plasmid 1 μL (320 ng / μL), ddH2O 21 μL. Reaction program: 94°C pre-denaturation 4 min, 1 cycle; 98°C denaturation 20 s, 52°C annealing 25 s, 68°C extension 30 s (1 min / 1000 bp), 35 cycles; 68°C final extension 10 min, 1 cycle; 4°C storage. The PCR amplification product was subjected to agarose gel electrophoresis (125 v, 20 min), and the correct size of the target fragment was selected and cut from the gel, and the gel recovery kit was used for recovery. The CRISPR / CAS9 vector plasmid was linearized by single enzyme digestion. The corresponding restriction enzymes were selected for double enzyme digestion, and the gene was connected by homologous recombination according to the ClonExpress II one Step Cloning Kit instructions. Then the centrifuge tube was placed in a 42°C water bath for 70-90 s, so that the plasmid entered the inside of E. coli, and then it was quickly taken out and placed in an ice box for cooling. 600-800 μL of LB liquid medium was added, and the culture was incubated at 37°C for 1 h, and then spread on LB solid medium with a concentration of 50 μg / L of kanamycin (Kan) resistance. Finally, the positive clones were picked up to extract the plasmid, that is, the correct sequencing edited vector CAS9-GhMRG (the sequence is shown as SEQ ID NO. 8, and the structure is shown as Figure 5 SEQ ID NO. 9), and it was transformed into the corresponding functional Agrobacterium. The specific method is as follows: 5 μL (about 1 ng) of positive plasmid was added to 50 μL of Agrobacterium EHA105 competent centrifuge tube, and mixed thoroughly. Ice for 30 min. Liquid nitrogen freezing 2 min, placed in 37°C water bath for 2 min, so that the plasmid entered the Agrobacterium through the cell membrane. Then, 28°C shaking culture for 1 h, the bacteria were spread on LB solid medium with a concentration of 100 μg / L of rifampicin (Rif) and a concentration of 50 μg / L of Kan resistance. After 2-3 days of culture, the Agrobacterium positive monoclonal was detected, and then the LB liquid medium containing 100 μg / L of antibiotic Rif and 50 μg / L of Kan was used for large-scale culture, and 50 % glycerol and 50 % bacteria were used for bacteria preservation, and stored in -80°C refrigerator.

[0038] At-26 plasmid core sequence: The CAS9-GhMRG vector sequence is: 3. Agrobacterium-mediated genetic transformation: (1) Seed germination: One day before the infection, 200-300 mature and dry cotton seeds (ZM24) without fiber were taken in a sterile glass bottle, 200 mL of 75% (v / v) alcohol was added and shaken for 30 s, the waste liquid was discarded, 200 mL of 6% H2O2 was added and shaken for 15 min, the waste liquid was discarded, and the seeds were washed with sterile water for 6-8 times, 200 mL of MSB medium was added, and the seeds were cultured at 28°C in the dark overnight for 18-24 h to absorb water.

[0039] (2) Preparation of Agrobacterium: The CAS9-GhMRG Agrobacterium stored at -80°C was inoculated on LB medium containing 50 μg / L kanamycin (Kanamycin), and was cultured in a 28°C incubator for 2 d to obtain activated Agrobacterium. One day before the infection, the Agrobacterium was inoculated again on LB medium containing 50 μg / L kanamycin, and was cultured in a 28°C incubator for 1 d. The next day, the Agrobacterium on the plate was collected with a sterile inoculation loop, and was suspended in the infection medium. The bacterial clumps were blown with a sterile pipette until the CAS9-GhMRG Agrobacterium cells were uniformly dispersed in the suspension. The optical absorption value A of the Agrobacterium suspension was measured with a spectrophotometer, and was about 0.9. 660

[0040] (3) Infection and co-culture: Healthy germinated seeds were selected for transformation. In a clean bench, the cotton seeds were clamped with a sterilized tweezers, and the seed coat was cut with a sterilized scalpel. Under a dissecting microscope, the cotyledon was cut to expose the embryo tip (explant). The explant was placed in a sterile conical flask containing 10 mL of the infection medium. After 100-150 explants were treated in each flask, the infection liquid was discarded, 15 mL of the prepared Agrobacterium suspension was added, and was placed in an ultrasonic cleaner for 40 s of ultrasonic treatment at 72 W. It was placed in a shaking bed for 50 min of shaking at room temperature. After the infection, it was blown dry on sterile filter paper for 10 min, and was placed in the co-culture medium (two layers of filter paper were placed on the medium, and 4 mL of the infection medium (AS was freshly added, and the concentration was 55 mg / mL cysteine 5 mL) was added), and was co-cultured in the dark at 23°C for 3-4 d.

[0041] (4) Primary screening culture: The co-cultured explants were transplanted to the primary screening medium with a sterile tweezers, and the radicles were inserted into the medium. 50 explants were transferred to a culture dish, and were cultured at 35°C under a light cycle of 16 h light / 8 h dark for 3 d, and then were further cultured at 25°C under the same light cycle for 4 d.

[0042] (5) Bud induction and secondary screening culture:​ After the initial screening culture, the explants were cut off the roots and transferred to bud induction screening medium (secondary screening medium) and cultured under a photoperiod of 16 h light / 8 h dark at 25 °C. About 2 weeks, subculture was needed once, and a total of 3-4 times of subculture was performed.

[0043] (6) Plant regeneration: The positive explants screened on the secondary screening medium were inserted into rooting medium and cultured under 25 °C, 16 h light / 8 h dark. Subculture was performed once every 2 weeks until resistant green buds and transgenic cotton roots were grown. Healthy transgenic lines were transplanted into flower pots in the greenhouse for growth. The greenhouse environment was set to 25 °C and a photoperiod of 14 h light / 10 h dark.

[0044] (7) Resistance identification: Healthy growing lines were subjected to plant resistance identification by smearing the back of young cotton leaves with spectinomycin at a concentration of 2.5 g / L, and phenotypic identification was performed 3-4 days later. Positive lines were selected and named KO-GhMRG.

[0045] 4. DNA extraction of KO-GhMRG: Healthy transgenic lines KO-GhMRG T1 generation seedlings were selected, and young leaves were used as samples for DNA extraction using the CTAB method.

[0046] Extraction of cotton leaf DNA: ① Sampling: Fresh cotton true leaves required for extraction were taken into a sterile 2 mL centrifuge tube, and the centrifuge tube was already equipped with a high-temperature baked steel ball. The tube was quickly frozen in liquid nitrogen and then ground into powder using a crusher under low temperature.

[0047] ② Lysis: 800 μL of CTAB lysis buffer preheated to 65 °C was added to the centrifuge tube and shaken well. Lysis was performed at 65 °C for 30 min, with shaking every 10 min to ensure complete lysis.

[0048] ③ Purification: This step was performed in a fume hood. An equal volume of 800 μL chloroform-isoamyl alcohol (volume ratio 24:1) was added, and the mixture was slowly inverted 60-80 times until the liquid did not separate into layers. Centrifugation was performed at 12000 rpm for 10 min.

[0049] (4) Extraction of crude DNA: The supernatant separated after centrifugation was transferred to a new 1.5 mL centrifuge tube, and an equal amount of anhydrous ethanol was added. At this time, white flocculent material can be observed, which is DNA. After standing at room temperature for 10 min, centrifugation was performed at 12000 rpm for 10 min.

[0050] (5) Purification: The supernatant after centrifugation in the previous step was discarded, and the DNA was washed twice with 75% (v / v) ethanol solution. The ethanol solution was discarded, and the DNA was left to stand at room temperature overnight until the ethanol evaporated.

[0051] (6) Dissolution: 50-100 μL of double-distilled water was added to the centrifuge tube to dissolve the DNA. After concentration detection, the DNA was stored in a refrigerator at -20°C for subsequent experiments.

[0052] 5. Identification of KO-GhMRG gene editing materials: PCR amplification was performed using the extracted DNA of the transformed T1 generation seedlings, the corresponding plasmid CAS9-GhMRG (positive control), ZM49 (negative control), and sterile water (blank control) as templates. According to the target information of GhMRG, the primers KO-MRG-A-F / R, KO-MRG-D-F / R, and the positive identification primers AADA-F / R were designed for detection of gene editing. The CAS9 detection protein primers CAS9-F / R were also used. The KO-GhMRG transgenic material seedlings obtained were identified and sequenced to confirm the GhMRG gene editing situation. Finally, the homozygous stable KO-GhMRG transgenic lines KO-GhMRG-3 (KO-GhTT2A07-3), KO-GhMRG-5 (KO-GhTT2A07-5), KO-GhMRG-27 (KO-GhTT2A07-27), KO-GhMRG-37 (KO-GhTT2A07-37), and KO-GhMRG-39 (KO-GhTT2A07-39) were obtained.

[0053] KO-MRG-A-F: 5'-AGTCTAGGGACTTAAATGAAAACTT-3' (SEQ ID NO. 9) KO-MRG-A-R: 5'-CCTCTCTTAATGTCCGGCCTCAAG-3' (SEQ ID NO. 10) KO-MRG-D-F: 5'-CAGTCTAGGGATTGAATGGTTCG-3' (SEQ ID NO. 11) KO-MRG-D-R: 5'-CCTCTCTTAATGTCCGGCCTCAAA-3' (SEQ ID NO. 12) AADA-F: 5'-ATCTCTACGGGTCAGCGGTTG-3' (SEQ ID NO. 13) AADA-R: 5'-GTCAGGAGGATAGCCAGATCA-3' (SEQ ID NO. 14) CAS9-F: 5'-gtggtggccaaagtggaaaag-3' (SEQ ID NO. 15) CAS9-R: 5'-tgatattctcggcctgctctc-3' (SEQ ID NO. 16) The identification and results of the edited material directly determine the phenotype of the material, so the identification and screening of the material is a key process. Because cotton is an allopolyploid, part of the GhMRG gene (GhTT2A07-A) of the strain is also edited. The results show that in the screened homozygous KO-GhMRG transgenic strain, the editing of KO-GhTT2A07-3 strain at target point 1 is that D07 subgenome increases one base and A07 subgenome lacks 3 bases; the editing at target point 2 is that A07 and D07 subgenomes are not edited. The editing of KO-GhTT2A07-5 strain at target point 1 is that D07 subgenome increases one base and A07 subgenome lacks 1 base; the editing at target point 2 is that A07 and D07 subgenomes are not edited. Figure 6 ).

[0054] Example 2: KO-GhMRG improves the content of cottonseed oil and fatty acid In order to further determine the content of oil and fatty acid in the edited material, full ZM49, KO-GhMRG (KO-GhTT2A07) cotton seeds were selected, among which KO-GhMRG strains KO-GhMRG-3 (KO-GhTT2-7A-3), KO-GhMRG-5 (KO-GhTT2-7A-5), KO-GhMRG-27 (KO-GhTT2-7A-27), KO-GhMRG-37 (KO-GhTT2-7A-37), KO-GhMRG-39 (KO-GhTT2-7A-39) and other GhMRG knockout strains were selected for determination of cottonseed oil and fatty acid content. The content of cottonseed oil and fatty acid was determined by the method of national standard (NY / T 1285-2007). The results show that the oil content of GhMRG knockout strains KO-GhMRG-3, KO-GhMRG-5, KO-GhMRG-27, KO-GhMRG-37 and KO-GhMRG-39 is significantly higher than that of ZM49 P<0.01), wherein the oil content of KO-GhMRG-5, KO-GhMRG-27 and KO-GhMRG-37 lines was significantly increased, and the highest oil content was increased by about 10% (Table 2) Figure 7 ), which indicated that KO-GhMRG promoted the synthesis of cottonseed oil, and implied that GhMRG gene negatively regulated the synthesis of cottonseed oil.

[0055] Oil is composed of fatty acids of each component, so the determination of the fatty acid content of each component is crucial. At the same time, according to the fatty acid content of each component, it is also conducive to create cottonseed oil with different proportions of short-chain and long-chain fatty acids, and to create functional cottonseed oil cotton varieties. The determination results of the fatty acid content of each component showed that (Table 3) Figure 8 ), except for individual single plants, the contents of short-chain fatty acid myristic acid (C14:0), long-chain fatty acid stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and total fatty acid in the KO-GhMRG cottonseed were significantly increased compared with the control ZM49, which indicated that KO-GhMRG significantly promoted the synthesis of fatty acids such as oleic acid (C18:1) and linoleic acid (C18:2) in cottonseed; and in the KO-GhMRG lines, except for individual single plants, the contents of palmitoleic acid (C16:1), heptadecanoic acid (C17:0), a-linolenic acid (C18:3) and behenic acid (C22:0) in the cottonseed were significantly lower than those in the control ZM49 cottonseed, which indicated that KO-GhMRG inhibited the synthesis of fatty acids such as palmitoleic acid, heptadecanoic acid, a-linolenic acid and behenic acid in cottonseed.

Claims

1. Application of GhMRG gene in regulating cottonseed oil synthesis.

2. The application according to claim 1, characterized in that, The GhMRG gene negatively regulates the synthesis of cottonseed oil.

3. The application according to claim 1 or 2, characterized in that, The encoding nucleotide sequence of the GhMRG gene is shown in SEQ ID NO.

1.

4. The application according to claim 1 or 2, characterized in that, The GhMRG gene was obtained by amplification using the genome of upland cotton ZM49 as a template and primers shown in SEQ ID NO.5 and SEQ ID NO.

6.

5. The application according to claim 1 or 2, characterized in that, The amino acid sequence encoded by the GhMRG gene is shown in SEQ ID NO.

2.

6. The application according to claim 1 or 2, characterized in that, The GhMRG gene was constructed into the plant editing vector CAS9 to obtain the editing vector CAS9-GhMRG. Cotton was transformed using Agrobacterium-mediated transformation to obtain homozygous edited GhMRG lines, which increased the oil content of cottonseed.

7. The application according to claim 6, characterized in that, The core sequence of the editing vector CAS9-GhMRG is shown in SEQ ID NO.

8.

8. The application according to claim 1 or 2, characterized in that, The cottonseed variety is upland cotton ZM49.

9. A high-oil transgenic strain KO-GhMRG.

10. The transgenic strain KO-GhMRG according to claim 9, characterized in that, Based on the DNA sequence of the CDS fragment of the GhMRG gene, the editing target sites SG-RNA 1 and SG-RNA 2 of the CRISPR / Cas9 system were designed, and the gene editing vector CAS9-GhMRG was constructed. Then, it was introduced into Agrobacterium and transformed into cotton using the Agrobacterium-mediated transformation method to obtain the transgenic line KO-GhMRG.

Images