Application of GhBCCP1 gene in regulating plant oil accumulation

Abstract

The present invention relates to the field of genetic breeding technology, and in particular to the application of the GhBCCP1 gene in regulating the accumulation of plant oils and fats. The present invention provides the application of the GhBCCP1 gene in regulating the accumulation of plant oils and fats, and the nucleotide sequence of the GhBCCP1 gene is shown in SEQ ID NO: 1. The present invention constructs an overexpression vector driven by a seed-specific promoter and transforms Arabidopsis thaliana to find that, compared with wild control plants, the expression of the GhBCCP1 gene in two transgenic Arabidopsis thaliana T3 generation strains is significantly increased, the oil content is increased by 5.6% and 7.5% respectively, and the levels of C18:1d9+C18:1d11, C18:2 and C20:1d11 in the fatty acid components are significantly increased, while the levels of C18:0 and C18:3 are significantly decreased.

Description

Technical Field

[0001] The invention relates to the technical field of genetic breeding, and in particular to the application of a GhBCCP1 gene in regulating the accumulation of plant oils and fats. Background Art

[0002] Cotton is an important cash crop and oil crop in my country. Cotton seeds are rich in fatty acids and proteins and are widely used in the processing of edible oils, industrial raw materials and biodiesel. Cotton breeding research has always focused on the yield and quality of fiber, while research on the oil content in cotton seeds has lagged far behind.

[0003] The rate of oil synthesis is closely related to the expression efficiency of genes involved in oil metabolism. Increasing or regulating the expression of any of these genes may affect the rate of oil synthesis. This feature has also become the metabolic basis for increasing cottonseed oil content and improving fatty acid composition. Heterotype acetyl-CoA carboxylase (ACCase) is a rate-limiting enzyme and key enzyme for de novo synthesis. It consists of biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and two subunits of carboxyltransferase (CT), α-CT and β-CT. BCCP is responsible for transferring the carboxyl group from BC to CT, and is the link and bridge connecting the other three subunits of ACCase. It plays a very important role in the biosynthesis of fatty acids in plant seeds. At present, although the oil synthesis pathway and the regulation of seed size have been recognized, the regulatory mechanism of GhBCCP1 in oil biosynthesis is still poorly understood.

[0004] In the prior art, a gene named GhBCCP1 (Gh_D06G1228) is disclosed and its role in regulating plant oil accumulation and seed size is studied. However, the above gene has only a 58.4% match rate with the GhBCCP1 (GH_A05G3915) DNA sequence of the present application, and the amino acid sequences encoded by the two genes also have only a 53.06% match rate, which shows that the two are not the same gene. In addition, the article Hub Genes in Stable QTLs Orchestrate the Accumulation of Cottonseed Oil in Upland Cotton via Catalyzing Key Steps of Lipid-Related Pathways (Alam B, Liu R, Gong J, et al. Hub Genes in Stable QTLs Orchestrate the Accumulation of Cottonseed Oil in Upland Cotton via Catalyzing Key Steps of Lipid-Related Pathways [J]. International Journal of Molecular Sciences, 2023, 24 (23):) disclosed the role of BCCP2 (GH_A05G3915) in regulating TAG (triacylglycerol) accumulation. However, TAG belongs to oil, and its synthesis is carried out in the endoplasmic reticulum through the Kendey pathway, while fatty acid synthesis is carried out in the plastid, such as Figure 1 Therefore, it is impossible to predict whether the GH_A05G3915 gene can regulate the synthesis of fatty acids, and it is necessary to study the use of the GhBCCP1 (GH_A05G3915) gene in regulating fatty acid synthesis. Summary of the invention

[0005] The purpose of the present invention is to provide application of GhBCCP1 in regulating plant oil accumulation.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides application of GhBCCP1 gene in regulating plant oil accumulation. The nucleotide sequence of the GhBCCP1 gene is shown in SEQ ID NO:1.

[0008] Preferably, the amino acid sequence of the GhBCCP1 protein encoded by the GhBCCP1 gene is shown in SEQ ID NO:2.

[0009] Preferably, the method of regulating the accumulation of plant oils and fats is to regulate the level of fatty acids in plant seeds.

[0010] Preferably, regulating the fatty acid level in plant seeds refers to increasing the content of one or more fatty acids among oleic acid, linoleic acid and arachidonic acid in plant seeds; and / or reducing the content of stearic acid and / or linolenic acid in plant seeds.

[0011] The invention also provides the use of the GhBCCP1 gene in cultivating transgenic plants with increased seed oil content.

[0012] Beneficial Effects

[0013] The present invention cloned the cotton GH_A05G3915 (GhBCCP1) gene from the upland cotton 'Zhongmian Institute 24' (specific link: https: / / cri.caas.cn / kycg / ycpz / mhpz / 267399.htm) material, successfully constructed a seed-specific promoter-driven overexpression vector and transformed Arabidopsis. The results showed that the two transgenic Arabidopsis T3 generation strains constructed by the present invention had significantly increased GhBCCP1 expression compared with the wild control plants, and the oil content increased by 5.6% and 7.5% respectively. The levels of C18:1d9+C18:1d11, C18:2 and C20:1d11 in the fatty acid components increased significantly, while the levels of C18:0 and C18:3 decreased significantly. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the in vivo synthesis pathway of GH_A05G3915 gene.

[0015] Figure 2 The agarose electrophoresis results of the GhBCCP1 gene cloning product.

[0016] Figure 3 These are the results of screening for T1 generation Arabidopsis resistant plants.

[0017] Figure 4 This is the electrophoresis diagram of PCR identification of T1 generation Arabidopsis resistant plants.

[0018] Figure 5 This is an analysis chart of the expression levels of the GhBCCP1 gene in different tissues of T1 generation Arabidopsis resistant plants.

[0019] Figure 6 The graph shows the expression level of GhBCCP1 in the siliques of two transgenic Arabidopsis T3 plants.

[0020] Figure 7 Nuclear magnetic resonance imaging was used to detect the oil content in GhBCCP1 transgenic Arabidopsis seeds.

[0021] Figure 8 Determination of fatty acid composition in seeds of GhBCCP1 transgenic Arabidopsis thaliana. DETAILED DESCRIPTION

[0022] The sources of the enzymes, reagents, drugs, vectors and competent cells required for the present invention are:

[0023] Enzymes and kits: Plant total RNA extraction kit (DP432) was purchased from Tiangen Biochemical (Technology) Beijing Co., Ltd.; RNA reverse transcription kit TransScript One-Step gDNARemoval and cDNA Synthesis SuperMix (AT311-02), fluorescent quantitative enzyme TransStart Top Green qPCR SuperMix (AQ131-04), cloning vector pEASY-T5 Zero Cloning Kit (CT501-01) and plasmid extraction kit EasyPure HiPure PlasmidMiniPrep Kit (EM111-01) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; high-fidelity PCR amplification enzyme KOD-Plus-Neo (KOD-401) was purchased from TOYOBO Biological Company; DNA Fragment Purification Kit ver4.0 gel recovery kit and DNAPurification Kit PCR product purification kits were purchased from TaKaRa Biotechnology Co., Ltd.; infusion ligase was purchased from Novozyme Biotechnology Co., Ltd.; Escherichia coli competent bacteria DH5α was purchased from Shanghai Bioengineering; Agrobacterium competent LBA4404 was purchased from Beijing Biomed Gene Technology Co., Ltd.; restriction endonucleases XbaI and SacI were purchased from NEB.

[0024] Other drugs: agarose was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. were domestic analytical grade; ampicillin (IA0340), kanamycin (YZ-130556), rifampicin (IR0110), streptomycin (IS0360), etc. were purchased from Beijing Solebow Technology Co., Ltd.

[0025] Solution preparation: All reagents mentioned but not listed in this article were prepared according to the methods in the third edition of the Molecular Cloning Experiment Guide, and the biochemical reagents were of analytical grade or above.

[0026] LB medium: 5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride (NaCl), and finally fixed to 1 L with ddH2O; LB solid medium: 5 g / L yeast extract, 10 g / L sodium chloride (NaCl), 15 g / L agar powder, 10 g / L tryptone, and finally fixed to 1 L with ddH2O.

[0027] Main instruments: PCR amplification instrument (BIO-RAD), electrophoresis equipment (BIO-RAD), fluorescence quantitative PCR instrument (ABI7500), gel imaging system (BIO-RAD), high-speed centrifuge (Hettich MIKRO 200R), microplate reader (BIO-TEC KC4), vacuum freeze dryer (ALPHAI-5), seed oil content nuclear magnetic resonance instrument (NMI20-Analyst) and Shimadzu fatty acid composition analysis gas chromatography instrument (GC-2030). The instrument for determining the oil content of Arabidopsis seeds is the Newmai nuclear magnetic resonance instrument (NMI20-Analyst).

[0028] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0029] Example 1 GhBCCP1 gene cloning process

[0030] (1) Experimental material 'Zhongmian Institute 24' was planted at the Anyang Institute of Technology experimental base and managed as normal field. The tissues obtained were mixed ovules at 3, 5, 10 and 20 days after flowering. The materials were quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for later use. Plant total RNA was extracted using the Tiangen Company kit.

[0031] (2) 1000 ng RNA was reverse transcribed into cDNA, and the cDNA solution of the reverse transcription product was diluted 4 times as a PCR reaction template. The mixed total RNA of cotton ovules extracted at different stages was used as a template and reverse transcribed into cDNA using a reverse transcription kit of Quanshijin. The cDNA synthesis system was as follows:

[0032] Random Primer (0.1ug / μl) 1μl 2×TSReactionMix 10μl TranScriptRT / RIEnzymeMix 1μl Total RNA (160 ng / μl) 6μl gDNARemover 1μl AnchoredOilgo(dT)Primer(0.5ug / μl) 1μl Total volume 20μl

[0033] Note: Incubate at 42℃ for 30min in a PCR instrument, then inactivate at 85℃ for 5s. Store at 4℃ for later use.

[0034] (3) Perform PCR reaction to amplify the target gene

[0035] The full-length CDS sequence of the GH_A05G3915 (GhBCCP1) gene was retrieved from the cotton functional genomic database (https: / / cottonfgd.org / ) using the local Blast method, and the primer sequences were designed using primer 5.0 software. The primer sequences are as follows: GhBCCP1-F (SEQ ID NO: 3): 5′-ATGGCTTCTTCACTCTCTTTTC-3′ GhBCCP1-R (SEQ ID NO: 4): 5′-TCACCCCACTATTACAAAT-3′

[0036] The synthetic primers GhBCCP1-F and GhBCCP1-R were used to perform PCR amplification of the cotton GhBCCP1 gene using the cDNA obtained by reverse transcription as a template. The target gene amplification system is as follows:

[0037]

[0038] The conditions required for the PCR reaction are set as follows:

[0039] Step 1 Predenature 94℃,2min Step 2 Denature 98℃,10s Step 3 Annealing 59℃,30s Step 4 Estension 68℃,30s Step 5 Estension 68℃,5min

[0040] Note: Perform 28 cycles of amplification from Step 2 to Step 4.

[0041] (4) After the reaction was completed, the product was stored at 4°C and detected by 0.8% agarose gel electrophoresis to obtain a PCR amplification product of 873 bp in size ( Figure 2 ), the nucleotide sequence of the product is shown in SEQ ID NO: 1, encoding 290 amino acid residues (as shown in SEQ ID NO: 2).

[0042] Nucleotide sequence SEQ ID NO: 1:

[0043]

[0044]

[0045] The amino acid sequence is (SEQ ID NO: 2):

[0046] MASSLSFPCP KISSFLKTNQ QTQTHKGSLS LPPTSNFNSK SCLSFGSSIRVPASSAPQWPNDGKQTTVFA SHTQLNEVAA EKSSNSVAVVDMKPKVALPE EDDKKSAEKA IPDAAAISEFMAQVSDLVKLVDSRDITELQ LKQSDCELVI RKKEALQQLE SASPIVMQQYMPQPTFQTPA PAAPVAAPAPANPAPPAAAP SSPPPAKAVG SSHPPLKCPMAGTFYRSPAP GEPPFVKVGD KVQKGQVVCIIIEAMKLMNEIEADQSGTVTE ILVEDGKPVS VDMPLFVIVG

[0047] (5) The reaction product of the target fragment was purified and recovered using TaKaRa MiniBEST DNA Fragment Purification Kit ver4.0.

[0048] (6) The product recovered from the gel was connected to the pEASY-T5 cloning vector and transformed into Escherichia coli competent DH5α.

[0049] (7) Heat shock in a 42°C water bath for 45 seconds, resuscitate on ice for 90 seconds, then add 600 μl of LB liquid medium without resistance, and culture at 37°C at a shaking speed of 160 rpm / min for 1 hour. Then take all the culture liquid and spread it on LB solid medium (containing kan resistance). Culture at 37°C overnight, pick a single clone from the kan resistance LB medium and culture it in 500 μl LB medium containing kan at 37°C overnight with shaking.

[0050] (8) PCR verification of bacterial solution: the bacterial solution containing the target gene fragment size band was sent for sequencing, and the clone with correct sequencing was named pEASY-GhBCCP1.

[0051] Example 2 Construction of p2301Mα-GhBCCP1 plant seed specific expression vector

[0052] 1. Plasmid extraction and enzyme digestion

[0053] The p2301Mα plasmid vector was preserved in this laboratory (the construction process can be referred to Section 3.2 of "Construction of seed-specific expression vector and genetic transformation of heteroplasmic ACCase gene in upland cotton"). The plasmid was extracted using the plasmid extraction kit of Quanshijin Company. The plasmid concentration was tested to be 120 ng / μl. Agarose gel electrophoresis showed no protein contamination, which met the test requirements. The endonucleases used were XbaI and SacI for double digestion.

[0054] Construction of 2p2301Mα-GhBCCP1 expression vector

[0055] (1) Amplification of the target gene ORF sequence: Based on the multiple cloning site on the final vector p2301Mα, XbaI-SacI was designed as the insertion site and primers were synthesized.

[0056] The primer sequences are as follows:

[0057] GhBCCP1-XbaI-F (SEQ ID NO: 5):

[0058] 5′- GCTCTAGA ATGGCTTCTTCACTCTCTTTTC-3' (the underlined part contains the recognition sequence of restriction endonuclease XbaI)

[0059] GhBCCP1-SacI-R (SEQ ID NO: 6):

[0060] 5′- CGAGCTC TCACCCCACTATTACAAAT-3' (the underlined part contains the recognition sequence of restriction endonuclease SacI)

[0061] KOD-Plus-Neo high-fidelity enzyme was used to amplify the GhBCCP1 target gene sequence containing XbaI and SacI restriction sites. The reaction system is as follows.

[0062]

[0063]

[0064] The conditions required for the PCR reaction were set as follows: pre-denaturation at 94°C for 2 min, denaturation at 98°C for 10 s, annealing at 60°C for 30 s, extension at 68°C for 30 s, 30 cycles; and extension at 68°C for 5 min.

[0065] (2) Construction of fusion expression vector:

[0066] 1) The plant-specific promoter expression vector p2301Mα was digested with restriction endonucleases XbaI and SacI, and the vector backbone was recovered.

[0067] 2) Recovering and purifying the GhBCCP1 amplification product containing XbaI and SacI restriction sites, and digesting the recovered product with restriction endonucleases XbaI and SacI.

[0068] 3) Connecting the vector skeleton obtained in step 1) and the recovered product obtained in step 2) to obtain a connection product.

[0069] 4) The ligation product of step 3 was heat-shock transformed into Escherichia coli DH5α, cultured inverted in a 37°C incubator overnight, and the positive clones were selected for sequencing. The bacteria with correct sequencing results were named p2301Mα-GhBCCP1 / DH5α, and the plasmid extracted from the correct bacteria was named p2301Mα-GhBCCP1 plasmid.

[0070] 3. Transformation of vector plasmid into LBA4404 competent cells

[0071] a. Take 100 μl of LBA4404 competent cells melted on ice and put them into a centrifuge tube. Under sterile conditions, add 2 μl (about 500 μg) of the target plasmid (p2301Mα-GhBCCP1 plasmid) to the competent cells, mix gently, and let stand in an ice water bath for 5 minutes;

[0072] b. Quick-freeze the centrifuge tube in liquid nitrogen for 5 minutes;

[0073] c. Then quickly place the centrifuge tube in a 37°C water bath and incubate for 5 minutes. Return the centrifuge tube to an ice water bath and keep it in an ice bath for 5 minutes.

[0074] d. Take 100 μL of bacterial solution and spread it on LB screening medium containing kanamycin, streptomycin and rifampicin, culture it at 28℃ for about 3 days, select the positive clone (p2301Mα-GhBCCP1 / LBA4404), culture it in LB liquid medium at 28℃ for 48 hours until the final glycerol concentration is about 15%, and store it at -20℃ for later use.

[0075] Example 3. Genetic transformation, identification and gene expression analysis of p2301Mα-GhBCCP1 seed-specific expression vector in Arabidopsis

[0076] First, use 1 ml of LB medium containing kan to culture the Agrobacterium liquid transformed with p2301Mα-GhBCCP1 / LBA4404, then take 100 μl of the overnight culture liquid and add it to 100 ml of fresh liquid LB medium containing kan, and culture it at 28°C with shaking for 16 hours. Centrifuge at 130000 rpm / min for 5 minutes, discard the supernatant, and then resuspend the bacteria in 100 ml of infiltration solution (10% sucrose + 400 μl / L Silwet-77) to an OD of 600 =about 0.8.

[0077] Columbia-type Arabidopsis was used as the recipient and genetic transformation was carried out by Arabidopsis floral infection. T0 transgenic seeds were harvested and positive transformants were screened on MS medium containing kanamycin (50 mg / L) to obtain T1 resistant plants ( Figure 3), and seeds were collected after flowering and maturity. Five weeks after the T1 generation resistant plants were transplanted, DNA was extracted from leaves of Arabidopsis plants, and the resistance marker NPTII gene was amplified by PCR to further screen positive Arabidopsis plants.

[0078] Results Figure 4 As shown, the target band can be obtained near 795 bp in the agarose gel electrophoresis diagram, which preliminarily proves that the seed-specific expression vector of the gene has been successfully transformed.

[0079] At the same time, 7 weeks after the T1 generation resistant plants were transplanted, RNA was extracted from the siliques, leaves and stems of Arabidopsis plants, and cDNA was synthesized. Specific primers (GhBCCP1-QRT-F (SEQ ID NO: 7): TTGGTTCTTCTCATCCGCCC and GhBCCP1-QRT-R (SEQ ID NO: 8): GCCTCGATGATGCACACAAC) were designed, and fluorescent quantitative PCR was performed using cDNAs from three different tissue parts (siliques, leaves and stems) of the plants transformed with the above vector as templates and internal reference primers AtactinqF (SEQ ID NO: 9): CCATGAACCCACCTATAACTCC and AtactinqR (SEQ ID NO: 10): TACTCTGCCTTTGCGATCCAC.

[0080] The results are as follows Figure 5 As shown, in T1 transformed plants, the GhBCCP1 gene can only be expressed at a high level in siliques, while the expression level of the GhBCCP1 gene in leaves and stems is very weak or not expressed, indicating that the GhBCCP1 gene transformed in Arabidopsis can achieve the effect of seed-specific expression.

[0081] T1 generation individual seeds were harvested separately, and about 50 seeds were spread on MS+Kan (50 mg / L) medium. After the cotyledons of the seedlings were fully expanded (about 10 days), the yellow-green seedling separation ratio was counted, and a chi-square test was performed. Plants that met the green seedling: yellow seedling = 3:1 separation were preliminarily predicted to be single-copy insertion strains, and seeds were harvested separately according to the strains for subsequent analysis.

[0082] Example 4. Determination of oil content and fatty acid composition of GhBCCP1 transgenic Arabidopsis seeds

[0083] Two single-copy T3 lines with high expression of GhBCCP1 gene in siliques were selected and grown on MS+Kan (50 mg / L) medium (GhBCCP1 gene expression level was as follows: Figure 6 The two lines were named GhBCCP1 OE6 and OE13, and 25 plants were transplanted from each line. After maturity, T3 seeds were harvested for later use and the oil content and fatty acid composition were determined.

[0084] 1. Using the NMI20-Analyst, the oil content of seeds of two GhBCCP1 transgenic T3 generation Arabidopsis lines and wild-type Arabidopsis was determined. The oil content of each transgenic line was determined three times. The average oil content of wild-type Arabidopsis seeds was 25.89% for three times; the average oil content of GhBCCP1 transgenic T3 generation OE6 for three times was 27.31%; the average oil content of GhBCCP1 transgenic T3 generation OE13 for three times was 27.84%; compared with the control strain, the average oil content of seeds increased by 1.42% and 1.95% ( Figure 7 ), indicating that the GhBCCP1 gene can regulate the oil content of Arabidopsis seeds.

[0085] 2. Extract total fatty acids, separate each fatty acid by gas chromatography and identify its components.

[0086] The fatty acid compositions of two GhBCCP1 transgenic Arabidopsis seeds and wild-type Arabidopsis samples were analyzed using a gas chromatograph (GC-2030, Shimadzu, Japan) equipped with a flame ionization detector (FID) and a SH-Rtx-65 capillary column (30 m × 0.25 mm × 0.50 μm). Samples (1 μl), including fatty acids and n-hexane solution, were injected in a 30:1 split mode at 280 °C. The significance of all fatty acid composition data was calculated by the Student t-test method. p < 0.05 (*) showed statistically significant differences compared with the control group.

[0087] The fatty acids in mature seeds of two GhBCCP1 transgenic Arabidopsis thaliana lines and wild-type plants were detected using a NMI20 nuclear magnetic resonance instrument. Figure 8 As shown in the figure, compared with the control, the levels of C18:1d9+C18:1d11 (oleic acid), C18:2 (linoleic acid) and C20:1d11 (arachidonic acid) in the seeds of the GhBCCP1 transgenic line increased significantly, while the levels of C18:0 (stearic acid) and C18:3 (linolenic acid) decreased significantly. These results indicate that overexpression of the GhBCCP1 gene can change the level of fatty acid components in seeds.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. GhBCCP1 The use of a gene in regulating the fatty acid composition level in plant seeds is characterized in that: Said GhBCCP1 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; The method for regulating the fatty acid composition level in plant seeds is: GhBCCP1 Gene overexpression increases the content of oleic acid, linoleic acid, and arachidonic acid in plant seeds, and decreases the content of stearic acid and linolenic acid in plant seeds; Wherein, the oleic acid is C18:1d9 and C18:1d11; the linoleic acid is C18:2; the arachidic acid is C20:1d11; the stearic acid is C18:0; the linolenic acid is C18:3; The plant is cotton or Arabidopsis thaliana.

2. The use according to claim 1, characterized in that Said GhBCCP1 The amino acid sequence of the GhBCCP1 protein encoded by the gene is shown in SEQ ID NO:2.

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