CN118127042A - GhBC1 gene and application of related biological material thereof in regulation and control of vegetable oil accumulation - Google Patents
GhBC1 gene and application of related biological material thereof in regulation and control of vegetable oil accumulation Download PDFInfo
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Abstract
The invention provides GhBC gene and application of related biological materials thereof in regulating and controlling accumulation of vegetable oil and fat, belonging to the technical field of biology. The GhBC gene or the biological material related to the GhBC gene can regulate and control the accumulation of vegetable oil, and the nucleotide sequence of the GhBC gene is shown as SEQ ID NO. 3. The GhBC gene can be successfully over-expressed in plants except cotton, can regulate and control the content of plant oil components and the composition of fatty acid components, and provides a new theoretical basis for researching the regulation and control mechanism of cotton BC participating in oil biosynthesis.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to GhBC gene and application of related biological materials thereof in regulating and controlling accumulation of vegetable oil.
Background
Cotton is an important commercial crop and oil crop. Cotton seeds are increasingly playing a role as their most prominent by-products in cotton production. The yield is 1.5-2 times of the yield of the ginned cotton. The shelled cotton seed accounts for 55 to 60 percent of the weight of the seed, and the oil content accounts for 30 to 40 percent. The cotton seeds contain rich fatty acid and protein, and are widely applied to processing edible oil, industrial raw materials and biodiesel. Cotton breeding studies have been focused mainly on the study of fiber yield and quality, while the study of cotton seed improvement has been severely retarded. Therefore, it is important to study the oil related genes and improve the cotton seed oil content and fatty acid components by transgenic means.
Lipid anabolism plays an important role in the growth, development and reproduction of plants, and is present throughout the life of plants. Vegetable oil is mainly present in plant seeds, is a main source of edible oil in daily life of human beings, and is widely applied in industries such as food, feed, pharmacy, biological energy sources and the like. To date, more than 200 fatty acids have been isolated from organisms. Fatty acids are one of the essential components of plant cells. De novo synthesis of fatty acids is an essential process necessary for the biological production of cell membranes and stored lipids. acetyl-CoA (acetyl-CoA) production of malonyl-CoA is the first step in fatty acid biosynthesis, followed by sequential condensation of two carbon units derived from malonyl-CoA. The synthesis of the grease firstly needs the synthesis of fatty acid in plastids, and the fatty acid is transported into cytoplasm to form a fatty acyl pool, so that the fatty acid can be used for the synthesis of the grease of an endoplasmic reticulum. Oil synthesis is limited by fatty acid production, which in turn is regulated by acetyl-coa carboxylase (ACCase) activity. Heterogeneous acetyl-coa carboxylase (ACCase) is the rate-limiting and key enzyme for the de novo synthesis of various fatty acids, and consists of 2 subunits α -CT and β -CT of Biotin Carboxylase (BC), biotin Carboxyl Carrier Protein (BCCP) and Carboxytransferase (CT). Where BC is the tie and bridge linking the other three subunits of ACCase. At present, although the grease synthesis pathway is known, little is known about the control mechanism of cotton BC involved in grease biosynthesis.
Disclosure of Invention
The invention aims to provide GhBC gene and application of related biological materials thereof in regulating and controlling accumulation of vegetable oil.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of GhBC gene or a biological material related to GhBC gene in regulating and controlling accumulation of vegetable oil, wherein the nucleotide sequence of GhBC gene is shown as SEQ ID NO. 3.
Preferably, the biological material related to GhBC gene comprises:
A1 GhBC 1a protein encoded by the gene;
A2 A recombinant vector comprising GhBC gene;
A3 A recombinant microorganism comprising GhBC gene;
A4 A) a recombinant microorganism comprising the protein of A1);
A5 A recombinant microorganism comprising the recombinant vector of A2).
Preferably, the amino acid sequence of the protein encoded by the GhBC gene is shown as SEQ ID NO. 4.
Preferably, the method for regulating and controlling the accumulation of vegetable oil comprises the following steps: the GhBC gene was introduced into plants to overexpress the GhBC gene.
Preferably, the plant comprises cotton, arabidopsis thaliana.
The invention provides GhBC gene and application of related biological materials thereof in regulating and controlling accumulation of vegetable oil. The invention clones cotton GhBC1 (ID: GH_A02G1009) gene from cotton institute 24 in upland cotton material, successfully constructs seed specific promoter driven recombinant expression vector and transforms Arabidopsis. Compared with wild control plants, the expression level of the T3 generation lines of 2 transgenic arabidopsis thaliana is obviously increased, the oil content is respectively increased by 9.5 percent and 11.4 percent, the C18:1d9+C18:1d11 and C20:1d11 levels in fatty acid components are obviously increased, and the C16:0, C18:0 and C18:2 levels are obviously reduced. It is shown that GhBC gene can be successfully over-expressed in plants other than cotton, and can regulate the oil content of plants and the composition of fatty acid components. The invention provides a new theoretical basis for researching the regulation and control mechanism of cotton BC participating in grease biosynthesis.
Drawings
FIG. 1 is a graph showing the result of electrophoresis of GhBC gene amplification products.
FIG. 2 shows the results of selection of transgenic plants of T0 generation on MS medium containing kanamycin.
FIG. 3 shows the results of PCR screening for T1 generation transgenic plants. Lanes 1-2 are false positive plants, target bands are not amplified, lanes 3-8 are positive plants, target bands are obtained near 795bp, and the success of the transformation of the seed-specific expression vector of the gene is preliminarily proved.
FIG. 4 shows GhBC gene expression levels in different tissues of transgenic plants.
FIG. 5 shows the amount of GhBC gene expression in transgenic lines OE1, OE2 and wild-type strain (WT) Hordeum vulgare.
FIG. 6 shows the results of the oil content determination in the seeds of transgenic lines OE1, OE2 and wild-type line (WT).
FIG. 7 shows the results of fatty acid composition analysis in transgenic strain OE1, OE2 and wild-type strain (WT) seeds.
Detailed Description
The sources of the kits and biological materials used in the examples of the present invention are as follows:
The total plant RNA extraction kit (DP 432) was purchased from Tiangen Biochemical (technology) Beijing Co., ltd; RNA reverse transcription Kit TRANSSCRIPT ONE-Step gDNA Removal AND CDNA SYNTHESIS Supermix (AT 311-02), fluorescence quantitative enzyme TRANSSTART TOP GREEN QPCR Supermix (AQ 131-04), cloning vector pEASY-T5 Zero Cloning Kit (CT 501-01) and plasmid extraction Kit EasyPure HiPure PLASMID MINIPREP KIT (EM 111-01) were purchased from Beijing full gold Biotechnology Co., ltd; the high-fidelity PCR amplification enzyme KOD-Plus-Neo (KOD-401) was purchased from TOYOBO BioCo; DNAFragmentPurificationKit ver 4.0.0 gel recovery kit and DNAPurificationKitPCR product purification kit were purchased from TaKaRa biosystems; an infusion ligase was purchased from nuprandial biotechnology limited; coli competent cells DH 5. Alpha. Were purchased from Shanghai Biotechnology Co., ltd; agrobacterium competent LBA4404 was purchased from Beijing Bomaide Gene technologies Co., ltd; restriction enzymes XbaI and SacI were both purchased from NEB company.
The sources of the medicines used in the embodiment of the invention are as follows:
Agarose was purchased from Beijing full gold biotechnology Co., ltd; peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. are domestic analytical purities; ampicillin (IA 0340), kanamycin (YZ-130556), rifampicin (IR 0110), streptomycin (IS 0360) and the like were purchased from Beijing Soy Bao technology Co.
The manufacturer information of the instrument used in the embodiment of the invention is as follows:
PCR amplification apparatus (BIO-RAD), electrophoresis apparatus (BIO-RAD), fluorescent quantitative PCR apparatus (ABI 7500), gel imaging system (BIO-RAD), high-speed centrifuge (Hettich MIKRO R), enzyme-labeled apparatus (BIO-TEC KC 4), vacuum freeze-dryer (ALPHAI-5), seed oil content nuclear magnetic resonance analyzer (NMI 20-analysis) and Shimadzu fatty acid composition analysis gas chromatograph (GC-2030); the Arabidopsis seed oil content measuring instrument is a Newmey nuclear magnetic resonance instrument (NMI 20-Analyst).
The various reagents mentioned in the examples of the present invention but not listed are all prepared according to the method on the third edition of the guidelines for molecular cloning experiments, and the biochemical reagents are analytically pure or superior.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The embodiment provides a cloning method of GhBC gene, which comprises the following specific steps:
The CDS full-length sequence of the GH_A02G1009 (GhBC 1) gene was called from cotton functional genomic database (https:// cottonfgd. Org /) using the local Blast method, and the primer sequence was designed using primer 5.0 software. The full open reading frame ORF was cloned from ovules of cotton 24 in upland cotton using PCR (Polymerase Chain Reaction) technology.
The specific cloning procedure is as follows:
1. Cotton stations 24 in the test materials were planted in the Anyang institute of technology test base and managed as a general field. The obtained tissue is ovule 3 and 5 days after flowering, and the obtained material is rapidly frozen in liquid nitrogen and stored in a refrigerator at-80 ℃ for standby. The total RNA of the plants is extracted by using a Tiangen company kit.
2. 1000Ng of RNA was reverse transcribed into cDNA, and the reverse transcription product cDNA solution was diluted 5-fold as a template for PCR reaction. The total RNA of the cotton ovule mixture extracted at each period was used as a template, and was reverse transcribed into cDNA using a full Jin Fanzhuai recording kit, and the cDNA synthesis system is shown in Table 1.
TABLE 1cDNA Synthesis System
Remarks: incubation was performed in a PCR apparatus at 42℃for 30min, and finally at 85℃for 5 s. Preserving at 4 ℃ for standby.
3. Primer 5.0 software designed the primer sequences as follows:
GhBC1-F：5′-ATGGGTATTTGCTATTGTAG-3’(SEQ ID NO.1)
GhBC1-R：5′-CTAAGCAGTTGCACTTGTTAATTC-3’(SEQ ID NO.2)
The PCR amplification of cotton GhBC gene was performed using the above primers and cDNA obtained by reverse transcription as a template. The target gene amplification system is shown in Table 2.
TABLE 2 Gene amplification System of interest
| 10×PCRBufferforKOD-PLUS-Neo | 5μl |
| KOD-Plus-Neo | 1μl |
| Template | 2μl |
| Forward primer GhBC-F (10. Mu.M) | 2μl |
| Reverse primer GhBC-R (10. Mu.M) | 2μl |
| 2mMdNTPs | 5μl |
| 25mMMgSO4 | 3μl |
| ddH2O | 30μl |
| Total volume of | 50μl |
The PCR reaction conditions are shown in Table 3.
TABLE 3PCR reaction conditions
| Step1 | Predenature | 94℃，2min |
| Step2 | Denature | 98℃，10s |
| Step3 | Annealing | 57℃，30s |
| Step4 | Estension | 68℃，45s |
| Step5 | Estension | 68℃，5min |
Remarks: 30 cycles of amplification were performed from Step2 to Step 4.
4. After the completion of the reaction, the mixture was stored at 4℃and detected by agarose electrophoresis at 0.8% to obtain a PCR amplification product of 1647bp in size, as shown in FIG. 1.
5. Purification and recovery of PCR amplification products were performed using TaKaRa MiniBEST DNA FRAGMENT Purification Kit ver 4.0. The recovered product was ligated into cloning vector pEASY-T5 cloning vector and E.coli competent DH 5. Alpha. Transformed. The culture was carried out overnight at 37℃and the single clone was picked from kan-resistant LB medium and cultured overnight at 37℃in 500. Mu.l of LB medium containing kan. And (3) performing PCR (polymerase chain reaction) verification on bacterial liquid, and sequencing the bacterial liquid containing the size strips of the target gene fragments, wherein the sequencing result is shown as SEQ ID NO.3:
ATGGGTATTTGCTATTGTAGGCATTTTCTGTTTTTCTCAATGGCAATGGATGCTTCACTGACTATGTGCAAATCGGTCACATCACCTCCTGGCTTATTCTTGGGGAGAAGTAGAGTTATTAGGAGTTCCCAGTGTACCTTTATGGTGGGAAGCAGGATCAACTTTCCTAGGCAGAAAGCTCAGTCAACCCAAGTTAAATGTAAATCTAGCAGGTGTGGAGTAGCTCTTGGTGCTAAATGCCGTGCTGAGAAAATTTTGGTGGCAAATAGAGGAGAAATTGCTGTTCGTGTTATCCGAACAGCTCATGAGATGGGAATACCATGTGTTGCTGTTTACTCTACAATTGATAAGGATGCACTTCATGTGAAGCTGGCTGATGAATCAGTTTGCATAGGCGAAGCACCAAGCAGTCAATCGTATTTATTGATTCCAAATGTCCTATCTGCTGCAATTAGCCGTAACTGTACTATGCTTCATCCTGGGTATGGTTTCCTTGCTGAGAATGCGGTTTTTGTAGAAATGTGCAGAGATCACAGGATCAACTTTATTGGGCCTAACCCTGACAGTATTCGTGTTATGGGTGACAAATCAACTGCACGAGAAACAATGAAGAACGCGGGTGTTCCTACTGTTCCGGGAAGTGATGGATTGTTACAGAGTACAGAGGAAGCAATCAAGCTTGCCCATGAGATTGGCTTTCCTGTGATGATCAAGGCCACAGCCGGTGGTGGAGGGCGTGGAATGCGTCTTGCTAAAGAGCCTGATGAGTTTGTGAAGTTACTGCAGCAAGCCAAAAGTGAAGCTGCAGCTGCATTTGGAAATGATGGAGTTTATTTGGAGAAGTACATCCAAAATCCAAGGCACATTGAGTTCCAGGTTCTTGCGGATAAATATGGTAATGTTGTTCACTTTGGTGAGCGAGACTGCAGCATCCAGAGACGTAATCAAAAACTTCTTGAAGAGGCTCCTTCTCCAGCATTGACACCAGAGTTGCGGAAGGCCATGGGTGATGCAGCAGTAGCTGCAGCAGCATCTATTGGTTACATTGGTGTAGGAACTGTTGAGTTCCTATTGGATGAAAGAGGTTCCTTCTACTTCATGGAAATGAACACTAGAATCCAGGTGGAGCATCCTGTAACTGAAATGATTTCCTCTGTTGACTTGATTGAAGAACAAATTCGTGTAGCTATGGGGGAAAAACTGCGCTACAAACAGGAAGATATTGTGCTCAGAGGGCATTCCATTGAATGTCGTATCAATGCAGAAGATGCATTTAAAGGGTTCAGACCCGGACCAGGGAGAATAACATCGTACTTGCCATCTGGAGGCCCATTTGTTCGAATGGATAGCCATGTTTATTCTGATTATGTAGTTCCTCCAAGCTATGATTCATTACTTGGAAAGCTTATTGTATGGGCTCCAACAAGGGAAAAAGCAATTGAGCGCATGAAAAGGGCTCTTGATGACACTGTAATTACAGGGGTTCCTACAACAATTGAATACCATAAACTCATCCTTGATATTGAGGACTTCAGAAACGGAATAGTTGACACTGCTTTCATTCCAAAACATGAAGAAGAGTTGGCAGCACCACAGAAAATGTTAGTAGCAAGCCCAACCAAAGAATTAACAAGTGCAACTGCTTAG.
The amino acid sequence of the protein encoded by the target gene is SEQ ID NO.4:
MGICYCRHFLFFSMAMDASLTMCKSVTSPPGLFLGRSRVIRSSQCTFMVGSRINFPRQKAQSTQVKCKSSRCGVALGAKCRAEKILVANRGEIAVRVIRTAHEMGIPCVAVYSTIDKDALHVKLADESVCIGEAPSSQSYLLIPNVLSAAISRNCTMLHPGYGFLAENAVFVEMCRDHRINFIGPNPDSIRVMGDKSTARETMKNAGVPTVPGSDGLLQSTEEAIKLAHEIGFPVMIKATAGGGGRGMRLAKEPDEFVKLLQQAKSEAAAAFGNDGVYLEKYIQNPRHIEFQVLADKYGNVVHFGERDCSIQRRNQKLLEEAPSPALTPELRKAMGDAAVAAAASIGYIGVGTVEFLLDERGSFYFMEMNTRIQVEHPVTEMISSVDLIEEQIRVAMGEKLRYKQEDIVLRGHSIECRINAEDAFKGFRPGPGRITSYLPSGGPFVRMDSHVYSDYVVPPSYDSLLGKLIVWAPTREKAIERMKRALDDTVITGVPTTIEYHKLILDIEDFRNGIVDTAFIPKHEEELAAPQKMLVASPTKELTSATA.
The correctly sequenced cloning vector was designated pEASY-GhBC1.
Example 2
The embodiment provides a method for regulating and controlling accumulation of plant oil, which comprises the following specific processes:
1. construction of recombinant expression vectors
1.1.A p2301M alpha plasmid vector is selected, which is transformed and stored in the laboratory, the transformation process is recorded in literature Liu Zhengjie, zhang Yuan, wang Yanxia, and the like, seed specific expression vector construction and genetic transformation [ J ]. Molecular plant breeding, 2011,9 3): 8.DOI:10.3969/mpb.009.000270. The concentration of the plasmid vector is 189ng/μl, and the agarose gel electrophoresis detection shows that the plasmid vector has no protein pollution and meets the test requirement. The vector p2301Mα was expressed by restriction enzymes XbaI and SacI for cleavage of the plant specific promoter, and the vector backbone was recovered.
1.2 XbaI-SacI was designed as an insertion site according to the multiple cloning site on the p2301M alpha plasmid vector, and primers were synthesized. The primer sequences were as follows:
GhBC1-XbaI-F:5'-GCTCTAGAATGGGTATTTGCTATTGTAG-3' (the underlined part contains the recognition sequence of the restriction enzyme XbaI) (SEQ ID NO. 5)
GhBC1-SacI-R:5'-CGAGCTCCTAAGCAGTTGCACTTGTTAATTC-3' (the underlined part contains the recognition sequence for the restriction enzyme SacI) (SEQ ID NO. 6)
The GhBC-mesh gene sequence containing XbaI and SacI cleavage sites was amplified using KOD-Plus-Neo high-fidelity enzyme, and the reaction system is shown in Table 4.
TABLE 4 reaction system
| 25mMMgSO4 | 3μl |
| 10×PCRBufferforKOD-PLUS-Neo | 5μl |
| KOD-Plus-Neo | 1μl |
| Template | 1μl |
| Forward primer GhBC-XbaI-F (10. Mu.M) | 2μl |
| Reverse primer GhBC-SacI-R (10. Mu.M) | 2μl |
| 2mMdNTPs | 5μl |
| ddH2O | 31μl |
| Total volume of | 50μl |
The conditions required for the PCR reaction were set as follows: pre-denaturation at 94℃for 2min, denaturation at 98℃for 10s, annealing at 60℃for 30s, elongation at 68℃for 45s,30 cycles; extending at 68℃for 5min. The GhBC1 amplified product containing XbaI and SacI cleavage sites was recovered and purified, and the recovered product was digested with restriction enzymes XbaI and SacI.
1.3 Connecting the vector skeleton recovered in 1.1 with the recovered product after 1.2 enzyme digestion to form a plant specific promoter expression vector p2301M alpha-GhBC 1. Converting the connection product into escherichia coli DH5 alpha by adopting a heat shock method, culturing the connection product in an incubator at 37 ℃ for inversion overnight, and sequencing positive clones; the recombinant expression vector with the correct sequencing result is named as p2301Mα -GhBC1/DH5 α.
2. Construction of recombinant microorganisms
2.1 Taking 100 mu l of agrobacteria LBA4404 competent cells melted on ice, adding p2301M alpha-GhBC 1/DH5 alpha 2 mu g into the competent cells under aseptic conditions, gently mixing, and standing in an ice water bath for 5 minutes;
2.2, placing the centrifuge tube in liquid nitrogen for quick freezing for 5 minutes;
2.3, rapidly placing the centrifuge tube in a water bath at 37 ℃ for incubation for 5 minutes, and placing the centrifuge tube back in an ice water bath for 5 minutes;
2.4, 100 μl of bacterial liquid is coated on LB screening medium containing kanamycin, streptomycin and rifampicin, cultured for 2.5d at 28 ℃, positive clones are selected, cultured for 48h at 28 ℃ on LB liquid medium, recombinant agrobacterium is obtained, and transferred to 15% glycerol with final concentration, and preserved at-20 ℃ for standby.
3. Construction of transgenic plants
The method comprises the steps of taking Columbia type arabidopsis thaliana as a receptor, inoculating recombinant agrobacterium with a flower dipping method for genetic transformation, harvesting T0 generation transgenic seeds, screening positive transformants on MS culture medium containing kanamycin (50 mg/L), selecting positive transformants at the position indicated by an arrow as shown in fig. 2, culturing the positive transformants to obtain T1 generation resistant plants, and collecting seeds after flowers are mature.
After T1 generation resistant plants are transplanted for 40 days, leaves of the Arabidopsis plants are taken to extract DNA, and a resistance marker NPTII is amplified by PCR, so that positive Arabidopsis plants are further screened (the resistance marker NPTII gene and a target gene GhBC are both in a carrier T-DNA region, and due to homology between BC genes in Arabidopsis and BC genes in cotton, false positive and other problems easily occur during amplification, the amplified NPTII gene can more indicate that GhBC1 genes are successfully transformed).
The results are shown in FIG. 3. Lanes 1-2 are false positive plants, target bands are not amplified, lanes 3-8 are positive plants, target bands are obtained near 795bp, and the success of the transformation of the seed-specific expression vector of the gene is preliminarily proved.
4. Determination of oil content in transgenic plants
With the result of the measurement shown in FIG. 3, false positive plants are discarded, RNA of the horn, leaf and stem of the T1 generation positive resistant plants is extracted respectively after 45 days of transplanting the T1 generation positive resistant plants, cDNA is synthesized, specific primers (GhBC-QRT-F: TCCTTGCTGAGAATGCGGTT (SEQ ID NO. 7) and GhBC1-QRT-R: CCGGAACAGTAGGAACACCC (SEQ ID NO. 8)) are designed, cDNA of different tissue parts are used as templates, and fluorescent quantitative PCR is carried out, wherein the internal reference primers are ATACTINQF respectively: CCATGAACCCACCTATAACTCC (SEQ ID NO. 9) and ATACTINQR: TACTCTGCCTTTGCGATCCAC (SEQ ID NO. 10). The results are shown in FIG. 4. As a result, the GhBC gene in the T1 transformed plant can only have higher expression level in the Horn fruit, while the GhBC gene expression level in the leaves and stems is weak or not. It was demonstrated that the GhBC gene transformed in Arabidopsis achieved seed-specific expression.
And respectively harvesting T1 generation single plant seeds, taking 100 seeds to be distributed on an MS+Kan (50 mg/L) culture medium, counting the separation ratio of yellow-green seedlings after the cotyledons of the seedlings are completely unfolded, and carrying out chi-square test to meet the requirements of green seedlings: yellow seedlings = 3:1 isolated plants were initially predicted as single copy insert lines, which were individually seed harvested for subsequent analysis.
The GhBC gene expression level in each strain of Hordeum vulgare was examined, compared with the Wild Type (WT), and 2 single copy T3 generation strains with higher expression level were selected and named GhBC1 OE1 and OE2, respectively. The GhBC gene expression levels are shown in FIG. 5. Each strain was transplanted into 20 plants, after maturation, T3 generation seeds were harvested to extract total fatty acids, and the seed oil content of 2 GhBC transgenic T3 generation arabidopsis lines (OE 1, OE 2) and wild arabidopsis (WT) was determined using a neumey nuclear magnetic resonance instrument (NMI 20-analysis). The oil content of each strain was measured in 3 replicates, and the average was taken and the results are shown in FIG. 6. From the results, the average oil content of 3 times of the wild type Arabidopsis seeds was 25.89%; whereas the GhBC1 transgenic T3-generation OE1 had an average oil content of 28.40% for 3 times; the GhBC1 transgenic T3 generation OE2 had a 3-time average oil content of 28.80%; compared with a control strain, after GhBC gene is over-expressed, the average oil content of seeds is increased by 9.5% and 11.4%, which shows that GhBC gene can regulate and control the oil content of arabidopsis seeds.
5. Determination of fatty acid Components in transgenic plants
Fatty acid components of GhBC a transgenic Arabidopsis seeds and control samples were analyzed using a gas chromatograph (GC-2030, shimadzu, japan) equipped with a Flame Ionization Detector (FID) and SH-Rtx-65 capillary column (30 m. Times.0.25 mm. Times.0.50 μm). Samples (1 μl) comprising fatty acid and n-hexane solutions were injected in a 30:1 split mode at 280 ℃. The significance of all fatty acid component data was calculated by the Student t-test method. p <0.05 (x) compared to the control, the difference was statistically significant. As a result, as seen in FIG. 7, C18:1d9+C18:1d11 and C20:1d11 levels were significantly increased in the GhBC1 transgenic line seeds and significantly decreased in C16:0, C18:0 and C18:2 levels compared to the control. These results indicate that GhBC gene overexpression can alter the levels of fatty acid components in seeds.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (5)
- The application of GhBC1 gene or biological material related to GhBC gene in regulating and controlling the accumulation of vegetable oil is characterized in that the nucleotide sequence of GhBC gene is shown as SEQ ID NO. 3.
- 2. The use of claim 1, wherein the GhBC gene-related biological material comprises:A1 GhBC 1a protein encoded by the gene;A2 A recombinant vector comprising GhBC gene;A3 A recombinant microorganism comprising GhBC gene;A4 A) a recombinant microorganism comprising the protein of A1);A5 A recombinant microorganism comprising the recombinant vector of A2).
- 3. The use according to claim 1, wherein the GhBC gene encodes a protein having the amino acid sequence shown in SEQ ID No. 4.
- 4. The use according to claim 1, wherein the method of regulating the accumulation of vegetable oils is: the GhBC gene was introduced into plants to overexpress the GhBC gene.
- 5. The use according to claim 1, wherein the plant comprises cotton, arabidopsis thaliana.
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