CN116855506A - BnaC05.UK gene for regulating oil content of rape and application thereof - Google Patents
BnaC05.UK gene for regulating oil content of rape and application thereof Download PDFInfo
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- CN116855506A CN116855506A CN202310688856.1A CN202310688856A CN116855506A CN 116855506 A CN116855506 A CN 116855506A CN 202310688856 A CN202310688856 A CN 202310688856A CN 116855506 A CN116855506 A CN 116855506A
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- rape
- oil content
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- crispr
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Medicinal Chemistry (AREA)
- Nutrition Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Botany (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a BnaC05.UK gene for regulating and controlling oil content of rape, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the invention also discloses a protein encoded by the gene, a CRISPR/Cas9 gene editing vector and recombinant bacteria. The CRISPR/Cas9 gene editing vector of the BnaC05.UK gene is transformed into the genome of rape by using an agrobacterium-mediated genetic transformation method, so that the variety of rape with the BnaC05.UK gene function deleted is obtained, and the oil content of a mutant material is measured, and as a result, the accumulation of the oil content of the rape is negatively regulated by the BnaC05.UK gene, and the oil content of the mutant material is obviously increased compared with that of a wild type. The invention has very important significance in rape high-oil breeding.
Description
Technical Field
The invention belongs to the field of genetic engineering and biotechnology, and particularly relates to an unexplored gene BnaC05.UK for regulating and controlling oil content of rape and application thereof.
Background
During the evolution of the last hundred million years, plants evolved the ability to synthesize various metabolites that play an important role in plant growth, propagation and adaptation to the environment. There are about 10 to 100 tens of thousands of metabolites in the plant kingdom, indicating that plants have a rich metabolic diversity. In recent years, more and more researchers have utilized whole genome association analysis (Genome Wide Association Studies, GWAS) to resolve metabolome diversity, which in combination with metabolome is called metabolite whole genome association analysis (Metabolite Genome Wide Association Studies, GWAS) mGWAS, which is capable of better resolving genetic variation behind metabolic diversity. Cabbage type rape (Brassica napus l.) is one of the most important oil crops in the world, with rapeseed oil accounting for about 50% of the vegetable oil supply in our country. The oil content and thousand kernel weight are important yield traits of rape, and the way and mechanism by which metabolites regulate the oil content and thousand kernel weight in rape seeds are not completely clear.
In organisms, fatty Acids (FA) are synthesized in plastids, and there are a number of transcription factors in plants that regulate the synthesis, transport, storage, and degradation of Fatty acids. The first step of fatty acid synthesis is to catalyze carboxylation of acetyl-coa to malonyl-coa by acetyl-coa carboxylase (ACCase), and then the fatty acid synthase synthesizes an acyl carbon chain by adding two carbons per cycle with malonyl-coa as a substrate to synthesize a saturated fatty acid of 16 to 18 carbons. WRINKLED 1 (WRI 1) is a transcription factor directly regulating FA metabolism during seed maturation and is involved in regulating genes in the late stages of glycolysis and early stages of FA biosynthesis, overexpression of WRI1 results in increased fatty acids in seeds and developing seedlings, and overexpression of WRI1 upregulates a range of gene expression levels in plastids involved in FA synthesis, including PYRUVATE KINASE (P1-PKbeta 1), BIOTIN CARBOXYL CARRIER PROTEIN (BCCP 2), ACYL CARRIER PROTEIN 1 (ACP 1), 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE I (KAS 1). In addition, FUS3 and ABI, etc. are also capable of inducing many genes involved in photosynthesis and fatty acid biosynthesis pathways.
There is a close relationship between flavonoids and FA biosynthesis. malonyl-CoA is a substrate for flavonoid biosynthesis and reacts with 4-coumarin-CoA to form chalcone under the catalysis of TRANSPARENT TESTA (TT 4). TT4 is the first step in catalyzing flavonoid biosynthesis and mutations in TT4 lead to a deficiency in flavonoid biosynthesis. Xuan et al describe in detail the effect of a hindered flavonoid synthesis on Arabidopsis seed lipid synthesis in Arabidopsis TT4 mutants. Specifically, in TT4 mutants, plants increase the availability of soluble sugars during seed development, activate transcription factors associated with glycolysis and fatty acid synthesis, such as WRI1, ABI3, etc., and increase auxin accumulation. TRANSPARENT TESTA 2 (TT 2) regulates FA biosynthesis early in seed development by targeted regulation of FUS3, TT2 also affects seed FA composition by related genes in late stages of seed development. The deletion of TT8 leads to abnormal flavonoid synthesis in the brassica napus seeds and simultaneously causes accumulation of fatty acid, the oil content and the protein content of BnTT8 double mutant seeds are obviously increased, and the fatty acid composition is also obviously changed. Zhang et al used 382 parts of Brassica napus germplasm, analyzed natural variation of seed coat content by TWAS and GWAS, combined with related networks of TWAS and seed coat content related gene modules, found a new gene BnaC07.CCR-LIKE (CCRL) regulating seed coat content, which determined seed coat thickness and seed coat lignin content by regulating lignin biosynthesis.
The invention clones an unannotated gene BnaC05.UK, creates a mutant of BnaC05.UK by using CRISPR/Cas9 technology, the oil content of the seed of the obtained rape mutant is obviously increased, and the result shows that the BnaC05.UK gene plays an important role in regulating the oil content of the rape seed, and has a quite large application prospect in creating new germplasm of rape with high oil content.
Disclosure of Invention
The invention aims to provide an unannotated gene BnaC05.UK for regulating and controlling oil content of rape and application thereof in creating a rape variety with high oil content.
Obtaining complete metabolic group data of mature seeds of the brassica napus through measuring 382 parts of core germplasm metabolic groups; constructing a comprehensive agronomic trait (oil content) ternary relationship network (metabolite-QTL-gene) by using genome and developing seed transcriptome data existing in the laboratory through association analysis (GWAS and TWAS); through the combination of metabolic markers and a plurality of groups of analytical data, novel genes related to the oil content of rape seeds are mined and cloned. A gene which is obviously related to the metabolite related to the oil content is found in the whole genome related analysis and the whole transcriptome related analysis of the seed metabolome of the brassica napus, and the gene is named as BnaC05.UK gene by the applicant. The gene exists on a cabbage type rape C05 chromosome (BnaC 05g 43050D), and the nucleotide sequence of the gene is shown as SEQ ID NO:1, wherein BnaC05.UK is composed of 276bp, and the protein sequence coded by the gene is shown in a sequence table SEQ ID NO:2, a total of 91 amino acids.
When the mutant of the gene is obtained, the CRISPR/Cas9 gene editing vector of the BnaC05.UK gene is transformed into the genome of rape by using an agrobacterium-mediated genetic transformation method, and the rape variety with the gene BnaC05.UK function deleted is obtained by using a CRISPR/Cas9 gene editing technology.
The oil content of the mutant material is found to be obviously increased compared with the wild type through measuring the oil content of the mutant material to find that the BnaC05.UK gene negatively regulates the accumulation of the oil content of the rape.
Rape yield is critical to global vegetable oil supply, and oil content belongs to one of three factors of rape yield. Comprehensive research on secondary metabolome and multiple groups of chemical analysis such as genome, transcriptome, agronomic characters and the like have great promotion effect on the research on brassica napus. A large number of novel genes involved in oil content accumulation can be mined through multiple groups of chemical analysis, and the novel genes have an important effect on the subsequent genetic improvement of the oil content of rape. The genetic resource created by the invention has very important significance in rape high-oil breeding.
Drawings
FIG. 1 is a phenotypic characterization of the gene BnaC05.UK in canola, using near-infrared analysis of oil content in canola seed. L1 and L5 are homozygous mutants edited by bnac05.Uk, with 3 different individuals per strain, representing P <0.05 in Student' st test.
FIG. 2 is a phylogenetic tree constructed using the cDNA sequence of BnaC05.UK.
FIG. 3 sequence analysis of BnaC05.UK in the Pan genome of Brassica napus in BnIR.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods not specified in detail in the following examples are generally followed by conventional methods or by the instructions "molecular cloning: the methods described in the laboratory guidelines (New York: cold Spring Harbor Laboratory, 1989) or according to the methods suggested in the operating manual provided by the manufacturer.
Plant metabolites can affect the growth and development of plants themselves and environmental adaptation, and plant metabolites play an important role in maintaining human food safety. Rape is the third largest oil crop in the world, and it accounts for about 50% of the vegetable oil supply in our country. Oil content is an important agronomic trait of rape, and increasing oil content of rape is an important breeding goal of rape. The experimental object of the research is a natural population of 382 inbred cabbage type rape, the genetic basis of the oil content of the rape seeds is comprehensively analyzed from the metabolism angle by using genome, transcriptome and metabolome data as the basis and by multi-group chemical combination analysis, and a batch of candidate genes influencing the oil content are excavated. The invention verifies the function of the candidate gene BnaC05.UK for specifically influencing the oil content of seeds through experiments, and the main contents are as follows:
mature seeds are selected as research materials, 2173 metabolites are detected in total by using a liquid chromatography-triple quaternary lever combination instrument and a method of widely targeting metabolite analysis, 131 metabolites which are obviously related to the oil content are identified through analysis, and the metabolites are used as metabolic markers of the oil content. Using these marker metabolites, a genome-wide association analysis (mGWAS) was performed, targeting 446 quantitative trait loci (Metabolite Quantitative Trait Loci, mQTL) of the metabolites associated with oil content. At the same time, a metabolite full transcriptome association analysis (Metabolite Transcriptome-Wide Association Study, mTMWAS) was performed in combination with population transcriptome data from 40 day post-flowering seed development, correlating to 7316 genes for oil content related metabolites. And analyzing the metabolite related gene expression network to obtain 19 gene expression modules of the metabolite related to the oil content. And combining weighted correlation network analysis, constructing a ternary relation network comprising metabolites, mQTL and modular genes for the oil content, and mining 240 candidate genes for regulating and controlling the oil content of the rape seeds.
Based on the results of the multi-set chemical combination analysis, one mQTL hotspot mQTL3947 co-localized with 11 oil content related metabolites was found on the C05 chromosome in the mGWAS results, and this hotspot was co-localized with the oil content and seed coat content GWAS localized QTL (oil content QTL: qSOC.C05, husk rate QTL: qSCC.C05). One unknown gene, bnac05.Uk, was screened in mQTL3947 for a significant association with 42 metabolites mTWAS. Mutant materials of BnaC05.UK are created through CRISPR/Cas9 technology, and the metabolome and transcriptome and oil content of the mutant materials are analyzed to find that the oil content of the BnaC05.UK mutant materials is improved by 2.6-3.1% compared with that of wild seeds. Meanwhile, mGWAS, mTMAS, gene expression level whole genome association analysis (Expression Genome-Wide Association Study, eGWAS) and seed oil content negative correlation metabolic markers (catechin, naringenin, S21-1612 and S21-4865) positioned by a co-expression network are remarkably reduced in the mutant. Seed transcriptome analysis and ruthenium red staining experiments of mutants in development show that mutation of the gene can cause change of plastid RNA level and influence accumulation of fatty acid, flavone and pectin in seeds. The above results indicate that BnaC05.UK is a novel gene for regulating the oil content of cabbage type rape seeds.
EXAMPLE 1 creation of rape BnaC05.UK mutant Material
Strains used in this study: coli Escherichia coli (dh5α) was used for gene cloning, vector construction and plasmid extraction; agrobacterium GV3101 is used for genetic transformation of canola; subcellular localization observations the vector used was pMDC83; the system for editing rape genes is a Chinese agricultural university Chen Jijun CRISPR/Cas9 gene editing system, the sgRNA framework template is pCBC-DT1T2 plasmid, and the Cas9 protein expression and genetic transformation vector is pKSE-401. Except that the system used for editing rape genes is derived from the China agricultural university Chen Jijun laboratory, other vectors are all derived from the laboratory.
Construction of Bnac05.UK-CRISPR vector
Rape BnaC05.UK mutants were created using the sgRNA-Cas9 system of the university of agricultural university, team Chen Jijun. The experimental operation steps are as follows:
(1) Logging in to a websitehttp://www.genome.arizona.edu/crispr/CRISPRsearch.htmlScreening target sgRNA1 according to the target position and the score: AAACGGAGCATCAGGCAGT and sgRNA2: CCTAATGCGTACTAATGGGT, all located in the first exon region of the gene BnaC05.UK.
(2) Designing primers
DT1-BsF:ATATATGGTCTCGATTGAAACGGAGCATCAGGCAGTGTT
DT1-F0:TGAAACGGAGCATCAGGCAGTGTTTTAGAGCTAGAAATAGC
DT2-R0:AACACCCATTAGTACGCATTAGCAATCTCTTAGTCGACTCTAC
DT2-BsR:ATTATTGGTCTCGAAACACCCATTAGTACGCATTAGCAA
(3) And (3) PCR amplification: four-primer PCR amplification was performed using 100-fold diluted pCBC-DT1T2 as a template. DT1-BsF and DT2-BsR are normal primer concentrations; DT1-F0 and DT2-R0 were diluted 20-fold. The amplification system is as follows:
PCR amplification procedure: total denaturation at 98℃for 1min; 15sec for denaturation at 98 ℃, 25sec for annealing at 56 ℃, 25sec for extension at 72 ℃,34 cycles; total extension at 72℃for 5min.
(4) The PCR product was purified and recovered, and the following cleavage-ligation system was established:
reaction conditions: 5hours at 37 ℃,5 minutes at 50 ℃,10 minutes at 80 ℃.
(5) Transformation of E.coli DH 5. Alpha: mu.L of plasmid containing the target cloning fragment is taken, competent E.coli is transformed, kan plate screening, positive clone PCR identification and sequencing are carried out. The vector with correct sequencing is the CRISPR vector of BnaC05.UK.
2. Agrobacterium-mediated genetic transformation
(1) The recombinant plasmid vector with correct construction is introduced into an agrobacterium strain GV3101, and positive monoclonal is selected and stored in a refrigerator at the temperature of minus 80 ℃. The method for introducing the medicine comprises the following steps:
a. cleaning an electric rotating cup: washing with pure water, washing with ultrapure water, pouring, washing with absolute ethyl alcohol (blowing with 1ml gun head), pouring absolute ethyl alcohol, and air drying in an ultra clean bench;
b. taking agrobacteria competent GV3101 20 μl;
c. taking 0.8 mu l of recombinant plasmid with correct construction, adding the recombinant plasmid into 20 mu l of competence, gently sucking and beating the recombinant plasmid, and uniformly mixing the recombinant plasmid to avoid generating bubbles;
d. placing the electric rotating cup with washed and dried drying in ice for precooling, and then driving the mixed liquid into the cup by the wall of the cup;
e. adjusting the electric converter to 1800V;
f. taking the electric rotating cup out of the ice, and wiping the outer wall of the electric rotating cup with water absorbing paper;
g. placing the electric rotating cup into an instrument, continuously pressing two push keys, and hearing a sound of a drop after a few seconds is successful;
h. after the electric shock is successful, 400 mu l of antibiotic-free LB is added into the electric rotating cup, and the electric rotating cup is transferred into a sterile centrifuge tube after being sucked for a few days;
i.28 ℃ for about 2 hours, 100 mul of the mixture is coated on the double antibody, the double antibody is sealed by a sealing film, the double antibody is inverted and cultured in a 28 ℃ incubator for 2 days, and spot picking detection is carried out.
(2) Agrobacterium colony detection
Colony is selected and cultured in double-antibody LB for 2 hours at 28 ℃, a proper amount of bacterial liquid is taken for PCR detection, and positive agrobacterium bacterial liquid is preserved.
(3) Genetic transformation of rape
The receptor for rape transformation is brassica napus Westar, and the specific operation flow is described in the reference literature: an efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus (1) seed of Brassica napus line 'Westar' is sown in a culture box of MS by sterilization and disinfection, and dark culture is carried out in a culture chamber; (2) culturing the constructed agrobacterium tumefaciens bacterial liquid expressing the CRISPR/Cas9 gene editing vector at 28 ℃ overnight in a shaking table, collecting bacterial liquid, and infecting hypocotyls; (3) the obtained transformed material was identified by PCR and planted in the experimental field.
(4) Identification of CRISPR transformed individuals
And sequencing the obtained rape CRISPR transformed single plant to screen rape mutants. First, the primers Cas9-570-F (5'-AGACCGTGAAGGTTGTGGAC-3') and Cas9-570-R (5'-TAGTGATCTGCCGTGTCTCG-3') are used for identifying Cas9 protein, and specific amplification and sequencing identification of target genes are carried out on Cas9 protein positive single plants. The specific amplification method of the target gene comprises the following steps: bnaC05.UK was specifically amplified with primers BnaC05.UK-C-F (5'-ATGGGGAAAGTGAGCTTGCT-3') and BnaC05.UK-C-R (5'-GAGTTGCAGATTCATACGAAATC-3'), respectively, by the following method:
the PCR system is as follows: easy taq polymerase 0.15. Mu.L, 10mM dNTP 0.4. Mu.L, 10 Xbuffer 2. Mu.L, DNA template 2. Mu.L, F primer 2. Mu.L, R primer 2. Mu.L, ddH 2 O to 20. Mu.L. PCR conditions: total denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 55℃for 30sec, extension at 72℃for 30sec,32 circulations; total extension at 72℃for 5min.
Sequencing the amplified target fragment by PCR product, and using DSDecode on-line website to obtain sequencing resulthttp:// skl.scau.edu.cn/dsdecode/)Analyzing the editing condition of the target site. Sequencing results showed that multiple mutant independent lines L1 and L5 were obtained (table 4) with the bnac05.Uk edited, where the L1 strain was inserted with one base a at target 1 and target 2 of the L5 strain were deleted.
TABLE 4 genotype of T2 generation BnaC05.UK mutant line
Example 2 functional verification and bioinformatic analysis of mutant materials
1. Oil content determination
Maturation ofThe oil content of the seeds was measured with a near infrared instrument and the fatty acid composition of mature and developing seeds was quantified with a gas chromatograph-flame ionization detector (Gas chromatography-flame ionization detector, GC-FID). The method of methyl esterification of fatty acids and their compositional analysis were according to the methods in the article and were slightly modified (Lu et al 2016). Weighing 10-15mg of dried mature seed into a glass spiral cover lipid extraction tube, adding 4mL of 5% H 2 SO 4 Methanol extract (containing 0.01% BHT) and gently crushing seed coat with glass rod; 100. Mu.L of heptadecanoic acid (C17:0) at a concentration of 16.2. Mu. Mol was added and the cap was screwed. The fat extraction tube was placed in a water bath at 85℃for 2 hours, then cooled to room temperature, followed by the addition of 3mL H 2 O and 3mL of n-hexane. After sufficient vortexing, centrifugation at 1000rpm for 10 minutes. 1.0mL of the supernatant was taken into a brown sample bottle. The organic reagents used in the fatty acid extraction process are all chromatographic grade reagents.
Determination of oil and fatty acid content by GC-FID, gas chromatography with hydrogen flame ionization detector and capillary RESTEK Rtx R -a wax column (0.25 mmx30 m), helium carrier 20mL/min. The instrument parameters are described as follows: and 1 mu L of sample is injected, and the split ratio is 20:1. The oven temperature was maintained at 170℃for 1min, then gradually increased to 210℃at a rate of 3℃per minute. Finally, the fatty acid species was identified based on retention time and its peak area was calculated. Fatty acids are calculated as nmol%. The oil content of the seeds was measured using heptadecanoic acid (17:0) as an internal standard, and the percentage of oil content on a dry weight basis was calculated.
The oil content results showed that the oil content of the two BnaC05-UK mutant lines was increased by 2.6% (L1) and 3.1% (L5), respectively, compared to WT (FIG. 1), with a significant difference (P < 0.05).
Bnac05.UK bioinformatic analysis
First, the BnaC05.UK was subjected to genetic bioinformatic analysis. A phylogenetic tree was constructed using the cDNA sequence of BnaC05.UK, 7 out of the 12 sequences retrieved belonging to Brassica (FIG. 2), and the results indicate that BnaC05.UK might have evolved mainly from Brassica and played a role. Basic information of BnaC05.UK, whose only one exon contains 276 bases and codes for 91 amino acids, was analyzed. The sequence of BnaC05.UK in the wild-type rape genome of BnIR was analyzed (http:// yanglab. Hzau. Edu. Cn/BnIR, FIG. 3), and the analysis of the wild-type genome sequence showed that most of the reference genome had only one BnaC05.UK except for one artificially synthesized wild-type rape variety No 2127. BnaC05.UK for InterPro analysis (http:// www.ebi.ac.uk/Interpro /) also has no typical domain. Furthermore, no study was reported on the function of BnaC05.UK and its homologous genes.
Multiple sets of chemical analyses showed that bnac05.Uk plays an important role in affecting many metabolite levels and may affect seed oil content. And the metabolite catechin (r= -0.47) with highest oil content correlation has obvious haplotype typing at the mQTL3947 locus. Metabolome analysis showed that catechin, S21-1612, naringenin and S21-4865 metabolite levels were significantly reduced compared to WT in the mutants, and that the increase in oil content of the BnaC05-UK mutant lines was probably due to the decrease in these metabolite levels, since these metabolites were significantly inversely correlated with oil content.
To explore transcriptional level changes in developing seeds, RNA sequencing was performed on seeds in WT and L5 development, with 706 genes up-regulated and 62 genes down-regulated in BnaC05.UK mutant relative to WT. The differential expression gene is enriched in pectin biosynthesis process, mucus pectin biosynthesis process, chloroplast RNA modification process and the like.
In the seed transcriptome during WT and L5 development, the gene BnaA06.ELI, bnaC07.ELI, SLO involved in chloroplast RNA modification was significantly elevated in the mutant and BnaA05.AXX17, bnaC05.AXX17 was significantly reduced in the mutant; genes GAUT3, GAUT5, QAUT3, ARAD1, bnaC05.RRT4, bnaCnn.RRT4 involved in pectin and seed coat mucus formation were significantly elevated in the mutants; ACT, ABI4, FATB, a gene associated with oil accumulation, was significantly elevated in the mutants. These results indicate that pectin and seed coat mucus and oil accumulation in seeds is significantly increased after the mutation bnac05.Uk and that chloroplasts act as the first sites of fatty acid synthesis, where changes in RNA modification-related gene expression levels may cause changes in photosynthetic product accumulation, fatty acid synthesis and transport.
Subcellular localization results indicate that the BnaC05.UK protein is localized in chloroplasts, and it is speculated that mutation of this gene may lead to changes in chloroplast RNA modification levels and affect accumulation of fatty acids, flavones and pectins. And the mutants had more leakage of seed coat mucus in mature seeds than WT. The phenotype of the mutant is consistent with the transcriptome result, which proves that BnaC05.UK is truly involved in the distribution of carbon sources in brassica napus seeds, and the biosynthesis of fatty acids and flavonoids is affected.
Claims (6)
1. A gene for regulating and controlling oil content of rape has a nucleotide sequence shown in SEQ ID No. 1.
2. The protein encoded by the gene of claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID NO. 2.
3. The CRISPR/Cas9 gene editing vector of the gene of claim 1.
4. A recombinant bacterium comprising the CRISPR/Cas9 gene-editing vector of claim 3.
5. Use of the gene of claim 1, the protein of claim 2, the CRISPR/Cas9 gene editing vector of claim 3, the recombinant bacterium of claim 4 for creating a high oil content canola variety.
6. A method of creating a high oil content canola variety, characterized by: transforming the CRISPR/Cas9 gene editing vector of the gene of claim 1 into the genome of rape by using an agrobacterium-mediated genetic transformation method, and obtaining the rape variety with the gene function deletion by using a CRISPR/Cas9 gene editing technology.
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