CN112048515B - Rape S-adenosine-L-methionine dependent methyltransferase gene BnPMT6 and application thereof - Google Patents

Abstract

The invention discloses a rape S-adenosine-L-methionine dependent methyltransferase gene BnPMT6, the nucleotide sequence of which is shown in SEQ ID NO. 1 or SEQ ID NO. 2. By obtaining over-expressed and mutant materials, the oil content of the transformed material was analyzed and it was found that BnPMT6 negatively regulates the accumulation of oil content while also regulating fatty acid composition. The invention further provides a method for obtaining a high oil content crop: the CRISPR/Cas9 gene editing vector containing rape S-adenosine-L-methionine dependent methyltransferase gene BnPMT6 is transformed into the genome of crop by using agrobacterium-mediated genetic transformation method, and the CRISPR/Cas9 gene editing technology is used to obtain the crop variety with gene BnPMT6 function deletion.

Description

Rape S-adenosine-L-methionine dependent methyltransferase gene BnPTM 6 and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and biology, and particularly relates to an S-adenosine-L-methionine-dependent methyltransferase gene BnPMT6 related to oil content of rape and application thereof.

Background

Vegetable oils are an important source of nutrition and energy for human beings, and oil crops mainly include soybeans, rapes, peanuts, and the like. Brassica napus (Brassica napus l.; AACC,2n ═ 38) is one of the main sources of vegetable edible oil, and plays an important role in oil crops in China. The correlation analysis data of the oil content and the yield of the rape seeds shows that the increase of 1 percent of the oil content of the rape seeds is equivalent to the increase of 2 to 3 percent of the yield of the rape seeds, so that the increase of the oil content of the rape seeds has important significance for increasing the oil yield of the rape seeds. The rapeseed oil mainly comprises various saturated fatty acids and unsaturated fatty acids, wherein the oleic acid content in the unsaturated fatty acids is the highest and accounts for more than 50% of the total fatty acids. Researches show that the high oleic acid vegetable oil has good health care effect, and the high oleic acid vegetable oil with reasonable ratio of fatty acids can effectively reduce the occurrence of human cardiovascular diseases. Meanwhile, the high-oleic acid vegetable oil has strong thermal stability, is not easily oxidized at high temperature, greatly reduces the risk of generating harmful substances due to high temperature, and meets the requirements of the traditional high-temperature cooking habit in China. Therefore, increasing the oil content of rapeseed and improving the oil quality are important targets for rape breeding.

The oil content of the seeds is closely related to various biological pathways such as plant photosynthesis, seed development, substance transport, lipid synthesis, accumulation and degradation. Studies have shown that over 700 genes are involved in this process in arabidopsis thaliana. The oil content of the rape can be improved by over-expressing acetyl coenzyme A carboxylase ACCase genes, TAG synthesis pathway genes (LPAAT, DGAT1), transcription factors (LEC1, WRI1) and the like in a fatty acid synthesis pathway. In addition, the tall oil lettuce mutants can also be created by physicochemical mutagenesis technology, RNAi technology, T-DNA insertion method, transposon tagging method, CRISPR Cas9 technology and the like. At present, the oil content gene of rape is determined and regulated by referring to the oil metabolism approach of arabidopsis thaliana, so that the development of high oil content materials is the molecular basis of rape high oil breeding, and the novel materials created by the method are widely applied to the rape high oil breeding.

The invention clones an S-adenosine-L-methionine dependent methyltransferase gene BnPMT6 (the gene is not reported to participate in seed oil synthesis) from rape. Research shows that the BnPMT6 gene is over-expressed in Arabidopsis thaliana or rape seeds under the action of a seed specific expression promoter Napin, so that the oil content of the seeds can be obviously reduced. And the oil content of the BnPTM 6 mutant rape seeds created by the CRISPR/Cas9 technology is obviously increased. In addition, the oleic acid content in both the overexpressed and mutant canola seeds was significantly different from that of the corresponding Wild Type (WT). The result suggests that the BnPMT6 gene plays an important role in regulating and controlling the oil content and the fatty acid composition of seeds, and can provide a new theory and a new gene resource for rape high-oil breeding and oil quality improvement.

Disclosure of Invention

The invention aims to provide a rape oil content related S-adenosine-L-methionine dependent methyltransferase gene, which codes S-adenosine-L-methionine dependent methyltransferase, wherein each chromosome of rape A5 and C5 has a copy, namely BnaA05.PMT6(BnaA05g28570D) and BnaC05.PMT6(BnaC05g42890D), and the nucleotide sequences of the gene are respectively shown as a sequence table SEQ ID NO. 1 and a sequence table SEQ ID NO. 2 and consist of 1755bp and 1758 bp; the protein sequences of the gene codes are respectively shown in sequence tables SEQ ID NO. 3 and SEQ ID NO. 4, and 584 amino acids and 585 amino acids are respectively coded. The gene of the invention and any polynucleotide of interest or a polynucleotide homologous thereto can be amplified from genomic, mRNA and cDNA using PCR techniques.

Another object of the present invention is to construct a recombinant plant expression vector comprising a S-adenosyl-L-methionine-dependent methyltransferase gene Bnac05.PMT6 by adding a seed-specific promoter Napin to the transcription initiation nucleotide, and to add an antibiotic marker to the expression vector for screening transgenic plants.

The invention provides a method for obtaining crops with high oil content, which comprises the following steps: a CRISPR/Cas9 gene editing vector containing rape S-adenosine-L-methionine dependent methyltransferase gene BnPMT6 is transformed into the genome of crops by using an agrobacterium-mediated genetic transformation method, and a crop variety with the gene BnPMT6 function deletion is obtained by using a CRISPR/Cas9 gene editing technology.

One feature of the invention is that the oil content of the transformed material is analyzed by obtaining over-expressed and mutant material, and the accumulation of the oil content negatively regulated by BnPMT6 is found, while also regulating the fatty acid composition.

The expression vector of the present invention refers to any vector known in the art capable of expression in plants, for example, suitable for constructing the expression vector of the present invention include, but are not limited to, such as: PBILOxP-Napin (supplied by the institute of oil crops, academy of agricultural sciences, China), PHSE401 (supplied by Chen's army group, university of agriculture, China), and the like.

The high oil content mutant rape obtained by the invention has no obvious difference with normal plants in the vegetative growth and reproductive growth periods, and the genetic resource has potential application value in rape high oil breeding.

Drawings

FIG. 1: and (3) detecting the result of agarose gel electrophoresis of the BnPMT6 gene amplification product.

FIG. 2: plasmid map of the constructed plant expression vector Napin-PBILOxP-BnaC05. PMT6.

FIG. 3: obtaining and identifying rape BnPT 6 mutant. sgRNA is located in the second exon region of genes bnaa05.pmt6 and bnac05.pmt6, and the editing of the target sites of the 5 mutants obtained: l1, L20 and L21 are double-process in which bnaa05.pmt6 and bnac05.pmt6 are edited, L4 is single-process in which bnac05.pmt6 is edited, and L7 is single-process in which bnaa05.pmt6 is edited.

FIG. 4: and (3) identifying the oil content phenotype of the Arabidopsis transformed by the BnaC05 PMT6 gene. (a) Oil content analysis of Arabidopsis thaliana material WT and transgenic lines (OE1, OE 4); (b) analysis of fatty acid composition of Arabidopsis thaliana material WT and transgenic lines. 6 independent lines were used for each material. Denotes P <0.05 in Student's t test, denotes P <0.01 in Student's t test.

FIG. 5: BnPT 6 was identified in the phenotype of oilseed rape. The oil content of rape seeds is analyzed by near infrared, and L1, L20 and L21 are CRISPR double mutants of BnaA05.PMT6 and BnaC05.PMT6, L4 is a CRISPR mutant of BnaC05.PMT6, and L7 is a CRISPR mutant of BnaA05. PMT6. OE5, OE6 and OE15 are overexpression strains. Each strain had 6-8 different individuals, and represent P <0.01 and P <0.05 in Student's t test, respectively.

Detailed Description

The invention is further defined by reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as the Instrument book molecular cloning: conditions as described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory,1989), or according to the procedures suggested in the manufacturer's manual.

EXAMPLE 1 cloning of BnPT 6 Gene

The PMT6 gene encodes an S-adenosyl-L-methionine-dependent methyltransferase, with one copy on each of the chromosomes rape A5 and C5, and was designated BnaA05.PMT6(BnaA05g28570D) and BnaC05.PMT6(BnaC05g42890D), respectively. CDS sequences are shown in sequence tables SEQ ID NO:1 and SEQ ID NO:2 respectively, contain complete ORF reading frame and initiation codon ATG, respectively encode 584 and 585 amino acids (shown in sequence tables SEQ ID NO:3 and SEQ ID NO:4 respectively), and the homology of the 2 copy proteins is 94% through BLAST comparison, and the similarity is very high. Randomly selecting gene BnaC05.PMT6 for cloning.

(1) Extraction of RNA

Extracting total RNA by adopting TansZol (catalog number ET101) of the general gold company, grinding and crushing cabbage type rape leaves in liquid nitrogen, transferring 100mg of ground sample into a 1.5mL centrifuge tube, adding 1mL of TransZol, violently reversing the mixture for several times to fully mix the mixture, and standing the mixture for 5 minutes at room temperature; adding 0.2mL of chloroform, shaking vigorously for 15 seconds, and incubating at room temperature for 3 minutes; centrifuging at 10000xg and 4 ℃ for 15 minutes; transferring the colorless water phase into a new centrifuge tube, adding 0.5mL of isopropanol, reversing, uniformly mixing, and incubating for 10 minutes at room temperature; centrifuging at 10000xg 4 ℃ for 10 minutes, and removing a supernatant; add 1mL of 75% ethanol (made up in DEPC-treated water) and vortex vigorously; 7500Xg centrifuging at 4 deg.C for 5 min; discarding the supernatant, and airing and precipitating at room temperature; dissolving the precipitate in 50-100 μ l RNA dissolving solution; incubate at 55 ℃ for 10 min. Taking l of total extracted RNA to determine the RNA concentration under the Nanodrop, and identifying the RNA purity according to the conditions that OD260 is more than 1.8 and OD280 is less than 2.0. Mu.l of the suspension was subjected to 1% agarose electrophoresis and the integrity was checked.

(2) Synthesis of cDNA

The reverse transcription uses all-type gold

One-Step gDNA Removal and cDNA Synthesis SuperMix (catalog No. AE 311). Mu.l of adsorbed Oligo (dT)18Primer and 10. mu.l of 2 × ES Reaction Mix were sequentially added to 5. mu.g of total RNA as a template,

RT/RI Enzyme Mix 1. mu.l, gDNA Remover 1. mu.l, RNase-free Water supplemented to 20. mu.l. The above system was gently mixed and placed at 42 ℃ for 30min, this step was the first strand cDNA synthesis and gDNA removal. Heating at 85 deg.C for 5s to deactivate

RT/RI and gDNA Remover. The synthesized cDNA was dissolved by adding 180. mu.l of RNase-free Water for use.

(3) Amplification of BnPT 6 Gene

The cDNA was used as a template, and the sequence of the forward primer PMT6-F1 was 5'-GCGcccgggACCATGGCCAGAGGTTACAACGTGTT-3', and the sequence of the reverse primer PMT6-R1 was 5 '-GCGggcgcgccTCATTTGTCGTCGTCGTCCTTGTAGT-CCATG-ATGATTGCCCAGAAT-3', and a fragment containing BnaC05.PMT6 full-length CDS was amplified. By using I-5 ^TM 2 × High-Fidelity Master Mix (TSINGKE Biologica technology) was subjected to PCR amplification. The PCR amplification system is as follows:

PCR amplification procedure: total denaturation at 98 deg.C for 1 min; denaturation at 98 ℃ for 15sec, annealing at 55 ℃ for 15sec, extension at 72 ℃ for 30sec, 34 circles; total extension at 72 ℃ for 5 min.

The amplified product is detected by agarose gel electrophoresis (figure 1), 1758bp BnaC05.PMT6 full length is obtained by amplification, and a Tiangen agarose gel recovery kit (http:// www.tiangen.com /) is used for gel digging and recovering the product.

Example 2 construction of BnPT 6 Gene overexpression transformation vector

(1) Carrying out double digestion on the BnaC05.PMT6 fragment obtained by the method by using quick restriction enzymes XmaI and ASCI, wherein the double digestion system is as follows:

the enzyme digestion reaction was carried out in a 37 ℃ water bath for 1 hour. The digested product was recovered with the DNA purification kit from Tiangen.

(2) The enzyme digestion product is connected to a vector PBILOxP, the vector contains a Napin promoter of a seed specific expression gene and an antibiotic marker, and the specific references include: the BnGRF2 gene (GRF2-like gene from Brassica napus) enhanced oil production through regulating cell number and plant photosynthesis.

The connection method comprises the following steps:

(3) transforming Escherichia coli DH5 alpha, screening positive clones, carrying out quality-improving cutting identification, selecting 3 positive clones, sending samples and sequencing, and analyzing results show that the CDS sequence of BnaC05.PMT6 gene is successfully connected with a vector, namely, the plant expression vector Napin-PBILOxP-BnaC05.PMT6 of the transformed plant is successfully constructed (as shown in figure 2).

(4) Introducing the correctly constructed recombinant plasmid vector into agrobacterium strain GV3101, and selecting positive monoclone for preservation in a refrigerator at-80 deg.c. The introduction method is as follows:

a. cleaning the electric revolving cup: washing with pure water, washing with ultrapure water, pouring off, washing with anhydrous ethanol (blowing with 1ml gun head), pouring off anhydrous ethanol, and air drying in a super clean bench;

b. taking the agrobacterium-infected GV 310120 μ l;

c. taking 0.8 mu l of correctly constructed recombinant plasmid, adding the correctly constructed recombinant plasmid into 20 mu l of competence, and slightly sucking, beating and uniformly mixing to avoid generating bubbles;

d. placing the washed and dried electric rotating cup in ice for precooling, and then driving the mixed liquid into the electric rotating cup by the cup wall;

e. adjusting the electric transfer instrument to 1800V;

f. taking the electric rotating cup out of the ice, and wiping the outer wall of the electric rotating cup by using absorbent paper;

g. putting the electric rotating cup into the instrument, continuously pressing a push key for two times, and hearing the sound of dropping after a few seconds to succeed;

h. after the electric shock succeeds, 400 mul of nonreactive LB is added into the electric rotating cup, sucked and beaten for a few times, and then transferred into a sterile centrifuge tube;

activating at i.28 deg.C for about 2h, applying 100 μ l of the extract to a culture container containing double antibodies (gentamicin and kanamycin), sealing with sealing film, culturing in 28 deg.C incubator for 2 days, and detecting by picking spots.

(5) Agrobacterium colony detection

Selecting bacterial colony in double-antibody (gentamicin and kanamycin) LB, culturing for 2 hours at 28 ℃, taking proper amount of bacterial liquid for PCR detection, and preserving positive agrobacterium liquid.

Example 3 construction of BnPT 6-CRISPR vector

The sgRNA-Cas9 system of Chen army team of university of agriculture university biological school of China was used to create the rape BnPTM 6 mutant. The experimental procedure was as follows:

(1) and logging in a website http:// www.genome.arizona.edu/crispr search. html, and screening the target point. The target sequence is 5'-actagtcatacccgaaact-3', located in the second exon region of the gene.

(2) Design of primers

DT1-BsF:ATATATGGTCTCGATTGactagtcatacccgaaactGTT

DT1-F0:TGactagtcatacccgaaactGTTTTAGAGCTAGAAATAGC

DT2-R0:AACagtttcgggtatgactagtCAATCTCTTAGTCGACTCTAC

DT2-BsR:ATTATTGGTCTCGAAACagtttcgggtatgactagtCAA

(3) And (3) PCR amplification: four-primer PCR amplification was performed using 100-fold diluted pCBC-DT1T2 as a template. DT 1-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 deg.C for 1 min; denaturation at 98 ℃ for 15sec, annealing at 56 ℃ for 25sec, extension at 72 ℃ for 25sec, 34 circles; total extension at 72 ℃ for 5 min.

(4) Purifying and recovering PCR products, and establishing an enzyme digestion-ligation system (restriction-ligation):

reaction conditions are as follows: 5hours at 37 deg.C, 5min at 50 deg.C, 10min at 80 deg.C

(5) Transforming and transforming Escherichia coli DH5 alpha, taking 5 mu L of transformed Escherichia coli competence, and screening by Kan plate. Screening positive clones, carrying out PCR identification on the colony with 726bp of U626-IDF + U629-IDR, sequencing U626-IDF and U629-IDF, and obtaining a vector with correct sequencing, namely the CRISPR vector of BnPT 6.

(6) The correctly constructed recombinant plasmid vector was introduced into Agrobacterium strain GV3101, and positive single clones were selected and stored in a refrigerator at-80 ℃ and the transformation method was the same as that shown in example 2.

Example 4 genetic transformation experiments

(1) Genetic transformation of Arabidopsis and herbicide screening

The constructed BnPMT6 overexpression vector is used for transforming Arabidopsis. Sowing mature Arabidopsis (ecological Columbia) seeds in a greenhouse, planting at 22 ℃ under 16h of illumination/8 h of darkness until buds appear, pruning for about one week to generate buds, removing the bloomed flowers and preparing for infection. Inoculating the Agrobacterium strain stored at-80 deg.C into 50ml liquid LB, culturing at 28 deg.C 180r/min, shaking to OD ₆₀₀ Centrifuging at 4000r/min to collect thallus 0.3-0.5 (about 12-14h), adding an equal volume of suspension (MS basic culture medium + 5% sucrose + 0.05% Silwet-77, pH 5.8) for heavy suspension, infecting arabidopsis inflorescence with the heavy suspension, and performing dark treatment for 24 h; the infection was repeated once a week later. And harvesting mature T0 generation seeds, and airing for later use.

The harvested T0 generation Arabidopsis seeds are uniformly sown in moist soil and placed at 4 ℃ for 2 days; moving to a growth chamber under 16h illumination/8 h dark condition at 22 ℃; on day 10, 120mg/L Glufosinate Ammonium solution (Glufosinate Ammonium, CAS:77182-82-2) (0.05% Silwet-77) was sprayed; on day 12, spraying for the second time; on the 15 th day, spraying for the third time, wherein the non-transgenic seedlings are yellow and killed; on day 17, the last spray; and carrying out normal growth management on the screened seedlings, and harvesting the seeds after the seedlings are mature.

(2) Genetic transformation of oilseed rape

The constructed BnPMT6 overexpression vector and CRISPR vector are subjected to rape genetic transformation, an agrobacterium-mediated genetic transformation mode is used, and the receptor for rape transformation is Brassica napus Westar.

The transformation procedure was as follows: soaking mature and plump Brassica napus seeds 'Westar' in 75% alcohol for 1min, sterilizing with 0.15% mercuric chloride solution for 12min, and sterilizing with sterile ddH ₂ O cleaning for 5-6 times; sowing the sterilized seeds in a culture medium with the number of M0, and culturing for 6-7 days in a dark room at the temperature of 22 ℃; the transformed Agrobacterium of examples 2 and 3 were picked, inoculated into 50mL of liquid LB, cultured at 28 ℃ for 180r/min to OD ₆₀₀ Centrifuging at 4000r/min to collect thallus 0.3-0.5 (12-14 h), suspending with suspension DM, culturing at 28 deg.C at 200r/min for 1 hr, and pouring into culture dish; cutting seedling hypocotyls with sterile forceps and scalpel, each length being 0.8-1.0cm (cut off vertically as soon as possible when cutting explants); placing the cut explant in a dish containing a bacterial solution, dip-dyeing for 10min, and shaking once every one moment; sucking the infected explant by using sterile filter paper, transferring the explant into a culture medium with the number of M1, and carrying out dark culture at the temperature of 22 ℃ for 36-48h until cloudy bacterial colonies appear around the explant; transferring the hypocotyl explant after co-culture to an M2 culture medium for inducing callus, wherein the culture medium contains timentin (TMT) for inhibiting the growth of agrobacterium tumefaciens and screening antibiotics, and culturing for 21 days under illumination; eliminating some dead brown explants, transferring the explants with normal growth and two expanded ends to a differentiation medium M3 for illumination culture, and carrying out subculture once every 2-3 weeks until green buds appear; transferring the differentiated green buds into an M4 culture medium, transplanting the green buds into a field after rooting, and observing the growth phenotype of the plants in the field (the formula of the culture medium is shown in table 1).

TABLE 1 culture Medium recipe and numbering used in the genetic transformation procedure for Brassica napus

Description of table 1: MS is collectively referred to as Murashige and Skoog Stock.

(3) Identification of overexpression of transformed Individual

Extracting the genome DNA of the obtained over-expressed transformation individual strains of arabidopsis thaliana and rape, detecting the insertion of a foreign gene fragment by PCR, wherein an over-expressed skeleton vector is Napin-PBILOxP, a primer Napin-F (5 '-GCGTGCAT-GCATTATTACAC-3') is designed on the skeleton vector, PCR is carried out by matching the vector skeleton primer and the foreign fragment primer (the sequences of Napin-F and PMT6-R1 and PMT6-R1 are shown in example 1), and the transgenic seedling is detected at the PCR level. The PCR system is as follows: easy taq polymerase 0.15 μ L,10Mm dNTP 0.4 μ L,10 XBuffer 2 μ L, DNA template 2 μ L, Napin-F2 μ L, primer PMT 6-R12 μ L, and complement ddH ₂ O to 20. mu.L. PCR conditions were as follows: total denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 30sec, 34 circles; total extension at 72 ℃ for 5 min.

And (3) carrying out qRT-PCR on the transgenic positive seedlings of the arabidopsis thaliana and the rape obtained by the PCR to detect the gene expression quantity. RNA of the transformed individual seed was extracted and cDNA was synthesized (same procedure as in example 1), quantitative primers were designed using Primer 3 software, the size of the product was 100bp to 200bp, and BLAST alignment was performed using reference sequences after design to ensure the specificity of primers RT-PMT6-F1 (5'-GTGGTTCCCTGGTGGTGGTACT-3') and RT-PMT6-R1(5 '-T-CTCGAACAATGAACCATCTCAAA-3'). Tub4-1(5 '-AGAGGTTGACGAGCAGA-TGA) and tub4-2 (5'-CCTCTTCTTCCTCCTCGTAC-3') as reference genes for Arabidopsis thaliana (see Marks et al 1987: The relative large beta-tubulin gene family of Arabidopsis thaliana with an unused 5' mutation sequence), BnACT 7-L (5 '-CGCCTA-GCAGCATGAA-3') and BnACT N7-R (5'-GTTGGAAAGTGCTGAGAGATGCA-3') as reference primers for Brassica napus-RT-PCR (see Zhou et al 2012: BnMs3 is required for gene differentiation and differentiation, microspSeparration, and lens-biosynthesis in branched Brassica napus branched genes). The reaction system is as follows:

reaction procedure: 30s at 94 ℃; 94 ℃ for 10s, 60 ℃ for 15s, 72 ℃ for 30s, 45 cycles; and (5) drawing a dissolution curve. qRT-PCR was performed in the Bio-Rad CFX96 Real-Time System.

The quantification of the variation between the different replicates was calculated by the delta-delta threshold cycle relative quantification (2-. DELTA.CT) method, using internal reference primers for normalization. Finally, arabidopsis thaliana over-expression transformation individuals OE1 and OE4 and rape over-expression transformation individuals OE5, OE6 and OE15 are obtained through analysis.

(4) Identification of CRISPR transformed Individual

Sequencing the obtained CRISPR transformed individual rape to screen rape mutants. Firstly, primers Cas9-570-F (5'-AGACCGTGAAGGTTGTGGAC-3') and Cas9-570-R (5 '-TAGTGATCTGCCGTGTCTC-G-3') are used for identifying Cas9 protein, and a Cas9 protein positive single strain is subjected to specific amplification of a target gene and sequencing identification. The specific amplification method of the target gene comprises the following steps: BnaA05.PMT6 was specifically amplified with primers PMT6-AC5-F (5'-atgagaggttacaacgtgttc-3') and PMT6-A5-R (5'-ctatactatagcccctaaaata-3'), respectively; BnaC05.PMT6 was specifically amplified by PMT6-AC5-F (5'-atgagaggttacaacgtgttc-3') and PMT6-C5-R (5'-aaattaaagatccggccctgtg-3'). The amplification method was the same as that described in example 4 (3).

And (3) carrying out PCR product sequencing on the amplified target fragment, and analyzing the editing condition of the target site by using a DSDecode online website (http:// skl. scau. edu. cn/dsDecode /). Sequencing results showed that multiple independent mutant strains with edited BnPMT6 were obtained (L1, L4, L7, L20, and L21). Wherein L1, L20 and L21 are double-process in which bnaa05.pmt6 and bnac05.pmt6 are edited simultaneously, the editing causing premature termination of the gene; l4 is a single mutation where only bnac05.pmt6 was edited, causing premature termination of the C5 copy; l7 is a single mutation in bnaa05.pmt6 edited, causing premature termination of the a5 copy (fig. 3).

(5) Oil content analysis of transformed plants

Firstly, the oil content of arabidopsis is measured by adopting a gas chromatography-flame ionization detector method

Weighing 5-10mg of arabidopsis seeds and placing the seeds in a fat extraction tube; adding 2ml of 5% sulfuric acid extract (methanol, containing 0.01% BHT); heptadecanoic acid (17:0, moleculars) was added at 16.2. mu. mol/mlAmount 270.45) 25. mu.l, and cover tightly; water bath at 85 ℃, screwing the cover again after 10 minutes, and continuing the water bath for 2 hours; after cooling, 2.0ml of H2O and 2.0ml of n-hexane are added, and vortex mixing is carried out; centrifuging at 1000rpm for 5 minutes; 1.0ml of the supernatant was taken to a sample vial. The organic reagents used in the above fatty acid extraction process are of chromatographic grade. The oil content and fatty acid content were determined by Gas chromatography-flame ionization detector (GC-FID) equipped with hydrogen flame ionization detector and capillary RESTEK

Wax column (0.25 mm. times.30 m) with a helium carrier of 20 ml/min. The process parameters are described as follows: sample injection is carried out for 1 mul, and the split ratio is 20: 1. The column box temperature was maintained at 170 ℃ for 1min and then gradually increased to 210 ℃ at a rate of 3 ℃/min. Finally, the fatty acid species are identified according to the retention time, and the peak area is calculated. Fatty acids are calculated as nmol%. And measuring the oil content of the seeds by taking heptadecanoic acid (17:0) as an internal standard substance, and finally calculating the percentage of the oil content in dry weight.

The oil content results show a significant 1.2-5.4 percentage point reduction in oil content for the T1, T2, and T3 generation OE materials compared to WT (35.88 ± 2.05) (fig. 4 a). The fatty acid composition results show that the C18:1 and C18:2 content of the OE material was increased, while the C18:3 content was decreased compared to WT (fig. 4 b). The results indicate that BnPMT6 does play a role in regulating seed oil content and fatty acid composition.

② adopting near-infrared analyzer to measure oil content of rape seeds

The quality of the rape seeds harvested in the mature period is analyzed by utilizing a near infrared analyzer to obtain the oil content data of the seeds, and a measuring instrument is provided by the national rape engineering technical research center of China university of agriculture.

The oil content results show that the oil content of the acceptor background material Westar is 41.05 + -0.33, the oil content of 3 overexpression lines OE5, OE6 and OE15 is 39.21 + -0.74, 38.49 + -1.21 and 39.76 + -1.14 respectively, and is significantly reduced by 1.29-2.56 percentage points. The oil contents of mutant materials L1, L4, L7, L20 and L21 were 43.66 ± 0.96, 42.07 ± 0.55, 42.33 ± 0.78, 43.89 ± 0.34 and 44.01 ± 0.68, respectively, and it is not difficult to see that not only the oil contents of the double mutants (L1, L20 and L21) were significantly increased by 2.6-3.0 percentage points, but also the oil contents of the single mutants of bna05. pmt6 (L7) and bnac05.pmt6 (L4) were significantly increased by 1 percentage point (fig. 5).

In conclusion, the gene BnPMT6 plays an important role in regulating the oil content of rape.

Sequence listing

<110> university of agriculture in Huazhong

<120> rape S-adenosine-L-methionine dependent methyltransferase gene BnPMT6 and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1755

<212> DNA

<213> Brassica napus (Brassica napus L.)

<400> 1

atgagaggtt acaacgtgtt ccgtgcagcg agatcgggaa agacgatact agtagctctc 60

tttcttacgg ttggatcatt ttacgctggc tctctcttcg gcaacaacga acccatctac 120

gtctctcaat ctgctctctt caaattccca aacaaaatca acctcactca ccgagtctca 180

ccactagtca tacccgaaac tgggatgaat gtgtgtccct caaagttcaa cgagtacctc 240

ccttgtcaca acgtcagtta cgtgcaccag ctgagcttga acgtctctag aagagaagag 300

ctcgagagac actgcccgcc gctcgagcag cgtctcttct gcttggtgcc tccaccaaaa 360

gattacaaga tacctttgag atggccaacc agtagagact acgtgtggag aagcaatgtg 420

aatcatacgc atctcgctca agttaacggt ggacaaagct gggtgcagga gcatggagag 480

ttctggtggt tccctggtgg tggtactcat ttcaaacatg gtgcttcaga atacatacaa 540

aggttgggag gtatggtgac taatgaaaca ggtgacttgc gttcagccgg ggtggtacaa 600

gtacttgatg ttggatgtgg agttgcaagc tttgcggctt atctacttcc tctaggtata 660

cagacaatgt cctttgctcc taatgatgct aatgagaatc agattcagtt tgcattggag 720

agaggcgtca gtgcaatgat ctctgctgtt gccaccaaac aactgccata tccttcatct 780

tcttttgaga tggttcattg ttcgagatgt cgtgttgatt ggcatgcaaa cggtggcatc 840

ttacttaaag aagttcatag gcttcttaga cctaatggct actttgtgta tacgtcgcca 900

ccagcttata ggaatgataa agagtatcct atgatttggg ataagttggt tagtctagct 960

aactcaatgt gctggaagct tgtttcccgg aaggtacaaa ctgcaatatg gattaaagaa 1020

gaaaacgtgg agtgccttaa gaagaatgca gagctaaagg ttataagctt atgtgatgtg 1080

gaagatgctt tgaaaccgtc ctggcaagtt cctcttagag attgtgtgca aattagtgga 1140

gatacagaga tgagatcttc ttctttagct gaacgtctct ccaagtatcc agaaactctg 1200

agaaacaaag gtattagtga agatgaatat acatcagata ctgtcttctg gagagaacaa 1260

gttaaacagt attggcggtt tatgagtatt aatgagactg aagtgcggaa tgtgatggat 1320

atgaatgcat tcattggtgg atttgcttca gccatggact cgtatcctgt ttgggtgatg 1380

aacatagtac ctgctaccat gaatgaaact ttgtccggtg tttttgagag gggtttaact 1440

ggtgctttcc atgattggtg tgagccgttc tcaacatatc cgcggacgta tgatttgctg 1500

catgccaatc atgtaatctc tcactaccaa agccgtggag atggttgctt ggtagaggat 1560

atcatgcttg agatggaccg gatgattcgc cctcagggat tcatcattat aagggacgag 1620

gaacaggtaa tctcaagaat ccgagacttg gctcctaagt acctctggga agtggaaact 1680

catcagctgg aaaacaaatt caagaagata gaaactgttc tgttttgcag aaagagattc 1740

tgggcaatca tctga 1755

<210> 2

<211> 1758

<212> DNA

<213> Brassica napus (Brassica napus L.)

<400> 2

atgagaggtt acaacgtgtt cggtgcaacg agatcgggga agacgatact agttgctctc 60

tttctaacgg ttggatcatt ctacgctggc tctcttttcg gcaacaacga acccatctac 120

gtctcacaat ctgctctctt caaattccca aacaaaatca accttactca ccgagtctca 180

ccactagtca tacccgaaac tgggatgaat gtgtgtccct taaagttcaa cgagtatctc 240

ccttgtcaca acgtcactta cgttcaccag ctgagcttga acgtctctag aagagaagaa 300

ctcgagagac actgcccgcc gctcgagcag cgtctcttct gcttggtgcc tccaccaaaa 360

gattacaaga tacctttgag atggccaacc agtagagact acgtgtggcg aagcaatgta 420

aatcatacgc atcttgctca agttaacggt ggacaaagct gggtgcaaga acatggagag 480

ttttggtggt tccctggtgg tggtactcat ttcaaacatg gtgcttcaga atacatacag 540

aggttgggag gtatggtgac taatgaaaca ggtgacttgc gttcagctgg ggtggtacaa 600

gttcttgacg ttggatgtgg agttgcaagc tttgcggctt accttcttcc tttaggtata 660

cagacaatgt cctttgctcc taatgatgct catgagaatc agattcagtt tgcattggag 720

agaggcatcg gcggtgcaat gatctctgct gttgccacca aacaactgcc gtatccctca 780

gcttcttttg agatggttca ttgttcgaga tgccgtgttg attggcatgc aaacgatggg 840

atcttactta aagaagttca taggcttctt agacccaatg gatactttgt gtatacgtcg 900

ccaccagctt ataggaatga taaagagtat ccaatgattt gggataagtt ggttagtcta 960

actaactcaa tgtgctggaa gcttgtttcc cggaaggtac aaactgctat atggattaaa 1020

gaagagaacg tggagtgcct taagaaaaag gcagagctaa aggttataag cttatgtgat 1080

gtggaagatg ctttgaaacc gtcctggcaa gttcctttta gagattgtgt gcagattagt 1140

ggacatacag agaagagacc ctcttcttta gctgaacgtc tttccacata tccagaaact 1200

ctaagaaaca taggcatcag tgaagatgag tatgcatcag acacagtctt ctggagagaa 1260

caagttaaac agtactggcg gtttatgaat gtcaatgaga gtgaagtgcg gaatgtgatg 1320

gacatgaatg catttattgg tggatttgct tcggccatga actcgtcccc tgtttgggta 1380

atgaacgtag tacctgctac catgaatgaa actttgtctg gagtttttga gaggggctta 1440

actggtgctt tccatgattg gtgtgagccg ttctcaacat atccgcggac gtatgatttg 1500

ttgcacgcca atcatgtcat ctctcattac caaagccgtg gagatggttg tttggtagag 1560

gatatcatgc ttgagatgga ccggatgatt cgccctcagg gattcatcat tataagggac 1620

gaggaaccta taatctcaag gatccgagac ttggctccta agtacctctg ggaagtggag 1680

actcatcagc tggaaaacaa attcaagaag acagaaaccg ttctgttttg cagaaagaga 1740

ttctgggcaa tcatctga 1758

<210> 3

<211> 584

<212> PRT

<213> Brassica napus (Brassica napus L.)

<400> 3

Met Arg Gly Tyr Asn Val Phe Arg Ala Ala Arg Ser Gly Lys Thr Ile

1 5 10 15

Leu Val Ala Leu Phe Leu Thr Val Gly Ser Phe Tyr Ala Gly Ser Leu

20 25 30

Phe Gly Asn Asn Glu Pro Ile Tyr Val Ser Gln Ser Ala Leu Phe Lys

35 40 45

Phe Pro Asn Lys Ile Asn Leu Thr His Arg Val Ser Pro Leu Val Ile

50 55 60

Pro Glu Thr Gly Met Asn Val Cys Pro Ser Lys Phe Asn Glu Tyr Leu

65 70 75 80

Pro Cys His Asn Val Ser Tyr Val His Gln Leu Ser Leu Asn Val Ser

85 90 95

Arg Arg Glu Glu Leu Glu Arg His Cys Pro Pro Leu Glu Gln Arg Leu

100 105 110

Phe Cys Leu Val Pro Pro Pro Lys Asp Tyr Lys Ile Pro Leu Arg Trp

115 120 125

Pro Thr Ser Arg Asp Tyr Val Trp Arg Ser Asn Val Asn His Thr His

130 135 140

Leu Ala Gln Val Asn Gly Gly Gln Ser Trp Val Gln Glu His Gly Glu

145 150 155 160

Phe Trp Trp Phe Pro Gly Gly Gly Thr His Phe Lys His Gly Ala Ser

165 170 175

Glu Tyr Ile Gln Arg Leu Gly Gly Met Val Thr Asn Glu Thr Gly Asp

180 185 190

Leu Arg Ser Ala Gly Val Val Gln Val Leu Asp Val Gly Cys Gly Val

195 200 205

Ala Ser Phe Ala Ala Tyr Leu Leu Pro Leu Gly Ile Gln Thr Met Ser

210 215 220

Phe Ala Pro Asn Asp Ala Asn Glu Asn Gln Ile Gln Phe Ala Leu Glu

225 230 235 240

Arg Gly Val Ser Ala Met Ile Ser Ala Val Ala Thr Lys Gln Leu Pro

245 250 255

Tyr Pro Ser Ser Ser Phe Glu Met Val His Cys Ser Arg Cys Arg Val

260 265 270

Asp Trp His Ala Asn Gly Gly Ile Leu Leu Lys Glu Val His Arg Leu

275 280 285

Leu Arg Pro Asn Gly Tyr Phe Val Tyr Thr Ser Pro Pro Ala Tyr Arg

290 295 300

Asn Asp Lys Glu Tyr Pro Met Ile Trp Asp Lys Leu Val Ser Leu Ala

305 310 315 320

Asn Ser Met Cys Trp Lys Leu Val Ser Arg Lys Val Gln Thr Ala Ile

325 330 335

Trp Ile Lys Glu Glu Asn Val Glu Cys Leu Lys Lys Asn Ala Glu Leu

340 345 350

Lys Val Ile Ser Leu Cys Asp Val Glu Asp Ala Leu Lys Pro Ser Trp

355 360 365

Gln Val Pro Leu Arg Asp Cys Val Gln Ile Ser Gly Asp Thr Glu Met

370 375 380

Arg Ser Ser Ser Leu Ala Glu Arg Leu Ser Lys Tyr Pro Glu Thr Leu

385 390 395 400

Arg Asn Lys Gly Ile Ser Glu Asp Glu Tyr Thr Ser Asp Thr Val Phe

405 410 415

Trp Arg Glu Gln Val Lys Gln Tyr Trp Arg Phe Met Ser Ile Asn Glu

420 425 430

Thr Glu Val Arg Asn Val Met Asp Met Asn Ala Phe Ile Gly Gly Phe

435 440 445

Ala Ser Ala Met Asp Ser Tyr Pro Val Trp Val Met Asn Ile Val Pro

450 455 460

Ala Thr Met Asn Glu Thr Leu Ser Gly Val Phe Glu Arg Gly Leu Thr

465 470 475 480

Gly Ala Phe His Asp Trp Cys Glu Pro Phe Ser Thr Tyr Pro Arg Thr

485 490 495

Tyr Asp Leu Leu His Ala Asn His Val Ile Ser His Tyr Gln Ser Arg

500 505 510

Gly Asp Gly Cys Leu Val Glu Asp Ile Met Leu Glu Met Asp Arg Met

515 520 525

Ile Arg Pro Gln Gly Phe Ile Ile Ile Arg Asp Glu Glu Gln Val Ile

530 535 540

Ser Arg Ile Arg Asp Leu Ala Pro Lys Tyr Leu Trp Glu Val Glu Thr

545 550 555 560

His Gln Leu Glu Asn Lys Phe Lys Lys Ile Glu Thr Val Leu Phe Cys

565 570 575

Arg Lys Arg Phe Trp Ala Ile Ile

580

<210> 4

<211> 585

<212> PRT

<213> Brassica napus (Brassica napus L.)

<400> 4

Met Arg Gly Tyr Asn Val Phe Gly Ala Thr Arg Ser Gly Lys Thr Ile

1 5 10 15

Leu Val Ala Leu Phe Leu Thr Val Gly Ser Phe Tyr Ala Gly Ser Leu

20 25 30

Phe Gly Asn Asn Glu Pro Ile Tyr Val Ser Gln Ser Ala Leu Phe Lys

35 40 45

Phe Pro Asn Lys Ile Asn Leu Thr His Arg Val Ser Pro Leu Val Ile

50 55 60

Pro Glu Thr Gly Met Asn Val Cys Pro Leu Lys Phe Asn Glu Tyr Leu

65 70 75 80

Pro Cys His Asn Val Thr Tyr Val His Gln Leu Ser Leu Asn Val Ser

85 90 95

Arg Arg Glu Glu Leu Glu Arg His Cys Pro Pro Leu Glu Gln Arg Leu

100 105 110

Phe Cys Leu Val Pro Pro Pro Lys Asp Tyr Lys Ile Pro Leu Arg Trp

115 120 125

Pro Thr Ser Arg Asp Tyr Val Trp Arg Ser Asn Val Asn His Thr His

130 135 140

Leu Ala Gln Val Asn Gly Gly Gln Ser Trp Val Gln Glu His Gly Glu

145 150 155 160

Phe Trp Trp Phe Pro Gly Gly Gly Thr His Phe Lys His Gly Ala Ser

165 170 175

Glu Tyr Ile Gln Arg Leu Gly Gly Met Val Thr Asn Glu Thr Gly Asp

180 185 190

Leu Arg Ser Ala Gly Val Val Gln Val Leu Asp Val Gly Cys Gly Val

195 200 205

Ala Ser Phe Ala Ala Tyr Leu Leu Pro Leu Gly Ile Gln Thr Met Ser

210 215 220

Phe Ala Pro Asn Asp Ala His Glu Asn Gln Ile Gln Phe Ala Leu Glu

225 230 235 240

Arg Gly Ile Gly Gly Ala Met Ile Ser Ala Val Ala Thr Lys Gln Leu

245 250 255

Pro Tyr Pro Ser Ala Ser Phe Glu Met Val His Cys Ser Arg Cys Arg

260 265 270

Val Asp Trp His Ala Asn Asp Gly Ile Leu Leu Lys Glu Val His Arg

275 280 285

Leu Leu Arg Pro Asn Gly Tyr Phe Val Tyr Thr Ser Pro Pro Ala Tyr

290 295 300

Arg Asn Asp Lys Glu Tyr Pro Met Ile Trp Asp Lys Leu Val Ser Leu

305 310 315 320

Thr Asn Ser Met Cys Trp Lys Leu Val Ser Arg Lys Val Gln Thr Ala

325 330 335

Ile Trp Ile Lys Glu Glu Asn Val Glu Cys Leu Lys Lys Lys Ala Glu

340 345 350

Leu Lys Val Ile Ser Leu Cys Asp Val Glu Asp Ala Leu Lys Pro Ser

355 360 365

Trp Gln Val Pro Phe Arg Asp Cys Val Gln Ile Ser Gly His Thr Glu

370 375 380

Lys Arg Pro Ser Ser Leu Ala Glu Arg Leu Ser Thr Tyr Pro Glu Thr

385 390 395 400

Leu Arg Asn Ile Gly Ile Ser Glu Asp Glu Tyr Ala Ser Asp Thr Val

405 410 415

Phe Trp Arg Glu Gln Val Lys Gln Tyr Trp Arg Phe Met Asn Val Asn

420 425 430

Glu Ser Glu Val Arg Asn Val Met Asp Met Asn Ala Phe Ile Gly Gly

435 440 445

Phe Ala Ser Ala Met Asn Ser Ser Pro Val Trp Val Met Asn Val Val

450 455 460

Pro Ala Thr Met Asn Glu Thr Leu Ser Gly Val Phe Glu Arg Gly Leu

465 470 475 480

Thr Gly Ala Phe His Asp Trp Cys Glu Pro Phe Ser Thr Tyr Pro Arg

485 490 495

Thr Tyr Asp Leu Leu His Ala Asn His Val Ile Ser His Tyr Gln Ser

500 505 510

Arg Gly Asp Gly Cys Leu Val Glu Asp Ile Met Leu Glu Met Asp Arg

515 520 525

Met Ile Arg Pro Gln Gly Phe Ile Ile Ile Arg Asp Glu Glu Pro Ile

530 535 540

Ile Ser Arg Ile Arg Asp Leu Ala Pro Lys Tyr Leu Trp Glu Val Glu

545 550 555 560

Thr His Gln Leu Glu Asn Lys Phe Lys Lys Thr Glu Thr Val Leu Phe

565 570 575

Cys Arg Lys Arg Phe Trp Ala Ile Ile

580 585

Claims (2)

1. Rape S-adenosyl-L-methionine dependent methyltransferase geneBnPMT6The nucleotide sequence of the gene is shown as SEQ ID NO. 1 or SEQ ID NO. 2 in the application of regulating the oil content and fatty acid composition of the cabbage type rape.

2. A method for obtaining cabbage type rape with high oil content is characterized in that: using Agrobacterium-mediated genetic transformation method to contain rape S-adenosyl-L-methionine dependent methyltransferase geneBnPMT6The CRISPR/Cas9 gene editing vector is transformed into the genome of crops, and the CRISPR/Cas9 gene editing technology is utilized to obtain a Brassica napus variety with gene BnPMT6 function deletion, wherein the Brassica napus S-adenosine-L-methionine dependent methyltransferase geneBnPMT6The nucleotide sequence of the gene is shown as SEQ ID NO 1 or SEQ ID NO 2, and sgRNA is located in the geneBnPMT6The target sequence of the second exon region of (a) is 5'-actagtcatacccgaaact-3'.

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