CN110777164A

CN110777164A - Method for obtaining Brassica napus yellow seed germplasm based on CRISPR/Cas9 technology

Info

Publication number: CN110777164A
Application number: CN201911139555.3A
Authority: CN
Inventors: 谢涛; 王幼平; 蒋金金; 陈鑫; 戎浩; 郭图丽; 蒋波
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2019-11-20
Filing date: 2019-11-20
Publication date: 2020-02-11

Abstract

The invention belongs to the technical field of genetic engineering, and relates to a method for obtaining yellow-seed rape germplasm based on CRISPR/Cas9 technology, wherein a CRISPR/Cas9 system is used for carrying out gene editing on BnYB 123 gene of the cabbage type rape so as to obtain the yellow-seed cabbage type rape with BnYB 123 gene function deletion, the BnYB 123 has two copies in the cabbage type rape, the gene coding region sequences are shown as SEQ ID NO 1 and SEQ ID NO 2, and the sequence length is 786 bp. BnYB 123 can regulate the synthesis and accumulation of isorhamnetin and epicatechin, thereby regulating the seed coat color of the cabbage type rape. The invention provides a brand-new approach for the breeding work of the yellow-seed rape germplasm and provides a theoretical basis for the development of the cabbage type yellow-seed rape germplasm.

Description

Method for obtaining Brassica napus yellow seed germplasm based on CRISPR/Cas9 technology

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to a method for obtaining yellow seed germplasm of Brassica napus based on CRISPR/Cas9 technology, in particular to a method for carrying out gene editing on Brassica napus BnMYB123 by using a CRISPR/Cas9 system, so that the seed coat color of Brassica napus is changed, and the yellow seed germplasm of Brassica napus is obtained.

Background

Brassica napus (AACC), the second largest oil crop in the world after soybeans only, is a Brassica plant of the brassicaceae family and is widely planted all over the world. In view of the important economic status of rape and the demand of people for vegetable oil, how to improve the oil content of cabbage type rape and improve the quality of rapeseed oil and cake meal has become an important goal of rape breeding. Compared with the brown-seed cabbage type rape, the yellow-seed cabbage type rape has many excellent properties, such as thin seed coat, high oil content, high protein content, low cellulose and lignin content and the like.

The natural yellow seed materials are commonly found in cabbage type rapes, mustard type rapes and eruca carinata, natural yellow seed germplasm does not exist in the cabbage type rapes, the yellow seed materials of the cabbage type rapes mainly come from interspecific hybridization in brassica at present, and the yellow seed germplasm is very deficient.

The traditional crossbreeding makes great contribution to the quality improvement and yield improvement of Chinese rape, but has many restrictive factors, such as long breeding time, high labor intensity, capability of only hybridizing the species or species with close relativity, easy character separation after hybridization and the like, and the current plant genome engineering technology provides more and better choices for solving the problems. The CRISPR/Cas9 system is a gene editing technology newly developed in recent years, and has been widely applied to plants such as arabidopsis, rice, corn, wheat and the like at present due to the characteristics of high mutation efficiency, simple and convenient operation, low cost and the like. The technology not only provides a new thought for the function research of the gene, but also can be applied to the character improvement of the crops.

Disclosure of Invention

The invention aims to provide a method for knocking out a brassica napus BnYB 123 gene based on a CRISPR/Cas9 system so as to obtain a brassica napus yellow seed germplasm. By knocking out BnYB 123 gene, the accumulation of isorhamnetin and epicatechin substances in the brassica napus seed coat is inhibited, so that the content of procyanidine in the seed coat is reduced, the color of the seed coat is lightened, and the yellow seed phenotype is presented.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for obtaining yellow seed germplasm of Brassica napus based on CRISPR/Cas9 technology, which utilizes CRISPR/Cas9 technology to directionally edit Brassica napus BnYB 123 gene so as to obtain Brassica napus with function deletion of BnYB 123 gene, the method comprises the following steps:

selecting a DNA fragment ending with a base NGG as a target site in a brassica napus BnYB 123 exon region, wherein N represents any one of the bases A, G, C, T;

constructing a CRISPR/Cas9 recombinant vector for directional editing of BnYB 123 gene of the brassica napus by using the target site;

transforming the recombinant vector into agrobacterium tumefaciens GV3101 by an electrotransformation method;

transforming the cabbage type rape by the agrobacterium tumefaciens GV3101 containing the recombinant vector to obtain a transgenic regeneration plant;

amplifying and sequencing a DNA region of the BnYB 123 gene in the obtained transgenic regeneration plant, wherein the DNA region comprises the target site;

selecting a regeneration strain in which two copies and two alleles of BnYB 123 are edited, and carrying out phenotype observation to obtain a strain in which the seed coat is yellow.

Furthermore, the nucleotide sequence of the BnYB 123 gene coding region is SEQ ID NO.1 and SEQ ID NO. 2.

Further, one strand of the target site has a 5 '-A- (G) - (N) X-NGG-3' structure, wherein (N) X represents a base sequence of X in number, and N in (N) X represents any one of bases A, G, C, T.

Further, X is 19 or 20.

Further, the knockout target point of the rape yellow seed germplasm obtained in the step (1) is a target point 1, and the sequence of the target point is shown as SEQ ID NO. 3.

Further, the functional deletion is that the coding sequence of the BnYB 123 has large fragment deletion, frame shift mutation or terminator in the target site region.

Further, the method for constructing the CRISPR/Cas9 recombinant vector comprises the steps of designing and synthesizing a pair of single-stranded oligo DNA sequences according to a target site sequence, wherein the sequences are respectively shown as SEQ ID No.4 and SEQ ID No.5, obtaining a sgRNA expression cassette by a side-cut edge-connecting method, connecting the sgRNA expression cassette with a CRISPR-Cas9 vector, and then transforming escherichia coli Trans5 α to obtain the recombinant vector BnYB 123-Cas 9.

Further, the method for selecting a regeneration line in which two copies of BnYB 123 and two alleles are edited comprises the following steps: the two copies of the BnYB 123 gene are BnaA.MYB123 and BnaC.MYB123 respectively; the upstream and downstream nucleotide sequences of the identification primer of the chromosome A copy are shown as SEQ ID NO.8 and SEQ ID NO.9, and the upstream and downstream nucleotide sequences of the identification primer of the chromosome C copy are shown as SEQ ID NO.10 and SEQ ID NO. 9.

Furthermore, the transgenic positive plants are screened by the specific primers of the vector, and the upstream and downstream nucleotide sequences of the identified primers are shown as SEQ ID NO.6 and SEQ ID NO. 7.

The invention also provides application of the method for obtaining the yellow seed germplasm of the brassica napus based on the CRISPR/Cas9 technology in breeding.

The invention has the following beneficial effects:

(1) provides a target site of a CRISPR/Cas9 system capable of changing the seed coat color of Brassica napus, and the target site can edit the BnYB 123 gene at a fixed point; the yellow seed germplasm of the brassica napus is obtained through the CRISPR/Cas9 system, a brand new way is provided for the breeding work of the yellow seed germplasm of the brassica napus, and the application prospect is wide.

(2) According to the invention, a gene editing technology is utilized, the BnYB 123 gene is specifically knocked out in the brassica napus, and a series of experiments prove that compared with a receptor brassica napus, the contents of total phenols and flavonoids in the seed coat of a BnYB 123 knock-out mutant are obviously reduced, wherein the accumulation of isorhamnetin and epicatechin substances in the seed coat of the BnYB 123 knock-out mutant is less or not, which indicates that the BnYB 123 can change the color of the seed coat of the brassica napus by regulating and controlling the synthesis and accumulation of the isorhamnetin and epicatechin substances; the discovery not only enriches the understanding of the forming mechanism of the seed coat color of the cabbage type rape, but also provides a theoretical basis for the development of the cabbage type yellow seed germplasm.

Drawings

The sequence tables SEQ ID NO 1 and SEQ ID NO 2 are nucleotide sequences of BnMYB123 gene coding regions in the invention, and the sequence length is 786 bp;

FIG. 1: a BnYB 123-Cas9 target site schematic diagram;

FIG. 2: a T0 generation and T1 mutation site detection sequence diagram of a BnYB 123-Cas9 transformed plant;

FIG. 3: BnYB 123-Cas9 transformed plant T1 generation homozygous mutant strain seed phenotype;

FIG. 4: analyzing the content of total phenols and total flavonoids in seeds of T1 generation homozygous mutant lines of BnYB 123-Cas9 transformed plants;

FIG. 5: and (3) analyzing the content of flavonoids in T1 generation homozygous mutant strains of BnYB 123-Cas9 transformed plants.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following example defines the invention and describes the method for obtaining Brassica napus yellow seed germplasm by knocking out BnMY 123 gene through CRISPR/Cas9 system. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1

A method for specifically knocking out Brassica napus BnYB 123 gene by using a CRISPR-Cas9 system comprises the following steps:

(1) a target site is designed in a conserved region of two copies (BnaA.MYB123 and BnaC.MYB123) of the BnYB 123 gene, the sequence of the target site is shown as SEQ ID NO.3, and sgRNA is positioned on a second exon of the two copies of the BnYB 123 gene, as shown in figure I;

(2) designing and synthesizing a pair of single-stranded oligo DNA sequences according to the target site sequence, wherein the sequences are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5;

(3) obtaining an sgRNA expression cassette by a side-trimming connection method;

(4) connecting the sgRNA expression cassette with a CRISPR-Cas9 vector, and then transforming escherichia coli Trans5 α to obtain a plant expression vector BnYB 123-Cas 9;

(5) transforming a BnYB 123-Cas9 vector into a cabbage type rape receptor material (cabbage type rape J9712 strain) in an agrobacterium tumefaciens mediated mode, screening a transgenic positive plant by a vector-specific primer, and identifying the nucleotide sequences of the used upstream primer and downstream primer as shown in SEQ ID No.6 and SEQ ID No. 7;

(6) screening plants with edited two copies on the A chromosome and the C chromosome of the BnYB 123 in transgenic offspring, and judging whether the two copies of the BnYB 123 gene are successfully knocked out according to a sequencing result after identifying primer amplification;

the nucleotide sequences of the upstream and downstream primers identified by the chromosome A copy are shown as SEQ ID NO.8 and SEQ ID NO.9, and the nucleotide sequences of the upstream and downstream primers identified by the chromosome C copy are shown as SEQ ID NO.10 and SEQ ID NO. 9.

The BnYB 123 gene can be knocked out by adopting methods such as EMS mutagenesis, homologous recombination, T-DNA insertion and the like besides a CRISPR/Cas9 gene editing system; the plant genetic transformation method is not limited to agrobacterium-mediated transformation method, and the transgenic plant can be obtained by pollen tube channel method, gene gun method, microinjection method and other genetic transformation methods.

Example 2

Design and Synthesis of knock-out target sites

1. The principle of target site selection:

(1) the target sequence is 20bp in length. The downstream 3 bases of the sequence are NGG;

(2) the targeting efficiency can be improved by the GC% of the target sequence, so that the target preferably contains 11-14C/G;

(3) blast analysis is carried out on the rape genome by using the target site sequence, so as to avoid off-target.

2. CRISPR-P through online website ( http://cbi.hzau.edu.cn/cgi-bin/CRISPR) A target site is designed in a conserved region of two copies of the BnYB 123 gene (the position of the target site is shown in figure 1), and sgRNA is positioned on a second exon of the two copies of the BnYB 123 gene, as shown in figure I.

Example 3

Construction of knockout vectors

The construction of the knockout vector adopts an improved vector pYLCRISPR/Cas9-DH, and the construction method of the vector is as follows:

1. first, primers necessary for synthesis according to the vector construction method are shown in Table 1.

TABLE 1 primers necessary for vector construction

2. Formation of double-stranded target sequence

Respectively dissolving the forward and reverse target sequences into 100 mu M mother solution by using TE solution; taking 1 μ L of each of the forward target sequence (SEQ ID NO.4) and reverse target sequence (SEQ ID NO.5) solutions, adding 98 μ L of 0.5 × TE solution, and mixing well to prepare 1 μ M working solution; and (3) taking 10 mu L of the mixed solution to a PCR tube, placing the mixed solution in a PCR instrument, heating the mixed solution at 90 ℃ for 30sec, moving the mixed solution to room temperature, and cooling the mixed solution to finish annealing to form a double-stranded target sequence.

Enzyme digestion and linkage of gRNA expression cassette

(1) Constructing the sgRNA expression cassette by a side-cutting continuous method, wherein the reaction system is as follows:

placing the reaction system in a PCR instrument at 5cycles, 37 ℃ for 5 min; 20 ℃ for 5 min.

T4 DNA ligase was purchased from Beijing Quanjin Biotechnology, Inc., and restriction enzymes were purchased from NewEngland Biolabs.

gRNA expression cassette amplification

(1) First round amplification: mu.L of each ligation product was taken as template, and 0.2. mu.M of each was used for reaction 1, and forward target sequence primers, gRNA-R, respectively. The amplification enzyme is high Fidelity Taq enzyme (e.g., PhantaSopper-Fidelity DNA Polymerase by Vazyme). Rather than using Taq enzyme that attaches an A base 3' to the product, this A base renders the product non-pairing with the complementary strand and non-extension-filling in the second round of PCR. The reaction system is as follows:

reaction procedure: 28 cycles, 95 ℃,10 sec; 60 ℃ for 15 sec; pre-denaturation at 68 deg.C, 20sec, and 95 deg.C for 1min, and extending for 5min after circulation is over.

mu.L of each sample was subjected to electrophoresis (reaction 1 was about 360bp long, reaction 2 was about 135bp long, and 1.5% agarose gel electrophoresis was used). The second round of PCR can be continued even if the amplification product is weak.

(2) Second round PCR: the position-specific primers B1 'and BL were diluted to 10. mu.M in advance, and 10. mu.M each of B1' and BL primer solution (75. mu.L) was added to 350. mu.L of ddH ₂In O, 500. mu.L of PT1L was prepared. Taking 1. mu.L of the 1, 2 product of the first round of PCR reaction and using H ₂O was diluted 10-fold, and 1. mu.L of each was mixed as a template. 1/10 amounts of each primer combination working solution (final concentration 0.15. mu.M) were added. The reaction system is as follows:

PCR reaction procedure: 15-20 cycles (adjusted as the case may be), 95 ℃,10 sec; 58 ℃, 15 sec; 72 ℃ for 30 sec; pre-denaturation at 95 ℃ for 3min, and extension for 5min after circulation is finished.

Taking 2-3 muL electrophoresis to check whether the product length is consistent, and estimating the approximate concentration of the sample. The product size was: AtU6-29-gRNA has a size of 487 bp. The amplification product was recovered by cutting the gel and dissolved in 30. mu.L of Tris-HCl pH 8.5.

5. The sgRNA expression cassette was ligated to the Cas9 vector using a side-by-side approach. The reaction system is as follows:

and (3) uniformly mixing, and performing reaction on a PCR instrument, wherein the reaction procedure is as follows: 15 cycles, 37 ℃,2 min; 3min at 10 ℃; 20 ℃ for 5min

6. Purification of ligation products by ethanol precipitation

(1) Adding 1.5 μ L of 3M NaAc with pH 5.2 to the product, and mixing well;

(2) then adding 30 mu L of precooled absolute ethyl alcohol, uniformly mixing, and standing at-20 ℃ for 15-30 min;

(3) centrifuging at 12000rpm for 10min, and removing supernatant and all liquid drops on the tube wall;

(4) adding 100 μ L70% ethanol, centrifuging at 12000rpm for 2min, and removing supernatant;

(5) drying at room temperature for 10min, adding 10 μ L ddH ₂And dissolving the O.

Transformation of Escherichia coli by BnYB 123-Cas9 recombinant vector and identification of positive bacteria

The general transformation method comprises the steps of transferring the ligation product into an escherichia coli Trans5 α strain, coating a plate, selecting a single colony, carrying out PCR positive identification by using primers SEQ ID NO.6 and SEQ ID NO.7, carrying out shake bacteria sequencing, storing a strain after the sequencing is correct, and extracting a plasmid.

Example 4

Transformation of knock-out vector Agrobacterium (Electrolysis method)

The BnYB 123-Cas9 plasmid is introduced into competent cells of Agrobacterium tumefaciens GV3101 strain by electric shock transformation. A single colony is selected and inoculated in 25mL LB culture medium (containing 50mg/L rifampicin) for culture overnight, 5mL bacterial liquid is taken and transferred into 100mL LB culture medium (containing 50mg/L rifampicin) for culture until OD600 is 0.7-0.8, the bacterial liquid is placed on ice for 10min, centrifuged at 5000rpm and 4 ℃ for 10min to collect thalli, and then 100mL sterile double distilled water is added for washing twice. The cells were suspended in 4mL of 10% glycerol and transferred to a 50mL centrifuge tube. The cells were collected by centrifugation at 5500rpm at 4 ℃ for 10min, and 500. mu.L of 10% glycerol was added to resuspend the cells, which were transferred to a 1.5mL centrifuge tube.

50 mu L of competent cells are taken, 1 mu L of BnMYB123-Cas9 recombinant plasmid is added, and the mixture is uniformly mixed by a gun head and then transferred into a 0.1cm electrotransformation cup. Electrical conversion parameters: 200 Ω, 1.7KV, 2.5F, 500. mu.L of LB medium was added immediately after the shock. After culturing at 37 ℃ and 220rpm for 1 hour, 100. mu.L of the strain was applied to LB medium containing Kanamycin resistance Kanamycin to select transformants, and cultured at 28 ℃ for 16 hours. Selecting single colony, performing PCR identification with SP-DL/SP-R primer to obtain positive strain, and storing the positive strain with glycerol.

Example 5

Genetic transformation of brassica napus

The hypocotyl of the cabbage type rape sterile seedling is used as an explant, and the genetic transformation of the exogenous segment in the cabbage type rape is realized by utilizing an agrobacterium tumefaciens mediated method.

The formula of the culture medium is as follows:

inoculation medium (M0): MURASHIGE & SKOOG MEDIUM (Duchefa Biochemie Inc.) +30.0g/L Sucrose Surcross +8g/L Agar (pH 5.8-pH 6.0).

Co-cultivation medium (M1): m0+18.0g/L Mannitol +1.0 mg/L2, 4-dichlorophenoxyacetic acid 2,4-D +0.3mg/L Kinetin + 100. mu.M acetosyringone AS (pH 5.8).

Callus differentiation medium (M2): m1+300.0mg/L Timentin +25mg/L hygromycin B.

Shoot medium (M3): MURASHIGE & SKOOG MEDIUM (Duchefa Biochemie company) +10.0g/L Glucose +0.25g/L Xylose Xylose +0.6g/L morpholine ethanesulfonic acid MES +2.0mg/L Zeatin Zeatin +0.1mg/L indoleacetic acid IAA +300.0mg/L Timentin +25mg/L Hygromycin B.

Strong seedling rooting medium (M4): m0+300.0mg/L Timentin.

Murashige & Skoog media is simply referred to as MS MEDIUM.

The specific operation steps are as follows:

(1) and (3) sterilization:

a. firstly, soaking cabbage type rape seeds in 75% alcohol for 1min, wherein attention cannot be paid for too long time;

b. then sterilizing with 2% sodium hypochlorite for 20 min;

c. finally, the seeds are washed for 4-5 times by sterile water, and are cleaned as far as possible.

(2) Sowing:

a. seeding the sterilized seeds to M with sterile forceps ₀Inoculating 30 grains in each pot on the culture medium;

b. placing the inoculated culture tank into an incubator, and culturing at 24 ℃ in the dark for 6-7 d.

(3) Shaking the bacteria:

after 5-6 days of sowing, the agrobacterium is inoculated into a sterile triangular flask or a centrifuge tube containing LB liquid medium and placed in a shaker at 180-.

(4) Explants were prepared and infected:

a. cutting the seedling which grows for 6-7d after seeding by using a sterile forceps and a scalpel, and cutting the hypocotyl into explant segments with the length of 0.8-1.0 cm;

b. measuring OD of Agrobacterium ₆₀₀Value (OD in LB medium) ₆₀₀About 0.3 is preferable), the previously cultured cell suspension is centrifuged at 6000rpm for 10min, the supernatant is discarded, and the cell suspension is resuspended in an MS liquid medium containing 100. mu.M acetosyringone AS, which is the same volume AS the cell suspension, and the procedure is repeated once more. Finally, taking 2mL of bacterial liquid, and diluting the bacterial liquid with 20mL of MS liquid culture medium containing 100 mu M acetosyringone AS;

c. and (3) putting the cut explants into the bacterial liquid resuspension with the adjusted concentration, and carrying out dip dyeing for 10min, wherein the infection time is not too long, otherwise, the explants die. 150-200 explants are suitably impregnated in each 20mL of the bacterial solution;

(5) transfer of infected explants to M ₁Culturing on culture medium at 24 deg.C in dark for 36-48 hr with 20-25 explants per dish;

(6) explant from M ₁Go to M ₂On the culture medium, and transferred to a light incubator (24 ℃ for 16 h/8 h) for 3 weeks;

(7) transfer of explants to M ₃Subculturing on the culture medium every 2-3 weeks until green buds appear;

(8) finally transferring the explants to M ₄Rooting in culture medium for 2-4 weeks.

Example 6

Mutation detection and hygromycin marker-free screening of transgenic plants

1. Extracting genome DNA of the brassica napus by adopting a CTAB method, which comprises the following steps:

(1) selecting 0.5g of tender leaf tissue, putting the tender leaf tissue into a 2mL clean centrifugal tube, adding liquid nitrogen, and grinding by a sample grinder;

(2) adding 450 μ L of 65 deg.C preheated CTAB solution, placing in 65 deg.C water bath kettle, and reacting with tissue by gently mixing every 5min for 30 min;

(3) after the reaction, taking out the sample, cooling to room temperature, adding chloroform/isoamylol with the same volume, slightly reversing the mixture up and down, uniformly mixing for 15min, and then centrifuging for 5min by using a centrifuge at 15 ℃ and 10000 rpm;

(4) sucking 600 mu L of supernatant into a new EP tube, and repeating the steps;

(5) transferring the supernatant into a new tube, adding 0.7 times volume of precooled isopropanol, slightly reversing the mixture up and down, uniformly mixing to separate out and aggregate DNA into flocculent precipitate, and standing the flocculent precipitate in a refrigerator at the temperature of minus 20 ℃ for half an hour;

(6) centrifuging at 10000rpm for 5min at normal temperature by using a centrifuge, and pouring off the supernatant;

(7) adding 1mL of 75% absolute ethyl alcohol, washing twice, and airing in a fume hood;

(8) adding sterile ultrapure water containing RNase, placing the sample in a water bath kettle at 37 ℃ and standing for about 30min to accelerate dissolution;

(9) and after the precipitate is completely dissolved, detecting the purity and the concentration quality of the sample by a spectrophotometer and an agarose electrophoresis method.

The formula of the extraction buffer solution is as follows:

2×CTAB：Tris-HCl 100mmol/L(pH8.0)、EDTA 20mmol/L(pH8.0)、NaCl1.4mol/L、CTAB 2％。

2. detection of transgenic positive plants

1 μ L of DNA was used as a template, and PCR amplification was performed using primers SEQ ID NO:6 and SEQ ID NO:7 (2 XTAQQ Master Mix by Vazyme was used for amplification). The PCR system was as follows:

the amplification conditions were: 5min at 94 ℃; 35 cycles of 94 ℃ for 30sec, 58 ℃ for 30sec, and 72 ℃ for 30 sec; 10min at 72 ℃. By using the transgenic cabbage type rape DNA as a template, a specific target fragment can be amplified, and the target vector BnYB 123-Cas9 is proved to be successfully integrated into the cabbage type rape genome.

3. Detecting the knockout effect of transgenic plants

Taking 1 mu L of DNA as a template, and respectively carrying out PCR amplification by using an identification primer of the A chromosome copy and an identification primer of the C chromosome copy, wherein the upstream and downstream nucleotide sequences of the identification primer of the A chromosome copy are shown as SEQ ID NO.8 and SEQ ID NO.9, the upstream and downstream nucleotide sequences of the identification primer of the C chromosome copy are shown as SEQ ID NO.10 and SEQ ID NO.9, and the PCR system and the amplification conditions are the same as those in the step 2 of the example 5.

And after amplification, recovering the PCR product and sequencing, sequencing the amplification product of the A chromosome copy by using SEQ ID NO.8, and sequencing the amplification product of the C chromosome copy by using SEQ ID NO. 10. Determining the knockout condition of the BnYB 123 by comparing sequencing peak maps, and determining the mutation type of the sequencing peak maps by DSDecodeM analysis, wherein if no wild type BnYB 123 peak map exists, the copy is subjected to homozygous mutation. As shown in FIG. 2, BnYB 123-Cas9 transformed plants underwent gene editing at the target site of both A copy and C copy, and homozygous mutant individuals of BnYB 123 were screened in the T1 generation. It was found by phenotypic observation that seeds of homozygous mutant individuals of bnyb 123 exhibited the trait of yellow seed (as shown in fig. 3).

4. Screening of yellow seed germplasm without transgenic component

Selecting a T1 generation homozygous mutant single plant, taking 1 mu L DNA of the T1 generation homozygous mutant single plant as a template, taking SEQ ID NO.6 and SEQ ID NO.7 as primers to amplify, and carrying out agarose gel electrophoresis after amplification to obtain a banding-free yellow seed material without transgenic components, which can be directly used for breeding yellow seed germplasm.

Example 7

Content analysis of flavonoid substances in transgenic T2 generation homozygous mutant line seeds

1. Extraction of seed Total phenolic substances

100 seeds were taken, 5mL of methanol/acetone/water (40:40:20, v: v: v) were added, ground, sonicated for 10min, centrifuged (13000g) at 4 ℃ for 10min, the supernatant collected, 5mL of methanol/acetone/water (40:40:20, v: v: v) was added again to the pellet, resuspended, and resuspended at 4 ℃ (actually 18 ℃) overnight (250rpm) on a shaker. The supernatants were collected by centrifugation (4 ℃, 13000g, 10min), combined and concentrated to dryness by nitrogen-blown or rotary concentration. The volume is adjusted to 1mL by 80% methanol solution. Hereinafter, the extract is referred to as a crude extract, and the crude extract is used for the measurement of total phenols, total flavonoids and HPLC-MS.

2. Determination of the Total phenol content

50 μ L of the crude extract was taken, 350 μ L of water and 400 μ L of Folin-C reagent were added, 400 μ L of 1M sodium carbonate was added after reaction for 3min, diluted 10-fold with water after reaction for 45min, and then the absorbance was measured at 725 nm. Standard curves were made using sinapinic acid, 6 points were plotted and the concentrations were 50,100,200,400,800,1600 (ppm).

The results are shown in FIG. 4, where the total phenol content in the seeds of two homozygous mutant lines MYB123m-1 and MYB123m-2 of BnYB 123 was significantly lower than the control recipient material throughout the entire period of seed development.

3. Determination of Total flavonoid content

0.2mL of the crude extract was diluted with 2.8mL of water, 0.5mL of 5% sodium nitrite was added, 0.3mL of 10% aluminum chloride solution was added after 6min of reaction, 1mL of 1M sodium hydroxide was added after 5min of reaction, 0.55mL of water was added to a constant volume of 5mL, and the absorbance was immediately measured at 510 nm. Standard curves were made using epicatechin at concentrations of 25,50,100,200,400,800(ppm) in that order.

The results are shown in FIG. 4, where the total flavonoid content in the seeds of two homozygous mutant lines MYB123m-1 and MYB123m-2 of BnMY 123 was significantly lower than the control recipient material throughout the entire period of seed development.

4. HPLC determination of phenolics

After the extract was filtered through a Teflon filter (0.45 μm), 10 μ L of the extract was examined by high performance liquid chromatography Agilent6460Triple Quad LC/MC equipped with an on-line degasser (G1322A), a cascade pump (G1311A), an autosampler (G1329A) and a DAD detector (G1315D) (Agilent Technologies, Waldbronn, Germany). The extract was separated by passing through a C18 guard column (4X 2.0, 3.5 μm; Phenomenex) on an Ultimate XB-C18 column (2.1mm X150 mm, 3.5 μm) (Welch). The chromatographic separation conditions for the flavonoids were: air flow rate of 0.3ml.min ^-1And the column temperature is 30 ℃, and the DAD detection wavelength is 190-800 nm. The mobile phases were solution a (0.1% formic acid-water solution) and solution B (0.1% formic acid-acetonitrile solution).

The elution step is as follows:

tandem mass spectrometry was performed using an Agilent6460triple tandem quadrupole mass spectrometer with electrospray ion source (ESI) and detection in positive and negative ion mode. The instrument parameters were as follows: the temperature of the dry gas was 300 deg.C, the dry gas flow rate was 10l/min, the atomizer pressure was 15psi, and the sheath gas (N) ₂) The flow rate is 7l/min, the sheath vapor temperature is 250 ℃, the capillary voltage positive mode is 4000V, the negative mode is 3500V, the spray voltage is 500V, and the full-scanning mass-to-charge ratio (m/z) is 90-2000. Soluble phenolic compounds were first tested in full scan mode and, to further determine the structure of the compounds,and (3) selecting ions with a specific mass-to-charge ratio for secondary mass spectrometry, wherein collision gas is nitrogen, and collision voltage is set between 15 and 40V according to the size of molecules. The instrument control software is Agilent Master Workstation Data Acquisition, and the Data Analysis software is Agilent Master Qualitative Analysis. Molecular ion peak ([ M-H ] of compound]-and [ M + H]The contents of hydroxyphenyl acrylic acid derivatives and epicatechin are represented by equivalent epicatechin standards, the contents of epicatechin polymers are represented by equivalent procyanidin B2 standards, and the derivatives of isorhamnetin and kaempferol are represented by equivalent isorhamnetin-3-O-glucoside standards.

The results are shown in fig. 5, the total content of flavonoids in the seeds of the two homozygous mutant strains MYB123m-1 and MYB123m-2 of BnMYB123 was significantly lower than the control recipient material throughout the period of seed development, and the most significant of the differences were isorhamnetin and epicatechin, indicating that BnMYB123 mutation affected the synthesis and accumulation of isorhamnetin and epicatechin in the flavonoids, and thus the coloration of the seed coat.

The invention provides a method for obtaining cabbage type rape yellow seed germplasm by separation, the cabbage type rape yellow seed germplasm created by the method only changes one gene BnMY 123, and the yellow seed germplasm without transgenic ingredients is obtained by genetic separation, so that the method has little influence on the environment. The method for obtaining the yellow seed germplasm of the cabbage type rape provides a brand new approach for the breeding work of the yellow seed germplasm of the rape, and has wide application prospect.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

<110> Yangzhou university

<120> method for obtaining cabbage type yellow seed germplasm based on CRISPR/Cas9 technology

<160>10

<170>SIPOSequenceListing 1.0

<210>1

<211>786

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

atgatgagaa agagagaaag tagtaaggtg aagaaagagg agttaaacag aggagcttgg 60

accgatcaag aagacaagat ccttaaagac tatatcatgt tccacggcga aggaaaatgg 120

agcacactcc caaaccaagc tggtctcaag aggtgtggca aaagctgcag acttcggtgg 180

aagaactact tgagaccagg cataaagcgc ggaaacatct catctgatga agaagaactt 240

ataatccgcc tccataatct ccttggaaac agcagatggt cgttgatagc tgggaggctt 300

ccagggcgaa cagacaatga aataaagaac cactggaact caaacctccg caaaagactt 360

ccaaaatctc aaaccaacca acagaaaagt cgaaaacatt ccaacaacaa caacatgaat 420

aaagtatgtg ttatacgtcc aaaggcgatt aggttcccaa aggctctgac atttcagaat 480

cagagtagta ttggtagtac cagtcttctt actgtgaagg aaaacgtgat tgatcatcaa 540

gctggttctc cttcgttgtt gggagatctt aaaatcgatt ttgataaaat tcagtctgag 600

tatctcttct ctgatttgat ggactttgat ggtttgggtt gtggaaacgt aatgtctctt 660

gtttcatctg acgaggtgct gggagattat gtttcggctg atacttcttg tctgggtaat 720

cttgatctta atagaccttt cacttcttgt cttcaagaag attgtctctg ggactttaat 780

tgttag 786

<210>2

<211>786

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atgatgagaa agagagaaag tagtagggtg aagaaagagg agttaaacag aggagcttgg 60

accgatcaag aagacaagat ccttaaagac tatatcatgt tccacggcga aggaaaatgg 120

agcacactcc caaaccaagc tggtctcaag aggtgtggca aaagctgcag acttcggtgg 180

aagaactact tgagaccagg cataaagcgc ggaaacatct catctgatga agaagaactt 240

ataatccgcc tccataatct ccttggaaac agcagatggt cgttgatagc tgggaggctt 300

ccagggcgaa cagacaatga aataaagaac cactggaact caaacctccg caaaagactt 360

ccaaaatcac aaaccaacca acagaaaagt cgaaaacatt ccaacaacaa caacatgaat 420

aaagtatctg ttatacgtcc aaaggcgatt aggtgcccaa aggctctgac atttcagaag 480

cagagtagta ttggtagtac cagtcttctt actgtgaagg aaaacgtgat tgatcaacaa 540

gctagttctc cttcatcgtt gggagatctt aaaatcgatt ttgataaaat tcagtctgag 600

tatctcttct ctgatctgat ggactttgat ggtttgggtt gtggaaacgt aatgtctctt 660

gtttcatctg acgaggtgct gggagattat gtttcggctg atgcttcttg tctgggtaat 720

cttgatctta atagaccttt cacttcttgt cttcaagaag attgtctctg ggactttaat 780

tgttag 786

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<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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ttgagaccag gcataaagcg cgg 23

<210>4

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

attgttgaga ccaggcataa agcg 24

<210>5

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

aaaccgcttt atgcctggtc tcaa 24

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<213> Artificial Sequence (Artificial Sequence)

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gtcgtgctcc acatgttgac cgg 23

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<213> Artificial Sequence (Artificial Sequence)

<400>7

cccgacatag atgcaataac ttc 23

<210>8

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gtgccttttt aatgattgaa aa 22

<210>9

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gtaacctgtt tccaaggaga tta 23

<210>10

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<213> Artificial Sequence (Artificial Sequence)

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tcgccttttt aatgattgta ag 22

Claims

1. A method for obtaining Brassica napus yellow seed germplasm based on CRISPR/Cas9 technology is characterized by comprising the following steps: the method comprises the following steps of directionally editing a brassica napus BnYB 123 gene by using a CRISPR/Cas9 technology to obtain the brassica napus with function deletion of the BnYB 123 gene:

2. The method of claim 1, wherein the nucleotide sequence of the coding region of the BnYB 123 gene is SEQ ID NO 1 and SEQ ID NO 2.

3. The method according to claim 1, wherein one strand of the target site has a 5 '-A (G) - (N) X-NGG-3' structure, wherein (N) X represents a base sequence of X in number, and N in (N) X represents any one of bases A, G, C, T.

4. The method of claim 3, wherein X is 19 or 20.

5. The method of claim 1, wherein the target site sequence is set forth in SEQ ID No. 3.

6. The method of claim 1, wherein the functional deletion is a large fragment deletion, a frame shift mutation or a terminator of the coding sequence of BnYB 123 in the region of the target site.

7. The method of claim 1, wherein the CRISPR/Cas9 recombinant vector is constructed by designing and synthesizing a pair of single-stranded oligo DNA sequences according to a target site sequence, wherein the sequences are respectively shown as SEQ ID NO.4 and SEQ ID NO.5, obtaining a sgRNA expression cassette by a side-cut ligation method, connecting the sgRNA expression cassette with a CRISPR-Cas9 vector, and then transforming Escherichia coli Trans5 α to obtain a recombinant vector BnYB 123-Cas 9.

8. The method of claim 1, wherein the selection of a regeneration line in which editing of both copies and both alleles of BnYB 123 occurs is by: the two copies of the BnYB 123 gene are BnaA.MYB123 and BnaC.MYB123 respectively; the upstream and downstream nucleotide sequences of the identification primer of the chromosome A copy are shown as SEQ ID NO.8 and SEQ ID NO.9, and the upstream and downstream nucleotide sequences of the identification primer of the chromosome C copy are shown as SEQ ID NO.10 and SEQ ID NO. 9.

9. The method of claim 1, wherein the transgenic positive plants are screened by vector-specific primers, and the upstream and downstream nucleotide sequences of the identified primers are shown as SEQ ID No.6 and SEQ ID No. 7.

10. Use of the method of obtaining Brassica napus yellow seed germplasm according to claim 1 based on CRISPR/Cas9 technology in breeding.