Method for rapidly identifying and regulating rape oil content gene based on genomics approach and application thereof

文档序号：1856649 发布日期：2021-11-19 浏览：29次中文

阅读说明：本技术 一种基于基因组学途径快速鉴定调控油菜含油量基因的方法及其应用 (Method for rapidly identifying and regulating rape oil content gene based on genomics approach and application thereof ) 是由黄会斌周永明赵青穆罕默德·沙希徳柯昌森蔡光勤王学敏范楚川于 2021-08-20 设计创作，主要内容包括：本发明属于生物技术领域,尤其涉及一种基于基因组学途径快速鉴定调控油菜含油量基因的方法及其应用。技术要点包括通过田间多年多点试验筛选到高含油量近交系1L99和低含油量近交系1L363,然后对两个近交系6个不同发育时期的种子进行转录组测序分析；用转录组分析获得的差异基因在qOC-A5QTL位点进行扫描,发现BnECH.A5基因在高含油量近交系不同油菜种子发育时期的表达量较低,而在低含油量近交系中表达量较高,且在开花后30天-44天间的种子含油量积累的关键时期表达量呈现剧烈变化。构建该基因的突变体植株,结果发现,功能缺失突变植株的含油量与野生型植株相比显著升高。(The invention belongs to the technical field of biology, and particularly relates to a method for rapidly identifying and regulating a rape oil content gene based on a genomics approach and application thereof. The technical key points comprise that a high-oil-content inbred line 1L99 and a low-oil-content inbred line 1L363 are screened through years and multipoint tests in a field, and then transcriptome sequencing analysis is carried out on seeds of 6 different development periods of the two inbred lines; the difference gene obtained by transcriptome analysis is scanned at qOC-A5QTL locus, and the expression level of BnECH.A5 gene is lower in the development period of different rape seeds of high-oil-content inbred line, higher in the low-oil-content inbred line, and the expression level shows drastic change in the key period of oil content accumulation of seeds between 30 days and 44 days after flowering. The result of constructing the mutant plant of the gene shows that the oil content of the function-deletion mutant plant is obviously increased compared with that of the wild plant.)

1. A method for rapidly identifying and regulating rape oil content genes based on a genomics approach is characterized by comprising the following steps:

s1: carrying out transcriptome sequencing on the siliques of the high-oil-content rape material and the low-oil-content rape material in different development periods;

s2: analyzing the transcriptome sequencing result, and screening expression difference genes;

s3: scanning the difference gene at qOC-A5QTL locus to identify the gene for regulating and controlling the oil content of rape seeds;

s4: the function of the identified gene is verified.

2. The method for rapidly identifying and controlling the oil content of rape based on the genomics approach according to claim 1, which is characterized in that: the high oil content rape material comprises a high oil content inbred line 1L 99; the low oil content oilseed rape material comprises a low oil content inbred 1L 363.

3. The method for rapidly identifying and controlling the oil content of rape based on the genomics approach according to claim 1, which is characterized in that: in step S4, a gene mutant is constructed by using a CRISPR technology, and the change of the oil content of the mutant material is detected.

4. The method for rapidly identifying and controlling the oil content of rape based on the genomics approach according to claim 1, which is characterized in that: genes identified that regulate oil content in oilseed rape seeds include bnech.a 5.

5. A method for regulating and controlling the oil content of rape seeds is characterized in that: knocking out or reducing the expression of BnECH.A5 gene in rape material, and increasing the oil content of rape seeds.

6. The method for controlling oil content of rape seeds as claimed in claim 5, wherein: knocking out or reducing the expression of BnECH.A5 gene in rape material by using CRISPR/Cas9 mediated gene knockout technology.

7. The method for controlling oil content of rape seeds as claimed in claim 6, wherein: 2 target sequences are involved in the CRISPR/Cas9 technology, which are respectively: t1: GCTGGTGTTGAAAGAGGAGACGG, respectively; t2: TCGTCACTATCAAACCAAAACGG are provided.

8. The method of claim 7, wherein the oil content of the rape seeds is controlled by: the gene editing primers of the 2 target sequences are respectively as follows: T1-F: GTCAGCTGGTGTTGAAAGAGGAGA, respectively; T1-R: AAACTCTCCTCTTTCAACACCAGC, respectively; T2-F: GTCATCGTCACTATCAAACCAAAA, respectively; T2-R: AAACTTTTGGTTTGATAGTGACGA are provided.

9. The method of claim 7, wherein the oil content of the rape seeds is controlled by: constructing a target gene editing vector, and converting the target gene editing vector into a receptor material to obtain a mutant material with improved oil content of rape seeds.

The application of the BnECH.A5 gene in regulating and controlling the oil content of rape seeds.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for rapidly identifying and regulating a rape oil content gene based on a genomics approach and application thereof.

Background

Rape is an important oil crop in China and a main edible vegetable oil source. Increasing the oil content of rape is very important for improving the yield of the rape oil. Research shows that the increase of 1% of oil content is equivalent to the increase of 2.3% -2.5% of rape yield, and germplasm resources with high oil content are the key points for cultivating new varieties. Therefore, the genetic basis analysis for enhancing the oil content metabolism of the rape is of great significance for creating new germplasm resources with high oil content.

However, because the oil content is greatly influenced by the environment, the genetic regulation is complex, and the genome of the brassica napus is complex, the difficulty in developing the gene for regulating the oil content of the rape is great. In rape, the traditional QTL positioning method for mining new genes mainly comprises three stages of QTL initial positioning, fine positioning, candidate gene mining and verification. Different populations need to be constructed in the initial positioning and fine positioning stages of the QTL, and phenotype and molecular marker data are collected through multi-year and multi-point planting, and the two stages take at least 4-5 years or more because the growth and development period of the rape is long (6-7 months). When a certain interval is located, transgene complementation verification needs to be carried out on candidate genes in the interval, and the number of the candidate genes in the interval is often dozens or more. In addition, the rape has no whole genome mutant library so far, and only can be subjected to complementation verification by using an arabidopsis mutant and overexpression. Therefore, the traditional QTL positioning method is used for mining and regulating the oil content genes of the rape, which consumes long time and has large workload.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for rapidly identifying and regulating the oil content gene of rape based on a genomics approach and application thereof, aiming at solving part of the problems in the prior art or at least relieving part of the problems in the prior art.

The invention is realized in such a way that a method for rapidly identifying and regulating the oil content gene of rape based on a genomics approach comprises the following steps:

s1: carrying out transcriptome sequencing on the siliques of the high-oil-content rape material and the low-oil-content rape material in different development periods;

s2: analyzing the transcriptome sequencing result, and screening expression difference genes;

s3: scanning the difference gene at qOC-A5QTL locus to identify the gene for regulating and controlling the oil content of rape seeds;

s4: the function of the identified gene is verified.

Further, the high oil content canola material comprises a high oil content inbred line 1L 99; the low oil content oilseed rape material comprises a low oil content inbred 1L 363.

Further, in step S4, a gene mutant is constructed by using CRISPR technique, and the change of oil content in the mutant material is detected.

Further, the identified genes that regulate oil content of oilseed rape seeds include bnech.a 5.

The application also provides a method for regulating the oil content of the rape seeds, which can knock out or reduce the expression of the BnECH.A5 gene in rape materials and improve the oil content of the rape seeds.

Further, the expression of the BnECH.A5 gene in the rape material is knocked out or reduced by using a CRISPR/Cas9 mediated gene knockout technology.

Further, 2 target sequences are involved in the CRISPR/Cas9 technology, which are respectively: t1: GCTGGTGTTGAAAGAGGAGACGG, respectively; t2: TCGTCACTATCAAACCAAAACGG are provided.

Further, the gene editing primers of the 2 target sequences are respectively: T1-F: GTCAGCTGGTGTTGAAAGAGGAGA, respectively; T1-R: AAACTCTCCTCTTTCAACACCAGC, respectively; T2-F: GTCATCGTCACTATCAAACCAAAA, respectively; T2-R: AAACTTTTGGTTTGATAGTGACGA are provided.

Further, a target gene editing vector is constructed and is transformed into a receptor material, and a mutant material with the oil content of the rape seeds improved is obtained.

The application also provides application of the BnECH.A5 gene in regulating and controlling the oil content of rape seeds.

In summary, the advantages and positive effects of the invention are:

according to the invention, the time for years is not needed for constructing a population, the phenotypic data is examined, transcriptome sequencing is directly carried out on the siliques in different development periods of the high-low oil content inbred line, the known QTL sites are combined for scanning, the non-target genes are excluded, the genes participating in regulating and controlling the oil content of rape seeds are identified, the workload of candidate gene verification is reduced, and the method is more pertinent; the target sequences T1 and T2 of the CRISPR/Cas9 system capable of improving the oil content of rape seeds are provided, and the two targets can accurately edit two copies of the BnECH.A5 gene at fixed points; the oil content of rape seeds can be improved to different degrees through a CRISPR/Cas9 mediated gene knockout technology; the function of the new gene can be determined and a new material with high oil content can be obtained only after 1.5-2 years, and finally the new gene for regulating and controlling the oil content of the cabbage type rape seeds is obtained, so that the time for obtaining the new gene is greatly shortened, and the cultivation of a new germplasm resource is accelerated.

In addition, the verification of the gene function of BnECH.A5 shows that after the gene BnECH.A5 of the brassica napus is knocked out, the oil content of brassica napus seeds can be obviously improved, the amplification reaches 24.3 percent, and the extremely obvious level is reached.

The invention provides a new way for the discovery and verification of a new gene for regulating the oil content of the brassica napus seeds, provides a new germplasm resource and has good application prospect.

Drawings

FIG. 1 shows the expression level change trend of BnECH.A5 gene in different materials at different time periods;

FIG. 2 is a PCR electrophoretogram of mutant material;

fig. 3 is the result of oil content measurement of bnech.a5 mutant.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

In the following examples of the present invention, the temperature is not particularly limited, and all of the conditions are normal temperature conditions. The normal temperature refers to the natural room temperature condition in four seasons, no additional cooling or heating treatment is carried out, and the normal temperature is generally controlled to be 10-30 ℃, preferably 15-25 ℃.

The invention discloses a method for rapidly identifying and regulating rape oil content genes based on a genomics approach and application thereof. The applicant screened high oil content inbred 1L99(Liu et al, 2016) and low oil content inbred 1L363(Liu S, Fan C, Li J, Cai G, Yang Q, Wu J, Yi X, Zhang C, Zhou Y.A genome-with association study novel inorganic particulate in segmented oil content of Brassica. The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Example 1 Rapid identification of Gene regulating oil content of oilseed rape based on genomic approach

1. RNA sample preparation for high oil content inbred 1L99 and Low oil content inbred 1L363

The brassica napus high-oil-content inbred line 1L99 and the low-oil-content inbred line 1L363 are planted in the Huazhong agriculture university test field in the sowing season. At different developmental stages, siliques were taken 11, 16, 23, 30, 37 and 44 days after flowering, respectively, and 3 biological replicates were taken from samples at each stage. Sample RNA was extracted according to the instructions of the RNA extraction kit (TransZol Plant, BioTeke, China), treated with RNase-free DNase-I (Thermo Scientific, USA) to remove gDNA contamination, and used with a nucleic acid/protein analyzer730(Beckman) Quantification was performed.

2. Library construction, sequencing and analysis

The Sample was subjected to library construction using TruSeq Nano DNA HT Sample Preparation Kit (Illumina, USA), and the specific procedures are described in the Kit instructions. Followed by IlluminaHiseq^TM2500 sequencer (crop genetic improvement national focus laboratory, Huazhong agriculture university)Science) all libraries were double-ended sequenced at 2X 150 bp. And (3) performing quality control on raw reads by using an NGS QC tool kit, comparing the clean reads subjected to the quality control with HISAT2 to a reference genome (Brassica napus v4.1, Chalhoub et al 2014) of the Brassica napus Darmor-bzh (http:// www.genoscope.cns.fr/brasssicanaus/data /), and adopting default settings for parameters. Clean reads were aligned to genomic sequences using Bowtie2 and gene expression levels were calculated for each sample using RSEM.

3. Identification of genes involved in regulation and control of oil content of brassica napus

A series of differential genes were obtained by differential analysis of expression levels, and in this application, 14 differential genes were found in the QTL interval by scanning QTL sites of qOC-A5(Liu S, Fan C, Li J, Cai G, Yang Q, Wu J, Yi X, Zhang C, Zhou Y.A genome-side association novel genes in seed oil content of Brassica applying Genet,2016,129, (6): 1203-. Analysis of the expression levels of 14 genes in the high oil content inbred line 1L99 and the low oil content inbred line 1L363 shows that the expression level of the BnECH.A5 gene in the development period of different rape seeds is lower in the high oil content inbred line, higher in the low oil content inbred line, and the expression level shows a drastic change in the key period of oil content accumulation of the seeds between 30 days and 44 days after flowering (FIG. 1). Therefore, the BnECH.A5 gene is possibly involved in the negative regulation of the oil content metabolism of rape seeds.

Example 2 validation of gene function of bnech.a5 by CRISPR/Cas 9-mediated gene knockout technology

1. Screening target sequences and designing primers

The nucleotide sequence of the Brassica napus BnECH.A5 gene (as shown in SEQ ID NO.17) was obtained from the reference genome Darmor-bzh. Two targets, T1 and T2, were designed and screened (Table 1), and primers were designed against the target sequences (Table 2).

TABLE 1 target sequences

TABLE 2 target primer sequences

2. Construction of two-target Gene editing vector pBnECH vector

(1) Preparing a target joint: the adaptor primers of Table 2 were dissolved in ddH2O to 10. mu.M stock solution. Mu.l of each of the left and right primers was added to 8. mu.l of ddH2O, diluted to 1. mu.M, and vortexed and shaken to mix. Then putting the mixture into a water bath kettle for 90 ℃, processing the mixture for 30 ℃, and naturally cooling the mixture to room temperature.

(2) sgRNA expression cassette ligation reaction

1) The two plasmids AtU3d and AtU3b were digested with enzymes, and the digestion system is shown in Table 3.

TABLE 3 plasmid digestion System

2) The two plasmid plasmids AtU3d and AtU3b which are cut by enzyme are connected with each corresponding adaptor for reaction, the connection is carried out for about 10-15min at room temperature (20-28 ℃), the plasmid and each corresponding adaptor are shown in table 4, and the connection reaction system is shown in table 5.

TABLE 4 plasmids and respective linkers

Joint	Bas1 enzyme cutting plasmid
		T1	AtU3d
T2	AtU3b

TABLE 5 expression cassette ligation reaction System

Composition (I)	Amount of addition	Final concentration (amount)
			10×T4 DNA ligase Buffer	1μl	1×
pYLsgRNA-U # plasmid (Bas1)	0.5μl	10～20ng
			Target point joint	0.5μl	0.05μM
T4 DNA ligase(NEB)(400U/ul)	0.1μl	～40U
			ddH2O	7.9μl
total	10μl

(3) And amplifying the sgRNA expression cassette fragment. 2 nested PCR rounds are carried out, 2 reactions are carried out in the first round of PCR, and U-F/joint reverse primers and joint forward primers/gR-R are respectively used; the second round is Overlapping PCR, and the expression cassette product is amplified by using a position-specific primer. The primer sequences used are shown in Table 6, the first round PCR reaction system is shown in Table 7, and the reaction procedures are shown in Table 8. After the first round of reaction, 1. mu.l of each of the products of reaction 1 and reaction 2 was added to 8. mu.l of ddH2O, diluted and mixed by vortexing and shaking. Second round PCR, method of use of sgRNA expression cassette specific position primer pairs: Pps-GGL/Pgs-GG2(PT1), Pps-GG2/Pgs-GGR (PT2R), and corresponding U # -T1-gRNA and U # -T2-gRNA are amplified. Each primer was first dissolved in ddH2O to 10. mu.M stock solution. Taking 1.5 mu l of Pps-GGL and Pgs-GG2 primers respectively, adding the Pps-GGL and the Pgs-GG2 primers into 7 mu l of ddH2O, mixing and diluting to 1.5 mu M, uniformly mixing by vortex vibration, and sequentially configuring PT1 and PT 2R; the reaction system is shown in Table 9, and the procedure is shown in Table 10.

TABLE 6 nested PCR primer sequences

TABLE 7 first round PCR reaction System

Composition (I)	Amount of addition
		sgRNA ligation products	0.5μl
10UM U-F/gR-R	0.3μl
		10UM T-R/T-F	0.3μl
2mM dNTP	1.5μl
		25mM MgSO4	0.6μl
10*Buffer	1.5μl
		KOD plus	0.3μl
ddH2O	10μl
		total	15μl

TABLE 8 first round PCR reaction procedure

TABLE 9 second round reaction System

Composition (I)	Amount of addition
		U#-T-gRNA	2ul
Primer combination PT	3ul
		2mM dNTP	2μl
10*KOD Buffer	15μl
		KOD polymerase	0.3μl
ddH2O	7.7μl
		total	30μl

TABLE 10 second round reaction procedure

(4) And (5) purifying the nested PCR product. Mixing the five products of the second reaction, and purifying the PCR product by using EasyPure Quick Gel Extraction Kit of Beijing all-terrain gold biotechnologies; the concentration was measured spectrophotometrically and was 25 ng/. mu.l, and 75ng, corresponding to 3. mu.l, was required for the test.

(5) And carrying out enzyme digestion and connection reaction on the binary vector and the sgRNA expression cassette.

The reaction system is shown in Table 11, and the reaction procedure is shown in Table 12.

TABLE 11 restriction enzyme ligation reaction System of binary vector and sgRNA expression cassette

Table 12 restriction ligation procedure for binary vectors and sgRNA expression cassettes

(6) And E.coli sequencing to determine the ligation product. The operation steps are as follows:

a. mu.l of Trans1-T1 Phage resist chemical composition Cell Competent cells thawed on ice bath were taken, 10. mu.l of the ligation product was added, gently mixed and placed in ice bath for 30 minutes.

b.42 ℃ Water bath Heat shock for 30 seconds, then quickly transfer the tube into an ice bath for 2 minutes without shaking the centrifuge tube.

c. Mu.l of sterile liquid LB medium (containing no antibiotics) was added to each tube, mixed well and then placed on a shaker at 37 ℃ for 1 hour at 200rpm to resuscitate the bacteria.

d. 50. mu.l of the transformed competent cells were pipetted onto an agar medium containing LB (25g/L) and the cells were spread out uniformly. The plate was placed at 37 ℃ until the liquid was absorbed, inverted and incubated overnight at 37 ℃.

e. Selecting a single clone, carrying out PCR positive identification, and carrying out the following system, program and primer:

PB-L：GCGCGCgGTctcGCTCGACTAGTATGG，SEQ ID NO.13；

PB-R：GCGCGCggtctcTACCGACGCGTATCC，SEQ ID NO.14；

f. and selecting positive clone for sequencing, and determining that the pBnECH plasmid has no mutation and can be used for subsequent tests.

(8) The constructed vector is transferred into agrobacterium GV3101 electroconceptive cell. The method comprises the following specific steps: a tube of 50 mu lGV3101 electroporation competent cells is placed on ice, 0.1 mu g of the constructed vector plasmid is added after the electroporation competent cells are dissolved, and the mixture is gently sucked and beaten by a pipette and evenly mixed; the competence containing the vector plasmid was pipetted into an ice-precooled electroporation cuvette and electrically shocked at 1800V for 6ms using a Gene pulser electroporation apparatus (available from Bio-Rad, Inc.); adding 500 mul of liquid LB into an electric rotating cup, uniformly mixing, transferring the bacterial liquid into a 2mL centrifuge tube, and culturing for 2hrs in a shaking table at 28 ℃ and at 150 rpm; spreading 100 μ l of the bacterial liquid on a solid LB plate (containing gentamicin 25 μ g/ml and kanamycin 50 μ g/ml), drying, and culturing in an incubator at 28 ℃ for 2-3 days; selecting bacterial plaque to carry out PCR detection, carrying out shake culture and split charging on the positive clone at-80 ℃ for storage or directly using the positive clone for plant transformation, and obtaining the pBnECH vector agrobacterium strain.

3. Genetic transformation of hypocotyl of pBnECH vector

(1) Sterilization

a. Soaking the mature seeds of Jia 9707 in 75% alcohol, and sun drying for 1 min.

b. Transferring the washed seeds into a sterile container (culture box), pouring alcohol into a waste liquid tank, adding an appropriate amount of disinfectant (sterile water with the concentration of 84 disinfectant: 84 liquid: 1), and sterilizing for 10-15 min.

c. After disinfection, the disinfectant is poured into a waste liquid tank, and the seeds are washed for 4-5 times by sterile water (about 50 ml).

(2) Seeding

a. The sterilized seeds were sown to M0 with sterile tweezers, 10-12 seeds per dish.

b. The petri dish was placed in a sterile culture box and incubated in dark at 24 ℃ for 6 days.

(3) Shake the fungus

4-5 days after sowing, the pBnECH vector agrobacterium strain is cultured by using LB liquid medium (Kanamycin 50mg/L + Gentamicin 25 mg/L). The culture was carried out at 28 ℃ and 180 ℃ for about 15 hours on a shaker at 220 rpm. The OD value of the cultured broth is preferably about 0.4 (using a sterile flask or a centrifuge tube, 10/15. mu.l of the broth with two concentrations and 4ml of LB are prepared).

(4) Preparation and infection of explants

a. Preparing bacterial liquid, sucking 2ml of cultured bacterial strain into a sterile 2ml centrifuge tube, centrifuging for 3min at 6000 rpm in a centrifuge, and pouring off the supernatant; resuspend once with the same volume of DM (plus AS) AS the inoculum, centrifuge under the same conditions, discard the supernatant, and resuspend with the same volume of DM (plus AS). The suspension was diluted with 18ml of DM (in sterile petri dishes).

b. Cutting the explant, and cutting hypocotyls of the seedling 6 days after sowing with sterile forceps and dissecting knives, each length of 0.8-1.0 cm. The cutting effect is better in M1 liquid culture medium, and the explants are cut vertically as soon as possible.

c. Placing the cut explant into a culture dish with prepared bacterial liquid with concentration, dip-dyeing for 15min (the time cannot be overlong, the explant is prevented from being seriously dead due to water loss or the agrobacterium can not be inhibited in the later period), shaking once at intervals, and sucking the bacterial liquid when the difference is 3 min. The impregnation is suitably carried out with 150-200 explants per dish (20ml of inoculum).

(5) The infected explants are blotted dry with sterilized filter paper, transferred to M1 culture medium by tweezers, and transferred to M2 after 50-60 explants per dish are cultured in the dark at 24 ℃ for 2 days, and cultured in the white light at 24 ℃ for 16 h/8 h in the dark.

(6) After 20 days, the cells were transferred to M3 and subcultured every 20 days until green shoots appeared.

Transferred into M4 to take root, the rooting time is 2-4 weeks. Transplanting the seedlings into a greenhouse plug tray after the roots grow, covering a plastic film to prevent excessive water loss in the early stage, and uncovering the film after one week. And finally, moving to a field for growing according to the climate.

4. Obtaining mutant plants

Firstly, extracting plant leaf DNA by a CTAB method, amplifying by using a specific sequence primer on a carrier to carry out positive detection, and detecting a product by using 1% agarose gel electrophoresis. The reaction system is carried out on a PCR amplification instrument, and the PCR amplification reaction system comprises the following steps:

the primer sequences were as follows (5 '-3'):

PB-L：GCGCGCgGTctcGCTCGACTAGTATGG，SEQ ID NO.15；

PB-R：GCGCGCggtctcTACCGACGCGTATCC，SEQ ID NO.16。

the detection results are shown in FIG. 2, CK1 is a positive control, and CK2 is a negative control.

And (9) editing and detecting. And selecting a positive individual strain TA clone for sequencing and comparing whether the wild type is mutated or not.

Preparation of PCR products:

1) the following reaction systems were prepared in sterile PCR tubes and amplified separately, with the amplification primers shown in Table 14:

the PCR program was set to denaturation at 98 ℃ for 3 min; 15sec at 98 ℃, 15sec at 59 ℃, 2min at 72 ℃ and 32 cycles; 10min at 72 ℃; 10min at 25 ℃.

TABLE 14 amplification primers

2) Sucking 4ul of PCR product to detect the quality of the product by horizontal gel. If the amplification product has multiple bands, gel recovery of the target fragment is recommended.

3) Then, the target gene fragment is ligated into a PEASY-Blunt vector according to the PEASY-Blunt Simple Cloning kit instruction, and a sequencing intermediate vector is constructed and delivered to sequencing companies for sequencing.

Cloning reaction

1) Sequentially adding 1ul of PEASY-Blunt Simple cloning Vector into a micro-centrifuge tube, adding 4ul of PCR products (which can be properly increased and decreased according to the amount of the PCR products and does not exceed 4ul at most), gently mixing, reacting for 5 minutes at room temperature (27-37 ℃), and placing the centrifuge tube on ice after the reaction is finished.

2) The ligation product was added to 50ul Trans1-T1 competent cells (ligation product was added just after thawing the competent cells), mixed gently, ice-bathed for 20-30 min, heat-shocked in a water bath at 42 ℃ for 30sec, and immediately placed on ice for 2 min. Mu.l of LB medium equilibrated to room temperature was added, and the mixture was incubated at 37 ℃ for 1 hour at 200 rpm. 50 μ L of the aspirated and shaken bacterial solution (depending on the length of the connecting piece) was uniformly spread on an LB plate containing 50 μ g/ml Kan resistance, and cultured overnight in an incubator at 37 ℃ (for more clones, centrifugation at 4000rpm for 1min, discarding part of the supernatant, retaining 100-.

PCR identification of positive clones by bacterial liquid:

1) white single clones were picked into 10ul of sterile water and vortexed.

2ul of mixed solution is taken to be put into a 20ul PCR system, and is amplified by using M13F/M13R universal primers, and the PCR program is set to be 94 ℃ for 6min of denaturation; 30sec at 94 ℃, 30sec at 59 ℃, 30sec at 72 ℃, 35 cycles; 10min at 72 ℃; 10min at 25 ℃. Positive clones were identified by 1% agarose gel electrophoresis.

2) And (4) selecting positive clone bacteria liquid for sequencing. Sequencing was performed with the M13F/M13R universal primer. And after the sequencing is finished, comparing the sequencing result with the sequence analysis corresponding to the wild type by using the sequencher for analysis.

Obtaining mutant plants

A mutant single plant is obtained after editing and detecting the BnECH.A5 gene in a positive plant of a T0 generation strain, and the BnECH.a5 mutant lacks 22 bases in a T2 target point compared with a wild type, and comprises the following steps:

T2 GAAGGGTTGGAATCGAACCGGGG

a9707: TCTTCTAGAAGGGTTGGAATCGAACCGGGGAGTCG

BnECH.a5:TCTTCTAGA----------------------GTCG

Example 3 rape BnECH. A5 mutation can be used to increase oil content in seeds

The mutant plants obtained in example 2 were selfed and generation-added to obtain homozygous mutant plants.

The oil content of the mutant seeds was determined as follows.

Preparation of a sample:

1) drying the mutant and the wild seeds in a constant-temperature incubator at 37 ℃ for 48 hours to ensure the moisture content consistency of the seeds. Taking 50mg of seeds with uniform sizes from each individual plant, weighing and recording the weight for later use;

2) crushing the weighed rape seeds by using a mortar, placing the crushed rape seeds into a clean 10ml glass test tube, and centrifugally throwing the seeds to the bottom of the tube;

3) adding 1.5ml of 2.5% sulfuric acid in chromatographically pure methanol solution (0.01% BHT), 200 μ l of standard sample (heptadecanoic acid triglyceride, 2mg/ml),400 μ l of chromatographically pure toluene into the glass tube, screwing the screw cap, shaking and mixing uniformly;

4) putting the prepared sample into a water bath kettle at 95 ℃ for water bath for 1h (paying attention to checking whether the glass test tube leaks air), taking out, standing and cooling to room temperature;

5) adding 1.8ml 0.9% NaC1 solution, 1ml chromatographically pure hexane, carefully mixing, centrifuging at 1000rpm for 5 min;

6) carefully pipette 600. mu.l of the supernatant into a new vial, and the sample is ready for use.

7) GC analysis is carried out on an Agilent GC6890 gas chromatograph, the temperature rise program is 180 ℃ for 2min, then the temperature is raised to 220 ℃ according to the temperature of 10 ℃/min, and the temperature is kept for 7 min. The capillary column was HP 19091N-133, and the sample size was 1. mu.l per sample. The program was run directly on an autosampler gas chromatograph manufactured by agilent.

Setting parameters of a gas chromatograph: agilent HP7890A, wherein the detector is a hydrogen flame ionization detector, the sample injection is carried out automatically at 1 μ L, the split ratio is set to 1:45, the temperature of the detector is 250 ℃, the temperature of the sample injection chamber is 280 ℃, and the carrier gas is N₂Flow rate of 30mL/min, tail blowing of 40mL/min, H₂The speed is 30mL/min, the air flow rate is 300mL/min, the furnace temperature is set to be continuously increased, and 180 DEG CKeeping at 2min, then raising the temperature to 220 deg.C at 10 deg.C/min, and keeping at 7 min. The fatty acid component is determined by comparing the peak time of each fatty acid after gasification with the peak time of a standard fatty acid, and the fatty acid content is expressed by the peak area percentage.

The results are shown in fig. 3, and the results show that in the bnech.a5 gene mutant, the oil content is up to 39.4%, the oil content of the wild-type material a 9707 under the same conditions is 31.7%, the amplification is up to 24.3%, and the very significant difference is achieved (the P value is 1.69181E-30).

Test results prove that the oil content of the cabbage type rape seeds can be obviously improved after mutation of the cabbage type rape BnECH.A5 gene.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> method for rapidly identifying and regulating rape oil content gene based on genomics approach and application thereof

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