CN116515859A - Preparation and Application of Brassica napus BnaTT18 Gene and Its Mutants - Google Patents

Abstract

The invention discloses preparation and application of a BnaTT18 gene of brassica napus and mutants thereof, wherein 4 homologous copies BnaTT18.A01, bnaTT18.A03, bnaTT18.C01 and BnaTT18.C07 of the BnaTT18 gene are targeted and knocked out through a CRISPR/Cas9 gene editing technology, the brassica napus mutants of BnaTT18 gene mutation are obtained, and compared with a wild type, researches show that compared with the wild type, the brassica napus mutants of a tetrahomozygous mutant strain have reduced procyanidine content in seed coats, yellow and thinned seed coats and reduced lignin content in the seed coats, and can generate a yellow seed phenotype; and the oil content of mature seeds and the ratio of linoleic acid to linolenic acid are obviously increased. Therefore, the BnaTT18 gene can be applied to rape breeding to improve rape seed characters, has huge application potential and prospect, and provides new germplasm resources for rape quality breeding.

Description

Preparation and Application of Brassica napus BnaTT18 Gene and Its Mutants

technical field

The invention belongs to the technical field of molecular breeding of rapeseed, and in particular relates to the preparation and application of the BnaTT18 gene of Brassica napus and its mutant.

Background technique

Brassica napus is the third largest crop in the world after soybean and oil palm, accounting for about 16% of global vegetable oil production. It not only provides edible oil for human diet, but also provides high-quality feed protein for animals, and also provides industrial production Provide raw materials. Therefore, quality improvement has always been one of the important breeding goals of rapeseed. At present, most commercial rapeseed varieties have black seed coats. Compared with black seeds, yellow seeds generally have the advantages of short dormancy period, easy germination, thinner seed coats, and lower shell rate. According to the existing research, the color of the kernel is mainly determined by the color of the seed coat. Although the diploid ancestors of Brassica napus have relatively stable natural resources of yellow seeds, so far, no natural resources of yellow seeds in Brassica napus have been found. The yellow-seeded Brassica napus used in breeding is basically selected through interspecific hybridization of Brassica, which not only has a long cycle, but also may have unfavorable linkage drag. Since Brassica napus is the most important type planted in my country, it will be of great application value to identify important functional genes of yellow seeds in Brassica napus, to study the regulation mechanism of its formation, and to create germplasm resources of yellow seeds and prospects.

Flavonoids are ubiquitous plant secondary metabolites, which are red, blue and purple anthocyanin pigments in plant tissues. Seed coat color in Arabidopsis and Brassica species is mainly caused by the accumulation of flavonols and proanthocyanidins, metabolites of the flavonoid pathway. A series of structural genes encoding enzymes and transporters and regulatory genes encoding transcription factors are involved in the flavonoid metabolic pathway. However, there are no relevant reports on the number of members of TT18 gene in Brassica napus, its gene and cDNA sequence, its relationship with yellow seed traits, and its application in genetic engineering.

Contents of the invention

The purpose of the present invention is to provide the preparation and application of the Brassica napus BnaTT18 gene and its mutant. The present invention targetedly knocks out four homologous copies of the BnaTT18 gene (BnaTT18.A01, BnaTT18.A03, BnaTT18.C01 and BnaTT18.C07 genes) by CRISPR/Cas9 gene editing technology, and obtains Brassica napus with BnaTT18 gene mutation Mutants, the study found that the proanthocyanidin content in the seed coat of the four homozygous mutant lines decreased, the mature seed coat turned yellow, thinned and the lignin content decreased; and the oil content of mature seeds and the unsaturated fatty acid components in them The molar percentages of linoleic acid (C18:2) and linolenic acid (C18:3) were significantly higher than those of the wild type. Therefore, the BnaTT18 gene can be applied to rapeseed breeding to improve rapeseed traits, which has great application potential and prospects, and provides new germplasm resources for rapeseed quality breeding.

In order to achieve the above object, the technical scheme adopted in the present invention is:

One of the objectives of the present invention is to provide Brassica napus BnaTT18 gene, said BnaTT18 gene includes 2 homologous copy genes, respectively: the BnaTT18.A01 gene shown in SEQ ID NO.1, the BnaTT18.A01 gene shown in SEQ ID NO.2 The BnaTT18.A03 gene shown in , the BnaTT18.C01 gene shown in SEQ ID NO.3, and the BnaTT18.C07 gene shown in SEQ ID NO.4.

Further, the cDNA sequence of the BnaTT18.A01 gene is shown in SEQ ID NO.5, the cDNA sequence of the BnaTT18.A03 gene is shown in SEQ ID NO.6, and the cDNA sequence of the BnaTT18.C01 gene is shown in SEQ ID As shown in NO.7, the cDNA sequence of the BnaTT18.C07 gene is shown in SEQ ID NO.8.

The second object of the present invention is to provide a method for preparing a yellow seed mutant of Brassica napus, which is obtained by simultaneous mutation in the gene coding region of the four homologous copy genes.

Further, the four homologous copy genes were knocked out using CRISPR/CAS9 technology.

Further, the following steps are included:

Step 1: obtaining the BnTT18 gene fragment;

Step 2: design sgRNA for the nucleotide sequence of the BnTT18 gene, and use the sgRNA as the target sequence to design and synthesize the forward and reverse oligonucleotide sequences respectively, anneal to form double strands, and make Oligo dimers. The Oligo dimer is connected with the Cas9 vector to construct a plant expression vector;

Step 3: transforming the expression vector constructed in step 2 into a Brassica napus line to obtain a transgenic rape plant;

Step 4: Detecting the transgenic rapeseed plants to determine the genotype;

Step 5: selfing of the transgenic rapeseed plants to obtain four homozygous mutants in which BnaTT18.A01, BnaTT18.A03, BnaTT18.C01 and BnaTT18.C07 genes were simultaneously knocked out.

Further, the sgRNA includes: sgRNA1 as shown in SEQ ID NO.16, sgRNA2 as shown in SEQ ID NO.17 and sgRNA3 as shown in SEQ ID NO.18.

Further, the oligonucleotide sequence includes:

The oligonucleotide sequences sgRNA1-F and sgRNA1-R designed according to sgRNA1 are shown in SEQ ID NO.19-20;

The oligonucleotide sequences sgRNA2-F and sgRNA2-R designed according to sgRNA2 are shown in SEQ ID NO.21-22;

The oligonucleotide sequences sgRNA3-F and sgRNA3-R designed according to sgRNA3 are shown in SEQ ID NO.23-24.

Further, in step 2, the pYLCRISPR/Cas9 multiple genome targeting vector system is used to construct the plant expression vector.

Further, in step 3, the constructed expression vector is transformed into the pure line J9707 of semi-winter Brassica napus.

The third object of the present invention is to provide the application of the Brassica napus BnaTT18 gene in rapeseed breeding.

The fourth object of the present invention is to provide the application of the method for preparing the yellow seed mutant of Brassica napus in rapeseed breeding.

Further, the rapeseed breeding includes: Brassica napus breeding for yellow seeds, thin seed coats, and high oil content in seeds.

Further, the rapeseed breeding also includes: breeding for increasing the proportion of linoleic acid and/or linolenic acid in Brassica napus seeds.

Compared with the prior art, the beneficial effect of the present invention is: the present invention utilizes CRISPR/Cas9 technology to target 4 homologous genes (BnaTT18.A01, BnaTT18.A03, BnaTT18.C01 and BnaTT18.C07) of BnaTT18, through genetic Four homozygous mutants without T-DNA insertion were obtained through transformation and mutant progeny screening, and the phenotype identification and quality analysis of these mutants were carried out. The study found that the proanthocyanidin content in the seed coat of the four homozygous mutants of the BnaTT18 gene decreased, the seed coat turned yellow and thin, and the lignin content of the testa coat decreased, which can produce a yellow seed phenotype; and the oil content of mature seeds and its The proportions of unsaturated fatty acid components linoleic acid (C18:2) and linolenic acid (C18:3) increased significantly. Therefore, the BnaTT18 gene can be applied to rapeseed breeding to improve the traits of rapeseed, which has great application potential and prospect, and provides new germplasm resources for rapeseed quality breeding.

Description of drawings

Fig. 1 is the gene structure diagram of BnaTT18 in the embodiment of the present invention and the vector diagram constructed by CRISPR/CAS9 technology;

Fig. 2 is the nucleotides at the sgRNA1, sgRNA2 and sgRNA3 sites of the four homozygous mutants TT18-127 and TT18-144 of the transformed plant T3 generation in the embodiment of the present invention;

Fig. 3 is the phenotype diagram of wild type and BnaTT18 mutant in the embodiment of the present invention, Fig. 3-A is the mature seed phenotype of four homozygous mutants and wild type, Fig. 3-B is wild type, four homozygous mutants The testa staining results of the homozygous mutant, triple homozygous mutant and double homozygous mutant at different development stages, in which vanillin staining is on the left and DMACA staining is on the right; Fig. 3-C shows wild type, quadruple homozygous mutant, triple Determination results of the total amount of proanthocyanidins in the testa of the homozygous mutants and double homozygous mutants at different developmental stages.

Fig. 4 is the testa development and testa thickness measurement result of wild type and BnaTT18 mutant in the embodiment of the present invention, and Fig. 4-A is the paraffin section of four homozygous mutants and wild type safranin and fast green staining and mature seed Section, Figure 4-B is the testa thickness measurement results of the four-homozygous mutant and wild type, Figure 4-C is the statistical result of the four-homozygous mutant and wild-type seed coat rate, and Figure 4-D is the four-homozygous mutant Lignin content assay results in body and wild-type mature seeds and seed coats;

Fig. 5 is the oil content analysis result of four homozygous mutant BnaTT18 transformed strains without transgenic components in the embodiment of the present invention, Fig. 5-A is the oil content of transformed strains, Fig. 5-B is the fatty acid component mole of transformed strains mass percentage.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example

In this example, CRISPR/Cas9 technology is used to target knockout of four homologous genes of BnaTT18 (BnaTT18.A01, BnaTT18.A03, BnaTT18.C01 and BnaTT18.C07 genes). The principle is shown in Figure 1, where Figure 1A is gray Boxes represent exons, black solid lines represent introns; the four homologous copies of BnaTT18 gene contain 2 exons and 1 intron. Vertical lines in gene models indicate target sites. S1, S2 and S3 show the target sequence, and the underline font indicates the PAM region; Figure 1B is the construction diagram of the BnaTT18 vector. Mutant single plants are obtained through genetic transformation, and homozygous mutants are finally obtained through self-segregation; and the phenotype of the obtained mutants is identified, and the oil content and fatty acids are determined. The specific process is as follows.

1. Gene cloning:

Plant the pure line J9707 of semi-winter rapeseed (seeds come from Rapeseed National Engineering Research Center, Wuhan, China), extract genomic DNA from fresh leaves, use 1% agarose gel electrophoresis to detect DNA quality, and use UV spectrophotometer to detect DNA concentration . Genomic DNA and coding sequences of BnaTT18.A01, BnaTT18.A03, BnaTT18.C01 and BnaTT18.C07 were cloned and isolated from the extracted DNA.

The genome nucleotide sequences of BnaTT18.A01, BnaTT18.A03, BnaTT18.C01 and BnaTT18.C07 genes are respectively shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. The full-length cDNA sequences of BnaTT18.A01, BnaTT18.A03, BnaTT18.C01 and BnaTT18.C07 genes are respectively shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8. The primers used for gene cloning include:

The primer pair for cloning the nucleotide sequence of BnaTT18.A01 genome is BnLDOX-14/BnLDOX-15, and its sequence is shown in SEQ ID NO.9-10;

The primer pair for cloning the nucleotide sequence of BnaTT18.A03 genome is BnLDOX-18/BnLDOX-13, and its sequence is shown in SEQ ID NO.13-14;

The primer pair BnLDOX-16/BnLDOX-17 for cloning the nucleotide sequence of the BnaTT18.C01 genome, the sequence of which is shown in SEQ ID NO.11-12;

The primer pair BnLDOX-13/BnLDOX-19 for cloning the nucleotide sequence of the BnaTT18.C07 genome is shown in SEQ ID NO.14-15.

2. Carrier Construction

The cloned BnaTT18.A01, BnaTT18.A03, BnaTT18.C01 and BnaTT18.C07 genomic DNA and coding sequences were analyzed, and sgRNA1 and sgRNA2 were designed on the copies of BnaTT18.A01 and BnaTT18.C01 using the CRISPR-P program. The sequences were respectively As shown in SEQ ID NO.16 and SEQ ID NO.17; using the CRISPR-P program to design sgRNA2 and sgRNA3 on the copies of BnaTT18.A03 and BnaTT18.C07, the sequences are respectively as SEQ ID NO.17 and SEQ ID NO.18 shown.

Design corresponding forward and reverse oligonucleotide sequences based on sgRNA1, sgRNA2 and sgRNA3, respectively, including:

The oligonucleotide sequences sgRNA1-F and sgRNA1-R designed according to sgRNA1 are shown in SEQ ID NO.19-20;

The oligonucleotide sequences sgRNA2-F and sgRNA2-R designed according to sgRNA2 are shown in SEQ ID NO.21-22;

The oligonucleotide sequences sgRNA3-F and sgRNA3-R designed according to sgRNA3 are shown in SEQ ID NO.23-24.

Two pairs of single-stranded Oligo DNA were annealed to form a double strand to make an Oligo dimer, which was ligated with the CRISPR/Cas9 (pYLCRIPSR/Cas9) vector. The process of vector construction using the pYLCRIPSR/Cas9 multiple genome targeting vector system refers to the relevant literature: Ma (2015b) A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant, 2015b, 8: 1274-1284. The constructed vectors were verified by sequencing.

3. Genetic transformation

Using the Agrobacterium-mediated hypocotyl genetic transformation method, the constructed vector was transferred into the semi-winter Brassica napus pure line J9707. For the specific operation process, refer to: Wu Yudi (2015) Function of the multilocular gene BrCLV3 in Brassica napus Research.

4. Detection of mutants:

(1) Use specific primers PB-L/PB-R to carry out positive identification of transgenes on mutant individual plants, and select positive individual plants containing T-DNA insertion. The PCR system and procedures refer to Zhu Kaiyu (2017) who used CRISPR/CAS9 technology to create multi-locular mutants of Brassica napus [Master's Dissertation]. The primer sequences include:

PB-L: GCGCGCgGTctcGCTCGACTAGTATGG (as shown in SEQ ID NO.25);

PB-R: GCGCGCggtctcTACCGACGCGTATCC (shown in SEQ ID NO. 26).

(2) Use primer premier5 to design primers based on the sequences near the target fragment, and perform blast analysis after the primers are determined to ensure that there are no other homologous sequences.

(3) Perform PCR amplification with designed primers near the target fragment (as shown in SEQ ID NO.27-32). The PCR system and procedures refer to Zhu Kaiyu (2017) who used CRISPR/CAS9 technology to create multi-locular mutants of Brassica napus [Master's Thesis].

Wherein the primer sequence is as follows:

PSH36-11 ggagtgagtacggtgtgcTAATCACTTGGTGTCTTGGGC (SEQ ID

No.27)

PSH36-16 gagttggatgctggatggGCAGGAGAAGAGTTCTTCGG (SEQ ID No. 28)

PSH36-12 gagttggatgctggatggCAAGTCCCACCATCGAC (SEQ ID No. 29)

PSH36-15 ggagtgagtacggtgtgcAAACCGAAGAACTCTTCTCCTG (SEQ ID

No.30)

PSH36-13 ggagtgagtacggtgtgcACATGTAATCAGTTGGTGTCTTAGG (SEQ ID No. 31)

PSH36-17 gagttggatgctggatggCATCGAGTCAGAAGACGAAACC (SEQ ID No. 32)

(4) 1% agarose horizontal electrophoresis to detect the effect of PCR amplification.

(5) HI-TOM sequencing The PCR amplification product was sequenced to determine the genotype of the transgenic plant.

5. Homozygous selfing

The obtained edited single plant of the T0 generation was self-pollinated to produce the T1 generation and the T2 generation, and homozygous mutants were obtained by sequencing the PCR products near the target site, and these homozygous mutations would cause frameshift mutations to produce proteins with loss of function. After PCR sequencing verification, a batch of four homozygous mutant lines TT18-127 and TT18-144 without T-DNA insertion were obtained, and the nucleotides of the sgRNA1, sgRNA2 and sgRNA3 sites are shown in Figure 2.

6. Phenotype observation and determination

Phenotypic observations were performed on the seeds produced by the wild type and BnaTT18 mutants, and the results are shown in Figure 3-A. The testa color of the four homozygous mutants was found to be yellow. However, the seed coats of the triple homozygous mutant, double homozygous mutant and the wild type were all black, showing the black seed phenotype.

The seed coats of wild type and BnaTT18 mutant seeds at different development stages were stained with vanillin and DMACA, as shown in Figure 3-B, it was found that the seed coats of wild type, triple homozygous mutant and double homozygous mutant were 21 days after flowering. The skin was stained red (vanillin staining) and blue (DMACA staining), and the color became darker during seed development; while the seed coat of the four homozygous mutants was less stained.

The total amount of proanthocyanidins in the seed coat of the wild type and the BnaTT18 mutant was further determined at different developmental stages. The results are shown in Figure 3-C. Compared with the wild type, the four homozygous mutants (a ₁ a ₁ a ₃ a ₃ c ₁ c ₁ c ₇ c ₇ ) significantly reduced the accumulation of proanthocyanidins.

7. Microscopic observation of paraffin section

The flowers were marked at the flowering stage, and the seeds were collected 28-49 days after flowering for microscopic observation of paraffin sections. The results are shown in Figure 4-A, and the cross-sections of the seeds stained with saffron fast green at 4 developmental stages (28-49DAF) show that: The palisade layer and the first inner cortex of the BnaTT18 quadruple homozygous mutant were thinner, and the degree of lignification of the seed coat was reduced; the seed coat thickness and coat ratio of the BnaTT18 quadruple homozygous mutant in mature seeds were significantly lower than those of the wild type , the results are shown in Figure 4-B and 4-C; the lignin content was further measured, as shown in Figure 4-D, the lignin content in the seeds and seed coats of the four homozygous mutants of BnaTT18 was also significantly lower than Wild type.

8. Analysis of seed quality traits

The oil content and ratio of the seeds of the wild type and BnaTT18 four homozygous mutants were further analyzed, and the results are shown in Figure 5. The results showed that the oil content of the BnaTT18 four homozygous mutants was significantly increased compared with the wild type, and the 2.00±0.77%, indicating that the mutation of BnaTT18 gene can significantly increase the total oil content of Brassica napus.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention.

Claims (10)

1. The BnatT18 gene of the brassica napus is characterized in that the BnatT18 gene comprises 4 homologous copy genes, which are respectively: the BnaTT18.A01 gene shown in SEQ ID NO.1, the BnaTT18.A03 gene shown in SEQ ID NO.2, the BnaTT18.C01 gene shown in SEQ ID NO.3 and the BnaTT18.C07 gene shown in SEQ ID NO. 4.

2. The brassica napus Bnatto 18 gene of claim 1, wherein the cDNA sequence of the Bnatto 18.A01 gene is shown in SEQ ID NO.5, the cDNA sequence of the Bnatto 18.A03 gene is shown in SEQ ID NO.6, the cDNA sequence of the Bnatto 18.C01 gene is shown in SEQ ID NO.7, and the cDNA sequence of the Bnatto 18.C07 gene is shown in SEQ ID NO. 8.

3. A method for preparing a brassica napus yellow seed mutant, wherein the mutant is obtained by mutating the coding region of 4 homologous copy genes simultaneously occurring genes according to claim 1.

4. A method of preparing a brassica napus yellow seed mutant according to claim 3 wherein the 4 homologous copy genes are knocked out using CRISPR/CAS9 technology.

5. The method for preparing the brassica napus yellow seed mutant according to claim 4, comprising the following steps:

step one: obtaining the BnTT18 gene fragment of claim 1;

step two: designing sgRNA aiming at the nucleotide sequence of the BnTT18 gene, respectively designing and synthesizing forward and reverse oligonucleotide sequences by taking the sgRNA as a target sequence, annealing to form double chains, preparing an Oligo dimer, connecting the Oligo dimer with a Cas9 vector, and constructing to obtain a plant expression vector;

step three: transforming the expression vector constructed in the second step into a cabbage type rape strain to obtain a transgenic rape plant;

step four: detecting transgenic rape plants and determining genotypes;

step five: the transgenic rape plant is selfed to obtain tetrahomozygous mutants with BnaTT18.A01 genes, bnaTT18.A03 genes, bnaTT18.C01 genes and BnaTT18.C07 genes knocked out simultaneously.

6. The method for preparing a brassica napus yellow seed mutant according to claim 5, wherein the sgRNA comprises: an sgRNA1 shown in SEQ ID NO.16, an sgRNA2 shown in SEQ ID NO.17 and an sgRNA3 shown in SEQ ID NO. 18.

7. The method for preparing a brassica napus yellow seed mutant according to claim 6, wherein the oligonucleotide sequence comprises:

oligonucleotide sequences sgRNA1-F and sgRNA1-R designed according to sgRNA1 are shown in SEQ ID NO. 19-20;

oligonucleotide sequences sgRNA2-F and sgRNA2-R designed according to sgRNA2 are shown in SEQ ID NO. 21-22;

oligonucleotide sequences sgRNA3-F and sgRNA3-R designed based on sgRNA3 are shown in SEQ ID NO. 23-24.

8. The method for preparing brassica napus yellow seed mutant according to claim 5, wherein in step two, the plant expression vector is constructed by using a pYLCRISPR/Cas9 multiplex genome targeting vector system.

9. Use of the brassica napus BnaTT18 gene according to claim 1 or 2 in rape breeding.

10. Use of a method for the preparation of a brassica napus yellow seed mutant according to any one of claims 3-8 in rape breeding.