CA3211382A1

CA3211382A1 - Method for site-directed mutagenesis of bnhbbd gene of brassica napus l., and use

Info

Publication number: CA3211382A1
Application number: CA3211382A
Authority: CA
Inventors: Xiaoli Tan; Rui GENG; Keming Zhu; Zheng Wang; Lina DING; Jun Cao; Yulong Li; Yixuan XUE; Yue SHAN; Lei Li
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2021-09-14
Filing date: 2022-08-25
Publication date: 2023-03-23
Also published as: WO2023040623A1; CN113980964A; CN113980964B

Abstract

The present disclosure provides a method for site-directed mutagenesis (SDM) of a BnHBBD gene of Brassica napus L., and a use, and belongs to the technical field of plant gene editing and plant breeding. In the present disclosure, Targetl and Target? are designed and screened for a BnHBBD gene in Brassica napus L., gRNA sequences are designed, and then the Targetl and the Target? are ligated with two identical gRNA sequences respectively to construct a dual-target gene-editing vector pKSE401-BnHBBD-CRISPR', and the dual-target gene-editing vector is transformed into Brassica napus L. to allow SDM of the BnHBBD gene of the Brassica napus L. to obtain a transgenic plant with a long flowering stage, Sclerotinia sclerotiorum resistance, and a silique that is not easy to crack.

Description

METHOD FOR SITE-DIRECTED MUTAGENESIS OF BnHBBD GENE OF BRASSICA
NAPUS L., AND USE
TECHNICAL FIELD
The present disclosure belongs to the technical field of plant gene editing and plant breeding, and specifically relates to a method for site-directed mutagenesis (SDM) of a BnHBBD gene of Brassica napus L., and a use.
BACKGROUND
As one of the most widely planted oil crops in China, Brassica napus L. can be used not only for production of edible oil, but also for ornamentation. Brassica napus L. is one of the major cash crops in China. The biological breeding and seed engineering have developed rapidly. Currently, the breeding means and techniques in China pay more attention to biological breeding, and China will soon set up key special projects for the mining and innovative utilization of agricultural biological germplasm resources, which enhances the innovation ability and improves the independent research and development level.
In the modern society, with the improvement of people's living standards and due to the bright colorful flowers, wide distribution, simple management, and low investment of Brassica napus L., Brassica napus L. has naturally become a farmland landscape crop with a high ornamental value, and the Brassica napus L. tourism has been increasingly popular. The most famous Duotian Brassica napus L. Flower Scenic Area in Jiangsu Xinghua and Brassica napus L.
Flower Sea Scenic Area in Qinghai Menyuan have a ticket revenue of nearly one million in just one day and a comprehensive tourism income of billions (the data are derived from the Jiangsu Provincial People's Government and the Menyuan County People's Government).
Gene editing is an emerging genetic engineering technology that can accurately modify a specific gene in a genome of an organism. In recent years, studies have shown that a gene editing technique can be used to knock out an LNK2 gene in Glycine max L. to affect a flowering time of Glycine max L.; a CRISPR/Cas9 system can be used to acquire an Otyza sativa L.
mutant in which a relationship between pyruvate kinase (PK) and the expression of a cyclin protein is revealed to facilitate the improvement of a grain yield; and the knockout of a plurality of lysophosphatidic acid acyltransferase (LPAT) genes in an allotetraploid of Brassica napus L.
through multiplexed gRNA and single gRNA can change a fatty acid content. The CRISPR/Cas9 system-based SDM
technique has gradually matured, and can greatly shorten an acquisition cycle of a new germplasm.
Currently, when Brassica napus L. grows to a flowering stage in a natural environment, ascospores of Sclerotinia sclerotiorum (S. sclerotiorum) may be transmitted to various parts of Brassica napus L. Among various parts of Brassica napus L., withered petals have the highest bacterium-bearing rate, and hyphae will fall to stems and leaves with the abscission of petals and cause secondary infection to Brassica napus L., resulting in large-area attack of S. sclerotiorum.
In addition, Brassica napus L. also has problems such as easy silique cracking, large rapeseed loss due to mechanized harvest, low harvesting efficiency, and short suitable ornamental flowering stage.
SUMMARY
In view of the deficiencies in the prior art, the present disclosure provides a method for SDM
of a BnHBBD gene of Brassica napus L., and a use. In the present disclosure, a CIRSPR/Cas9 system is used to breed a transgenic plant with a long flowering stage, S.
sclerotiorum resistance, and a silique that is not easy to crack through SDM of a BnHBBD gene of Brassica napus L. In the gene name BnHBBD, Bn represents an English abbreviation of Brassica napus L., and H, B, 13, and D are Chinese pinyin initials of flower (Hua), petal (Ban), No (Bu), and abscission (Diao).
The present disclosure provides a CRISPR/Cas9 system sequence element set for SDM of a BnHBBD gene of Brassica napus L., including U6-26p-Targetl-gRNA, U6-26p-Target2-gRNA, and a Cas9 gene optimized according to a codon, where the U6-26p-Targetl-gRNA includes a promoter U6-26p, a gRNA backbone structure, and Target 1; the U6-26p-Target2-gRNA includes a promoter U6-26p, a gRNA
backbone structure, and Target2;
the BnHBBD gene of Brassica napus L. includes BnHBBD-006 and BnHBBD-A07; the Targetl is a target sequence of the gene BnHBBD-006; and the Target2 is a target sequence of the gene BnHBBD-A07.
The Targetl has a nucleotide sequence: 5'-TACGATGGTTCTGCTCTGTC-3' (SEQ ID NO:
1);
the Target2 has a nucleotide sequence: 5'-TGCAAGAATTGGAGCCACCG-3' (SEQ ID NO:

2); and the gRNA has a nucleotide sequence:
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA
AAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 3).
Further, the BnHBBD-006 corresponds to a nucleotide sequence shown in SEQ ID
NO: 4 and an amino acid sequence shown in SEQ ID NO: 6; and the BnHBBD-A07 corresponds to a nucleotide sequence shown in SEQ ID NO: 5 and an amino acid sequence shown in SEQ ID NO: 7.
The present disclosure also provides a gene-editing vector pKSE401-BnHBBD-CRISPR, including the CRISPR/Cas9 system sequence element set for SDM of a BnHBBD gene of Brassica napus L. described above.

The present disclosure also provides a genetically engineered bacterium (GEB) for SDM of a BnHBBD gene of Brassica napus L., and the GEB is obtained by transforming the gene-editing vector pKSE401-BnHBBD-CRISPR described above into a host bacterium.
The present disclosure also provides a kit for SDM of a BnHBBD gene of Brassica napus L., including the gene-editing vector described above or the GEB described above.
The present disclosure also provides a use of the sequence element set described above, the gene-editing vector pKSE401-BnHBBD-CRISPR described above, the GEB described above, or the kit described above, including:
A) a use in SDM of a gene BnHBBD-006 and/or a gene BnHBBD-A07 of Brassica napus L., where the gene BnHBBD-006 corresponds to a nucleotide sequence shown in SEQ ID
NO: 4 and an amino acid sequence shown in SEQ ID NO: 6, and the gene BnHBBD-A07 corresponds to a nucleotide sequence shown in SEQ ID NO: 5 and an amino acid sequence shown in SEQ ID NO:
7;
B) a use in breeding of Brassica napus L. with a long flowering stage; and/or C) a use in breeding of Brassica napus L. with S. sclerotiorum resistance;
and/or D) a use in breeding of Brassica napus L. with a silique that is not easy to crack.
The present disclosure also provides a method for SDM of a BnHBBD gene of Brassica napus L. with a CIRSPR/Cas9 system, including:
(1) designing and screening Targetl and Target2 for the BnHBBD gene in the Brassica napus L., designing gRNA sequences, and ligating the Targetl and the Target2 with the gRNA sequences respectively to construct a dual-target gene-editing vector pKSE401-BnHBBD-CRISPR;
(2) transforming the gene-editing vector pKSE401-BnHBBD-CRISPR into Agrobacterium GV3101 to obtain Agrobacterium carrying the gene-editing vector pKSE401-BnHBBD-CRISPR;

(3) conducting expanded cultivation to obtain an Agrobacterium bacterial solution, and mediating transformation of a hypocotyl of the Brassica napus L. with the Agrobacterium bacterial solution;

(4) cultivating the hypocotyl of the Brassica napus L., and conducting callus induction, redifferentiation, rooting cultivation, seedling exercise, and transplantation to obtain transgenic Brassica napus L.; and

(5) identifying the transgenic Brassica napus L. in which the BnHBBD gene has undergone a mutation.
The BnHBBD gene of Brassica napus L. includes BnHBBD-006 and BnHBBD-A07; the Target 1 is a target sequence of the gene BnHBBD-006; and the Target2 is a target sequence of the gene BnHBBD-A07;
the Targetl has a nucleotide sequence shown in SEQ ID NO: 1;
the Target2 has a nucleotide sequence shown in SEQ ID NO: 2;

the sgRNA has a nucleotide sequence shown in SEQ ID NO: 3;
the BnHBBD-006 corresponds to a nucleotide sequence shown in SEQ ID NO: 4 and an amino acid sequence shown in SEQ ID NO: 6; and the BnHBBD-A07 corresponds to a nucleotide sequence shown in SEQ ID NO: 5 and an amino acid sequence shown in SEQ ID NO: 7.
The mutation of the BnHBBD gene includes insertion of a T base.
The present disclosure further provides a use of a mutated BnHBBD gene obtained by the method described above in regulation of abscission of a floral organ of Brassica napus L.
Specifically, the use includes: inhibiting normal synthesis of an HBBD protein in Brassica napus L., breeding Brassica napus L. with a long flowering stage, breeding Brassica napus L.
with S. sclerotiorum resistance, and breeding Brassica napus L. with a silique that is not easy to crack.
Compared with the prior art, the present disclosure has the following advantages.
Inflorescence deficient in abscission (IDA) can bind to co-receptors HAE and HSL2 on a membrane, and through phosphorylation and a signaling cascade amplification reaction, an abscission signal is transmitted to an intracellular downstream regulation factor, such that cells in an abscission zone (AZ) are expanded to finally cause abscission of floral organs. However, cells in an AZ of a mutant are no longer expanded, and petals no longer fall off, such that a separation layer between a silique peel and a false dissepiment is affected to some extent, a silique is not easy to crack, and rapeseeds are not easy to fall off, which reduces a loss during mechanized harvest and improves the production efficiency of rapeseeds. In the present disclosure, among five homologous genes of Brassica napus L., two effective genes BnaA07g27400D and BnaC06g29530D in Brassica napus L. that have the highest expression level, are closest to Arabidopsis thaliana (A. thaliana), and can control the abscission of floral organs are identified and named BnHBBD-A07 and BnHBBD-006, and a CIRSPR/Cas9 system is used to conduct SDM
for the genes to obtain a Brassica napus L. germplasm without petal abscission. Since ascospore of S. sclerotiorum can only germinate to produce hyphae on petals and cannot germinate to produce hyphae when directly falling on Brassica napus L. leaves, the non-abscission of petals can prevent S. sclerotiorum from further infecting lower leaves, which can allow S.
sclerotiorum resistance.
The present disclosure successfully applies a gene editing technique to Brassica napus L., which greatly shortens an acquisition cycle of a new germplasm and provides a new idea for Brassica napus L. breeding. A transformant obtained after transforming the gene-editing vector pKSE401-BnHBBD-CRISPR constructed by the present disclosure into Brassica napus L. provides an experimental material for research on a function and an action mechanism of the gene BnHBBD, can also be used as a novel germplasm resource with a long flowering stage, S.
sclerotiorum resistance, and no abscission, and provides a novel gene source for Brassica napus L. breeding, which is conducive to promotion of a progress of agricultural science.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a comparison diagram illustrating differences in nucleotide and amino acid sequences between BnHBBD-A07 and BnHBBD-006.
FIG. 2 shows a schematic diagram illustrating locations of the screened Target 1 and Target2 on a gene (a) and a brief schematic diagram illustrating a pKSE401-BnHBBD-CRISPR plasmid between LB and RB (b), where LB: left boundary; RB: right boundary; Kan:
kanamycin resistance gene; P-CaMV35S: CaMV35 promoter; U6-26p-Targetl-gRNA: gRNA expression element set, including a promoter U6-26p, a gRNA backbone structure, and Targetl; U6-26p-Target2-gRNA:
gRNA expression element set, including a promoter U6-26p, a gRNA backbone structure, and Target2; and Cas9: a Cas9 gene optimized according to a codon.
FIG. 3 is a PCR identification gel pattern of leaf genomes extracted from two positive transformants, where WT: wild type; hbbd-1 and hbbd-2: mutant transgenic plants; +: positive control, pKSE401-BnHBBD-CRISPR plasmid; -: negative control, ddH20; and Marker: Takara DL2000 DNA Marker.
FIG. 4 is a schematic diagram illustrating sequencing results of a gene BnHBBD-A07 (a) and a gene BnHBBD-006 (b) in an hbbd mutant compared with WT.
FIG. 5 is a schematic diagram illustrating analysis results of frameshift mutations caused by T insertion at Targetl in an hbbd mutant, where (a) shows a change of a gene BnHBBD-A07 in the mutant compared with WT and (b) shows a change of a gene BnHBBD-006 in the mutant compared with WT.
FIG. 6 is a comparison diagram illustrating flowering stages of WT (a) and a non-floral organ abscission phenotype of an hbbd mutant (b).
FIG. 7 is a comparison diagram illustrating silique mature stages of non-floral organ abscission phenotypes (hbbd) of three different hbbd mutant strains and WT.
FIG. 8 is a comparison diagram illustrating inflorescence stages of a mutant (hbbd) and WT, where numbers in this figure represent position numbers of Brassica napus L.
inflorescences; and a first flower blossomed from a flower bud is numbered 1, a second flower is numbered 2, and so on.
FIG. 9 is a schematic comparison diagram illustrating pathogenesis pathways of an hbbd mutant and WT infected with S. sclerotiorum under natural conditions.
FIG. 10 is a schematic comparison diagram illustrating incidence of an hbbd mutant and WT
infected with S. sclerotiorum in an incubator environment, where short arrows in (a) and (c) represent inoculation positions of S. sclerotiorum; a long arrow in (b) represents abscission of petals of the WT to leaves; a long arrow + cross in (d) represents non-abscission of petals of the mutant to leaves; and 0 day of post-inoculation (dpi) and 4 dpi represent day 0 and day 4 after inoculation of S. sclerotiorum, respectively.
FIG. 11 is a statistical chart illustrating a number of diseased petals after inoculation with S.
sclerotiorum, where according to Hest, P < 0.001, which indicates a significant difference and is represented by three *.
FIG. 12 is a measurement chart of silique cracking forces of a mutant (hbbd) and WT, where according to t-test, P < 0.05, which indicates a significant difference and is represented by one *.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present disclosure will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present disclosure is not limited thereto.
In the following embodiments, various processes and methods that are not described in detail are conventional methods well known in the art. The sources, trade names, and components needing to be listed of reagents are indicated when the reagents appear for the first time, and unless otherwise specified, the same reagents appearing thereafter are the same as those indicated for the first time. The reagents, materials, or the like involved are commercially available unless otherwise specified.
Media and formulas thereof in the present disclosure are as follows.
Lysogeny broth (LB) liquid medium: 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride were weighed and dissolved in 80 mL of double distilled water (DDW), the resulting solution was diluted to 1 L and then dispensed into 10 Erlenmeyer flasks, and the Erlenmeyer flasks were sealed with a sealing film, autoclaved at 121 C for 15 min, cooled, and stored in a 4 C
refrigerator.
LB solid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of an agar powder were weighed and dissolved in 800 mL of DDW, the resulting solution was diluted to 1 L and then dispensed into 10 Erlenmeyer flasks, and the Erlenmeyer flasks were sealed with a sealing film, autoclaved at 121 C for 15 mm, cooled, and stored in a 4 C
refrigerator. When in use, the LB solid medium was heated and melted in a microwave oven, the resulting liquid was cooled to about 50 C, an antibiotic was added, and the resulting mixture was thoroughly shaken and then immediately poured into sterile petri dishes with about 10 mL for each petri dish.
MO medium: 4.4 g/L of an MS powder and 30 g/L of sucrose were mixed and diluted with DDW, a pH was adjusted to 5.84 to 5.88, 10 g/L of a coagulating agent Agar was added, and the resulting medium was sterilized and dispensed.
DM medium: 4.4 g/L of an MS powder and 30 g/L of sucrose were mixed and diluted with DDW, a pH was adjusted to 5.84 to 5.88, and the resulting medium was sterilized and then cooled;

6 and AS was added at 1 mL/1 L (stock solution: 100 gnol/mL) and the resulting medium was placed in a 4 C refrigerator for later use, or the AS could be added when in use.
M1 medium: 4.4 g/L of an MS powder, 30 g/L of sucrose, 18 g/L of mannitol, 1 mg/L of 2,4-D, and 0.3 mg/L of KT were mixed and diluted with DDW, a pH was adjusted to 5.84 to 5.88, 10 g/L of a coagulating agent Agar was added, and the resulting medium was sterilized and then cooled; and AS was added at 1 mL/1 L (stock solution: 100 iimol/mL) and the resulting medium was placed in a 4 C refrigerator for later use, or the AS could be added when in use.
M2 medium: 4.4 g/L of an MS powder, 30 g/L of sucrose, 18 g/L of mannitol, 1 mg/L of 2,4-D, and 0.3 mg/L of KT were mixed and diluted with DDW, a pH was adjusted to 5.84 to 5.88, 10 g/L of a coagulating agent Agar was added, and the resulting medium was sterilized and then cooled; and 300 mg/L of timentin (TMT), 150 !anion of STS (because a precipitate will appear in STS after being placed for a long time, STS should be prepared just before use), and 25 mg/L
of kanamycin were added, and the resulting medium was then dispensed into sterile petri dishes.
M3 medium: 4.4 g/L of an MS powder, 10 g/L of glucose, 0.25 g/L of xylose, and 0.6 g/L of MES were mixed and diluted with DDW, a pH was adjusted to 5.84 to 5.88, 10 g/L
of a coagulating agent Agar was added, and the resulting medium was sterilized and then cooled;
and then 2 mg/L
of ZT, 0.1 mg/L of IAA, 300 mg/L of TMT, 150 mon of AgNO3, and 25 mg/L of kanamycin were added, and the resulting medium was then dispensed into sterile petri dishes.
M4 medium: 4.4 g/L of an MS powder and 10 g/L of sucrose were mixed and diluted with DDW, a pH was adjusted to 5.84 to 5.88, 8 g/L of a coagulating agent Agar was added, and the resulting medium was sterilized and then cooled; and then 300 mg/L of TMT was added, and the resulting medium was dispensed.
PDA solid medium: 7.4 g of a potato glucose agar medium powder purchased from Sinopharm was weighed, 200 mL of distilled water was added, and the resulting medium was autoclaved at 121 C for 15 min, cooled, and stored in a 4 C refrigerator. When in use, the PDA
solid medium was heated and melted in a microwave oven, the resulting liquid was cooled to about 50 C, an antibiotic was added, and the resulting mixture was thoroughly shaken and then immediately poured into sterile petri dishes with about 20 mL for each petri dish.
Example 1 Identification and acquisition of a BnHBBD gene There are five members of HBBD in Brassica napus L. In the present disclosure, the five member genes were screened through an evolutionary tree and homology comparison according to transcriptome data and bioinformatics analysis to obtain two HBBD genes with the highest expression level and the highest homology, namely, BnaA07g27400D and BnaC06g29530D
(https://www.genoscope.cns.fr/brassicanapus/) (referred to as BnHBBD-A07 and BnHBBD-006 hereinafter). Because the two genes have a high degree of similarity and are different merely in a

7 few bases, it is difficult to distinguish the two genes by an ordinary PCR
method. In this example, BnHBBD-A07 and BnHBBD-006 were distinguished by a sequencing method.
Primers were designed according to coding sequences (CDS sequences, gene Nos.:

BnaA07g27400D and BnaC06g29530D) of the BnHBBD gene on the Brassica napus L.
website (https://www.genoscope.cns.fr/brassicanapus/), and sequences of the primers were as follows:
HBBD-F (SEQ ID NO: 13): ATGGCTCCGTGTCGTACG and HBBD-R (SEQ ID NO: 14): TCAATGAGGATGAGAGTC.
With leaf DNA of a Brassica napus L. variety Y127 (derived from Huazhong Agricultural University) as a template, a CDS sequence of a BnHBBD gene was amplified with a high-fidelity enzyme 2*Phanta MAX Master Mix (purchased from Nanjing Vazyme Biotech Co., Ltd.), and a PCR reaction was shown in Table 1.
Table 1 PCR amplification reaction system with the high-fidelity enzyme PCR system Volume ddH20 20 lit 2*Phanta Max Master Mix 25 pL
Upstream primer (10 [LM) 2 ii,L, Downstream primer (10 M) 2 lit Template DNA (50 ng to 400 ng) 14 A PCR procedure was as follows: predenaturation at 95 C for 3 min;
denaturation at 95 C
for 15 s, annealing at 52 C for 15 s, and extension at 72 C for 30 s, with 35 cycles in total; and final extension at 72 C for 5 min. After the PCR was completed, a PCR product was subjected to gel electrophoresis at 120 V for 30 min in a 2% (mass volume fraction) agarose gel and then imaged by an ultraviolet (UV) gel imager, and results were recorded. The results showed that target fragments amplified by the primers, namely, BnHBBD-A07 and BnHBBD-006 gene fragments, had a size of about 231 bp.
With reference to operating instructions of a UNIQ-10 column gel DNA recovery kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), a PCR amplification product BnHBBD
was recovered from an agarose gel and then ligated with a pMD19-T vector (purchased from Takara Biotechnology (Dalian) Co., Ltd.). A ligation system included 4.5 [tI., of a gel recovery product, 0.5 1AL of a pMD-19T vector, and 5 1.1L of a Solution I (purchased from Takara Biotechnology (Dalian) Co., Ltd.), and ligation was conducted overnight at 16 C to obtain a ligation product.
[tI., of the ligation product was added to 301.IL of competent Escherichia coil (E. coil) cells (purchased from Nanjing Vazyme Biotech Co., Ltd.), and then the ligation product was transformed into E. coil through heat shock; and then positive colonies were screened with an LB
medium including Amp at a final concentration of 30 mg/mL, 10 single colonies were picked and

8 cultivated under shaking for 12 h to 16 h, and 2 L of a bacterial solution was collected and used as a template to conduct PCR amplification for identification. Primers for the PCR were as follows:
M13-F (SEQ ID NO: 15): TGTAAAACGACGGCCAGT
M13-R (SEQ ID NO: 16): CAGGAAACAGCTATGACC.
A PCR amplification reaction system was shown in Table 2; and a PCR procedure was as follows: predenaturation at 94 C for 3 min; denaturation at 94 C for 30 s, annealing at 50 C for 30 s, and extension at 72 C for 1 min, with 28 cycles in total; and final extension at 72 C for 10 min.
Table 2 Bacterial solution PCR amplification reaction system PCR system Volume ddH20 6 pt rTaq 10 pL
Upstream primer (10 M) 1 pt Downstream primer (10 M) 1 [IL
Bacterial solution 2 pt PCR amplification results were obtained by testing on a 2% agarose gel, and test results showed that a DNA fragment obtained was of about 400 bp, indicating successful transformation.
bacterial solutions with successful transformation were selected, and 100 [IL
of each of the bacterial solutions was pipetted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.
Analysis of sequencing results showed that BnHBBD-A07 corresponded to a nucleotide sequence shown in SEQ ID NO: 4 and an amino acid sequence shown in SEQ ID NO: 6; and BnHBBD-006 corresponded to a nucleotide sequence shown in SEQ ID NO: 5 and an amino acid sequence shown in SEQ ID NO: 7.
According to comparison with reference to a sequence list, a nucleotide sequence of BnHBBD-006 was different from a nucleotide sequence of BnHBBD-A07 in a total of 4 bases, and the 4 bases in BnHBBD-006 and BnHBBD-A07 were as follows: position 59: G¨>A, position 129:
T¨>C, position 140: T¨>A, and position 159: C¨>G. The above sequence differences between the two nucleotide sequences led to changes in two amino acids, and the two amino acids in BnHBBD-006 and BnHBBD-A07 were as follows: position 20: N¨>S and position 47: H¨>L. A
schematic comparison diagram was shown in FIG. 1.
Example 2 Construction of a gene-editing vector for SDM of genes BnHBBD-A07 and BnHBBD-006 in Brassica napus L. based on a CRISPR/Cas9 system BnHBBD-A07 and BnHBBD-006 gene sequences were submitted to a website http://cbi.hzau.edu.cn/cgi-bin/CRISPR, targets were screened, and Targetl and Target2 were selected. The Targetl had a sequence of 5'-TACGATGGTTCTGCTCTGTC-3' (SEQ ID NO:
1), and the Target2 had a sequence of 5'-TGCAAGAATTGGAGCCACCG-3' (SEQ ID NO: 2).
The

9 above two target sequences were ligated with 5' termini of two identical gRNA
sequences respectively: [(20 bp target) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA
GTGGCACCGAGTCGGTGCTTTTTTT] (SEQ ID NO: 3). The (20 bp target) indicated a length of each of Targetl and Target2, such that the constructed dual-target gene-editing vector pKSE401-BnHBBD-CRISPR could knock out a target sequence twice to ensure effective editing.
CRISPR/Cas9 vector target primers were designed according to the screened targets, and sequences of the primers were shown in Table 3, which ensured that the designed 2 targets could knock out BnHBBD-A0 7 and BnHBBD-006 simultaneously.
Table 3 CRISPR/Cas9 vector target primers Primer Sequence 5'-3' HBBD-DT1-F0 (SEQ ID NO: 9) TGTACGATGGTTCTGCTCTGTCGTTTTAGAGCTAGAAATAGC
FIBBD-DT2-R0 (SEQ ID NO: 10) AACCGGTGGCTCCAATTCTTGCACAATCTCTTAGTCGACTCTAC
HBBD-DT1-Bs (SEQ ID NO: 11) ATATATGGTCTCGATTGTACGATGGTTCTGCTCTGTCGTT
HBBD-DT2-BsR (SEQ ID NO: 12) ATTATTGGTCTCGAAACCGGTGGCTCCAATTCTTGCACAA
Subsequently, a template entry vector pCBC-DT1T2 (from Professor Hong Dengfeng of Huazhong Agricultural University) was subjected to PCR amplification with the four primers in Table 3. A PCR reaction system was the same as in Table 1; and a PCR procedure was as follows:
predenaturation at 95 C for 3 min; denaturation at 95 C for 15 s, annealing at 52 C for 15 s, and extension at 72 C for 30 s, with 35 cycles; and final extension at 72 C for 5 min. The primers HBBD-DT1-BsF and HBBD-DT2-BsR had a normal concentration of 10 M; and the primers HBBD-DT1-F0 and HBBD-DT2-R0 were diluted 20 times to a concentration of 5 M.
A product of the PCR was purified and recovered, and the product of the PCR had a length of 626 bp. An enzyme digestion-ligation reaction system was established, a specific reaction system was shown in Table 4, and reaction conditions were as follows: keeping at 37 C for 5 h, keeping at 50 C for min, and keeping at 80 C for 10 min.
Table 4 Enzyme digestion-ligation reaction system Component Volume PCR product (626 bp) 2 I., pKSE401 2 [iL

10*NEB T4 Buffer 1.5 iaL
10*BSA 1.5 IAL
Bsa I (NEB) 1 [iL
T4 Ligase (NEB)/high concentration 1 I., ddH20 6 !IL
After the reaction was completed, 51AL of a ligation product was taken to transform competent E. coil DH5a, transformed E. coil was cultivated overnight at 37 C on a solid LB plating medium including 50 mg/mL Kan for screening, and then positive clones were picked and cultivated under shaking for 4 h to 6 h in 400 j.tL of a liquid LB medium including 50 mg/mL
Kan; and 2 tiL of the resulting bacterial solution was taken and used as a template to conduct PCR
amplification for identification. Identification primers were designed and identified with a sequence in a U6 promoter on a pKSE401 vector, an annealing temperature was changed to 57 C, and other PCR
amplification reaction systems and conditions were the same as that for the bacterial solution PCR
amplification reaction in Table 2. Specific primer sequences were as follows:
U626-IDF: TGTCCCAGGATTAGAATGATTAGGC (SEQ ID NO: 17) and U629-IDR: AGCCCTCTTCTTTCGATCCATCAAC (SEQ ID NO: 18).
A fragment obtained after electrophoresis in the PCR identification had a size of 726 bp. 100 1.t1_, of a positive clone bacterial solution with a correct fragment size was taken and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and then forward sequencing primers were designed with a sequence in the U6 promoter on the pKSE401 vector. Primer sequences were as follows:
U626-IDF: TGTCCCAGGATTAGAATGATTAGGC (SEQ ID NO: 17) and U629-IDF: TTAATCCAAACTACTGCAGCCTGAC (SEQ ID NO: 19).
According to sequencing results, a positive clone bacterial solution with the designed Targetl and Target2 was subjected to expanded cultivation, a plasmid was extracted to obtain apKSE401-BnHBBD-CRISPR plasmid, and finally the plasmid was transformed into Agrobacterium GV3101;
and transformed Agrobacterium was subjected to expanded cultivation and preserved for later use.
FIG. 2 shows a schematic diagram illustrating locations of the screened Targetl and Target2 on the gene (a) and a brief schematic diagram illustrating a pKSE401-BnHBBD-CRISPR plasmid between LB and RB (b), where LB: left boundary; RB: right boundary; Kan:
kanamycin resistance gene; P-CaMV35S: CaMV35 promoter; U6-26p-Targetl-gRNA: gRNA expression element set, including a promoter U6-26p, a gRNA backbone structure, and Targetl; U6-26p-Target2-gRNA:
gRNA expression element set, including a promoter U6-26p, a gRNA backbone structure, and Target2; and Cas9: a Cas9 gene optimized according to a codon. The Cas9 gene was derived from a sequence of Streptococcus pyogenes (S. pyogenes) and was optimized based on a Zea mays L.
codon, and the Cas9 gene optimized according to a codon was purchased from http://www.addgene.org,/62202/.
Example 3 Transformation of the gene-editing recombinant vectorpKSE401-BnHBBD-CRISPR into Brassica napus L.
A. Sowing:
In order to quickly acquire a required novel Brassica napus L. germplasm, seeds of Brassica napus L. Y127 that did not require vernalization and could grow fast (the seeds were from Professor Hong Dengfeng of Huazhong Agricultural University) were selected and placed in a 10

11 mL centrifuge tube, alcohol with a volume fraction of 75% was added, the centrifuge tube was inverted up and down, and the seeds were soaked for 1 min; the alcohol was removed by a pipette, and the seeds were rinsed with an appropriate amount of sterile water 3 to 5 times; and a 15%
bleach solution (which was prepared with 8.115 mL of sterile water, 1.875 mL
of sodium hypochlorite, and 10 [LL of Triton) was added, the centrifuge tube was inverted up and down, and the seeds were soaked for 6 min. For heavily-contaminated seeds, a time of the disinfection and sterilization with alcohol could be appropriately extended, but a too-long time would affect the germination of seeds. Then the disinfectant was removed, and the seeds were rinsed with an appropriate amount of sterile water 3 to 5 times, during which the centrifuge tube was inverted up and down each time and an inside of the centrifuge tube was kept in a sterile environment. Finally, the sterile water was removed, burned sterile forceps were used to sow sterilized seeds on an MO
medium with about 25 seeds per bottle, and the seeds were cultivated in the dark light at 24 C for 6 d to obtain Brassica napus L. hypocotyls each with a required length.
B. Bacterial solution preparation:
to 7 days after the sowing, Agrobacterium carrying the pKSE401-BnHBBD-CRISPR
plasmid obtained in Example 2 was cultivated with liquid LB, and a specific cultivation method was as follows: 20 III., of the Agrobacterium carrying the pKSE401-BnHBBD-CRISPR plasmid was added to 5 mL of resistant LB (50 mg/L Kan +50 mg/L Gen +50 mg/L Rif) and cultivated in a shaker at 28 C and 180 rpm to 220 rpm for about 14 h to 16 h.
Because a reproduction rate of Agrobacterium in a medium is related to an activity of Agrobacterium and Agrobacterium at a logarithmic propagation state has the optimal activity and is most likely to infect a plant, an inoculation time should be strictly calculated. The inoculation was repeatedly conducted at an interval of 2 h, for example, the inoculation was conducted at 18:00 and 20:00, and an appropriate concentration was selected at 8:00 the next morning, which could prevent a bacterial concentration from being too high. Before shaking cultivation of bacteria, positive single colonies were picked, inoculated on a resistant plate, and cultivated at 28 C for 48 h until single colonies grew on the plate, and then the single colonies were repeatedly pipetted up and down by a 101.IL pipette tip in the medium to make the bacteria grow evenly.
C. Infection and co-cultivation:
A co-cultivation medium M1 and a DM medium each were prepared. The M1 medium was sterilized at 121 C for 15 min and then quickly cooled (by about 50 C) during which acetosyringone (AS) was added (final concentration: 100 laM); and AS was also added (final concentration: 100jaM) to the DM medium, and the resulting medium was denoted as DM (AS+) for later use.
An OD value of bacteria in the LB medium obtained in step B was measured by a spectrophotometer, and a bacterial solution with an OD value of about 0.4 was selected and

12 generally subjected to shaking cultivation for 14 h to 16 h. 2 mL of a cultivated bacterial solution was pipetted to a sterile centrifuge tube and centrifuged at 3,000 rpm for 3 mm, and the resulting supernatant was discarded; then 2 mL of the DM (AS+) medium was added for suspending, the resulting suspension was centrifuged at 3,000 rpm for 3 min, and the resulting supernatant was discarded; and 2 mL of the DM (AS+) medium was added for suspending, and the resulting suspension was placed in a 4 C refrigerator for later use.
The Brassica napus L. hypocotyl grown after the sowing in step A was cut off by sterile dissecting scissors, cut into 0.8 cm to 1.0 cm segments, and placed in a petri dish with 18 mL of the DM medium; and after the hypocotyl was completely cut into segments, 2 mL
of a bacterial solution resulting from resuspending with the DM (AS+) medium was then poured into the petri dish to a liquid volume of 20 mL to allow infection for 10 min to 15 min (the infection time could not be too long, otherwise the explant was easy to die), during which the petri dish was shaken 4 to 5 times at a specified interval. After the infection was conducted for 8 min, the DM (AS') bacterial solution was removed by a pipette, the explant was transferred by sterile forceps to sterile filter paper and placed for a moment to absorb the excess bacterial solution on the explant, then transferred to an M1 solid medium, and placed in the dark at 24 C or at a dark place in a light cultivation chamber.
D. Selective cultivation and callus induction:
The explant cultivated in the M1 medium for 36 h to 48 h was transferred to an M2 medium, normally cultivated at 24 C under light, and then alternately cultivated with a 16 h light/8 h dark cycle to induce a callus within 2 to 3 weeks.
E. Redifferentiation:
The explant was transferred to an M3 medium and sub-cultivated every 2 to 3 weeks until green shoots appeared.
F. Rooting cultivation:
It took about 20 days for green shoots with intact growth points transferred to an M4 medium to grow and root. Rooted seedlings could be directly placed in a cultivation room for seedling exercise, and after a seedling state was stable, the seedlings were taken out from a medium without destroying a root system of a seedling, then transferred to a soil, and cultivated with the seedlings moisturized by a plastic wrap for 1 to 2 weeks to obtain transgenic Brassica napus L. for identification.
Example 4 Identification of transgenic Brassica napus L. and detection of gene editing sites After the growth of the transgenic Brassica napus L. plant in Example 3 was stable, DNA
was extracted by a cetyltrimethylammonium bromide (CTAB) method from leaves of the transgenic Brassica napus L., and specific steps were as follows:

13 A. A small amount of leaves was collected, added to a 1.5 mL centrifuge tube, and ground with liquid nitrogen into a dry powder, then 6001.tL of CTAB was added, and the resulting sample was incubated in a 65 C water bath for 60 min.
B. After the incubation was completed, 600 III, of a chloroforrn/isoamyl alcohol (in a volume ratio of 24:1) solution was added to the centrifuge tube, and the centrifuge tube was vigorously shaken to thoroughly remove proteins and then centrifuged in a centrifuge at 12,000 g for 10 min.
C. After the centrifugation was completed, the centrifuge tube was gently taken out, where a solution in the centrifuge tube was separated into three layers including an aqueous phase, a leaf fragment impurity phase, and an organic phase; 400 ttL to 500 ttL of the upper aqueous phase was pipetted and transferred to a new centrifuge tube, then 400 tit to 500 [d., of isopropyl alcohol (IPA) was added, and the centrifuge tube was gently inverted up and down for thorough mixing; and the resulting sample was placed in a -20 C refrigerator and cooled for at least 10 min to make the IPA
precipitate DNA effectively.
D. The centrifuge tube was centrifuged in a centrifuge for 10 min at room temperature and 12,000 g.
E. The resulting supernatant was discarded, 700 paL of a pre-cooled ethanol with a volume fraction of 70% was added for washing, a bottom of the centrifuge tube was flicked to make a precipitate floated, and the centrifuge tube was gently inverted up and down and then instantaneously centrifuged at 12,000 g.
F. The resulting supernatant was discarded, the ethanol solution was removed by a pipette, and then the resulting precipitate was air-dried in a clean bench to remove the volatile organic solution.
G. 50 ilL to 1001aL of ddH20 was added to the centrifuge tube to dissolve the precipitate, and the centrifuge tube was placed in a 37 C water bath for 30 min to obtain a genome sample.
H. lIAL of the genome sample was taken and tested for concentration, and if it was tested to be qualified, the genome sample was placed in a -20 C refrigerator for later use.
With the genome sample obtained in the above step as a template, the pKSE401-BnHBBD-CRISPR plasmid as a positive control, and DNA of a recipient material that was not genetically transformed and ddH20 as negative controls, PCR identification was conducted.
Identification primers were designed according to the U6 promoter and Cas9 protein sequence on the pKSE401 vector (2 pairs of primers were simultaneously used to identify a genome of transgenic Brassica napus L. to be identified to ensure a confidence level of a result), annealing temperatures were 57 C and 62 C, respectively, and other PCR amplification reaction procedures and conditions were the same as that for the bacterial solution PCR in Table 2. Primer sequences were as follows:
Primer set 1: length of an amplified fragment: 726 bp U626-IDF: TGTCCCAGGATTAGAATGATTAGGC (SEQ ID NO: 17) and

14 U629-IDR: AGCCCTCTTCTTTCGATCCATCAAC (SEQ ID NO: 18).
Primer set 2: length of an amplified fragment: 701 bp Cas9-F: TGCAGGAGATTTTCTCCAACGA (SEQ ID NO: 20) and Cas9-R: AGCCTTCGTAATCTCGGTGTTCA (SEQ ID NO: 21).
After the PCR was completed, an amplification product was subjected to electrophoresis in a 1% agarose gel and then imaged by a UV gel imager, and results were recorded.
FIG. 3 is a PCR
identification gel pattern of leaf genomes extracted from two positive transformants, where WT:
wild type; hbbd-1 and hbbd-2: mutant transgenic plants; +: positive control, pKSE401-BnHBBD-CRISPR plasmid; -: negative control, ddH20; and Marker: Takara DL2000 DNA
Marker The figure could confirm that the gene-editing vector constructed in Example 3 was successfully transformed into Brassica napus L. Successfully-transformed positive plants were acquired through a plant tissue cultivation process.
In order to further determine a gene editing situation of a positive plant, for a genome of a successfully-transformed positive plant, BnHBBD-A07 and BnHBBD-006 were subjected to PCR
amplification with a high-fidelity enzyme, electrophoresis, gel recovery, and ligation with a pMD19-T vector, the resulting plasmid was transformed into E. coli, transformed E. coli was picked for identification, and a monoclone bacterial solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Specific experimental operations and methods were the same as in Example 1.
Sequencing results were shown in FIG. 4. The sequencing results were analyzed and compared with actual sequencing results of WT BnHBBD-A07 and BnHBBD-006 obtained in Example 1, and it could be found that there was a T base insertion at Targetl in many monoclones.
Thus, the T base insertion was further analyzed, and analysis results were shown in FIG. 5.
FIG. 5 is a schematic diagram illustrating analysis results of frameshift mutations caused by T insertion at Targetl in an hbbd mutant, where (a) shows a change of a gene BnHBBD-A07 in the mutant compared with WT and (b) shows a change of a gene BnHBBD-006 in the mutant compared with WT. It can be seen from the figure that the BnHBBD-A07 and BnHBBD-006 genes of the hbbd mutant both are subjected to frameshift mutations, such that a translation process of an HBBD gene is terminated early, and thus an HBBD protein cannot be synthesized normally, which can confirm that the gene-editing vector successfully conducts its function at targets to successfully knock out the BnHBBD-A07 and BnHBBD-006 genes in Brassica napus L.
Example 5 Analysis of a non-floral organ abscission phenotype of transgenic Brassica napus L.
The qualified hbbd mutant in Example 4 was placed in an incubator with a 16 h light/8 h dark cycle and a relative humidity of 70% and cultivated to a flowering stage; and the WT (Brassica napus L. Y127, which was from Professor Hong Dengfeng of Huazhong Agricultural University) and the mutant were observed, and petal abscission conditions were recorded.
In this experiment, 3 biological replicates were set; and an attachment situation of floral organs referred to a natural abscission situation of the floral organs without being affected by external forces, and a specific attachment time referred to a time from blossoming of flower buds to complete abscission. Results were shown in Table 5.
Table 5 Statistical data of attachment of floral organs Number of Attachment of floral Plant name investigated floral organs (d) organs WT 10 5 0.5 hbbd-1 12 00 hbbd-2 10 oo hbbd-3 11 oo FIG. 6 is a comparison diagram illustrating flowering stages of WT (a) and a non-floral organ abscission phenotype of an hbbd mutant (b). It can be clearly seen that floral organs of the mutant are attached to an AZ. It can be seen from the statistical data in Table 5 that floral organs of the hbbd mutant can continuously exist at a flower bud stage, a first-blooming stage, a fully-blooming stage, a pollination stage, and a mature stage when not undergoing an action of an external force.
FIG. 7 is a comparison diagram illustrating silique mature stages of non-floral organ abscission phenotypes (hbbd) of three different hbbd mutant strains and WT. It can be seen from the figure that a color of floral organs gradually changes from yellow to white, and even at a silique growth stage and a silique mature stage, floral organs continue to exist in the non-floral organ abscission phenotype. FIG. 8 is a comparison diagram illustrating inflorescence stages of an hbbd mutant and WT, where numbers in this figure represent position numbers of Brassica napus L. inflorescences;
and a first flower blossomed from a flower bud is numbered 1, a second flower is numbered 2, and so on. It can be seen from the figure that, when flowers are numbered according to inflorescence positions, a non-floral organ abscission phenotype can be clearly identified.
When ascospores of S. sclerotiorum in nature fall on petals and then fall to leaves or stems with the abscission of WT floral organs, hyphae of S. sclerotiorum begin to grow to produce an infection environment, and in severe cases, sclerotia will be produced in a stem of Brassica napus L., such that the stem becomes hollow due to infection of S. sclerotiorum, which leads to death of the entire plant, causing a great economic loss. FIG. 9 is a schematic comparison diagram illustrating pathogenesis pathways of an hbbd mutant and WT infected with S.
sclerotiorum under natural conditions. It can be seen from the figure that floral organs of the hbbd mutant do not fall off, and thus ascospores falling on the floral organs do not grow.
In this example, the resistance of the non-floral organ abscission phenotype of the hbbd mutant to S. sclerotiorum was also tested in an incubator environment, and a specific test method was as follows: sclerotia of S. sclerotiorum isolated from a test field were inoculated in a PDA
solid petri dish and invertedly cultivated at 28 C for 6 d until hyphae grew to an edge of the petri dish, 0.3 cm * 0.3 cm mycelium at the edge were collected and inoculated to petals of 3 WT plants and 3 mutant plants with 6 petals per plant, and then the inoculated plants were cultivated in an artificial climate chest (purchased from Shanghai Yiheng Instrument Co., Ltd.). In order to simulate natural conditions, conditions for the cultivation were as follows:
temperature: 22 C, humidity: 90%, 12 h weak light/12 h dark cycle, and the growth of hyphae was observed every 12 h. Statistical data of incidence after petals were inoculated with S.
sclerotiorum were shown in Table 6.
Table 6 Statistical data of incidence after petals were inoculated with S.
sclerotiorum Plant name Number of inoculated petals Number of diseased petals after inoculation hbbd-1 6 1 hbbd-2 6 0 hbbd-3 6 0 Table 6 showed the statistical data of incidence after petals were inoculated with S.
sclerotiorum, and it could be seen that, after the hbbd mutant and the WT were infected with S.
sclerotiorum, inoculated petals of the WT were basically diseased, while inoculated petals of the hbbd mutant were basically not diseased.
FIG. 10 is a schematic comparison diagram illustrating incidence of an hbbd mutant and WT
infected with S. sclerotiorum in an incubator environment, where short arrows in (a) and (c) represent inoculation positions of S. sclerotiorum; a long arrow in (b) represents abscission of petals of the WT to leaves; a long arrow + cross in (d) represents non-abscission of petals of the mutant to leaves; and 0 dpi and 4 dpi represent day 0 and day 4 after inoculation of S. sclerotiorum, respectively. It can be seen from the figure that petals of the WT fall off, S. sclerotiorum that has begun to grow on the petals is very likely to fall off with the petals and is attached to leaves, and the continuous infection of S. sclerotiorum causes the rot of the leaves, resulting in a heavy disease;
and floral organs of the hbbd mutant do not fall off, and are at a top layer of a plant with low humidity and excellent ventilation, such that S. sclerotiorum is not easy to grow, the disease is not developed, and the plant grows normally and will not be infected by S.
sclerotiorum to die.
FIG. 11 is a statistical chart illustrating a number of diseased petals after inoculation with S.
sclerotiorum, where according to t-test, P < 0.001; and it can be seen that a number of diseased petals of the hbbd mutant is significantly lower than a number of diseased petals of the WT.
In this example, silique cracking forces of the hbbd mutant and WT were also tested, and specific test steps were as follows: 40 days after flowering of the WT and hbbd mutant, a total of mature siliques were collected, placed in an environment with a temperature of 25 C and a humidity of 50% for one week, and then glued by a glue to a thin plate, where a plane in which a false dissepiment of a Brassica napus L. silique was parallel to a plane of the thin plate, a tail of a silique was aligned with an edge of the thin plate, and a handle of a silique was outside the thin plate. With a TA.XT Plus texture analyzer (Stable Micro System, UK), an L-hook was fixed on a probe, and then used to hook and fix a base of a silique in a direction perpendicular to the thin plate at a junction between a silique and a handle of a silique on the thin plate. During a measurement, the thin plate was pressed by hands, the probe was allowed to move upwards at a uniform speed of 1 mrn/min and then move upwards at a uniform speed of 0.5 mm/min when touching a handle of a silique, the silique was pulled off, and pull crack force data of the WT and mutant were recorded.
Before cracking of a silique, a force received by the silique continues to increase; and after cracking of the silique, a force received by the silique suddenly decreases. A
peak of the force received by the silique is a maximum pull crack force of the silique; and the larger the peak, the greater the crack resistance of the silique. FIG. 12 is a measurement chart of silique cracking forces of the mutant (hbbd) and WT, and it can be seen from the figure that a maximum pull crack force of a silique of the WT is about 0.3 N to 0.5 N and a maximum pull crack force of a silique of the mutant is about 0.6 N to 0.8 N, where according to t-test, P < 0.05; and a pull crack force of the mutant is significantly larger than a pull crack force of the WT, that is, the cracking resistance of the silique of the mutant is enhanced.
The above experimental results can indicate that an HBBD protein in Brassica napus L. is also one of the important proteins to regulate the abscission of floral organs, which provides a resource for allowing flowering stage extension, S. sclerotiorum resistance, and mechanized harvest.
The basic principles, main features, and advantages of the present disclosure are shown and described above. It should be understood by those skilled in the art that the present disclosure is not limited by the above examples, and the above examples and the description only illustrate the principle of the present disclosure. Various changes and modifications may be made to the present disclosure without departing from the spirit and scope of the present disclosure, and such changes and modifications all fall within the claimed scope of the present disclosure.
The claimed protection scope of the present disclosure is defined by the appended claims and equivalents thereof.

Claims

What is claimed is:

1. A CRISPR/Cas9 system sequence element set for site-directed mutagenesis of a BnHBBD
gene of Brassica napus L., characterized by comprising U6-26p-Targetl-gRNA, U6-26p-Target2-gRNA, and a Cas9 gene optimized according to a codon, wherein the U6-26p-Target1 -gRNA
comprises a promoter U6-26p, a gRNA backbone structure, and Targetl; the U6-26p-Target2-gRNA comprises a promoter U6-26p, a gRNA backbone structure, and Target2; and the BnHBBD gene of Brassica napus L. comprises BnHBBD-006 and BnHBBD-A07; the Target 1 is a target sequence of the gene BnHBBD-006; and the Target2 is a target sequence of the gene BnHBBD-A07.

2. The CRISPR/Cas9 system sequence element set for the site-directed mutagenesis of the BnHBBD gene of the Brassica napus L. according to claim 1, characterized in that the Targetl has a nucleotide sequence shown in SEQ ID NO: 1;
the Target2 has a nucleotide sequence shown in SEQ ID NO: 2; and the gRNA has a nucleotide sequence shown in SEQ ID NO: 3.

3. The CRISPR/Cas9 system sequence element set for the site-directed mutagenesis of the BnHBBD gene of the Brassica napus L. according to claim 1, characterized in that, the BnHBBD-006 corresponds to a nucleotide sequence shown in SEQ ID NO: 4 and an amino acid sequence shown in SEQ ID NO: 6; and the BnHBBD-A07 corresponds to a nucleotide sequence shown in SEQ ID NO: 5 and an amino acid sequence shown in SEQ ID NO: 7.

4. A gene-editing vector pKSE401-BnHBBD-CRISPR, characterized by comprising the CRISPR/Cas9 system sequence element set for the site-directed mutagenesis of the BnHBBD gene of the Brassica napus L. according to any one of claims 1 to 3.

5. A genetically engineered bacterium for site-directed mutagenesis of a BnHBBD gene of Brassica napus L., characterized in that the genetically engineered bacterium is obtained by transforming the gene-editing vector pKSE401-BnHBBD-CRISPR according to claim 4 into a host bacterium.

6. A kit for site-directed mutagenesis of a BnHBBD gene of Brassica napus L., characterized by comprising the gene-editing vector according to claim 4 or the genetically engineered bacterium according to claim 5.

7. A use of the sequence element set according to any one of claims 1 to 3, the gene-editing vector pKSE401-BnHBBD-CRISPR according to claim 4, the genetically engineered bacterium according to claim 5, or the kit according to claim 6, characterized by comprising:
A) a use in site-directed mutagenesis of a gene BnHBBD-006 and/or a gene BnHBBD-A07 of Brassica napus L., wherein the gene BnHBBD-006 corresponds to a nucleotide sequence shown in SEQ ID NO: 4 and an amino acid sequence shown in SEQ ID NO: 6, and the gene BnHBBD-A07 corresponds to a nucleotide sequence shown in SEQ ID NO: 5 and an amino acid sequence shown in SEQ ID NO: 7;
B) a use in breeding of Brassica napus L. with a long flowering stage; and/or C) a use in breeding of Brassica napus L. with Sclerotinia sclerotiorum resistance; and/or D) a use in breeding of Brassica napus L. with a silique that is not easy to crack.

8. A method for site-directed mutagenesis of a BnHBBD gene of Brassica napus L. with a CIRSPR/Cas9 system, characterized by comprising:
(1) designing and screening Targetl and Target2 for the BnHBBD gene in the Brassica napus L., designing gRNA sequences, and ligating the Targetl and the Target2 with the gRNA sequences respectively to construct a dual-target gene-editing vector pKSE401-BnHBBD-CRISPR;
(2) transforming the gene-editing vector pKSE401-BnHBBD-CRISPR into Agrobacterium GV3101 to obtain Agrobacterium carrying the gene-editing vector pKSE401-BnHBBD-CRISPR;
(3) conducting expanded cultivation to obtain an Agrobacterium bacterial solution, and mediating transformation of a hypocotyl of the Brassica napus L. with the Agrobacterium bacterial solution;
(4) cultivating the hypocotyl of the Brassica napus L., and conducting callus induction, redifferentiation, rooting cultivation, seedling exercise, and transplantation to obtain transgenic Brassica napus L.; and (5) identifying the transgenic Brassica napus L. in which the BnHBBD gene has undergone a mutation.

9. The method according to claim 8, characterized in that the BnHBBD gene of the Brassica napus L. comprises BnHBBD-006 and BnHBBD-A07; the Targetl is a target sequence of the gene

BnHBBD-006; the Target2 is a target sequence of the gene BnHBBD-A07;
the Targetl has a nucleotide sequence shown in SEQ ID NO: 1;
the Target2 has a nucleotide sequence shown in SEQ ID NO: 2;
the gRNA has a nucleotide sequence shown in SEQ ID NO: 3;
the BnHBBD-006 corresponds to a nucleotide sequence shown in SEQ ID NO: 4 and an amino acid sequence shown in SEQ ID NO: 6; and the BnHBBD-A07 corresponds to a nucleotide sequence shown in SEQ ID NO: 5 and an amino acid sequence shown in SEQ ID NO: 7.10. The method according to claim 8, characterized in that the mutation of the BnHBBD gene comprises insertion of a T base.

11. A use of a mutated BnHBBD gene obtained by the method according to any one of claims 8 to 10 in regulation of abscission of a floral organ of Brassica napus L.

12. The use according to claim 11, characterized in by comprising: inhibiting normal synthesis of an HBBD protein in Brassica napus L., breeding Brassica napus L. with a long flowering stage, breeding Brassica napus L. with Sclerotinia sclerotiorum resistance, and breeding Brassica napus L. with a silique that is not easy to crack.