Rapeseed gene BnBPR1 and its application in clubroot disease control

Abstract

The present invention belongs to the field of plant biotechnology. Specifically, the present invention provides rapeseed BnBPR1 Gene and its application in rapeseed clubroot resistance. The present invention uses gene editing technology to knock out two homologous copies in rapeseed. BnaA10.PBR1 and BnaC09.PBR1 , obtained BnBPR1 The homozygous double mutant of the gene mutation and the analysis of its disease resistance function revealed that the gene had a negative regulatory effect on rapeseed resistance to clubroot, which clearly indicated that it could be used to BnBPR1 Two homologous copies of a gene BnaA10.PBR1 and BnaC09.PBR1 Gene editing is performed to obtain a new material of homozygous double mutant transgenic rapeseed with enhanced resistance to clubroot. The discovery of the present invention has important significance and application value for the prevention and treatment of rapeseed clubroot, and can be used in the breeding of new rapeseed varieties resistant to clubroot.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and particularly relates to a Brassica napus BnBPR1 gene and an application thereof in the prevention and control of rapeseed clubroot disease. Background Art

[0002] Clubroot is caused by Brassicae root knot fungi, which can specifically harm cruciferous crops and is a soil-borne disease. Clubroot mainly affects the normal growth of plant roots. Tumor-like swollen tubers form at the roots of diseased plants, causing a 20%-30% reduction in the yield of cruciferous crops each year, and up to 60% or more in severe cases. Compared with traditional agricultural prevention and control methods, breeding clubroot-resistant varieties is the most green, environmentally friendly and economically feasible measure. Among the Brassica germplasm resources, most of the resistant materials discovered so far are from turnip, and the earliest resistance loci used in Brassica napus also come from turnip. In the early days, researchers synthesized Brassica napus through artificial interspecific hybridization, and then introduced the disease-resistant loci in turnip (ECD04), and finally selected the clubroot-resistant variety Mendel. At present, domestic researchers have also bred new clubroot-resistant varieties such as Huayouza 62R, Kenyouza 741R and Huayouza 706R by introducing the disease-resistant loci in turnip into Brassica napus. The clubroot-resistant varieties of Brassica napus currently on the market are all bred by introducing exogenous clubroot-resistant genes. However, the introduction of exogenous genes may be accompanied by some adverse traits, which ultimately affect the quality and agronomic traits of the crop, and the breeding cycle is relatively long. Among the clubroot-resistant loci that have been reported, such as Crr1, Crr2, Crr4, CRd in cabbage, and CRb and Crr3 in turnip, only a very small number of genes, such as CRb, have been applied to the resistance improvement of Brassica napus. There is a lack of rapeseed disease-resistant genes and resistance source materials that can be used for resistance improvement. Therefore, it is crucial to discover new rapeseed clubroot-resistant genes and create new rapeseed clubroot-resistant materials for the prevention and control of rapeseed clubroot and the breeding of disease-resistant varieties.

[0003] Genetic variation is the driving force behind molecular crop breeding, and the rise of CRISPR gene editing technology provides a more efficient way to create genetic variation in crops. CRISPR technology provides a rapid way to produce ideal germplasm, either by deleting negative regulatory genetic elements that cause undesirable traits, or by introducing gain-of-function mutations through precise genome editing. In recent years, with the rapid development of genome editing technology, the progress of crop genetic breeding has been greatly accelerated, and this technology has been widely used in improving the yield, quality and resistance of various crops such as rice, wheat and corn. Combining gene editing technology with rapeseed transgenic technology, creating clubroot-resistant germplasm and resistant varieties is an economical, effective and safe way to green disease prevention and control. Summary of the invention

[0004] Identifying key clubroot resistance regulatory genes and creating and rationally utilizing highly resistant materials through biotechnology are crucial for the prevention and control of rapeseed clubroot and green development. Based on this, through extensive and in-depth research, the present invention discovered the disease resistance function application of rapeseed BnBPR1 gene.

[0005] In one aspect of the present invention, a Brassica napus clubroot resistance gene BnBPR1 is provided, wherein the BnBPR1 gene comprises two homologous copies, namely, BnaA10.PBR1 gene and BnaC09.PBR1 gene; wherein the nucleotide sequence of BnaA10.PBR1 is shown in SEQ ID NO.1, and the nucleotide sequence of BnaC09.PBR1 is shown in SEQ ID NO.2.

[0006] In one aspect of the present invention, the present invention provides an application of the BnBPR1 gene in the prevention and control of root knot fungi. The two homologous copies of the rapeseed BnBPR1 gene, BnaA10.PBR1 and BnaC09.PBR1, are knocked out through gene editing technology to form a homozygous double mutant, and the expression of the BnBPR1 gene is downregulated to prevent and control rapeseed root knot disease.

[0007] In one aspect of the present invention, a method for preparing clubroot-resistant rapeseed is provided, the method comprising knocking out two homologous copies BnaA10.PBR1 and BnaC09.PBR1 of the rapeseed BnBPR1 gene through gene editing technology to obtain a new homozygous double mutant transgenic rapeseed material with enhanced clubroot resistance.

[0008] In one embodiment of the present invention, a rapeseed material resistant to clubroot disease is obtained by obtaining a homozygous double mutation transgene in rapeseed, and the method is achieved by the following steps:

[0009] (1) Designing sgRNAs that can simultaneously target BnaA10.PBR1 and BnaC09.PBR1, and cloning the sequences into CRISPR / CAS9 vectors; wherein the sgRNA sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, the nucleotide sequence of BnaA10.PBR1 is shown in SEQ ID NO.1, and the nucleotide sequence of BnaC09.PBR1 is shown in SEQ ID NO.2;

[0010] (2) introducing the knockout vector CRISPR / CAS9-BnBPR1 containing sgRNA in step (1) into the Agrobacterium strain GV3101 with strong infection ability to rapeseed by electroporation;

[0011] (3) transferring the Agrobacterium vector into rapeseed by a genetic transformation method in which Agrobacterium infects the hypocotyl of rapeseed to obtain transgenic rapeseed plants;

[0012] (4) Testing the transgenic plants obtained above, screening and identifying transgenic plants with two copies successfully edited and homozygous genotypes.

[0013] The present invention uses the Westar variety as the background, and knocks out two homologous copy genes (BnaA10.PBR1 and BnaC09.PBR1) of BnBPR1 in rapeseed by CRISPR / CAS9 technology to obtain a homozygous double copy mutant strain, and the specific mutation types are shown in Table 1. Compared with the non-transgenic wild type, the homozygous double mutant has enhanced resistance to clubroot and is more resistant to root-knot fungi, indicating that BnBPR1 strongly negatively regulates rapeseed's resistance to root-knot fungi.

[0014] Advantages of the present invention

[0015] The rapeseed BnBPR1 gene provided by the present invention is a high-quality anti-clumproot disease regulating gene resource. The homozygous double mutant obtained by knocking out the two homologous copies of the gene, BnaA10.PBR1 and BnaC09.PBR1, has significantly enhanced resistance to root-root fungi. The BnBPR1 gene belongs to an endogenous gene of cabbage-type rapeseed. The disease-resistant material obtained by using the gene has the advantages of strong disease resistance and avoiding the introduction of exogenous resistance genes that may carry unfavorable gene fragments. Traditional breeding methods have the disadvantages that disease-resistant resources are limited by natural genetic isolation, the breeding cycle is long, and it is time-consuming and laborious. The genetic engineering breeding method using disease-resistant regulatory genes has the advantages of more extensive disease-resistant resources, simple operation, short cycle, economy, and suitable for cultivating broad-spectrum, durable, and highly disease-resistant varieties. Combining gene editing technology and rapeseed transgenic technology, using the BnBPR1 gene to create root-root disease-resistant germplasm and resistant varieties is an economical, effective and safe way to green disease prevention and control, which lays a foundation for the prevention and control of rapeseed root root disease and the breeding of rapeseed disease-resistant varieties.

[0016] The present invention utilizes the BnBPR1 gene of Brassica napus and adopts genetic engineering methods to create rapeseed materials with high resistance to clubroot, which has the characteristics of short cycle and rapid breeding, and at the same time lays the foundation for the research on the function of the BnBPR1 gene of rapeseed and the research on the immune and antibacterial mechanism of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The sgRNA sequences and their targeting sites in the embodiments of the present invention are capable of simultaneously targeting the two homologous copies of the rapeseed BnBPR1 gene, BnaA10.PBR1 and BnaC09.PBR1. sgRNA1 targets the third exon of BnaA10.PBR1 and the fourth exon of BnaC09.PBR1; sgRNA2 targets the fourth exon of BnaA10.PBR1 and the fifth exon of BnaC09.PBR1.

[0018] Figure 2 These are the results of inoculating the wild type and the BnBPR1 double copy mutant (pbr1#1 and pbr1#2) with root knot fungi in the examples of the present invention. Figure 2 A in the middle shows the root phenotype observation results after 30 days of inoculation with root knotweed bacteria; Figure 2 Middle B is the quantitative analysis of the amount of root knotweed bacteria in the roots of wild-type and BnBPR1 double-copy mutant plants 30 days after inoculation; Figure 2 Middle C shows the morphological structure of epidermal cells of wild type and BnBPR1 double copy mutant before and after inoculation. The results showed that compared with the wild type, the resistance of BnBPR1 double copy mutant plants to root knot fungi was significantly enhanced 30 days after inoculation, with no obvious swelling of the roots and the regular arrangement of epidermal cells intact, while the roots of the wild type material showed obvious swelling and irregular swelling of the root epidermal cells. DETAILED DESCRIPTION

[0019] Unless otherwise specified, the relevant reagents in the present invention are all conventional reagents, and the relevant operation steps are all conventional operations, which can be performed according to the operating instructions of the reagent supplier or the guidance of classic reference books such as "Molecular Cloning Experiment Guide".

[0020] Embodiment 1:

[0021] The present invention cloned a gene BnBPR1 related to clubroot resistance in Brassica napus. By obtaining transgenic rapeseed for the first time, the regulation function of the gene in clubroot resistance was clarified, and it was revealed that the gene had a negative regulation function on clubroot resistance in rapeseed. The mutation of the BnBPR1 gene significantly enhanced the resistance of rapeseed to clubroot. Therefore, a knockout vector of the gene can be constructed and a new material of rapeseed resistant to clubroot can be created and obtained by using genetic engineering technology. The main steps include:

[0022] 1.1 CRISPR / Cas9 vector construction

[0023] Combining the rapeseed BnPIR database and the CRISPR / P target design online website, sgRNAs (SEQ ID NO.3 and SEQ ID NO.4) that can simultaneously target BnaA10.PBR1 and BnaC09.PBR1 were designed. Two pairs of conservative primers were designed based on the target sequence information, and product fragments carrying the two target sequences were amplified from the intermediate vector pCBC-DT1DT2. After the fragments were recovered, they were cleaved by BsaI restriction endonuclease (Thermo Scientific TM ) and T4 ligase (Thermo Scientific TM ) was connected to the CRISPR / CAS9 vector, and after PCR verification and sequencing comparison, the knockout vector CRISPR / CAS9-BnBPR1 of the BnBPR1 gene was obtained.

[0024] 1.2. Agrobacterium transformation with CRISPR / CAS9-BnBPR1 vector

[0025] The CRISPR / CAS9-BnBPR1 vector carrying the sgRNA target sequence was transformed into the Agrobacterium strain GV3101, which has strong infectivity to rapeseed, by electroporation. Positive strains were screened on LB medium containing kanamycin and rifampicin antibiotics at 28°C. Positive single colonies were shaken and stored in a -80°C refrigerator with 30% glycerol for the next step of rapeseed genetic transformation.

[0026] 1.3. Obtaining the BnBPR1 mutant of Brassica napus

[0027] (1) Seed disinfection and sowing

[0028] Select full Westar rapeseed seeds, sterilize them with 75% alcohol under aseptic conditions, wash them with sterile water for 5-6 times, and discard the washing liquid. Sow the sterilized seeds in MS medium and culture them at 25℃ for 5-7 days.

[0029] (2) Agrobacterium infection and co-cultivation

[0030] Before infection, the target strain was cultured in kanamycin LB liquid medium, and cultured at 28℃ and 180r / min for about 20h-24h, so that the Agrobacterium cultured to the bacterial liquid OD600 = 0.6. Take out the rapeseed seedlings that have been cultured for 5-7 days, cut the seedling hypocotyls vertically with sterile tweezers and scalpels, cut off the cotyledons and roots of the seedlings, leaving only the middle part of the hypocotyls, and cut into small sections with a length of 0.8-1.0cm; place the cut explants in a dish containing bacterial liquid and immerse for about 8-15min. After the infected explants are dried with filter paper, the surface liquid is transferred to M1 medium and cultured in the dark at 25℃ for 36-48h.

[0031] (3) Selective culture and callus induction

[0032] The hypocotyl explants after co-cultivation were transferred to M2 medium for inducing callus, which contained timentin and selection antibiotics to inhibit the growth of Agrobacterium, and cultured under light in a tissue culture room at 25°C with 16 h light / 8 h darkness for 2-3 weeks.

[0033] (4) Induction of differentiation and bud regeneration

[0034] The explants with normal growth and swelling at both ends were transferred to differentiation M3 medium for light culture, and subcultured every 2-3 weeks until green buds appeared. The conditions of differentiation culture were the same as those of selection culture.

[0035] (5) Bud elongation and rooting

[0036] After the callus of the hypocotyl segment has differentiated and obvious growth points can be seen, the young shoots are carefully cut from the callus with sterile tweezers and scalpels and transferred to M4 medium for rooting culture. After rooting, the seedlings can be grown in a light incubator and then transplanted to the transgenic test field.

[0037] (6) Screening and identification of homozygous mutants of BnBPR1 in Brassica napus

[0038] Genomic DNA was extracted from leaves at the seedling stage, and positive plants were detected by PCR amplification. Primers for editing detection were designed, and positive plant DNA was used as a template for amplification. The editing situation was analyzed after sequencing to obtain BnBPR1 double-copy homozygous mutant plants.

[0039] 1.4. Disease resistance detection of BnBPR1 double-copy homozygous mutant

[0040] The double-copy homozygous mutant of the BnBPR1 gene obtained above was used as the material. By inoculating with root-knot fungi, its resistance to rapeseed clubroot was detected and analyzed, thereby clarifying the regulatory role of the BnBPR1 gene on rapeseed clubroot resistance and whether it is possible to knock out the two homologous copies of the rapeseed BnBPR1 gene, BnaA10.PBR1 and BnaC09.PBR1, through gene editing technology to obtain new transgenic rapeseed materials with homozygous double mutants and enhanced resistance to clubroot.

[0041] (1) Preparation of inoculum

[0042] Take an appropriate amount of clubroot-infected tumors stored in the laboratory, cut them into pieces, add an appropriate amount of sterile water, put them into a wall-breaking machine to break them, collect the zoospores, count them with a hemocytometer, and adjust the bacterial solution concentration to 1×10 7 / mL, stored at 4℃.

[0043] (2) Inoculation of root knot fungi

[0044] Take rapeseed with good growth and inoculate it at the 4-5 leaf stage. When inoculating, 6 mL of the bacterial solution prepared in step (1) is inoculated around the roots of each plant. Water the roots in a timely manner after inoculation with root-knot bacteria. After 30 days, wash the roots repeatedly with water to observe the phenotype, and quantitatively analyze the amount of root-knot bacteria at the roots of the material. At the same time, take the diseased roots and uninoculated roots after inoculation with wild-type and pbr1 mutant root-knot bacteria to prepare frozen sections, and observe cell changes with an inverted fluorescence microscope. The results showed that compared with the wild type, the BnBPR1 double copy mutant plants had significantly enhanced resistance to root-knot bacteria 30 days after inoculation, with no obvious swelling of the roots and the regular arrangement of the epidermal cells not damaged, while the roots of the wild-type material showed obvious swelling, and the root epidermal cells were irregularly swollen ( Figure 2). The deletion mutation of the BnBPR1 gene leads to a significant increase in rapeseed's resistance to clubroot. Therefore, BnBPR1 plays a strong negative regulatory role in rapeseed's resistance to clubroot.

[0045] See Table 1 for the detection results of the editing of the homozygous mutation of the double copy of BnBPR1 in the T1 generation of the transformed plant in the embodiment of the present invention. The results show that BnaA10.PBR1 of pbr1#1 has a deletion mutation of 8 bases at sgRNA1, BnaC09.PBR1 has an insertion of A base and T base at sgRNA1, and the sgRNA2 target has an insertion of A base at sgRNA2 of BnaA10.PBR1 and BnaC09.PBR1; BnaA10.PBR1 and BnaC09.PBR1 of pbr1#2 have an insertion of A mutation at sgRNA1, and BnaA10.PBR1 and BnaC09.PBR1 of pbr1#2 have an insertion of A base at sgRNA2. sgRNA targeted the genes BnaA10.PBR1 and BnaC09.PBR1, causing the changes in base insertion or deletion described above, resulting in changes in the encoded amino acids and causing functional changes in the BnPBR1 gene.

[0046] Table 1 Mutation types of T1 generation BnPBR1 mutants

[0047]

[0048] In summary, the present invention uses CRISPR / Cas9 technology to target two homologous genes (BnaA10.PBR1 and BnaC09.PBR1) of BnBPR1, and obtains double-copy homozygous mutants by genetic transformation, and identifies and analyzes the resistance phenotype of the mutants to root-root fungi. It is clarified that the BnBPR1 gene plays a strong negative regulatory role in the resistance to rapeseed root-root disease, and the two homologous copies of the rapeseed BnBPR1 gene BnaA10.PBR1 and BnaC09.PBR1 can be knocked out by gene editing technology to obtain a homozygous double mutant rapeseed new material with enhanced resistance to root-root disease, laying a foundation for breeding new rapeseed varieties resistant to root-root disease.

Claims

1. Brassica napus clubroot resistance gene BnBPR1 The application in the prevention and control of clubroot disease is characterized in that By knocking out BnBPR1 The two homologous copies of the gene were homozygous double mutants, downregulating BnBPR1 The expression of the gene is used to control rapeseed clubroot disease; the two homologous copies are BnaA10.PBR1 Genes and BnaC09.PBR1 Gene; among them, BnaA10.PBR1 The nucleotide sequence is shown in SEQ ID NO.1, BnaC09.PBR1 The nucleotide sequence is shown in SEQ ID NO.

2.

2. A method for screening the ability of Brassica napus to resist root knot fungi, characterized in that: By detecting the BnBPR1 Gene, if BnBPR1 Two homologous copies of the gene BnaA10.PBR1 Genes and BnaC09.PBR1 The gene function is lost, becoming a homozygous double mutant, and the Brassica napus has a strong resistance to root knot fungi; among them, BnaA10.PBR1 The nucleotide sequence is shown in SEQ ID NO.1, BnaC09.PBR1 The nucleotide sequence is shown in SEQ ID NO.

2.

3. A method for obtaining Brassica napus resistant to root knotweed, characterized in that: By knocking out the BnBPR1 The two homologous copies of the gene obtained a homozygous double mutant, and then obtained Brassica napus resistant to root knot fungus; the two homologous copies were BnaA10.PBR1 Genes and BnaC09.PBR1 Gene; among them, BnaA10.PBR1 The nucleotide sequence is shown in SEQ ID NO.1, BnaC09.PBR1 The nucleotide sequence is shown in SEQ ID NO.2.

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