CN-115838757-B - Method for creating cabbage type rape dwarf material by utilizing gene editing technology and application

Abstract

The invention belongs to the field of plant genetic engineering and biotechnology, and particularly relates to a method for creating a cabbage type rape dwarf material by using a CRISPR/Cas9 gene editing technology and application thereof. The method comprises the steps of editing a cabbage type rape BnaA G0083400WE gene by using a CRISPR/Cas9 gene editing technology to obtain a cabbage type rape dwarf material, wherein the nucleotide sequence of a coding gene mutant of the cabbage type rape dwarf material is SEQ ID NO.1. According to the invention, the CRISPR/Cas9 gene editing technology is used for carrying out gene editing on the cabbage type rape BnaA G0083400WE gene to obtain dwarf cabbage type rape, so that precious gene resources and germplasm resources are provided for cabbage type rape breeding; the method has strong characteristics and is easy to obtain, is an effective way for realizing target character improvement and cultivating new materials, and can be applied to oil tea breeding.

Inventors

• ZHI TIANTIAN
• ZHOU ZHOU

Assignees

• 宜春学院

Dates

Publication Date

20260512

Application Date

20220909

Claims (3)

1. 1. A method for creating a cabbage type rape dwarf material by utilizing a gene editing technology is characterized in that the cabbage type rape BnaA G0083400WE gene is edited by utilizing a CRISPR/Cas9 gene editing technology to obtain the cabbage type rape dwarf material; the nucleotide sequence of the encoding gene mutant of the cabbage type rape dwarfing material is SEQ ID NO.1; the method comprises the following specific steps: (1) Construction of cabbage type rape BnaA G0083400WE gene CRISPR/Cas9 expression vector: 1) selection of sgRNA target sites: The sgRNA target site is designed based on CRISPRDIRECT websites aiming at the structure and homology of different copies of BnaA G0083400WE genes in brassica napus westar, and the nucleotide sequence is as follows: SEQ ID NO.2:5’-agaggtcaaggccatccacaagg-3’; The nucleotide sequence of the sgRNA target site is selected from a sixth exon region of BnaA G0083400WE gene in brassica napus westar; 2) Primer design and PCR amplification: The primer sequences are as follows: SEQ ID NO.3:5’-cagtGGTCTCagtca agaggtcaaggccatccaca-3’; SEQ ID NO.4:5’-cagtGGTCTCaaaac tgtggatggccttgacctct-3’; The PCR amplification process comprises mixing the upstream and downstream primers according to 50 μl system, and performing denaturation annealing by a PCR instrument to obtain gRNA fragment; 3) Construction of CRISPR/Cas9 expression vector and agrobacterium transformation: T4 ligase is utilized to carry out T4 connection on the gRNA fragment obtained in the step 2) and the CRISPR/Cas9 plasmid subjected to BsaI/Eco31I digestion, then the connection product is transformed into escherichia coli DH5a, and after screening by antibiotics, colony PCR identification and sequencing verification are carried out, so as to obtain an expression vector; transforming the vector verified to contain sgRNA into agrobacterium, and performing colony PCR verification to obtain agrobacterium strain containing the expression vector; (2) Obtaining and identifying cabbage type rape BnaA G0083400WE gene CRISPR/Cas9 gene editing mutant: 1) Placing the pre-cultured hypocotyl into agrobacterium suspension containing an expression vector, performing callus induction, transferring the hypocotyl which generates the callus onto a culture medium which induces embryogenic callus generation, performing embryogenic cell induction, performing differentiation and screening of rape genetic transformation seedlings simultaneously, adding corresponding antibiotics into the differentiation culture medium, and performing primary screening and secondary screening to obtain positive seedlings, and performing seedling rooting culture; 2) Detecting the CRISPR/Cas9 expression vector in the positive brassica napus seedlings by utilizing PCR, and detecting the editing condition of BnaA G0083400WE genes in the CRISPR/Cas9 gene editing plants by sequencing; (3) The differences in plant height of brassica napus westar and BnaA G0083400WE gene CRISPR/Cas9 gene editing mutants were verified.
2. 2. The method for creating a dwarf brassica napus material using a gene editing technique as claimed in claim 1, wherein the antibiotic is chloramphenicol.
3. 3. Use of the method of creating brassica napus dwarf material according to any one of claims 1-2 in rape breeding by using gene editing technology.

Description

Method for creating cabbage type rape dwarf material by utilizing gene editing technology and application Technical Field The invention belongs to the field of plant genetic engineering and biotechnology, relates to a method and application of site-directed mutagenesis of a brassica napus BnaA G0083400WE gene, and in particular relates to a method and application for creating brassica napus dwarf material by using CRISPR/Cas9 gene editing technology. Background The plant height directly affects the lodging resistance and high yield potential of crops, and becomes one of important indexes of modern crop breeding and ideal plant type breeding. Dwarf breeding is an important component of the green revolution, and starts with the wheat breeding revolution initiated by Norman Borlaug at 1950 at the earliest, and the discovery and application of rice semi-dwarf gene sd1 and wheat Rht8 are the milestones of the revolution, and later, dwarf breeding work of crops such as rice, corn and cucumber is carried out successively. At present, more than 90% of rape varieties in rape main production areas in China are cabbage type rape, the plant height is more than 36% and is only 11% lower than 160cm, and the plant height is more than 180 cm. The plant height is too high, which is easy to cause the production problems of lodging, intolerance to fertilizer, unsuitable mechanical operation and the like, and is one of important factors restricting the mechanized production of rape. After the rape lodges, the number of grains per pod of the rape is reduced by 17.5%, the seed yield is reduced by 16.2%, and meanwhile, the difficulty and the mechanical damage rate of mechanical harvesting of the rape are increased, so that the harvestability is reduced. At present, a plurality of researchers obtain a plurality of rape dwarf intermediate materials through mutagenesis, spontaneous mutation and other ways, but the dwarf rape with excellent agronomic characters is not yet obtained for production and application due to factors such as too short plants, weak growth vigor before winter, self-incompatibility of dwarf plants, serious diseases and the like. Therefore, the cultivation of dwarf or semi-dwarf rape varieties and the genetic analysis and research of the dwarf or semi-dwarf rape varieties are of great significance for improving the mechanized harvesting of rape. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a method for creating a cabbage type rape dwarfing material by utilizing a gene editing technology and application thereof, which can provide precious gene resources and germplasm resources for rape breeding, in particular cabbage type rape breeding. The inventors have conducted extensive studies and summaries prior to conducting the experiments of the present invention. The simulated lesion mutant is a mutant which can spontaneously form programmed cell death after being infected by similar pathogenic matters on leaves without obvious stress, injury or pathogenic matters, and the mutant is widely existing in plants, such as Arabidopsis thaliana, rice, corn, sorghum, wheat, barley, peanut and the like. These mutants exhibit local and systemic resistance to many pathogens while undergoing programmed cell death. Many of the phenotype typical of the simulated lesion mutants is reported to be dwarf. Such as the simulated plaque mutant ssi4 exhibiting dwarfing and spontaneous cell death, mutations in chs3-2D triggering immune activation, the simulated plaque mutant chs3-2D leading to activation of extreme dwarfing and defensive responses, the simulated plaque mutant bir-1 exhibiting extreme dwarfing phenotype, spontaneous cell death and defensive responses, the T-DNA insertion mutant of calmodulin-binding transcriptional activator (CAMTA) 3 exhibiting enhanced disease resistance and dwarfing upon low temperature growth, and, in addition, the simulated plaque mutants bon1 and snc4-1D, snc2-1D, mkk1 mkk2, bir-1, ssi4, cpr22 and slh1 all exhibiting dwarfing phenotypes of varying degrees. The tyrosine degradation pathway was first found in animals and bacteria, and the metabolic pathway is degraded in five steps, the last step forming acetoacetate and fumarate into the tricarboxylic acid cycle for complete decomposition under the action of fumarylacetoacetase (fumarylacetoacetate hydrolase, FAH). Genes and enzymes of the classical tyrosine degradation pathway have been identified from arabidopsis thaliana and demonstrated to have their respective catalytic activity in vitro. The inventors screened and identified a mutant sscd (short-DAY SENSITIVE CELL DEATH 1) in Arabidopsis that forms a simulated lesion under short-day conditions, which was not significantly different from wild-type Arabidopsis under long-day conditions, but produced a necrotic-like lesion locally in the leaf under short-day conditions without pathogen infection. By means of map-based cloning, the SSCD1 gene has been isolated