A method for regulating the flowering period of brassica napus by gene editing and application thereof

Abstract

The application belongs to the technical field of plant genetic engineering, and discloses a gene editing method for regulating the flowering period of Brassica napus and application BnLWD1 After that, the CRISPR / Cas9 gene editing technology is used to carry out site-specific editing on the gene in Brassica napus, so that the gene function is lost or weakened, and finally the flowering of Brassica napus is 7-8 days earlier. The gene editing method provided by the application is accurate and efficient, and the edited Brassica napus has the advantages of controllable flowering period, adaptation to planting in different ecological regions (such as shortening the vegetative growth cycle in high-latitude regions, promoting flowering and fruiting, improving biomass or stress resistance, etc.), provides a new technical means and gene resource for Brassica napus variety improvement, and has important agricultural application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a gene editing method and application for regulating the flowering period of Brassica napus, which is particularly suitable for improving the flowering period of rapeseed varieties. Background Technology

[0002] Rapeseed is an important oilseed and economic crop in my country. The flowering period is a key agronomical trait for rapeseed growth and development, directly affecting its adaptability, yield, and quality. Different ecological regions have varying requirements for rapeseed flowering time: for example, in high-latitude or high-altitude areas, the suitable growing season for rapeseed is shorter, requiring rapeseed with an early flowering period and a short growth cycle; while in the rice-rapeseed rotation system in the Yangtze River basin and southern provinces of my country, early flowering allows rapeseed to mature earlier, better meeting the requirements of the "rice-rice-rapeseed" and "rice-rapeseed" three-crop planting patterns, which not only helps ensure the rapeseed's own yield but also avoids competition for land with staple crops such as rice.

[0003] Improving the flowering time of rapeseed mainly relies on traditional hybridization breeding, but this method suffers from problems such as long cycles, low breeding efficiency, and limited genetic background. The emergence of gene editing technologies (such as CRISPR / Cas9) has provided a new approach for the precise improvement of crop traits. By editing flowering-related genes at specific points, variants of the target trait can be quickly obtained. Currently, many flowering-time genes have been reported; however, existing technologies lack genes that can precisely regulate early flowering without adverse agronomical trait associations, as well as efficient editing methods, thus limiting the progress of targeted improvement of rapeseed flowering time. Therefore, discovering new flowering-time regulating genes and establishing corresponding gene editing technologies are of great significance for rapeseed germplasm innovation. Summary of the Invention

[0004] The purpose of this invention is to provide a gene that regulates the flowering period of Brassica napus. By performing site-specific editing on this gene, the flowering period of Brassica napus can be precisely advanced, solving the problems of long cycle and unstable effect in existing rapeseed flowering period improvement technologies. This provides new gene resources and technical means for the breeding of adaptable rapeseed varieties.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A gene that regulates the flowering period of Brassica napus. BnLWD1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0007] A method to regulate the early flowering of Brassica napus: using gene editing technology to... BnLWD1 Gene function loss, preferably, for the above BnLWD1Nucleotides 62-81 and 425-444 of the gene's coding region were edited.

[0008] BnLWD1 Gene editing vector, the vector containing two target genes BnLWD1 The sgRNA of the gene has the sequence shown in SEQ ID NO.3 and 4.

[0009] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0010] ① This invention is the first to identify and disclose a gene that regulates the flowering period of Brassica napus. BnLWD1 This provides new gene resources for the molecular improvement of rapeseed flowering time and expands the range of gene editing targets for advancing rapeseed flowering time in existing technologies.

[0011] ② The gene editing method is precise and efficient, using CRISPR / Cas9 technology for targeted gene editing. BnLWD1 The editing efficiency is over 35%, and there is no off-target effect.

[0012] ③ The edited rapeseed plants have a significantly earlier flowering period and maintain good agronomic traits. They can be adapted to planting in special ecological areas such as high latitudes and high altitudes, or used to optimize crop rotation systems, improve rapeseed yield and stress resistance, and have broad agricultural application prospects. Attached Figure Description

[0013] Figure 1: Candidate genes BnLWD1 Manhattan plot of genome-wide association analysis.

[0014] Figure 2: Gene editing vector pCas9-sgRNA- BnLWD1 A schematic diagram of its construction.

[0015] Figure 3 Sequencing results of the target genes of two homozygous gene-edited lines, KO #6 and KO #7, selected by T2 generation.

[0016] Figure 4 Comparison of flowering periods of gene-edited plants. a: Wild type, flowering period is 144-145 days after sowing; b: Gene-edited line, flowering period is 136-137 days after sowing. Detailed Implementation

[0017] Example 1: Acquisition and identification of candidate genes associated with flowering time of Brassica napus

[0018] The 350 rapeseed germplasm resources collected by the inventor's research group in the early stage were used as experimental materials and planted at the Yangluo Experimental Base of the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences.

[0019] In agricultural and phenological observations, the flowering period generally refers specifically to the initial flowering stage, which is the starting point when a certain flowering proportion (50%) is reached in the population. In this embodiment, during the flowering period survey, each rapeseed variety was considered to have flowered when its flowering proportion reached 50% or more, and the date of that day was recorded. The flowering period of the natural population was surveyed for two consecutive years, and the average value was used as the phenotypic data; Genotypic detection: Whole genome resequencing (WGS) technology was used to obtain the population genotypic data.

[0020] The initial stage involved the identification of SV loci within the associated population, comprising approximately 100,000 SV sites. Genome-wide association analysis (SV-GWAS) based on structural variation was then performed using rMVP software and the FarmCPU model, with a threshold of -log. 10 (1 / SV locus number) A significant SV associated with flowering time was identified in a natural rapeseed population. This SV is a large deletion of 3727 bp, located at 51844144 bp on chromosome C08, in the gene... BnLWD1 Upstream start-up sub-region, preliminary speculation BnLWD1 The gene is a candidate gene for flowering, its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. This was verified through gene expression analysis. BnLWD1 The gene is highly expressed during the flower bud differentiation stage of rapeseed. Knocking out the gene significantly advances the flowering period of rapeseed, confirming that it is a key gene regulating the flowering period of rapeseed.

[0021] Example 2 BnLWD1 Gene editing methods and phenotypic identification

[0022] 1. Construction of gene editing vectors

[0023] Designing targeted genes using CRISPR-P 2.0 software BnLWD1 Two sgRNAs in the coding region: sgRNA1 (SEQ ID NO. 3) targets nucleotides 62-81 of the coding region, and sgRNA2 (SEQ ID NO. 4) targets nucleotides 425-444 of the coding region. The pHK2-Cas9-U6 vector was digested with Eco31I, and the linearized vector fragment was recovered, as follows: Figure 2 As shown, two sgRNAs were ligated to a linearized vector using a homologous recombinase, transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing kanamycin, and single colonies were picked for sequencing verification, successfully constructing the recombinant editing vector pCas9-sgRNA- BnLWD1 .

[0024] 2. Rapeseed genetic transformation

[0025] The recombinant editing vector was transferred into the Brassica napus recipient material “Zhongshuang 11” using Agrobacterium-mediated transformation, including the following steps:

[0026] (1) Seed cleaning and germination: Disinfect with 75% ethanol for 30-60 seconds, rinse once with sterile water for 1 minute each time; disinfect with 0.15% mercuric chloride for 10 minutes, rinse twice with sterile water for 1 minute each time; rinse with sterile water for 30 minutes, then inoculate onto sterile filter paper and air dry. Inoculate the seeds into germination culture flasks and incubate in the dark at 23°C for 5-6 days.

[0027] (2) Pre-culture: Cut the hypocotyl of the germinating rapeseed seedlings into segments of 0.4-0.6cm, inoculate them into pre-culture medium, and culture at 23℃ under light for 2-3 days.

[0028] (3) Agrobacterium infection and co-culture: Agrobacterium was picked into the infection solution and OD was prepared. 600 The explants were inoculated into a 0.2 μL Agrobacterium resuspension and incubated for 10 min. After inoculation, the explants were inoculated onto sterile filter paper and air-dried, then inoculated onto co-culture medium and incubated in the dark at 23°C for 48-72 h.

[0029] (4) Destermination (delayed screening): The co-cultured explants were inoculated onto a destermination medium and cultured at 23°C under light for 6 days.

[0030] (5) Screening / differentiation: The sterilized explants were inoculated onto the screening / differentiation medium, with 30 explants per plate. The medium was cultured at 23°C under light and the plates were changed every 15 days.

[0031] (6) Rooting culture: Inoculate the differentiated buds into the rooting medium and culture them under light at 23°C until they root.

[0032] 3. Editing plant identification and phenotypic analysis

[0033] Molecular identification: DNA was extracted from the leaves of regenerated plants and amplified by PCR using sgRNA-specific primers (primers for target 1: SEQ ID NO. 5 and 6; primers for target 2: SEQ ID NO. 7 and 8). The amplified products were verified by HI-TOM sequencing. Six gene-edited plants (KO #3, KO #6, KO #7, KO #11, KO #15, KO #16) were obtained in the T0 generation, representing three gene-editing types. In the T2 generation, two homozygous lines with gene-editing types, KO #6 and KO #7, were selected. Target gene sequencing results are shown below. Figure 3 As shown.

[0034] Phenotypic identification: Gene-edited positive plants and wild-type plants were planted in the same experimental field and managed under routine field conditions. Flowering time (the time when 50% of the population reached flowering) was recorded. The average flowering time of wild-type Brassica napus was 144-145 days after sowing, while the average flowering time of gene-edited plants was 136-137 days after sowing. Compared with the wild type, the flowering time of gene-edited positive plants was on average 7-8 days earlier. Other agronomic traits of gene-edited plants, such as plant height, number of branches, and number of siliques, showed no significant adverse variations, indicating that the gene-edited plants were genetically modified. BnLWD1 The gene specifically regulates the flowering period.

Claims

1. A gene regulating the flowering period of Brassica napus. BnLWD1 Its characteristics are, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The gene according to claim 1 BnLWD1 The encoded protein is characterized by, The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. The version of claim 1 BnLWD1 The application of genes in promoting early flowering in Brassica napus is characterized by, Using gene editing technology to BnLWD1 Gene function loss.

4. BnLWD1 Gene editing vector, characterized in that, The carrier contains two targets. BnLWD1 The sgRNA of the gene, the sequence of which is shown in SEQ ID NO.3 and 4.

5. A method for regulating the early flowering of Brassica napus, characterized in that, Using gene editing technology to BnLWD1 The gene is missing or absent, and its nucleotide sequence is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that, Regarding the BnLWD1 Nucleotides 62-81 and 425-444 of the gene's coding region were edited.

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