阅读说明：本技术 一种快周期油菜在油菜功能基因研究领域的应用及方法 (Application and method of fast-cycle rape in rape functional gene research field ) 是由 杨梅 杨铭浩 邓扬菲 杨峻辉 孟冬林 杨峻烨 莫从古 许兴旺 钱汇海 于 2020-07-02 设计创作，主要内容包括：本发明属于植物基因工程领域,具体涉及一种快周期油菜在油菜功能基因组学研究领域的应用,并进一步公开一种基于快周期甘蓝型油菜进行油菜功能基因编辑的方法以及通过化学诱变制备突变体的方法。本发明方案基于快周期油菜进行油菜功能基因突变的相关研究,具体包括油菜基因的编辑和/或油菜基因突变体的制备,基于所述生育期短且植株小的优势,可以替代拟南芥实现短周期内对油菜基因的研究。(The invention belongs to the field of plant genetic engineering, particularly relates to application of fast-cycle rape in the field of rape functional genomics research, and further discloses a method for carrying out rape functional gene editing based on fast-cycle cabbage type rape and a method for preparing mutants by chemical mutagenesis. The scheme of the invention is based on the fast-cycle rape to carry out the related research of rape functional gene mutation, particularly comprises the editing of rape gene and/or the preparation of rape gene mutant, and can replace arabidopsis thaliana to realize the research of rape gene in short cycle based on the advantages of short growth period and small plant.)

1. An application of fast-cycle rape in the research field of rape functional genomics is characterized in that:

the fast-cycle rape is a cabbage type rape (Brassica napus) which has a short life cycle and only has a period from emergence to maturation of about 60 days under the conditions of 22 ℃ and 16 hours of light/8 hours of dark culture;

the functional gene research comprises gene editing and/or preparation of gene mutants.

2. A method for carrying out rape gene editing based on fast-cycle rape is characterized in that the fast-cycle rape is adopted as a donor to carry out target gene editing, and gene editing filial generation can be harvested about 80 days after explant inoculation, and the method specifically comprises the following steps

(1) Taking the seeds of the fast-cycle rape, performing surface disinfection, and then sowing to prepare an explant for later use;

(2) constructing a gene editing carrier according to the sequence structure of a target gene, and converting escherichia coli competent cells;

(3) after the obtained escherichia coli competent cells are fused with agrobacterium by electric shock, the explant obtained in the step (1) is impregnated by the obtained agrobacterium liquid;

(4) transferring the explants soaked with the agrobacterium liquid into an M1 culture medium, and co-culturing for 36-48h in a dark room at 22 ℃;

(5) transferring the explant cultured in the step (4) from the M1 medium to the M2 medium, and performing illumination culture at 22 ℃ for 2 weeks;

(6) transferring the cultured explants into an M3 culture medium, and forming plantlets through conventional culture;

(7) sequencing the selected editing sites, transplanting the plantlets determined to be positive single plants into a greenhouse for culturing, and managing the plantlets according to the conventional method after survival.

3. The method for gene editing in oilseed rape of claim 2, wherein the gene editing is performed in a single step,in the step (4), the M1 culture medium comprises the following components in percentage by weight: 4.4g of MS finished product powder, 30g of cane sugar, 18g of D-mannitol, 1mg of 2,4-dichlorophenoxyacetic acid, 0.3mg of kinetin, 5.5g of Phytagel and ddH₂O is subjected to constant volume to 1L, and the pH value is adjusted to 5.88; sterilizing at high temperature, adding 100 μmol acetosyringone, and mixing.

4. The method for rape gene editing based on fast cycle rape as claimed in claim 2 or 3, wherein in the step (5), the M2 culture medium comprises the following components: 4.4g of MS finished product powder, 30g of cane sugar, 18g of D-mannitol, 1mg of 2,4-dichlorophenoxyacetic acid, 0.3mg of kinetin, 5.5g of Phytagel and ddH₂O is subjected to constant volume to 1L, and the pH value is adjusted to 5.86; after high temperature sterilization, 150. mu.l of silver thiosulfate, 300mg of timentin and 25mg of kanamycin sulfate are added.

5. The method for gene editing in rape of fast cycle duration as claimed in claim 4, wherein in the step (5), the light culture is performed under the control of 16 hours light/8 hours dark.

6. The method for gene editing in rape of fast cycle duration as claimed in any one of claims 2 to 5, wherein in step (6), the M3 medium comprises the following components: 4.4g of MS finished product powder, 30g of cane sugar, 5.5g of Phytagel and ddH₂O is metered to 1L, and the pH value is adjusted to 5.84-5.88; after high temperature sterilization, 300mg of timentin is added.

7. The method for gene editing in oilseed rape (Brassica napus) as claimed in any one of claims 2 to 6, wherein in step (7), the cultivation step is carried out under the conditions of 22 ℃ and 16 hours light/8 hours dark.

8. A method for preparing functional gene mutants based on fast-cycle rape is characterized in that the method adopts the fast-cycle rape as a donor to carry out mutagenesis treatment, and specifically comprises the following steps:

(1) taking the seeds M0 of the fast-cycle rape for immersion and imbibition pretreatment for later use;

(2) carrying out mutagenesis treatment on the fast-period rape to obtain fast-period M1 seeds for later use;

(3) sowing the obtained fast-cycle M1 seeds in a culture room, screening mutant phenotype, and selfing to obtain fast-cycle M2 seeds for later use;

(4) continuously sowing the fast-period M2 seeds in a culture room, verifying whether the mutant phenotype is heritable or not, collecting mutant strains, and continuously selfing to obtain an M3 mutant strain;

(5) and (3) breeding the M3 mutant strain into an inbred line, and continuously selfing the mutation strain without separation to obtain the homozygous M4 mutant.

9. The method for preparing functional gene mutants based on fast cycle rape as claimed in claim 8, wherein the mutagenesis step in the step (2) comprises chemical mutagenesis and/or physical mutagenesis.

10. The method for preparing rape gene mutants based on fast cycle rape as claimed in claim 8, wherein the culture conditions in the steps (3), (4) and (5) are controlled as follows: the temperature was 22 ℃ and the light was 16 hours light/8 hours dark.

Technical Field

The invention belongs to the field of plant genetic engineering, particularly relates to application of fast-cycle rape in the field of functional genomics research, and further discloses a method for performing functional gene editing based on the fast-cycle cabbage type rape and a method for preparing a functional gene mutant through chemical mutagenesis.

Background

Cabbage type rape (Brassica napus L.) is an important oil crop widely planted in China, large-area planting is carried out in Yangtze river basin, Yunobu plateau and northwest climate cold areas in China, and the total annual planting area is about 1 hundred million mu. The genome is AACC, 19 pairs of chromosomes, the size is about 1200-1280Mb (Song Jia-Ming et al, 2020), the structure is complex, and more homologous fragments exist. Rape likes cold and requires a certain time of low-temperature treatment to bloom (usually 4-10 ℃), the growth period of south autumn sowing reaches more than 200 days, northwest summer sowing also reaches about 120 days, and the plant type is high. These biological characteristics determine that high-throughput culture (i.e., large-scale planting in limited time and space) of rape is difficult to realize in the researches of tissue culture, gene mutagenesis, gene localization, omics, particularly functional genomics and the like. Therefore, under the prior art conditions, when researchers verify the functions of rape genes, if a transgenic method is adopted, the results can be obtained only from the T0 generation to the T2 generation in 2-3 years; even with gene editing techniques, phenotypic validation was obtained from the T0 generation to T1, which was 1-2 years old. Therefore, in order to save time, researchers often have to use arabidopsis thaliana, which is also a brassicaceae species, as a model plant instead of brassica napus for gene cloning or functional verification. This is mainly because arabidopsis thaliana has a short growth period under long-day conditions, short plants are favorable for high-throughput culture (6 generations can be grown every year in a large scale in a laboratory), and the simple genome structure is also favorable for gene cloning, as compared with brassica napus.

However, with the development of rape breeding and basic research, the limitation of using Arabidopsis as a substitute is more and more obvious because (1) the Arabidopsis genome is small, and a plurality of genes which are not available in Arabidopsis exist in the rape, especially the genes related to yield or heterosis utilization, such as rape dominant genic male sterile gene MS5(Xin Qiang et al 2016), can be cloned or verified only when the rape is used as a donor; (2) the flower bud of arabidopsis is smaller than that of rape, and the hybridization test has certain difficulty; (3) more importantly, the arabidopsis thaliana is a weed, has no direct utilization value in field production practice, and a positive test product cannot be put into production practice. However, when rape is used as a receptor for researches such as gene mutation, transgenosis or gene editing, the positive product may have direct field popularization value.

Therefore, the development of a high-throughput method for directly utilizing rape to research gene functions has positive significance for the development of the field of rape genetic engineering.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide the application of the fast-cycle cabbage type rape serving as a donor in the aspects of functional genomics gene editing and functional gene mutagenesis of the rape;

the first technical problem to be solved by the invention is to provide a method for rape gene editing based on fast-cycle rape;

the second technical problem to be solved by the present invention is to provide a method for preparing functional gene mutants based on fast cycle oilseed rape.

In order to solve the technical problems, the application of the fast-cycle rape in the field of functional gene research is as follows:

the fast-cycle rape is a cabbage type rape (Brassica napus) which has a short life cycle and only has a period from emergence to maturation of about 60 days under the conditions of 22 ℃ and 16 hours of light/8 hours of dark culture;

the functional gene research comprises gene editing and/or preparation of gene mutants.

The invention discloses a method for carrying out rape gene editing based on fast-cycle rape, which adopts the fast-cycle rape as a donor to carry out directional editing of a target gene, and can harvest gene editing filial generation about 80 days after explant inoculation, and specifically comprises the following steps

(1) Taking the seeds of the fast-cycle rape, performing surface disinfection, and then sowing to prepare an explant for later use;

(2) constructing a gene editing carrier according to the sequence structure of a target gene, and converting escherichia coli competent cells;

(3) fusing the obtained escherichia coli competent cells with agrobacterium by electric shock, and impregnating the explant obtained in the step (1) with the obtained agrobacterium liquid;

(4) transferring the explants soaked with the agrobacterium liquid into an M1 culture medium, and co-culturing for 36-48h in a dark room at 22 ℃;

(5) transferring the explant cultured in the step (4) from the M1 medium to the M2 medium, and performing illumination culture at 22 ℃ for 2 weeks;

(6) transferring the cultured explants into an M3 culture medium, and forming plantlets through conventional culture;

(7) sequencing the selected editing sites, transplanting the plantlets determined to be positive single plants into a greenhouse for culturing, and managing the plantlets according to the conventional method after survival.

Specifically, in the step (4), the M1 medium comprises the following components in parts by weight: 4.4g of MS finished product powder, 30g of cane sugar, 18g of D-mannitol, 1mg of 2,4-dichlorophenoxyacetic acid, 0.3mg of kinetin, 5.5g of Phytagel and ddH₂O is subjected to constant volume to 1L, and the pH value is adjusted to 5.88; sterilizing at high temperature, adding 100 μmol acetosyringone, and mixing.

Specifically, in the step (5), the M2 cultureThe nutrient comprises the following components in percentage by weight: m S finished product powder 4.4g, sucrose 30g, D-mannitol 18g, 2,4-dichlorophenoxyacetic acid 1mg, kinetin 0.3mg, Phytagel5.5g, ddH₂O is subjected to constant volume to 1L, and the pH value is adjusted to 5.86; after high temperature sterilization, 150. mu.l of silver thiosulfate, 300mg of timentin and 25mg of kanamycin sulfate are added.

Specifically, in the step (5), in the light culture, the light condition is controlled to be 16 hours of light/8 hours of dark.

Specifically, in the step (6), the M3 medium comprises the following components in parts by weight: 4.4g of MS finished product powder, 30g of cane sugar, 5.5g of Phytagel and ddH₂O is metered to 1L, and the pH value is adjusted to 5.84-5.88; after high temperature sterilization, 300mg of timentin is added.

Specifically, in the step (6), the culture time is controlled to be 2 weeks, namely the test-tube plantlet is in the 2-leaf stage, otherwise, the plantlet flowers in the test tube for more than 24 days.

Specifically, in the step (7), the culture conditions in the cultivation step are 22 ℃ and 16 hours of light/8 hours of darkness, the flowering can be performed without season limitation and low-temperature vernalization, and only about 40 days are required from the transplanting survival to the harvest of the T1 seeds.

The invention also discloses a method for preparing a functional gene mutant based on the fast-cycle rape, which adopts the fast-cycle rape as a donor to carry out mutagenesis treatment and specifically comprises the following steps:

(1) taking the seeds M0 of the fast-cycle rape for immersion and imbibition pretreatment for later use;

(2) carrying out mutagenesis treatment on the fast-period rape to obtain fast-period M1 seeds for later use;

(3) sowing the obtained fast-cycle M1 seeds in a culture room, screening mutant phenotype, and selfing to obtain fast-cycle M2 seeds for later use;

(4) continuously sowing the fast-period M2 seeds in a culture room, verifying whether the mutant phenotype is heritable or not, collecting mutant strains, and continuously selfing to obtain an M3 mutant strain;

(5) and (3) breeding the M3 mutant strain into an inbred line, and continuously selfing the mutation strain without separation to obtain the homozygous M4 mutant.

Specifically, in the step (2), the mutagenesis step includes chemical mutagenesis and physical mutagenesis.

Specifically, in the steps (3), (4) and (5), the culture conditions are controlled as follows: the temperature is 22 ℃, the illumination is 16 hours, the illumination is dark per 8 hours, the method is not limited by seasons, and the flowers can bloom without low-temperature vernalization, thereby realizing large-scale indoor culture.

The scheme of the invention is based on the fast-cycle rape to perform related research of functional genomics, particularly, but not limited to the editing of rape genes and/or the preparation of rape gene mutants, and based on the advantages of short growth period and smaller individual of the fast-cycle rape, the fast-cycle rape can replace arabidopsis thaliana to realize the research of the rape genes in a short cycle.

The method for gene editing based on the fast-cycle cabbage type rape as the donor uses the fast-cycle rape to replace arabidopsis thaliana or traditional rape as the donor for tests such as functional verification and the like to edit the target gene, so that the gene functional verification result can be quickly obtained. Although the gene editing method is similar to the common rape gene editing technology, the whole process from inoculation of the fast-period explant to harvest of the T1 positive seedling seeds is only about 80 days, and the short and small plants occupy little space and are not limited by seasons any more; and the common rape can only obtain test-tube seedlings in 80 days at the fastest, and can bloom only after being transplanted and low-temperature vernalization treatment is needed. Therefore, the use of fast-cycle rape as a donor can save research time and culture space, realize high-throughput research, thereby accelerating the basic research process of rape, and the experimental product has the potential of being directly used for field production.

According to the method for preparing the functional gene mutant based on the fast-cycle rape, the fast-cycle rape is adopted to replace arabidopsis thaliana or traditional rape as a donor and is used as a tool for basic research and breeding practice to carry out mutagenesis treatment, the mutant can be rapidly identified indoors on a large scale, and the new fast-cycle rape strain obtained by mutagenesis of important character related genes can meet high-throughput experiment requirements such as gene positioning and Tilling, and an experiment product has the potential of being directly applied to breeding practice.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 is a structural schematic diagram of a double-target CRISPR/Cas9 binary vector in example 1 of the invention;

FIG. 2 is a test result of Cas9 element in fast cycle rape horizontal gel detection Fad2 gene editing seedlings as described in example 1 of the present invention.

Detailed Description

In the following examples, the fast cycle brassica napus is obtained according to the method described in chinese patent CN110100723A (a method for cross breeding fast cycle brassica napus and the application thereof), and a "perceival AR-41L" type artificial incubator manufactured by perceival Scientific company is selected, the variety flowers 27 days after sowing (22 ℃, 16h light/8 h dark) in the artificial incubator, and does not need to vernalize at low temperature, the plant height is only about 50 cm, the fruit setting is normal, and the whole growth period is as short as 60 days.