CN120519495A

CN120519495A - A method for efficient genetic transformation and gene editing of cabbage

Info

Publication number: CN120519495A
Application number: CN202510595758.2A
Authority: CN
Inventors: 吕红豪; 张语伦; 刘金晖; 张扬勇; 杨丽梅; 庄木; 王勇; 季家磊; 关慧明; 张一凡; 刘晓霞; 白晓龙; 靳永胜
Original assignee: Helinger National Agricultural Science And Technology Park Service Center; Wulanchabu Science And Technology Commission Business Association; Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Current assignee: Helinger National Agricultural Science And Technology Park Service Center; Wulanchabu Science And Technology Commission Business Association; Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Priority date: 2024-11-13
Filing date: 2025-05-09
Publication date: 2025-08-22

Abstract

The invention relates to a cabbage high-efficiency genetic transformation and gene editing method, which belongs to a biological breeding technology and comprises the steps of (1) introducing a CRISPR/Cas9 gene editing vector into a callus of a target cabbage material, (2) screening the cabbage callus successfully introduced with the CRISPR/Cas9 gene editing vector for regeneration culture, and (3) screening T0 generation transgenic cabbage subjected to gene editing, wherein the CRISPR/Cas9 gene editing vector contains sgRNA of a target editing gene, and the PAM sequence of the CRISPR/Cas9 gene editing vector is 5'-' NGGT '-3', wherein N is one of A, C and G, and the method can remarkably improve the gene editing efficiency and type.

Description

Efficient genetic transformation and gene editing method for cabbage

Technical Field

The invention relates to the technical field of biology, in particular to a cabbage efficient genetic transformation and gene editing method.

Background

Brassica oleracea is an important biennial herb species in the cruciferae family. Cruciferous crops of 377 hectares, such as cabbage, broccoli and cauliflower, have been statistically cultivated worldwide and constitute an important agricultural resource (Li et al 2024).

In recent years, CRISPR/Cas9 technology has been widely used in major crops such as rice, wheat, and potato. The technology can be used to develop high-yield, disease-resistant or stress-resistant crops by inhibiting gene expression (Gao, 2021; he et al, 2022).

However, the current regeneration efficiency of cabbage transgenes is lower, resulting in limited agrobacterium-mediated transformation efficiency, below 1.0%, thereby affecting CRISPR/Cas9 gene editing efficiency, which is only 12.9% lower than 68% of rice editing efficiency (Li et al 2021).

Disclosure of Invention

In view of the requirements and the current situation in the field, the invention provides a cabbage efficient genetic transformation and gene editing method according to research results, which comprises the following steps:

in a first aspect of the present invention, there is provided a method for efficient genetic transformation and gene editing of cabbage, characterized in that,

(1) Introducing a CRISPR/Cas9 gene editing vector into the callus of the target cabbage material,

(2) Screening cabbage callus which is successfully introduced into the CRISPR/Cas9 gene editing vector, and carrying out regeneration culture on the cabbage callus;

(3) Screening T0 generation transgenic cabbage with gene editing;

wherein the CRISPR/Cas9 gene editing vector contains the sgRNA of the target editing gene and has a PAM sequence of 5'-' NGGT '-3', where N is one of a, C, G.

Preferably, the method is characterized by further comprising introducing an expression vector expressing fusion protein GRF5-GIF1-GRF5 either before or after introducing a CRISPR/Cas9 gene editing vector into the cabbage material of interest;

the amino acid sequence of the fusion protein GRF5-GIF1-GRF5 has any one of the following characteristics:

(1) From N to C end, the polypeptide is obtained by linear fusion of GRF5-1 polypeptide shown in Seq ID No.33, GIF1 polypeptide shown in Seq ID No.37 and GRF5-2 polypeptide shown in Seq ID No.34, and the polypeptides are connected through 3-5 alanine residues;

(2) The amino acid sequences of the polypeptide from N to C are obtained by linear fusion of a GRF5-2 polypeptide shown in a Seq ID No.34 and a GIF1 polypeptide shown in a Seq ID No.37 and a GRF5-1 polypeptide shown in a Seq ID No.33, and the polypeptides are connected through 3-5 alanine residues.

Preferably, the method, wherein the introducing CRISPR/Cas9 gene editing vector is introduced by agrobacterium-mediated genetic transformation, and the introducing expression vector expressing fusion protein GRF5-GIF1-GRF5 is also introduced by agrobacterium-mediated genetic transformation.

In another aspect of the present invention, there is provided a cabbage growth regulator fusion protein having the structure GRF5-GIF1-GRF5, the amino acid sequence of which has any one of the following characteristics:

Preferably, the cabbage growth regulator fusion protein is characterized in that its amino acid sequence is as shown in Seq ID No. 38.

In yet another aspect of the invention, the nucleotide sequence encoding the cabbage growth regulator fusion protein is provided.

Preferably, the nucleotide sequence is as shown in Seq ID No. 41.

In yet another aspect of the invention, an expression vector is claimed which carries the nucleotide sequence.

The invention further provides a kit for improving the efficient genetic transformation and gene editing efficiency of cabbage, which is characterized by comprising a CRISPR/Cas9 gene editing vector, wherein the PAM sequence of the gene editing vector is 5'-' NGGT '-3', and N is one of A, C and G.

Preferably, the kit further comprises an expression vector for expressing the cabbage growth regulator fusion protein.

The invention improves the cabbage gene editing and genetic transformation method. Experimental data show that taking the PDS gene editing as an example, the latter position of the PAM sequence 5'-NGG-3' in the CRISPR/Cas9 vector is selected as 'T', the editing type of the CRISPR/Cas9 gene editing system can be obviously increased and the editing efficiency can be improved no matter in dicotyledonous plants cabbage or monocotyledonous rice, and meanwhile, the expression vector for expressing fusion protein GRF5-GIF1-GRF5 is transformed, so that the average regeneration efficiency of cabbage is improved by 55.2%, and the improvement can obviously improve the efficiency of cabbage molecular breeding. In the breeding application experiment of cabbage susceptibility genes (BoDMR, boBPM) discovered by the inventor at the same time, the invention adopts the improved gene editing technology and genetic transformation method to carry out first-generation sequencing on the T0 generation positive plants, the transformation efficiencies of bobpm6 and bodmr6 are respectively 5.5% and 8.2%, the editing efficiencies are respectively 62.0% and 62.5%, and the method is remarkably higher than the technical level reported in the prior art (the genetic transformation efficiency of cabbage is lower than 1% and the editing efficiency is only 12.9%).

Drawings

FIG. 1 is a schematic diagram of a PDS gene editing vector constructed for researching the influence of different PAM sequences on the gene editing efficiency and type;

FIG. 2 is a graph showing experimental results of effects of different PAM sequences on editing efficiency and type of rice gene, wherein,

The upper panel shows rice using NGGN and NGGT as PAM sequence knocks out OsPDS gene, the red circle shows plants where no gene editing occurred, and the lower panel shows rice OsPDS gene editing efficiency (bar graph) and editing type (line graph).

FIG. 3 is a graph showing experimental results of the effect of different PAM sequences on the editing efficiency and type of cabbage gene, wherein,

The upper panel shows the case of using NGGN and NGGT for PAM sequence knockout BoPDS gene, mock shows the cabbage plant where no gene editing has occurred, and the lower panel shows the cabbage gene editing efficiency (bar graph) and editing type (line graph).

FIG. 4 is a result of investigation of the effect of the growth factor on the regeneration efficiency of genetic transformation according to the present invention, wherein the average regeneration efficiency of cabbage is improved by 55.2% in the treatment in which GRF5-GIF1-GRF5 fusion protein is expressed.

FIG. 5 shows the structure of BoDMR gene of the clone of the invention and the case of gene editing, wherein light pink represents the gene coding region (exon), dark pink represents the intron region, red short underlined target 1 at the bottom of the first exon region represents the sgRNA target region, the lower nucleotide sequence represents target region 1, wherein the wild-type target nucleotide sequence is shown as underlined, the remaining four T0 generation mutants from which gene editing occurs, blue broken lines represent deletions, yellow letters represent insertions, and pre-target ACCG represents the PAM sequence.

FIG. 6 shows the structure of BoBPM gene cloned by the present invention and the case of gene editing, wherein light pink represents the gene coding region (exon), dark pink represents the intron region, red short underlined target 1 at the bottom of the first exon region represents the sgRNA target region, the lower nucleotide sequence represents target region 1, the wild-type target nucleotide sequence is shown as underlined, the remaining four T0 generation mutants from which gene editing occurs, blue broken lines represent deletions, yellow letters represent insertions, and pre-target ACCG represents the PAM sequence.

FIG. 7 shows the results of a disease resistance study of bodmr plants subjected to BoDMR gene editing, in which the disease index of the inoculated black rot fungus was reduced from 79.3 to 55.1 and the disease index of the inoculated clubroot was reduced from 90.7 to 57.6 (significant).

FIG. 8 shows the results of a disease resistance study of bobpm plants in which BoBPM gene editing occurred, in which the disease index of inoculated blight, black rot, clubroot was reduced from 65.4 to 14.5 (significant), from 53.8 to 20.9 (significant), and from 63.1 to 55.7, respectively.

Detailed Description

The present invention will be further illustrated by the following specific embodiments and drawings, but the present invention is not limited to the following examples. In the following examples, unless otherwise indicated, all experimental procedures were as routine in the art using conventional commercially available reagents.

Experimental materials used in this study:

the genetic transformation material of the rice in the test is Japanese sunny;

Wild cabbage variety 'M1', which is an inbred material, has a preservation in applicant units and can be provided for a verification test;

the CRISPR/Cas9 vector for rice is pVS1, purchased from ADDGENETM;

The CRISPR/Cas9 vector for cabbage is PYLCRISPR/Cas9-35S-B, purchased from adedge ^TM;

EXAMPLE 1 construction of CRISPR/Cas9 editing vector and Effect of PAM sequence on Gene editing efficiency and editing type

The inventors have observed in the study that Cas9 (ScCas) from Streptococcus canis requires a spacer adjacent motif (PAM) sequence such as 5'-NNG-3' after which any base can be selected, however when the base after the PAM sequence is "T", the editing efficiency is higher (http:// crispor. Tefor. Net /).

To test whether "T" following SpCas9 PAM sequence can enhance the editing efficiency of CRISPR/Cas9 technology, the inventors constructed CRISPR/Cas9 gene editing vectors (OsPDS and BoPDS) for rice and cabbage, respectively, to knock out phytoene dehydrogenase gene PDS.

S1, constructing a PDS gene editing vector

In order to construct a PDS editing vector for knocking out cabbage and rice, 5'-NGGN-3', 5'-NGGT-3' are respectively selected as PAM sequences of rice and cabbage, wherein 'N' represents three bases except 'T'.

According to the selected different target sites and PAM sequences, designing target introduced sequence sgRNA and a joint primer thereof (table 1), respectively connecting the target introduced sequence sgRNA and the joint primer thereof to pVS1 after synthesis to obtain a rice PDS gene editing vector OsPDS;

schematic of CRISPR/Cas9 construct as shown in figure 1, panel a is schematic of CRISPR/Cas9 construct OsPDS for rice PDS editing, NGGN (N1-N3) and NGGT (T1-T3) represent positions of target sites, panel B is schematic of CRISPR/Cas9 construct BoPDS for cabbage PDS editing, NGGN (N1-N2) and NGGT (T1-T2) represent positions of target sites.

TABLE 1 Rice PDS Gene sgRNA sequence

TABLE 2 cabbage rice PDS Gene sgRNA sequence

Influence of S2.5'-NGGT-3' PAM sequence on CRISPR/Cas9 editing efficiency and diversity of editing types

The rice-editing vector OsPDS and the cabbage-editing vector BoPDS constructed in S1 are respectively introduced into a rice variety Nipponbare and a wild cabbage variety M1 through agrobacterium-mediated genetic transformation.

According to the standard procedure, agrobacterium is transformed by adding 1 μg of plasmid into 100 μl of agrobacterium competent cells, mixing, sequentially standing on ice, liquid nitrogen, 37 ℃ water bath and ice for 5min, adding 800 μl of LB liquid medium (without antibiotics), and shake culturing at 200rpm and 28 ℃ for 3 hours. Centrifuging at 5000rpm for 1 min, removing part of supernatant, gently sucking the rest, beating, spreading on LB solid medium containing kanamycin and rifampicin, and culturing at 28deg.C for 2-3 days to obtain Agrobacterium plasmid containing PDS gene editing vector BoPDS and OsPDS.

Genetic transformation of rice is well known in the art and is exemplified below.

(1) And in the co-culture stage, the agrobacterium cultured on the AB medium in advance is diluted to an OD600 of about 0.2 by using an AAM liquid medium containing a proper amount of AS, and the co-culture stage is started, and the diluted bacterial liquid and the rice callus which is cultured in advance and grows vigorously are co-cultured for 3 days.

(2) And in the screening culture stage, the callus subjected to co-culture is cleaned by using sterile water containing antibiotics Tm, which is killed in advance, and the surface moisture is dried under the room temperature condition, and the operations are carried out in an ultra clean bench under the sterile condition. Transferring the completely dried callus to a screening culture medium added with a proper amount of antibiotics by using tweezers, replacing the culture medium once after illumination culture for about 10 days, and setting a gradient for reducing the concentration of the antibiotics for screening for about 2-3 times.

(3) And in the differentiation stage, transferring the well-activated callus subjected to screening into a differentiation medium, wherein the well-activated callus gradually changes to green until seedlings are differentiated.

(4) And in the rooting stage, the rice seedlings with a few calluses are transferred into a rooting culture medium and are subjected to illumination culture for about 2 weeks until most of roots grow.

Extracting genome DNA of rice seedlings, identifying the transformed seedlings by adopting a resistance primer, primarily screening out rice positive strains, and further identifying the transformed seedlings after the rice positive strains are moved to the field.

Genetic transformation of cabbage is a well known technique in the art, and the following is an exemplary procedure.

(1) Acquisition of cabbage explants

Selecting mature, full and round cabbage seeds without mildew spots, sterilizing for 3 minutes by using 75% alcohol, sterilizing for 8-10 minutes by using 8-10% sodium hypochlorite solution, washing for 3 times by using sterilized water, using sterile filter paper for the sterilized seeds, sucking redundant water in an ultra-clean workbench, and uniformly placing the seeds on a solid MS culture medium. Culturing under 16h light/8 h dark condition for 5-7 days, cutting hypocotyl of caulis et folium Brassicae Capitatae into 0.8-1cm long, and using as receptor for Agrobacterium-mediated transformation.

(2) Genetic transformation of cabbage

Agrobacteria plasmids containing PDS gene editing vector were cultured to od600=0.4-0.6, respectively, and centrifuged at 6000rpm for 10 minutes, and resuspended in liquid MS medium as an invader solution.

The cabbage explants are infected for 8-10 min, placed in co-culture medium for dark culture at 25 ℃ for 36-48 h, then the explants are transferred to selection medium containing 10mg/L Basta concentration, and the selection culture is replaced every two weeks under 16h light/8 h dark condition. When the resistant buds grow to about 2-3cm in length, cutting off the resistant buds, transferring the cut resistant buds to a long seedling culture medium containing timentin, culturing for 20-30 days under 16h illumination/8 h darkness, and culturing for 20 days in a rooting culture medium. And transferring the plants with developed root growth into vermiculite for hardening for 7 days, and then transplanting the plants into nutrient soil.

And (3) carrying out PCR amplification by using Bar primers, and detecting by using 1.0% agarose gel electrophoresis to obtain the T0 generation positive plant.

The Bar primer sequences were as follows:

BarH-F:5’-AAACCCACGTCATGCCAGTT-3’;SEQ ID No.31

BarH-R:5’-GTCTGCACCATCGTCAACCAC-3’;SEQ ID No.32

And carrying out first-generation high-throughput sequencing on the T0-generation positive plants subjected to screening, and carrying out statistics on sequencing results.

The results show that:

For rice, the average rise in editing efficiency was 13.8% with the 'NGGT' compared to the 'NGGN' for the OsPDS knocked-out rice positive editing plants, which was 28.6%.

TABLE 3 statistical data on the types of editing and efficiency of Rice Gene Using different PAM sequences

For cabbage, knockout BoPDS caused the cabbage positive editing plants to exhibit a albino phenotype, with the 'NGGN' editing plants essentially appearing as chimeras, the 'NGGT' exhibited a higher degree of editing on the cabbage plants than the 'NGGN', and the editing efficiency increased from an average of 20.4% to an average of 68.7%, as shown in fig. 3 and table 4.

TABLE 4 cabbage Gene editing type, efficiency statistics

The results in both rice and cabbage show that when the PAM sequence 5'-NGG-3' is later selected as 'T', the CRISPR/Cas9 gene editing system editing type can be significantly increased and editing efficiency can be improved.

Example 2 Effect of fusion expression of GRF5-GIF1-GRF5 on cabbage B.oleracea regeneration efficiency

The vector used in the genetic system test was an Empty pBWA (V) BS-Empty engineered from the pCAMBIA1301 vector (purchased from Addgene ^TM) which replaced Hyg resistance in the original vector with Basta resistance.

The test material was cabbage inbred line 'M1'.

It has been found that overexpression of some plant growth regulators significantly increases plant regeneration efficiency in transgenic tissue culture (Debernardi et al, 2020).

The research and development team of the present invention identified 19 Growth Regulator (GRF) proteins and 1 GRF Interacting Factor (GIF) protein in the cabbage genome via bioinformatic analysis.

Further studies were performed on 2 GRF5 proteins, 2 GRF4 proteins and 1 GIF1 protein, and two GRF5 proteins (Seq ID No.33 and Seq ID No. 34), two GRF4 proteins (Seq ID No.35 and Seq ID No. 36) and GIF1 protein (Seq ID No. 37) were respectively fusion-expressed by 4 alanine residues to GRF5-GIF1-GRF5 (Seq ID No.38 or Seq ID No. 39), GRF4-GIF1-GRF4 (Seq ID No. 40).

Four proteins GRF5-GIF1-GRF5, GRF4-GIF1-GRF4, GRF5, GIF1 and a blank were tested for their effect on the efficiency of cabbage regeneration, see FIG. 4, i.e., the nucleotide sequences encoding the four proteins (Seq ID No. 41-44) were each constructed into the expression vector pBWA (V) BS-Empty, and introduced into the wild-type cabbage variety M1 via Agrobacterium-mediated genetic transformation.

Genetic transformation was performed as in S2 of example 1.

The results showed that the average regeneration efficiency of the cabbage transformant expressing GRF5-GIF1-GRF5 was increased by 55.2%, as shown in FIG. 4.

Table 5 nucleotide or amino acid sequences referred to in examples

Example 3 discovery and cloning of Gene BoBPM and construction of Gene editing vector

The inventors found a novel differential expression gene BTB/POZ (Broad complex, TRAMTRACK, bric-a-brac/Pox virus and Zinc finger) -MATH 6 (BPM 6) in the study, reported that the gene is induced to express by fusarium wilt and black rot, and speculated that the gene is a disease-sensitive gene inducing the onset of various diseases.

It has been demonstrated by studies that inactivation of the DMR6 gene as a conserved disease (S) gene, tomato DMR6 gene, results in a broad spectrum of disease resistance (Thomazella et al., 2021), and the inventors hypothesized that inactivation or inhibition of expression of DMR6 in cabbage may also result in a broad spectrum of disease resistance.

To test whether the cabbage BPM6 and DMR6 genes can be used to generate a broad spectrum of disease resistance in cabbage, the inventors constructed a gene editing vector to knock out it.

By designing primers (Table 6)

TABLE 6

The DNA of wild type M1 of cabbage is used as template to obtain the genome segment of BPM6 and DMR6 in cabbage, boBPM6 is full-length 3526bp (Seq ID No. 49), and its coding region is formed from 2326bp bases (Seq ID No. 50). BoDMR6 full-length 6757bp (Seq ID No. 51), and a coding region consisting of 1026 bases (Seq ID No. 52). The sequences are shown in Table 7.

I. construction BoBPM of CRISPR/Cas9 editing vector of 6:

(1) PYLCRISPR/Cas9-35S-B was selected as the gene editing vector framework;

(2) Selecting 5'-' NGGT '-3' as PAM sequence of the cabbage BPM6 editing vector;

(3) The following sgrnas and their adaptor primers were then designed for BoDMR, boBPM6 genes,

BoDMR6 sgRNA ACCGTCCACGTCTCTCCCAAGTTT, seq ID No.53, see FIG. 5

BD6-F:cagtGGTCTCatgcaTCCACGTCTCTCCCAAGTTTgttttaga,Seq ID No.54

BD6-R:cagtGGTCTCaaaacAAACTTGGGAGAGACGTGGACGGT,Seq ID No.55

BoBPM6 sgRNA CTCCAAGTCCGTGACGCAGACGG, seq ID No.56, see FIG. 6

BB6-F:cagtGGTCTCatgcaCTCCAAGTCCGTGACGCAGAGttttaga,Seq ID No.57

BB6-R:cagtGGTCTCaaaacTCTGCGTCACGGACTTGGAG,Seq ID No.58

After annealing the synthetic adapter primer to double strand, the primer is digested and connected to PYLCRISPR/Cas9-35S-B vector to obtain BoDMR gene editing vector and BoBPM gene editing vector.

EXAMPLE 4 BoBPM Gene editing on cabbage Material

The BPM6 gene editing vector constructed in example 3 was co-transferred with the expression vector of the GRF5-GIF1-GRF5 fusion protein (Seq ID No. 38) constructed in example 2 into wild-type cabbage variety M1 by Agrobacterium-mediated genetic transformation:

According to the standard procedure, agrobacterium is transformed by adding 1 μg of plasmid into 100 μl of agrobacterium competent cells, mixing, sequentially standing on ice, liquid nitrogen, 37 ℃ water bath and ice for 5min, adding 800 μl of LB liquid medium (without antibiotics), and shake culturing at 200rpm and 28 ℃ for 3 hours. Centrifuging at 5000rpm for 1 min, removing part of supernatant, gently sucking the rest, stirring, smearing on LB solid medium containing kanamycin and rifampicin, and culturing at 28deg.C for 2-3 days.

The genetic transformation method is the same as S2 in example 1.

Carrying out first-generation sequencing on the T0-generation positive plants subjected to screening, wherein the genetic transformation efficiencies of bobpm < 6 > and bodmr < 6 > are respectively 5.5% and 8.2%, and the editing efficiencies are respectively 62.0% and 62.5%;

wherein transformation efficiency = number of positive plants/number of infected explants, edit efficiency = number of edit plants/number of positive plants.

The conversion efficiency is significantly higher than the 1% cabbage conversion efficiency reported in the prior literature, and the editing efficiency is 12.9%.

Obtaining T1 generation seeds through selfing reserved seeds, randomly selecting the seeds for first generation sequencing, wherein bodmr and bobpm6 are respectively used for obtaining 4 editing types and 3 editing types (figures 5 and 6);

example 5 disease resistance test of BoBPM6 Gene editing Material

The T1-generation cabbage material obtained in example 4 was inoculated with pathogenic bacteria of the wilt, black rot, clubroot (three main diseases of cabbage).

The black rot is artificially inoculated by adopting a spray method:

(1) The preparation of bacterial liquid comprises streaking and activating preserved black rot germ (Xanthomonas campestris pv. Campestris), adding the activated black rot germ into liquid PSA culture medium in the form of bacterial clusters by using an inoculating loop, shading and culturing for 16h in a shaking table at 28 ℃ and 200rpm, adjusting OD ₆₀₀ value to 0.2 by using sterile water, and preparing for inoculation.

(2) And (3) in the inoculation process, after the seedlings grow to 4-5 true leaves, preparing for inoculation, moving the seedlings to be inoculated to an inoculation place one day before inoculation, watering the seedling raising matrix thoroughly, spraying water on the leaves by using a sprayer, and moisturizing the leaves until inoculation, wherein the water content in the leaves is required to be kept to be more than 90%, so that the water holes at the edges of the leaves before inoculation are in an open state. The bacterial liquid is uniformly sprayed on the blades by using a sprayer until the blades are fully covered with the bacterial liquid, and the temperature is controlled to be about 28 ℃.

(3) The resistance investigation and the resistance level are divided into a resistance evaluation standard of 0 level, no symptoms of inoculated leaves, 1 level which is less than 5% of leaf area, 3 level which is 5-15% of leaf area, 5 level which is 15-30% of leaf area, 7 level which is 30-50% of leaf area and 9 level which is more than 50% of leaf area. Di=Σ (number of leaves at disease level x extreme value of disease)/(total number of leaves investigated x highest disease level) ×100. High disease resistance (HR), 0< DI < 10 >, disease resistance (R), 10< DI < 30%, moderate disease resistance (MR), 30< DI < 50%, disease susceptibility (S), 50< DI < 70%, high disease susceptibility (HS), DI >70.

The fusarium wilt bacteria are inoculated manually by a root dipping method:

(1) Preparation of bacterial liquid preserved fusarium wilt bacteria (Fusarium oxysporum f.sp.Conglutans) were added to liquid CM medium, shading cultured for 3 days in a shaker at 28℃and the mycelia were filtered using gauze and the remaining spores were adjusted to a concentration of 1X10 ⁶/mL and then prepared for inoculation.

(2) The inoculation process is that after the seedlings grow to 3 true leaves, the seedlings are pulled out from the plug seedling matrix and the roots are washed before inoculation, the roots are completely soaked in bacterial liquid, the seedlings are taken out after 15 minutes, transplanted into a seedling bowl filled with soil and moved to a temperature control greenhouse, and the temperature is controlled at 23-29 ℃.

(3) The resistance investigation and the resistance level are divided into a resistance evaluation standard, namely 0 level, no symptoms, 1 level, 1 leaf slight yellowing, 2 level, 1-2 leaf moderate yellowing, 3 level, half leaf severe yellowing or wilting, 4 level, all leaves except heart leaf severe yellowing or wilting, and 5 level, all leaves severe yellowing or plant death. Leaf spot grade was investigated for material inoculation, average Disease Index (DI) was calculated, and resistance grade was divided according to Disease index. Di= [ Σ (each disease level×number of disease plants at the corresponding level)/(total number of investigated plants×highest disease level) ]×100. High disease resistance (HR), 0< DI < 10 >, disease resistance (R), 10< DI < 30%, moderate disease resistance (MR), 30< DI < 50%, disease susceptibility (S), 50< DI < 70%, high disease susceptibility (HS), DI >70.

The clubroot is inoculated manually by root irrigation:

(1) Preparation of bacterial liquid, namely placing the cabbage swelling root stored in a refrigerator at-20 ℃ for activation for 12 hours at room temperature in advance, adding three times of sterile water, fully crushing the cabbage swelling root by using a juicer, filtering the cabbage swelling root by using gauze, collecting filtrate into a 50mL centrifuge tube, centrifuging the cabbage swelling root at 10min at 600rpm, collecting supernatant, then collecting the cabbage swelling root by using 10min at 2500 rpm, re-suspending the cabbage swelling root by using sterile water, and regulating the spore concentration to 2x10 ⁷ cfu/mL under a microscope.

(2) Inoculating when the cabbage seedling grows to 2 true leaves, scratching 2 knives on the root of each plant by a knife, sucking 2mL of bacterial liquid by a pipetting gun to inject the bacterial liquid into the root of the seedling, and moving the seedling to a temperature control greenhouse, wherein the temperature is controlled at 18-25 ℃.

(3) The resistance investigation and the resistance level are divided into a resistance evaluation standard that 0 grade = root is asymptomatic, 1 grade = main root is asymptomatic, lateral root has small tumor, 2 grade = main root slightly swelled, lateral root tumor is bigger, 3 grade = main root swelled is more serious and has obvious lateral root, and 4 grade = main root swelled is very serious and almost has no lateral root. Di= Σ (number of individual stages of disease × number of corresponding stages of disease)/(total number of test × highest stage of disease) ×100. The resistance evaluation criteria include immunity (I) DI=0, high Resistance (HR) 0< DI <5, disease resistance (R) 5< DI < 20, medium Resistance (MR) 20< DI < 30, disease susceptibility (S) 30< DI < 60, and High Susceptibility (HS) DI >60.

The investigation result shows that:

the disease index of bodmr plants inoculated with wilt, black rot, clubroot decreased from 79.0 to 78.4, from 79.3 to 55.1 (significant) and from 90.7 to 57.6 (significant), respectively, compared to wild-type cabbage (fig. 7);

The disease index of bobpm plants inoculated with wilt, black rot, clubroot decreased from 79.0 to 78.4, from 79.3 to 55.1 (significant) and from 90.7 to 57.6 (significant), respectively, compared to wild-type cabbage (fig. 8);

The experimental results show that BoDMR gene and BoBPM gene are sensitive genes for inducing the attack of various cabbage diseases, and knocking off or inhibiting the expression of the genes can obtain novel germplasm with broad-spectrum disease resistance. The matched optimized gene editing and genetic transformation system provided by the invention provides powerful technical support for cabbage disease-resistant breeding.

TABLE 7

Claims

1. A cabbage high-efficiency genetic transformation and gene editing method is characterized in that,

(3) Screening T0 generation transgenic cabbage with gene editing;

2. The method of claim 1, further comprising introducing an expression vector expressing the fusion protein GRF5-GIF1-GRF5 either before or after introducing a CRISPR/Cas9 gene editing vector into the cabbage material of interest;

3. The method according to claim 2, wherein the introduced CRISPR/Cas9 gene editing vector is introduced by agrobacterium-mediated genetic transformation, and the introduced expression vector expressing the fusion protein GRF5-GIF1-GRF5 is also introduced by agrobacterium-mediated genetic transformation.

4. A cabbage growth regulator fusion protein, characterized in that it has the structure GRF5-GIF1-GRF5, and the amino acid sequence has any one of the following characteristics:

5. The cabbage growth regulator fusion protein according to claim 4, wherein the amino acid sequence is as set forth in Seq ID No. 38.

6. A nucleotide sequence encoding the cabbage growth regulator fusion protein of claim 4 or 5.

7. The nucleotide sequence according to claim 6, as set forth in Seq ID No. 41.

8. An expression vector for expressing the cabbage growth regulator fusion protein of claim 4 or 5, which is loaded with the nucleotide sequence of claim 6 or 7.

9. A kit for improving the efficiency of efficient genetic transformation and gene editing of brassica oleracea, characterized in that it comprises a CRISPR/Cas9 gene editing vector having a PAM sequence of 5'-' NGGT '-3', wherein N is one of a, C, G.

10. The kit of claim 9, further comprising an expression vector of claim 8.