Common head cabbage mutant gene and application thereof in orange heart common head cabbage breeding

Abstract

The invention relates to the field of crop genetic breeding and crop new resource innovation. The invention discloses creation of a common head cabbage mutant gene CRTISO-TP and application of the common head cabbage mutant gene CRTISO-TP in orange-core common head cabbage breeding. Specifically, A1) application of the common head cabbage mutant gene CRTISO-TP in preparation of orange-core cabbage and / or production and preparation of orange-core cabbage products; a2), application in orange-core cabbage breeding and / or production and preparation of orange-core cabbage breeding products; the protein is any one of the following proteins: B1) a protein with an amino acid sequence as shown in a sequence 20; b2) a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues of the protein B1), has 80% or more of identity with the protein B1) and has the same function as the protein B1); and B3) a fusion protein obtained by connecting the N terminal or / and the C terminal of B1) or B2) with a protein tag. Orange-core cabbages can be obtained through the method and can be used for agricultural production.

Description

Technical Field

[0001] The invention relates to the field of crop genetic breeding and new crop resource innovation, and in particular to a cabbage mutant gene and application thereof in breeding of orange-heart cabbage. Background Art

[0002] Cabbage belongs to the genus Brassica of the family Brassicaceae. It is a biennial or perennial head vegetable that originated from the Mediterranean to the North Sea coast of Europe. Because cabbage has a crisp and tender taste and is rich in nutrients, it can be eaten cold, fried, pickled, and processed into dehydrated vegetables. It has become an indispensable dish on the tables of Chinese residents. At present, the cultivated area of ​​cabbage in my country has reached more than 900,000 hectares, and it plays an extremely important role in my country's vegetable export trade and year-round supply. In recent years, with the comprehensive construction of a well-off society in my country and the improvement of residents' living standards, my country's vegetables are changing from quantity guarantee to nutrition quality, and urban and rural residents have put forward higher requirements for vegetable quality. Therefore, improving the nutritional quality traits of cabbage has gradually become the future breeding direction.

[0003] The quality traits of cabbage include appearance quality, taste quality and nutritional quality, among which nutritional quality refers to a series of substances with nutritional and health functions, including carotenoids, dietary fiber, vitamins, carbohydrates, mineral elements, etc. Carotenoids are an important type of nutrient in cabbage and the main source of vitamin A in the human body. They have health benefits such as antioxidant, immunomodulatory, anti-cancer, and anti-aging. The more the color of the edible organs of vegetables is yellow, orange or red, the higher the content of carotenoids. For example, orange-heart Chinese cabbage is rich in pro-lycopene, lycopene and β-carotene. So far, there has been no report on the application of orange-heart cabbage germplasm and commercial varieties in China or even in the world, which has seriously affected the genetic improvement of the carotenoid content of cabbage.

[0004] The main methods for creating germplasm resources are hybrid breeding, mutagenesis breeding, and gene editing breeding. Compared with the first two methods, gene editing can efficiently and accurately insert, delete, or replace genes, thereby changing their genetic information and traits. Therefore, gene editing can efficiently create new orange-heart cabbage germplasm. However, the use of gene editing to create orange-heart cabbage requires solving the problem of target genes. At present, due to the lack of orange-heart cabbage germplasm, it is impossible to identify the orange-heart gene of cabbage through forward genetics. At present, the orange-heart gene of Chinese cabbage has been identified as the CRTISO gene. The loss of this gene function will lead to the obstruction of pro-lycopene isomerization, thereby causing the orange-heart phenotype of Chinese cabbage. The previous study of this research group showed that there is only one CRTISO gene in the whole genome of cabbage, and the in vitro enzyme activity experiment verified that CRTISO has the activity of pro-lycopene isomerase, which is a potential orange-heart gene in cabbage. Therefore, by using gene editing to knock out the CRTISO gene of cabbage, it is very likely to obtain new cabbage germplasm with orange-red heart, which is of great significance for promoting the rapid development of breeding of cabbage for carotenoid content traits. Summary of the invention

[0005] The technical problem to be solved by the invention is to regulate the color of cabbage heart.

[0006] In order to solve the above technical problems, the present invention provides the following applications.

[0007] Use of a substance for reducing the expression of a gene encoding the protein in any of the following;

[0008] A1) Application in preparing orange-red-heart cabbage and / or producing orange-red-heart cabbage products;

[0009] A2) Application in breeding of orange-red-heart cabbage and / or producing breeding products of orange-red-heart cabbage;

[0010] The protein is any of the following:

[0011] B1) the amino acid sequence is the protein shown in SEQ ID NO: 20;

[0012] B2) a protein having more than 80% identity with the protein shown in B1) and having the same function as the protein shown in B1) obtained by substitution and / or deletion and / or addition of amino acid residues of the protein shown in B1);

[0013] B3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of B1) or B2) to a protein tag.

[0014] In the above application, the protein is derived from cabbage.

[0015] In the above application, the substance that reduces the expression of the gene encoding the protein is any one of the following:

[0016] B1), a nucleic acid molecule that reduces the expression of the gene encoding the above protein;

[0017] B2), expressing the coding gene of the nucleic acid molecule described in B1);

[0018] B3), an expression cassette containing the gene described in B2);

[0019] B4, a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3);

[0020] B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

[0021] In the above application, the target gene of the nucleic acid molecule described in B1) is shown in sequence 22.

[0022] In order to solve the above technical problems, the present invention also provides a method for cultivating orange-heart cabbage.

[0023] The method comprises the step of reducing the expression level of a gene encoding any of the above proteins in the target cabbage to obtain orange-heart cabbage.

[0024] In order to solve the above technical problems, the present invention also provides a method for reducing the brightness of cabbage heart.

[0025] The method comprises reducing the expression level of a gene encoding any of the above proteins in the target cabbage to obtain low-brightness cabbage heart, wherein the lightness of the cabbage heart of the low-brightness cabbage heart is lower than that of the target cabbage.

[0026] In the above method, reducing the expression of the gene encoding any of the above proteins in cabbage comprises introducing the nucleic acid molecule described in B1), the expression cassette of the gene described in B2), the expression cassette described in B3) or the recombinant vector described in B4) into the target cabbage.

[0027] In order to solve the above technical problems, the present invention also provides a method for cultivating orange-heart cabbage.

[0028] The method comprises the steps of performing deletion mutation on the thymine deoxyribonucleotide residue at position 324 of sequence 22 in the cabbage genome to obtain orange-heart cabbage.

[0029] In any of the above uses or any of the above methods, the cabbage is cabbage.

[0030] In any of the above uses or methods, the heart of the orange-red heart cabbage is orange-red during the heading period, or the lightness of the heart of the low-lightness heart cabbage during the heading period is lower than that of the target cabbage.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0032] 1) The present invention realizes the site-directed mutation of the CRTISO gene of cabbage for the first time through gene editing technology, and then obtains a new cabbage germplasm with no exogenous T-DNA fragments and stable inheritance of orange-red heart phenotype in the T1 generation through screening of T1 plants. In addition to the orange-red heart trait, the new germplasm has no obvious changes in other phenotypes. Compared with conventional mutagenesis breeding and hybrid breeding, gene editing breeding only takes 2-3 years to complete, has the advantages of high efficiency and precision, shortens the breeding cycle, and has a very broad application prospect.

[0033] 2) The mutation of the CRTISO-TP gene at the InDel-C08:50542693 site created by gene editing in the present invention is the first of its kind in this patent. By comparing with the polymorphic sites of the cabbage pan-genome, it was found that the mutation of InDel-C08:50542693 did not exist in the cabbage pan-genome composed of 119 cabbage materials.

[0034] 3) Based on the sequence difference between the wild type and the mutant at the InDel-C08:50542693 position, this patent developed the KASP molecular marker that can specifically distinguish the wild type (the 324th base of the CRTISO coding sequence is T) and the mutant (the 324th base of the CRTISO coding sequence is G), which can be applied to molecular marker-assisted breeding of cabbage.

[0035] 4) The cabbage germplasm created by the present invention using CRISPR / Cas9 gene editing technology shows orange-red at the growth point during the rosette stage, and the leaves become greener as they go outward. During the heading stage, the outer leaves of this germplasm are normal green, but the closer the leaves are to the growth point, the color becomes orange-yellow. After cutting the head, the color inside the head becomes orange-red. The present invention is the first to create a new cabbage germplasm with orange-red heart. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the base variation map of the cabbage mutant gene CRTISO-TP.

[0037] Figure 2 It is a high-generation inbred line material of cabbage, sgRNA expression cassettes of T0 and T1 plants, Blp RGene and Cas9 gene detection results; Marker is DL2000, G1-10 is a high-generation inbred line material of cabbage (negative control), T0-1 and T0-2 are T0 generation transgenic plants (positive control), T1-1 to T1-8 are T generation transgenic plants containing exogenous T-DNA fragments, and T1-9 and T1-10 are T1 generation plants without exogenous T-DNA fragments.

[0038] Figure 3 This is a comparison of the center color of the head of the wild-type cabbage 09-4-3 and the CRTISO knockout homozygous line T1-10 during the heading period.

[0039] Figure 4 Comparison of plant phenotypes of wild-type and orange-heart cabbage at the seedling, rosette and heading stages.

[0040] Figure 5 For the development of KASP molecular markers for the CRTISO-TP gene, the materials tested included high-generation inbred lines of cabbage, T0 generation, T1 generation and F2 population plants of T1-10×09-4-3. DETAILED DESCRIPTION

[0041] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0042] The background material selected by the present invention is cabbage 09-4-3, which is a high-generation inbred line material bred by Zhejiang Academy of Agricultural Sciences, has good bolting resistance, excellent taste quality, and strong disease resistance, but has a common yellow heart after heading. The present invention uses CRISPR / Cas9 gene editing technology to perform site-directed mutation on the CRTISO gene of cabbage 09-4-3 to obtain cabbage with an orange-red heart phenotype, so as to meet consumers' urgent needs for nutritious quality cabbage and promote the progress of cabbage carotenoid content trait breeding.

[0043] Example 1: Creation process of orange-heart cabbage mutant

[0044] 1. Cloning of the cabbage 09-4-3CRTISO gene and design of its gene editing target site

[0045] Referring to the original read data of the transcriptome sequencing of cabbage 09-4-3 leaves, the coding sequence of CRTISO was obtained by read splicing. The total RNA of cabbage 09-4-3 leaves was extracted and reverse transcribed into cDNA. The cDNA template was PCR amplified with primers CRTISO-cDNA-F: 5'-ATGAATCTCTGTCTCCGCAACCC-3' and CRTISO-cDNA-R: 5'-TGCGAGTGTCCTTAACCAACCAA-3', and the PCR product was sent to Qingke Biotechnology Co., Ltd. (Hangzhou Sequencing Department) for sequencing. The sequencing results were compared with the BRAD (http: / / brassicadb.cn / # / ) database, and it was found that the CRTISO coding region sequence of cabbage 09-4-3 was different from the cabbage reference genome (Brassica oleraceacv.JZS V2.0).

[0046] Based on the CRTISO coding sequence of cabbage 09-4-3 and the design principle of target sites, target sites 1: CAGCACACGTTACGGGGTTG, target site 2: TCTTTCTTCTACTACAGCAG, target site 3: GTCATCGGGTCTGGGATTGG, and target site 4: AATAAACCAGAGCTCCCACCA were selected as target sites for gene editing on the first exon of the CRTISO gene. Mutations at these sites are expected to cause the loss of CRTISO function, thereby obtaining new cabbage germplasm with orange-red heart.

[0047] 2. Construction of CRISPR / Cas9 gene editing vector

[0048] (1) Synthesis of sgRNA expression cassette

[0049] Four sets of 539bp sgRNA expression cassette sequences consisting of tRNA-target site-guide RNA sequences were designed and sent to a primer synthesis company for direct synthesis.

[0050] (2) Amplification of sgRNA expression cassette

[0051] Dissolve the synthesized sgRNA expression cassette in ultrapure water to a concentration of 50 ng / μl, take 1 μl as a template, and use primers with BsaI restriction sites to perform PCR amplification on sgRNA-F: 5'-cagtGGTCTCatgcaCAGCACACGTTACGGGGTTG-3' and sgRNA-R: 5'-cagtGGTCTCaaaacTGGTGGGAGCTCTGGTTATT-3'. The amplified product is cleaned and recovered using a DNA gel recovery kit. The recovered product is the sgRNA expression cassette ligation product. The PCR amplification system and amplification procedure are as follows: 50 μL PCR amplification system

[0052] Element volume Synthetic Template 1μL sgRNA-F (100 μM) 2μL sgRNA-R (100 μM) 2μL 2×PfuPCRMix 25μL <![CDATA[ddH2O]]> 20μL

[0053] The PCR amplification procedure is as follows

[0054] step Number of cycles 94℃for5min 1 94℃for30sec 30 50℃for45sec 30 72℃for54sec 30 72℃for10min 1 16℃for30min 1

[0055] (3) sgRNA expression cassette connected to pBSbdcas9i-tRNA vector

[0056] The recovered product after amplification of the sgRNA expression cassette obtained in step 2) and the pBSbdcas9i-tRNA vector were subjected to restriction enzyme ligation reaction using Bsa I endonuclease and T4 DNA ligase at the same time, and the sgRNA expression cassette was inserted into the pBSbdcas9i-tRNA vector. The restriction enzyme ligation reaction system and amplification procedure are as follows:

[0057] 20 μL of enzyme digestion and ligation system

[0058]

[0059]

[0060] BsaI and T4 DNA ligase were purchased from NEB, and the pBSbdcas9i-tRNA (k6) vector was provided by Wuhan Boyuan Biological Company.

[0061] Restriction Digestion and Ligation Procedure

[0062] step Number of cycles 37℃for1hour 1 16℃for30min 1 60℃for5min 1

[0063] (4) Transformation of E. coli DH5α and verification

[0064] The enzyme-digested ligation product in 3) was transformed into E. coli by heat shock and cultured overnight at 37°C on LB medium containing 50 μg / ml kanamycin until a single colony grew. A single colony was picked and cultured at 37°C by shaking, and PCR amplification was performed using the sgRNA-F and sgRNA-R primer pairs for verification.

[0065] 20 μL PCR amplification system

[0066] Element volume Bacterial liquid 1μL sgRNA-F (100 μM) 1μL sgRNA-R (100 μM) 1μL 2×TaqPCRStarMix 10μL <![CDATA[ddH2O]]> 7μL

[0067] 2×Taq premix was purchased from Genstar.

[0068] Amplification was performed in a PCR amplifier. The PCR program is shown in the table below. The PCR product was loaded into an agarose gel for electrophoresis, and the length of the PCR product was detected using a gel imager. The plasmid of the bacterial solution with the correct PCR band was extracted using a gel recovery kit (omega) and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced recombinant plasmid was named pBSbdcas9i-tRNA-sgRNA knockout vector. The pBSbdcas9i-tRNA-sgRNA knockout vector was obtained by opening the restriction endonuclease BsaI recognition site of the pBSbdcas9i-tRNA (k6) vector, replacing the sequence between fragment 1 (5'-TTGCTATTTCTAGCTCTAAAAC-3') and fragment 2 (5'-TGCACCAGCCGGGAATCGA-3') of the pBSbdcas9i-tRNA (k6) vector with a DNA fragment (sequence 23) with four target linker sequences, and keeping the other sequences of the pBSbdcas9i-tRNA (k6) vector unchanged. The resulting recombinant vector was named pBSbdcas9i-tRNA-sgRNA knockout vector.

[0069] Bacterial liquid PCR reaction procedure

[0070] step Number of cycles 94℃for5min 1 94℃for30sec 32 50℃for45sec 32 72℃for54sec 32 72℃for10min 1 16℃for30min 1

[0071] (5) Obtaining a mutant of orange-heart cabbage

[0072] The above positive plasmid was transformed into Agrobacterium tumefaciens EHA105. EHA105 / pBSbdcas9i-tRNA-sgRNA was obtained. EHA105 / pBSbdcas9i-tRNA-sgRNA is Agrobacterium tumefaciens EHA105 containing the pBSbdcas9i-tRNA-sgRNA knockout vector.

[0073] Cabbage 09-4-3 (the cabbage material is derived from the self-segregating population of the F1 hybrid of Chunfeng cabbage, and is a high-generation inbred line material obtained through multi-generation breeding and self-pollination purification) was transformed by conventional Agrobacterium-mediated method. A total of 2 T0 transgenic plants were obtained.

[0074] The genomic DNA of the two T0 transgenic plants was used as PCR templates, and the primer combination CRTISO-F: 5'-CTCTGTCTCCGCAACCCC-3' and CRTISO-R: 5'-TCTCACGACCAACTGCCTTC-3' was used to amplify and sequence the two T0 transgenic plants. Figure 1 As shown in the figure, the CRTISO gene of T0-1 has not mutated, and the CRTISO allele of T0-2 has a frameshift mutation due to the deletion of a thymine deoxyribonucleotide residue (T) at position 324. The corresponding protein product contains 136 amino acids, while the wild-type CRTISO protein has 590 amino acids, as shown in SEQ ID NO: 20. The CRTISO nucleotide sequence of the orange-heart cabbage is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO: 21. The CRTISO mutant gene in the T0-2 plant was named CRTISO-TP.

[0075] The CRTISO-TP gene created for the first time by the present invention lacks the 324th base T and the frameshift mutation caused by it is reported for the first time.

[0076] The following mutations occurred in T0-2 plants:

[0077] In one chromosome set, the thymine deoxyribonucleotide residue at position 324 of the CRTISO gene (nucleotide sequence is SEQ ID NO.22) is missing, resulting in a frameshift mutation in the CRTISO gene, causing the encoded protein (SEQ ID NO.20) to terminate prematurely at the 137th amino acid, thereby knocking out the CRTISO gene.

[0078] In another chromosome set, the thymine deoxyribonucleotide residue at position 324 of the CRTISO gene (nucleotide sequence is SEQ ID NO.22) is not deleted, resulting in no frameshift mutation in the CRTISO gene, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.20.

[0079] Example 2 Screening of orange-heart cabbage mutants without exogenous T-DNA fragments

[0080] The binary T-DNA vector for directional editing of the CRTISO gene constructed by the present invention mainly comprises an sgRNA expression cassette, a nuclease gene Cas9 and a herbicide resistance gene Blp R , the above three genes are mainly responsible for the site-specific editing of the target gene and the screening of regenerated seedlings, and are all exogenous genes relative to the cabbage genome. If the sgRNA expression cassette and the Cas9 gene continue to be retained, there is a possibility of continuing to edit the CRTISO gene and other genes; in addition, the continued retention of the T-DNA fragment may lead to random insertions, which in turn cause other unpredictable gene mutations. Therefore, after achieving the goal of gene editing, it is often necessary to remove the exogenous T-DNA fragments. Since the exogenous T-DNA and the target gene will be separated after the transgenic plants are self-pollinated, PCR detection can be used to screen and obtain plants without exogenous T-DNA fragments. In order to obtain orange-heart cabbage plants that do not carry the above-mentioned exogenous T-DNA fragments, the inventors simultaneously detected the sgRNA expression cassette, Cas9 gene and Blp of the T1 generation plants of the T0-2 strain by PCR. R Genes were expressed in 247 genes, and each gene was repeated 3 times to screen T1 generation plants that did not contain these 3 gene fragments.

[0081] The genomic DNA of the cabbage high-generation inbred line materials (selected and bred by the Cabbage Breeding Laboratory of the Vegetable Research Institute of Zhejiang Academy of Agricultural Sciences, Table 1), T0 generation plants (including T0-1 and T0-2) and T1 generation plants from the T0-2 line (including T1-1-T1-10) were extracted, and the sgRNA expression cassette was detected by PCR using primers Target1-F: 5'-CAGCACACGTTACGGGGT-3' and Target4-R: 5'-TGGTGGGAGCTCTGGTTATT-3'. The PCR product was 539 bp; the Cas9 gene and Blp were simultaneously detected by primers Cas9-TF: 5'-CAGTCGATTACCGGCCTCTACG-3' and BLP-SR: 5'-GGTGGCACGGCGGATGTC-3'. R The gene was tested by PCR. Figure 2 As shown, no sgRNA expression cassette, Cas9 gene and Blp were detected in any non-transgenic cabbage. R Gene, sgRNA expression cassette, Cas9 gene and Blp were detected in T0 generation strains T0-1 and T0-2. R Gene, no sgRNA expression cassette, Cas9 gene and Blp were detected in the T1 generation plants numbered T1-9 and T1-10 from the T0-2 line. R gene, indicating that these two strains did not carry exogenous T-DNA fragments.

[0082] Table 1 Variety characteristics of cabbage high-generation inbred lines

[0083]

[0084]

[0085] The genome sequencing results showed that the following mutations occurred in the T1 generation plant T1-10:

[0086] In both chromosome sets, the thymine deoxyribonucleotide residue at position 324 of the CRTISO gene (nucleotide sequence is SEQ ID NO.22) is missing, resulting in a frameshift mutation in the CRTISO gene, causing the encoded protein (SEQ ID NO.20) to terminate prematurely at the 137th amino acid, thereby knocking out the CRTISO gene.

[0087] Cabbage 09-4-3 and T1-10 were planted in pots (28 cm in diameter, 20 cm in diameter, and 22 cm in height) in the glass greenhouse of the Zhejiang Academy of Agricultural Sciences (120° east longitude, 30° north latitude). Reasonable water and fertilizer management and pest and disease control were carried out during the growth period to ensure that the cabbage would form heads normally. The cabbage must be harvested after the heads are formed. The results of cutting and observing the heads showed that the center and central column of the T1-10 plants were orange-red during the heading period ( Figure 3 ).

[0088] Example 3 Phenotypic investigation of the crtiso mutant of cabbage and the inheritance of the orange-heart phenotype

[0089] CRTISO protein is a pro-lycopene isomerase, which can isomerize pro-lycopene into lycopene. At the same time, light can also isomerize pro-lycopene into lycopene, which can compensate for the function of the CRTISO gene. At this time, pro-lycopene cannot be enriched, so the leaves of the orange-heart cabbage that can receive light are still green, such as rosette leaves and outer leaves. Only the leaves near the growth point in the seedling and rosette stages, and the heart of the head (cabbage heart) in the head stage can show orange ( Figure 3 and Figure 4 ).

[0090] T1-10 and 09-4-3 were used as parents for hybridization to obtain F1 generation seeds, and F1 was self-pollinated to obtain F2 seeds. The orange-red heart phenotype of T1-10, 09-4-3, F1 and F2 was investigated. The phenotypic results showed that T1-10 had an orange-red heart, while 09-4-3 and F1 had yellow hearts. In the F2 population, the number of yellow-hearted plants and orange-red-hearted plants was 92 and 28, respectively. The chi-square test X 2 <X 2 (P＝0.05,df＝1), which is consistent with the segregation ratio of 3:1 (as shown in Table 2), indicating that the orange red heart trait is controlled by the recessive nuclear gene CRTISO-TP.

[0091] Table 2 Investigation on the inheritance of orange-red heart trait in cabbage

[0092] Hybrid combination Offspring Huang Xin Co., Ltd. Orange Heart Co., Ltd. Expected Value <![CDATA[X 2 ]]> <![CDATA[X 2 (P＝0.05,df＝1) ]]> T1-10×09-4-3 F2 92 28 3:1 0.178 3.841

[0093] Example 4 Development and application of functional markers of CRTISO-TP gene

[0094] According to the characteristics of InDel-C08:50542693, targeted PCR primers were designed to amplify the marker, including three primers:

[0095] 50542693-FAM (SEQ ID NO: 17):

[0096] 5'-GAAGGTGACCAAGTTCATGCTTGGAGAAGTATCTCATCCCTGGT-3'.

[0097] 50542693-HEX (SEQ ID NO: 18):

[0098] 5'-GAAGGTCGGAGTCAACGGATTTGGAGAAGTATCTCATCCCTGGG-3'.

[0099] 50542693-Common(SEQ ID NO:19):

[0100] 5'-CACATCGAATGTGTACCCATCT-3'.

[0101] Using primers 50542693-FAM, 50542693-HEX and 50542693-Common, specific PCR amplification was performed on the DNA of individual plants of the F2 population of cabbage high-generation inbred line materials, T0 generation (including T0-1 and T0-2), T1 generation (derived from the self-pollinated progeny of T0-2 line) and T1-10×09-4-3.

[0102] The preferred specific PCR amplification reaction system in the present invention is as follows:

[0103] 1.6 μL PCR amplification system

[0104] Element volume DNA to be tested (20ng / μL) 0.7μL Primer mix (100 μM) 0.05μL 2 × KASPMastermix 0.8μL <![CDATA[ddH2O]]> 0.05μL

[0105] 2×KASP Master mix was purchased from LGC, Biosearch Technologies. The primer mixture consisted of 50542693-FAM, 50542693-HEX and 50542693-Common

[0106] The specific PCR reaction procedure for detecting the genotype of the InDel-C08:50542693 allele in the present invention is as follows: PCR reaction procedure

[0107] step Number of cycles 94℃for15min 1 94℃for20sec 10 61-55℃for60sec(-0.6℃ / cycle) 10 94℃for20sec 26 55℃for60sec 26

[0108] The fluorescence data was read and analyzed by the IntelliQube machine to obtain the genotyping results of the InDel-C08:50542693 allele; when the InDel-C08:50542693 allele was homozygous T / T, the cabbage material had a common yellow heart phenotype (red dots). When the InDel-C08:50542693 allele was heterozygous T / G, the plant had a common yellow heart phenotype (purple dots). When the InDel-C08:50542693 allele was homozygous G / G (blue dots), the phenotype of the plant after heading was orange-red heart (results as shown in Figure 2). Figure 5 The method for detecting the InDel-C08:50542693 site based on the KASP technology provided by the present invention can identify two homozygous genotypes and a heterozygous genotype, and the KASP genotyping result is completely consistent with the phenotypic identification result (Table 3).

[0109] Table 3 Genotypes and ball center color phenotypes of some individuals in the F2 population of T1-10×09-4-3 at position C08:50542693 of the cabbage genome

[0110] serial number genotype Phenotype serial number genotype Phenotype serial number genotype Phenotype 1 G / G Orange Heart 13 G / G Orange Heart 25 T / T Yellow Heart 2 G / G Orange Heart 14 G / G Orange Heart 26 T / T Yellow Heart 3 G / G Orange Heart 15 G / G Orange Heart 27 T / G Yellow Heart 4 G / G Orange Heart 16 G / G Orange Heart 28 T / G Yellow Heart 5 G / G Orange Heart 17 G / G Orange Heart 29 T / G Yellow Heart 6 G / G Orange Heart 18 G / G Orange Heart 30 T / T Yellow Heart 7 G / G Orange Heart 19 G / G Orange Heart 31 T / G Yellow Heart 8 G / G Orange Heart 20 G / G Orange Heart 32 T / G Yellow Heart 9 G / G Orange Heart 21 T / G Yellow Heart 33 T / G Yellow Heart 10 G / G Orange Heart 22 T / G Yellow Heart 34 T / T Yellow Heart 11 G / G Orange Heart 23 T / T Yellow Heart 35 T / G Yellow Heart 12 G / G Orange Heart 24 T / G Yellow Heart 36 T / G Yellow Heart

[0111] The above specific examples show that the CRTISO gene is knocked out and mutated by gene editing technology. Then, by screening the T1 generation plants, a new cabbage germplasm with no exogenous T-DNA fragments and stable inheritance of the orange-red heart trait can be obtained in the T1 generation. Compared with breeding methods such as hybridization and mutagenesis, gene editing has the advantages of precision, speed, and efficiency. Combined with the developed functional markers for auxiliary selection, it will shorten the breeding cycle and improve the efficiency of cabbage breeding.

[0112] The sequence information is as follows:

[0113] SEQ ID NO: 1:

[0114]

[0115] SEQ ID NO:2(CRTISO-cDNA-F):

[0116] 5’-ATGAATCTCTGTCTCCGCAACCC-3’。

[0117] SEQ ID NO:3(CRTISO-cDNA-R):

[0118] 5’-TGCGAGTGTCCTTAACCAACCAA-3’。

[0119] SEQ ID NO:4:CAGCACACGTTACGGGGTTG。

[0120] SEQ ID NO:5:CCTTTCTTCTACTACAGCAG。

[0121] SEQ ID NO:6:GTCATCGGGTCTGGGATAGG。

[0122] SEQ ID NO:7:AATAACCAGAGCTCCCACCA。

[0123] SEQ ID NO:8:

[0124] CAGCACACGTTACGGGGTTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCACCTTTCTTCTACTACAGCAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGTCATCGGGTCTGGGATAGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAAATAACCAGAGCTCCCACCA。

[0125] SEQ ID NO:9(sgRNA-F)

[0126] 5’-cagtGGTCTCatgcaCAGCACACGTTACGGGGTTG-3’。

[0127] SEQ ID NO:10(sgRNA-R):

[0128] 5’-cagtGGTCTCaaaacTGGTGGGAGCTCTGGTTATT-3’。

[0129] SEQ ID NO:11(CRTISO-F):

[0130] 5’-CTCTGTCTCCGCAACCCC-3’。

[0131] SEQ ID NO:12(CRTISO-R):

[0132] 5’-TCTCACGACCAACTGCCTTC-3’。

[0133] SEQ ID NO:13(Target1-F):

[0134] 5’-CAGCACACGTTACGGGGT-3’。

[0135] SEQ ID NO:14(Target4-R):

[0136] 5’-TGGTGGGAGCTCTGGTTATT-3’。

[0137] SEQ ID NO:15(Cas9-T-F):

[0138] 5’-CAGTCGATTACCGGCCTCTACG-3’。

[0139] SEQ ID NO:16(BLP-S-R):

[0140] 5’-GGTGGCACGGCGGATGTC-3’。

[0141] SEQ ID NO:17(50542693-FAM):

[0142] 5’-GAAGGTGACCAAGTTCATGCTTGGAGAAGTATCTCATCCCTGGT-3’。

[0143] SEQ ID NO:18(50542693-HEX):

[0144] 5’-GAAGGTCGGAGTCAACGGATTTGGAGAAGTATCTCATCCCTGGG-3。

[0145] SEQ ID NO:19(50542693-Common):

[0146] 5’-CACATCGAATGTGTACCCATCT-3’。

[0147] SEQ ID NO:20:

[0148] 。

[0149] SEQ ID NO: 21:

[0150] MNLCLRNPVTCADRSSLFSALKTSNNKLGTSKFGFLKNPKKNHVVAVRSVSSTAVVEERTKRESGGESKVYDAIVIGSGIGGLVAATQLAVKEAKVLVLEKYLIPGGALVITKEMGTHSMWVPLSCLVSAIRGI.

[0151] SEQ ID NO: 22:

[0152]

[0153] SEQ ID NO:23:

[0154] TGGTGGGAGCTCTGGTTATTTGCACCAGCCGGGAATCGAACCCGGGTCTGTACCGTGGCAGGGTACTATTCTACCACTAGACCACTGGTGCTTTGTTGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCCTATCCCAGACCCGATGACTGCACCAGCCGGGAATCGAACCCGGGTCTGTACCGTGGCAGGGTACTATTCTACCACTAGACCACTGGTGCTTTGTTGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCTGCTGTAGTAGAAGAAAGGTGCACCAGCCGGGAATCGAA

[0155] CCCGGGTCTGTACCGTGGCAGGGTACTATTCTACCACTAGACCACTGGTGCTTTGTTGCACCGACTCGGTGCCACTTT

[0156] TTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCAACCCCGTAACGTGTGCTG。

Claims

1. Use of a substance for reducing the expression of a gene encoding the protein in any of the following; A1) Application in preparing orange-red-heart cabbage and / or producing orange-red-heart cabbage products; A2) Application in breeding of orange-red-heart cabbage and / or producing breeding products of orange-red-heart cabbage; The protein is any of the following: B1) the amino acid sequence is the protein shown in SEQ ID NO: 20; B2) a protein having more than 80% identity with the protein shown in B1) and having the same function as the protein shown in B1) obtained by substitution and / or deletion and / or addition of amino acid residues of the protein shown in B1); B3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of B1) or B2) to a protein tag.

2. The use according to claim 1, characterized in that: The protein is derived from cabbage.

3. The use according to claim 1 or 2, characterized in that: The substance that reduces the expression of the gene encoding the protein is any one of the following: B1), a nucleic acid molecule that reduces the expression of a gene encoding the protein according to claim 1 or 2; B2), expressing the coding gene of the nucleic acid molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

4. The use according to claim 3, characterized in that: B1) The target gene of the nucleic acid molecule is shown in Sequence 22.

5. A method for cultivating orange-heart cabbage, characterized in that: The method comprises the step of reducing the expression level of the gene encoding the protein of any one of claims 1 to 3 in the target cabbage to obtain orange-heart cabbage.

6. A method for reducing the brightness of cabbage heart, characterized in that: The method comprises reducing the expression level of the gene encoding the protein of any one of claims 1 to 3 in the target cabbage to obtain low-brightness cabbage heart, wherein the lightness of the cabbage heart of the low-brightness cabbage heart is lower than that of the target cabbage.

7. The method according to claim 5 or 6, characterized in that The inhibition, reduction or down-regulation of the expression of the gene encoding the protein of any one of claims 1 to 3 in cabbage comprises introducing the nucleic acid molecule described in B1) of claim 3, the expression cassette of the gene described in B2), the expression cassette described in B3) or the recombinant vector described in B4) into the target cabbage.

8. A method for cultivating orange-heart cabbage, characterized in that: The method comprises the steps of performing deletion mutation on the thymine deoxyribonucleotide residue at position 324 of sequence 22 in the cabbage genome to obtain orange-heart cabbage.

9. The use according to any one of claims 1 to 5 or the method according to any one of claims 6 to 8, characterized in that: The cabbage is cabbage.

10. The use according to any one of claims 1 to 3 or the method according to any one of claims 5 to 9, characterized in that: The orange-heart cabbage has an orange-red heart during the heading period, or the low-brightness cabbage has a lower brightness than the target cabbage during the heading period.

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