CN114317561A

CN114317561A - Broccoli gene fixed-point editing method based on CRISPR/Cas9

Info

Publication number: CN114317561A
Application number: CN202210155687.0A
Authority: CN
Inventors: 马存发; 巫水钦; 武婷; 赵辉; 肖建成; 赵立坚
Original assignee: Zhejiang Beauty Seed Industry Ltd By Share Ltd
Current assignee: Zhejiang Beauty Seed Industry Ltd By Share Ltd
Priority date: 2021-08-17
Filing date: 2022-02-21
Publication date: 2022-04-12
Anticipated expiration: 2042-02-21
Also published as: CN114317561B

Abstract

The invention discloses a broccoli gene fixed-point editing method based on CRISPR/Cas9, and relates to the technical field of genetic engineering. According to the method provided by the invention, the broccoli BoMYB101 gene is subjected to site-specific knockout editing by using a CRISPR/Cas9 technology, 2 sgRNA target sites are designed according to a broccoli BoMYB101 gene sequence, a knockout expression vector of the BoMYB101 gene is constructed, the cauliflower hypocotyl is subjected to agrobacterium-mediated transformation, and a mutant plant is screened by molecular detection, so that a high-efficiency broccoli gene editing plant is obtained, and an application support is provided for character improvement and gene function research of the broccoli plant such as disease resistance, cold resistance, breeding and the like.

Description

Broccoli gene fixed-point editing method based on CRISPR/Cas9

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a broccoli gene fixed-point editing method based on CRISPR/Cas 9.

Background

Broccoli (A. B. Moench)Brassica oleracea L. var. iTalica) also known as broccoli, the Mediterranean region of origin. Broccoli is a variety of brassica species in brassica of brassicaceae and is known as "vegetable crown" due to abundant nutrition. Also rich in thioglycoside and having anticancer effect, and particularly, the anticancer mechanism of the metabolic component of the component, namely, the mustard (indole-3-carbinol), has been scientifically demonstrated from the molecular level. The broccoli is also rich in the linear thioglycoside sulforaphane, so that the low-fat-density cholesterol can be effectively reduced, the incidence rate of heart diseases is reduced, and the broccoli is popular with consumers at home and abroad. The broccoli is one of the important large vegetable varieties in China, is planted all over the country, is mainly concentrated in the places of Yunnan, Hebei, Zhejiang, Hubei, Jiangsu, Guangdong and the like, and the planting area is more than 8.7 million hectares (130 million mu). Broccoli is also an important export-earning vegetable, and the annual export amount exceeds 12 million tons.

The back of the dominant industry of broccoli in China is the weak breeding industry, the core breeding industry mainly depends on import from Japan and other countries, the foreign variety occupancy once exceeds 95%, and the monopoly is formed for the broccoli seed supply in China. The research and breeding of broccoli in China are late, the excellent resources are lack, the existing excellent resources can not be reused due to the wide use of the male sterility technology, and the research and breeding of broccoli in China are still different from those of international enterprises and research institutions. The molecular breeding development of broccoli is very lagged because the restriction genetic transformation system such as the characteristics, the genotype and the like of broccoli materials is difficult to establish all the time.

Disclosure of Invention

The invention mainly aims to provide a broccoli gene fixed-point editing method based on CRISPR/Cas9, which is used for solving the technical problem of poor comprehensive properties of broccoli plants such as disease resistance, cold resistance, breeding and the like in the prior art, thereby achieving the technical effect of improving the comprehensive properties of broccoli varieties. The technical scheme of the invention is as follows:

according to an aspect of the embodiment of the invention, a method for fixed-point editing of broccoli genes based on CRISPR/Cas9 is provided, which is characterized by comprising the following steps:

obtaining a BoMYB101 gene sequence according to broccoli inbred line 2M genome sequencing analysis, selecting 5'-GCCAAATCTAAGGACAGGAT-3' sequence as a target site sgT1 from a first exon of the BoMYB101 gene sequence, selecting 5'-GATTCACCTGCGGGGAACAG-3' sequence as a target site sgT2 from a second exon of the BoMYB101 gene sequence, and respectively synthesizing complementary primer pairs corresponding to the target sites, wherein the complementary primer pairs corresponding to the target sites sgT1 comprise:

sgT1-F：5’-TGCAGCCAAATCTAAGGACAGGAT-3’、sgT1-R：5’-AAACATCCTGTCCTTAGATTTGGC-3’；

the complementary primer pair corresponding to target site sgT2 includes:

sgT2-F：5’-TGCAGATTCACCTGCGGGGAACAG-3’、sgT2-R：5’-AAACCTGTTCCCCGCAGGTGAATC-3’；

for each complementary primer pair, according to primer F and primer R, ddH₂Preparing a reaction system of the complementary primer pair with the volume ratio of O being 1:1:8, carrying out 95 ℃ denaturation on the reaction system of the complementary primer pair for 5min by using a PCR (polymerase chain reaction) amplification instrument, then cooling to 25 ℃ at the cooling rate of 0.2 ℃/s, and using ddH (ddH) to react products₂Diluting by 250 times through O to obtain DNA fragments of each target site, wherein the concentrations of the primer F and the primer R are both 100 mu Mol/L;

connecting each target site DNA fragment to a sgRNA expression frame respectively to construct a sgRNA expression frame plasmid containing two target site DNA fragments;

according to the primer FW and the primers RV and ddH₂Preparing a reaction system of a linker primer pair with the volume ratio of O being 1:1:8, carrying out denaturation on the reaction system of the linker primer pair for 5min at 95 ℃ by using a PCR (polymerase chain reaction) amplification instrument, then cooling to 25 ℃ at the cooling rate of 0.2 ℃/s, and using ddH (ddH) to react products₂Diluting by 250 times through O to obtain a linker DNA fragment, wherein the concentration of the primer FW and the concentration of the primer RV are both 100 mu Mol/L, and the linker primer pair comprises:

FW：5’-CGTACCTGCAGGAAAGCGGCCGCGTCAGGCGCGCCTAACT-3’、RV：5’-CTAGAGTTAGGCGCGCCTGACGCGGCCGCTTTCCTGCAGGTACGAGCT-3’；

the linker DNA fragment was ligated to the expression vector pCAMBIA2301 plasmidSacI andXbaconstructing pCAMBIA2301 plasmid containing linker DNA fragment between enzyme cutting sites I;

using restriction endonucleasesNotI andAsci, connecting Cas9 expression frame to pCAMBIA2301 plasmid containing linker DNA fragmentNotI andAscobtaining pCAMBIA2301 plasmid containing linker DNA fragment and Cas9 expression frame between enzyme cutting sites;

using restriction endonucleasesNotI andSbfi, connecting the sgRNA expression frame plasmid containing the DNA fragments with the two target sites to the pCAMBIA2301 plasmid containing the linker DNA fragment and the Cas9 expression frameNotI andSbfi, obtaining an expression vector plasmid edited by a BoMYB101 gene between enzyme cutting sites;

transforming the expression vector plasmid edited by the BoMYB101 gene into an EHA105 agrobacterium-competent cell by using a freeze-thaw method to obtain an EHA105 agrobacterium strain containing the expression vector plasmid;

and infecting the broccoli hypocotyl explant by the EHA105 agrobacterium strain containing the expression vector plasmid, and obtaining a transgenic plant of which the expression vector is introduced into broccoli cells through resistance screening culture.

Preferably, the step of constructing a sgRNA expression frame plasmid containing two target site DNA fragments by separately ligating each target site sequence fragment into a sgRNA expression frame includes:

according to the expression frame plasmid of 10 XNEB Buffer 2.1 and sgRNA and endonucleaseBsaⅠ、ddH₂Preparing a sgRNA expression frame enzyme digestion reaction system corresponding to sgT1 target sites with the volume ratio of O being 3:4:1:22, and using the endonuclease of type II endonuclease site for the sgRNA expression frame plasmidBsaI, enzyme digestion, carrying out agarose gel electrophoresis on the enzyme digestion product, cutting the gel and recycling to obtain the productBsaI, digesting a linearized sgRNA expression frame plasmid;

according toBsaI, preparing a sgT1 target site assembly and connection reaction system by carrying out enzyme digestion on a linearized sgRNA expression cassette plasmid, an sgT1 target site DNA fragment, a T4 DNA ligase Buffer and a T4 DNA ligase in a volume ratio of 15:2:2:1, and carrying out enzyme digestion on the sgRNA expression cassette plasmid and the DNA fragment of the target site sgT1BsaI, connecting the linearized sgRNA expression frame plasmid subjected to enzyme digestion at 4 ℃ overnight under the action of T4 DNA ligase Buffer and T4 DNA ligase, then transforming a connecting product into an escherichia coli DH5 alpha competent cell, coating the competent cell on an LB solid culture medium with 50 mu g/mL kanamycin, culturing at 37 ℃ overnight, screening positive clones, and extracting to obtain a sgRNA expression frame plasmid containing a sgT1 target site DNA fragment;

10 XNEB Buffer 2.1, sgRNA expression frame plasmid containing sgT1 target site DNA fragment and endonucleaseBbsⅠ、ddH₂Preparing a sgRNA expression frame enzyme digestion reaction system corresponding to sgT2 target sites with the volume ratio of O being 3:4:1:22, and carrying out endonuclease treatment on the sgRNA expression frame plasmid containing the DNA fragment with the sgT1 target sites by using type II endonuclease sitesBbsI, enzyme digestion, carrying out agarose gel electrophoresis on the enzyme digestion product, cutting the gel and recycling to obtain the productBbsI, digesting a linearized sgRNA expression frame plasmid;

according toBbsI, preparing a sgT2 target site assembly and connection reaction system by carrying out enzyme digestion on a linearized sgRNA expression cassette plasmid, an sgT2 target site DNA fragment, a T4 DNA ligase Buffer and a T4 DNA ligase in a volume ratio of 15:2:2:1, and carrying out enzyme digestion on the sgRNA expression cassette plasmid and the DNA fragment of the target site sgT2BbsI after enzyme digestion, linearized sgRNA expression frame plasmids are connected at 4 ℃ overnight under the action of T4 DNA ligase Buffer and T4 DNA ligase, then the connection products are transformed into escherichia coli DH5 alpha competent cells, the competent cells are coated on LB solid culture medium with 50 mu g/mL kanamycin and cultured at 37 ℃ overnight, and positive grams are screenedAnd (4) extracting to obtain sgRNA expression frame plasmids containing two target site DNA fragments.

Preferably, the linker DNA fragment is ligated to the expression vector pCAMBIA2301 plasmidSacI andXbathe method comprises the following steps of constructing pCAMBIA2301 plasmid containing linker DNA fragment between enzyme cutting sites, wherein the pCAMBIA2301 plasmid comprises the following steps:

according to the method, 10 XNEB Buffer, pCAMBIA2301 plasmid and restriction enzymeSacI, restriction enzymeXbaⅠ、ddH₂Preparing a first pCAMBIA2301 linear enzyme digestion reaction system with the volume ratio of O being 5:5:1:1:38, and using restriction enzyme for pCAMBIA2301 plasmidSacI andXbacarrying out double digestion, carrying out agarose gel electrophoresis on a digestion product, cutting gel, and recovering a linearized pCAMBIA2301 plasmid;

preparing a Linker DNA fragment connection reaction system according to the volume ratio of the Linker DNA fragment to the linearized pCAMBIA2301 plasmid, T4 DNA ligase Buffer and T4 DNA ligase of 6:20:3:1, connecting the Linker DNA fragment and the linearized pCAMBIA2301 plasmid at 4 ℃ under the action of T4 DNA ligase Buffer and T4 DNA ligase overnight, transforming a connection product into an Escherichia coli DH5 alpha competent cell, coating the Escherichia coli DH5 alpha competent cell on an LB solid culture medium with 50 mu g/mL kanamycin, culturing at 37 ℃ overnight, screening positive clones, and extracting to obtain the pCAMBIA2301 plasmid containing the Linker DNA fragment.

Preferably, the use of restriction enzymesNotI andAsci, connecting Cas9 expression frame to pCAMBIA2301 plasmid containing linker DNA fragmentNotI andAscthe method comprises the following steps of I, obtaining pCAMBIA2301 plasmid containing linker DNA fragment and Cas9 expression frame between enzyme cutting sites, wherein the pCAMBIA2301 plasmid comprises the following steps:

according to the method, 10 XNEB Buffer is mixed with pCAMBIA2301 plasmid containing linker DNA fragment and restriction enzymeNotI, restriction enzymeAscⅠ、ddH₂Preparing a second pCAMBIA2301 linear enzyme digestion reaction system with the volume ratio of O to 5:5:1:1:38, and utilizing restriction enzymeNotI andAscthe pCAMBIA2301 plasmid containing the linker DNA fragment and the Cas9 expression frame are subjected to double enzyme digestion simultaneously, and the enzyme digestion product is subjected to agarose gel electrophoresis, gel cutting, recovery and linearization, and containspCAMBIA2301 plasmid with linker DNA fragment and linearized Cas9 expression cassette fragment;

preparing a Cas9 expression frame fragment connection reaction system according to the volume ratio of a linearized Cas9 expression frame fragment to the linearized pCAMBIA2301 plasmid containing the linker DNA fragment, T4 DNA ligase Buffer and T4 DNA ligase being 6:20:3:1, connecting the linearized pCAMBIA2301 plasmid containing the linker DNA fragment and the linearized Cas9 expression frame fragment at the action of T4 DNA ligase Buffer and T4 DNA ligase at 4 ℃ overnight, transforming a connection product into Escherichia coli DH5 alpha competent cells, coating the cells on LB solid medium with 50 mu g/mL kanamycin at 37 ℃ for overnight, screening positive clones, and extracting the pCAMBIA2301 plasmid containing the linker DNA fragment and the Cas9 expression frame.

Preferably, the use of restriction enzymesNotI andSbfi, connecting the sgRNA expression frame plasmid containing the DNA fragments with the two target sites to the pCAMBIA2301 plasmid containing the linker DNA fragment and the Cas9 expression frameNotI andSbfthe method comprises the following steps of I, obtaining an expression vector plasmid edited by a BoMYB101 gene between enzyme cutting sites, wherein the steps comprise:

mixing 10 XNEB Buffer with pCAMBIA2301 plasmid containing linker DNA fragment and Cas9 expression cassette, restriction endonucleaseNotI, restriction enzymeSbfⅠ、ddH₂Preparing a third pCAMBIA2301 linear enzyme digestion reaction system with the volume ratio of O to 5:5:1:1:38, and utilizing restriction enzymeNotI andSbfcarrying out double enzyme digestion on the pCAMBIA2301 plasmid containing the linker DNA fragment and the Cas9 expression frame and the sgRNA expression frame plasmid containing the two target site DNA fragments, carrying out agarose gel electrophoresis on the digestion products, cutting gel, and recovering a linearized pCAMBIA2301 plasmid containing the linker DNA fragment and the Cas9 expression frame and a linearized sgRNA expression frame fragment containing the two target site DNA fragments;

preparing a sgRNA expression frame fragment connection reaction system according to the volume ratio of the sgRNA expression frame fragment which is linearized and contains two target site DNA fragments to the pCAMBIA2301 plasmid which is linearized and contains a linker DNA fragment and a Cas9 expression frame, the T4 DNA ligase Buffer and the T4 DNA ligase are 6:20:3:1, connecting the sgRNA expression frame fragment which is linearized and contains two target site DNA fragments and the pCAMBIA2301 plasmid which is linearized and contains the linker DNA fragment and the Cas9 expression frame at the action of the T4 DNA ligase Buffer and the T4 DNA ligase at 4 ℃ overnight, transforming a connecting product into Escherichia coli 5 alpha competent cells, coating the cells on LB solid medium with 50 mu g/mL of kanamycin, culturing at 37 ℃ overnight, screening positive clones, and extracting expression vector plasmids edited by the BoB 101 gene.

Preferably, the efficiency of editing the BoMYB101 gene by the expression vector plasmid is 75-80%.

Compared with the prior art, the broccoli gene site-directed editing method based on CRISPR/Cas9 provided by the invention has the following advantages:

the invention provides a CRISPR/Cas 9-based broccoli gene site-directed editing method, which is characterized in that a CRISPR/Cas9 technology is utilized to perform site-directed knockout editing on a broccoli BoMYB101 gene, 2 sgRNA target sites are designed according to a broccoli BoMYB101 gene sequence, a knockout expression vector of the BoMYB101 gene is constructed, a cauliflower hypocotyl is subjected to agrobacterium-mediated transformation, and a mutant plant is screened by molecular detection, so that a high-efficiency broccoli gene editing plant is obtained, and application support is provided for character improvement and gene function research of disease resistance, cold resistance, breeding and the like of the broccoli plant.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

FIG. 1 is a schematic diagram of an expression vector plasmid edited by a BoMYB101 gene provided by the invention.

FIG. 2 is a schematic diagram of the transgene detection of broccoli provided by the present invention.

FIG. 3 is a direct sequencing peak diagram of a PCR product of a target site of a broccoli BoMYB101 gene provided by the invention.

FIG. 4 is a statistical chart of monoclonal sequencing analysis of broccoli BoMYB101 gene target site sgT2 provided by the invention.

Detailed Description

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

The genotype of broccoli determines that the broccoli has great difficulty in character improvement and molecular biology research, and in order to avoid the technical problems, the inventor provides a method for editing a broccoli gene at a fixed point based on CRISPR/Cas9 through a large amount of creative experimental research and thinking, which is specifically shown as follows.

A method for fixed-point editing of broccoli genes based on CRISPR/Cas9, comprising:

a step (100): obtaining a BoMYB101 gene sequence according to broccoli inbred line 2M genome sequencing analysis, selecting 5'-GCCAAATCTAAGGACAGGAT-3' sequence as a target site sgT1 from a first exon of the BoMYB101 gene sequence, selecting 5'-GATTCACCTGCGGGGAACAG-3' sequence as a target site sgT2 from a second exon of the BoMYB101 gene sequence, and respectively synthesizing complementary primer pairs corresponding to the target sites, wherein the complementary primer pairs corresponding to the target sites sgT1 comprise:

the complementary primer pair corresponding to target site sgT2 includes:

sgT2-F：5’-TGCAGATTCACCTGCGGGGAACAG-3’、sgT2-R：5’-AAACCTGTTCCCCGCAGGTGAATC-3’。

it should be noted that MYB97, MYB101 and MYB120, which are pollen transcription factors, may participate in information exchange between a pollen tube and a helper cell through the expression of transcription regulation downstream genes, and a MYB97, MYB101 and MYB120 ternary mutant pollen tube cannot stop growing after entering a embryo sac, cannot burst to release sperm cells, and cannot perform double fertilization, so that the study on the MYB101 gene function is important for analyzing the molecular mechanism accepted by the pollen tube.

Step (200): for each complementary primer pair, according to primer F and primer R, ddH₂Preparing a reaction system of the complementary primer pair with the volume ratio of O being 1:1:8, carrying out 95 ℃ denaturation on the reaction system of the complementary primer pair for 5min by using a PCR (polymerase chain reaction) amplification instrument, then cooling to 25 ℃ at the cooling rate of 0.2 ℃/s, and using ddH (ddH) to react products₂Diluting by 250 times through O to obtain DNA fragments of each target site, wherein the concentration of the primer F and the concentration of the primer R are both 100 mu Mol/L.

In one possible embodiment, the reaction system of the complementary primer pair has primer F and primer R, ddH₂The volume of O was 1. mu.L, and 8. mu.L, respectively.

Step (300): and respectively connecting the DNA fragments of the target sites to the sgRNA expression frame to construct a sgRNA expression frame plasmid containing the DNA fragments of the two target sites.

step (310): 10 XNEB Buffer 2.1 and sgRNA expression frame plasmid, endonuclease BsaI, ddH₂Preparing a sgRNA expression frame enzyme digestion reaction system corresponding to sgT1 target sites with the volume ratio of O being 3:4:1:22, and using the endonuclease of type II endonuclease site for the sgRNA expression frame plasmidBsaI, enzyme digestion, carrying out agarose gel electrophoresis on the enzyme digestion product, cutting the gel and recycling to obtain the productBsaI restriction enzyme digestion of linearized sgRNA expression frame plasmid.

In one possible embodiment, the expression frame plasmid of 10 XNEB Buffer 2.1 and sgRNA and the endonuclease in the sgRNA expression frame enzyme digestion reaction system corresponding to the sgT1 target siteBsaⅠ、ddH₂The volumes of O were 3. mu.L, 4. mu.L, 22. mu.L, and 1. mu.L, respectively.

A step (320): preparing a sgT1 target site assembly and connection reaction system according to the volume ratio of the linearized sgRNA expression frame plasmid after Bsa I digestion to the sgT1 target site DNA fragment, the T4 DNA ligase Buffer and the T4 DNA ligase of 15:2:2:1, and mixing the sgT1 target site DNA fragment and the DNA fragment of the sgT1 target siteBsaI the linearized sgRNA expression cassette plasmid after digestion is linked at 4 ℃ under the action of T4 DNA ligase Buffer and T4 DNA ligaseInoculating overnight, then transforming the ligation product into escherichia coli DH5 alpha competent cells, coating the cells on LB solid culture medium with 50 ug/mL kanamycin, culturing overnight at 37 ℃, screening positive clones, and extracting sgRNA expression frame plasmid containing sgT1 target site DNA fragment.

In one possible embodiment, sgT1 target site assembly is connected in the reaction systemBsaThe volumes of the linearized sgRNA expression cassette plasmid after digestion, sgT1 target site DNA fragment, T4 DNA ligase Buffer and T4 DNA ligase are 15 muL, 2 muL, 22 muL and 1 muL respectively.

Step (330): 10 XNEB Buffer 2.1, sgRNA expression frame plasmid containing sgT1 target site DNA fragment and endonucleaseBbsⅠ、ddH₂Preparing a sgRNA expression frame enzyme digestion reaction system corresponding to sgT2 target sites with the volume ratio of O being 3:4:1:22, and carrying out endonuclease treatment on the sgRNA expression frame plasmid containing the DNA fragment with the sgT1 target sites by using type II endonuclease sitesBbsI, enzyme digestion, carrying out agarose gel electrophoresis on the enzyme digestion product, cutting the gel and recycling to obtain the productBbsI restriction enzyme digestion of linearized sgRNA expression frame plasmid.

In one possible embodiment, 10 XNEB Buffer 2.1 and sgRNA expression frame plasmid containing sgT1 target site DNA fragment and endonuclease in sgRNA expression frame enzyme digestion reaction system corresponding to sgT2 target siteBbsⅠ、ddH₂The volumes of O were 3. mu.L, 4. mu.L, 1. mu.L, and 22. mu.L, respectively.

Step (340): according toBbsI, preparing a sgT2 target site assembly and connection reaction system by carrying out enzyme digestion on a linearized sgRNA expression cassette plasmid, an sgT2 target site DNA fragment, a T4 DNA ligase Buffer and a T4 DNA ligase in a volume ratio of 15:2:2:1, and carrying out enzyme digestion on the sgRNA expression cassette plasmid and the DNA fragment of the target site sgT2BbsI after enzyme digestion, linearized sgRNA expression frame plasmids are connected at 4 ℃ overnight under the action of T4 DNA ligase Buffer and T4 DNA ligase, then a connection product is transformed into escherichia coli DH5 alpha competent cells, the escherichia coli DH5 alpha competent cells are coated on an LB solid culture medium with 50 mu g/mL kanamycin and cultured at 37 ℃ overnight, positive clones are screened, and sgRNA expression frame plasmids containing two target site DNA fragments are extracted.

In one possible embodiment, sgT2 target site assembly is connected in the reaction systemBbsI after digestionThe volumes of the linearized sgRNA expression cassette plasmid, sgT2 target site DNA fragment, T4 DNA ligase Buffer and T4 DNA ligase are 15. mu.L, 2. mu.L and 1. mu.L, respectively.

Wherein the gene sequence table of the sgRNA expression cassette plasmid containing the DNA fragments of the two target sites is shown as SEQ ID NO. 1 in the specification.

Step (400): according to the primer FW and the primers RV and ddH₂Preparing a reaction system of a linker primer pair with the volume ratio of O being 1:1:8, carrying out denaturation on the reaction system of the linker primer pair for 5min at 95 ℃ by using a PCR (polymerase chain reaction) amplification instrument, then cooling to 25 ℃ at the cooling rate of 0.2 ℃/s, and using ddH (ddH) to react products₂Diluting by 250 times through O to obtain a linker DNA fragment, wherein the concentration of the primer FW and the concentration of the primer RV are both 100 mu Mol/L, and the linker primer pair comprises:

FW：5’-CGTACCTGCAGGAAAGCGGCCGCGTCAGGCGCGCCTAACT-3’、RV：5’-CTAGAGTTAGGCGCGCCTGACGCGGCCGCTTTCCTGCAGGTACGAGCT-3’。

in one possible embodiment, the primer FW and the primers RV and ddH in the reaction system of the linker primer pair₂The volume of O was 1. mu.L, and 8. mu.L, respectively.

A step (500): the linker DNA fragment was ligated to the expression vector pCAMBIA2301 plasmidSacI andXbaand constructing pCAMBIA2301 plasmid containing linker DNA fragment between the enzyme cutting sites.

step (510): according to the method, 10 XNEB Buffer, pCAMBIA2301 plasmid and restriction enzymeSacI, restriction enzymeXbaⅠ、ddH₂Preparing a first pCAMBIA2301 linear enzyme digestion reaction system with the volume ratio of O being 5:5:1:1:38, and using restriction enzyme for pCAMBIA2301 plasmidSacI andXbathe product is electrophoresed through agarose gel, and then the linearized pCAMBIA2301 plasmid is recovered.

In one possible embodiment of the method according to the invention,10 XNEB Buffer and pCAMBIA2301 plasmid, restriction enzyme in the first pCAMBIA2301 linear enzyme digestion reaction systemSacI, restriction enzymeXbaⅠ、ddH₂The volumes of O were 5. mu.L, 1. mu.L, and 38. mu.L, respectively.

Step (520): preparing a Linker DNA fragment connection reaction system according to the volume ratio of the Linker DNA fragment to the linearized pCAMBIA2301 plasmid, T4 DNA ligase Buffer and T4 DNA ligase of 6:20:3:1, connecting the Linker DNA fragment and the linearized pCAMBIA2301 plasmid at 4 ℃ under the action of T4 DNA ligase Buffer and T4 DNA ligase overnight, transforming a connection product into an Escherichia coli DH5 alpha competent cell, coating the Escherichia coli DH5 alpha competent cell on an LB solid culture medium with 50 mu g/mL kanamycin, culturing at 37 ℃ overnight, screening positive clones, and extracting to obtain the pCAMBIA2301 plasmid containing the Linker DNA fragment.

In one possible embodiment, the volumes of the Linker DNA fragment, the linearized pCAMBIA2301 plasmid, the T4 DNA ligase Buffer and the T4 DNA ligase in the Linker DNA fragment ligation reaction system are respectively 6. mu.L, 20. mu.L, 3. mu.L and 1. mu.L.

A step (600): using restriction endonucleasesNotI andAsci, connecting Cas9 expression frame to pCAMBIA2301 plasmid containing linker DNA fragmentNotI andAsci, getting pCAMBIA2301 plasmid containing linker DNA fragment and Cas9 expression frame.

step (610): according to the method, 10 XNEB Buffer is mixed with pCAMBIA2301 plasmid containing linker DNA fragment and restriction enzymeNotI, restriction enzymeAscⅠ、ddH₂Preparing a second pCAMBIA2301 linear enzyme digestion reaction system with the volume ratio of O to 5:5:1:1:38, and utilizing restriction enzymeNotI andAsci the pCAMBIA2301 plasmid containing linker DNA fragment andand carrying out double enzyme digestion on the Cas9 expression frame, carrying out agarose gel electrophoresis on the digestion product, cutting gel, and recovering a linearized pCAMBIA2301 plasmid containing a linker DNA fragment and a linearized Cas9 expression frame fragment.

In one possible embodiment, the second pCAMBIA2301 linearized reaction system is 10 XNEB Buffer, pCAMBIA2301 plasmid containing linker DNA fragment, restriction enzymeNotI, restriction enzymeAscⅠ、ddH₂O is 5. mu.L, 1. mu.L, 38. mu.L, respectively.

Step (620): preparing a Cas9 expression frame fragment connection reaction system according to the volume ratio of a linearized Cas9 expression frame fragment to the linearized pCAMBIA2301 plasmid containing the linker DNA fragment, T4 DNA ligase Buffer and T4 DNA ligase being 6:20:3:1, connecting the linearized pCAMBIA2301 plasmid containing the linker DNA fragment and the linearized Cas9 expression frame fragment at the action of T4 DNA ligase Buffer and T4 DNA ligase at 4 ℃ overnight, transforming a connection product into Escherichia coli DH5 alpha competent cells, coating the cells on LB solid medium with 50 mu g/mL kanamycin at 37 ℃ for overnight, screening positive clones, and extracting the pCAMBIA2301 plasmid containing the linker DNA fragment and the Cas9 expression frame.

In one possible embodiment, the volumes of the linearized Cas9 expression frame fragment and the linearized pCAMBIA2301 plasmid containing the linker DNA fragment, T4 DNA ligase Buffer and T4 DNA ligase in the Cas9 expression frame fragment ligation reaction system are 6. mu.L, 20. mu.L, 3. mu.L and 1. mu.L, respectively.

Step (700): using restriction endonucleasesNotI andSbfi, connecting the sgRNA expression frame plasmid containing the DNA fragments with the two target sites to the pCAMBIA2301 plasmid containing the linker DNA fragment and the Cas9 expression frameNotI andSbfand I, obtaining an expression vector plasmid edited by the BoMYB101 gene between enzyme cutting sites.

a step (710): mixing 10 XNEB Buffer with pCAMBIA2301 plasmid containing linker DNA fragment and Cas9 expression cassette, restriction endonucleaseNotI, restriction enzymeSbfⅠ、ddH₂Preparing a third pCAMBIA2301 linear enzyme digestion reaction system with the volume ratio of O to 5:5:1:1:38, and utilizing restriction enzymeNotI andSbfthe pCAMBIA2301 plasmid containing a linker DNA fragment and a Cas9 expression frame and the sgRNA expression frame plasmid containing two target site DNA fragments are subjected to double enzyme digestion, the enzyme digestion products are subjected to agarose gel electrophoresis and then gel cutting to recover a linearized pCAMBIA2301 plasmid containing the linker DNA fragment and a Cas9 expression frame and a linearized sgRNA expression frame fragment containing the two target site DNA fragments.

In one possible embodiment, the third pCAMBIA2301 linearized reaction system is 10 XNEB Buffer, and pCAMBIA2301 plasmid containing linker DNA fragment and Cas9 expression cassette, restriction endonucleaseNotI, restriction enzymeSbfⅠ、ddH₂O is 5. mu.L, 1. mu.L, 38. mu.L, respectively.

Step (720): preparing a sgRNA expression frame fragment connection reaction system according to the volume ratio of the sgRNA expression frame fragment which is linearized and contains two target site DNA fragments to the pCAMBIA2301 plasmid which is linearized and contains a linker DNA fragment and a Cas9 expression frame, the T4 DNA ligase Buffer and the T4 DNA ligase are 6:20:3:1, connecting the sgRNA expression frame fragment which is linearized and contains two target site DNA fragments and the pCAMBIA2301 plasmid which is linearized and contains the linker DNA fragment and the Cas9 expression frame at the action of the T4 DNA ligase Buffer and the T4 DNA ligase at 4 ℃ overnight, transforming a connecting product into Escherichia coli 5 alpha competent cells, coating the cells on LB solid medium with 50 mu g/mL of kanamycin, culturing at 37 ℃ overnight, screening positive clones, and extracting expression vector plasmids edited by the BoB 101 gene.

In one possible embodiment, the sgRNA expression frame fragment connecting the sgRNA expression frame fragment linearized in the reaction system and containing the two target site DNA fragments and the pCAMBIA2301 plasmid, T4 DNA ligase Buffer, T4 DNA ligase linearized containing the linker DNA fragment and the Cas9 expression frame have volumes of 6. mu.L, 20. mu.L, 3. mu.L, 1. mu.L, respectively.

In one possible embodiment, a schematic representation of an expression vector plasmid edited by the BoMYB101 gene can be shown in FIG. 1, where LB, RB are the left and right borders in FIG. 1; p 2X 35-spCas9-Tcamv is a Cas9 expression cassette; pBoU6-trna-sgRNA1-trna-sgRNA2-Tsp is broccoli U6 start sgRNA expression cassette; kan is kanamycin resistance gene.

Preferably, the method further comprises:

step (800): and transforming the expression vector plasmid edited by the BoMYB101 gene into an EHA105 agrobacterium-competent cell by using a freeze-thaw method to obtain the EHA105 agrobacterium strain containing the expression vector plasmid.

Step (900): infecting the broccoli hypocotyl explant by the EHA105 agrobacterium strain containing the expression vector plasmid, and obtaining a transgenic plant of which the expression vector is introduced into broccoli cells through resistance screening culture, wherein the transgenic plant specifically comprises the following steps:

(a) preparing the hypocotyls of the broccoli:

selecting 2M broccoli inbred line seeds with full and uniform seeds, sterilizing the 2M broccoli inbred line seeds for 45 s by using 75% alcohol, then sterilizing the 2M broccoli inbred line seeds for 15 min by using 10% sodium hypochlorite, and then rinsing the broccoli inbred line seeds for 5-6 times by using sterile water; uniformly sowing the disinfected seeds in 1/2 MS solid culture medium (preferably 30 seeds per bottle); culturing at 25 deg.C for 7 days under 16h illumination, cutting hypocotyl of aseptic seedling into small segments of about 1cm, and pre-culturing in broccoli differentiation medium (MS +30g/L sucrose +7g/L agar powder + 3.0 mg/L6-BA + 0.05 mg/L NAA pH5.8) for 2 days.

(b) And (3) agrobacterium culture and infection:

the EHA105 Agrobacterium strain containing the expression vector plasmid was picked up with an inoculating needle and streaked on a YEB solid medium plate containing Kan (50 mg/L) and Rif (50 mg/L), and placed in an oven at 28 ℃ for 2 days; picking single colony of positive clone in 5 ml liquid culture medium containing Kan (50 mg/L) and Rif (50 mg/L), and culturing for 2 days at 28 ℃ on a shaker with 225 rpm; 600 plus 700 mu L of bacterial liquid is taken on the day of infection and added to50 mL of YEB liquid without antibiotics, shaking and culturing at 28 ℃ and 225 rpm for 4 h to OD₆₀₀Centrifuging at 4 deg.C and 4000 rpm for 10 min to obtain a supernatant, and suspending the thallus in 20 ml of ice-cold MS liquid culture medium (MS +20g/L sucrose pH 5.8); transferring the pre-cultured hypocotyl into a sterile bottle, and adding the suspension for infection for 15 min; transferring the hypocotyl to sterile filter paper, and blotting excess bacteria liquid on the hypocotyl, transferring to differential culture medium, and dark culturing for 2 days to obtain infected broccoli hypocotyl.

(c) Screening culture and callus induction:

transferring the infected cauliflower hypocotyls to a cauliflower screening culture medium (MS + 3.0 mg/L6-BA + 0.05 mg/L NAA +30g/L cane sugar +7g/L agar powder +400mg/L Cb +30mg/L Kan) for culture, and then replacing the culture medium every 14 days; cutting the adventitious bud which normally grows in the screening culture medium, inserting the cut adventitious bud into a rooting culture medium (MS + 0.2 mg/L NAA +30g/L sucrose +7g/L agar powder +400mg/L Cb +30mg/L Kan) for rooting; transplanting into soil when the root system grows to 1-2 cm to obtain Kan-resistant transgenic plants.

(d) Detecting transgenic plants:

a genomic DNA of a Kan-resistant transgenic plant is extracted by using a CTAB method, primers Cas9-F and Cas9-R (Cas 9-F: 5'-CGCAAGAACCGCATCTGCTACCTC-3', Cas 9-R: 5'-TTCTCGAGACGCCTGGACTTGGAG-3') are used for PCR amplification, and agarose gel electrophoresis is carried out to obtain a broccoli transgene detection schematic diagram as shown in figure 2, wherein in figure 2, a letter M is a DNA marker; the number "1-46" is Kan resistant plants; "+" is an expression vector plasmid as a positive control; "-" is the untransformed broccoli inbred line 2M as a negative control.

In order to further detect whether the BoMYB101 gene of the obtained Kan-resistant transgenic plant is mutated or not, synthetic primer pairs BoMYB101-F1+ BoMYB101-R1 and BoMYB101-F2+ BoMYB101-R2 (BoMYB 101-F1: 5'-ATGGACGGTGGTGAAGAGACGTC-3', BoMYB 101-R1: 5'-GAATAAAGGCAAACCAGCTCG-3', BoMYB 101-F2: 5'-CGTCGAATGAGTTATGTAATCC CGACC-3' and BoMYB 101-R2: 5'-GATTGATGAGAAGAGCTTAGATG-3') are designed at the upstream and downstream of target sites T1 and T2 respectively for PCR amplification, PCR products of 43 strains of transgenic materials are sequenced, the sequences of the target sites are compared with the sequences of corresponding wild-type plant PCR products, and the mutation of the genes is judged when the sequences of the target sites are changed or the sequencing peak images show the phenomenon. Comparing and analyzing the sequencing result, and finding that the target site sequence of the 43T 1 strain is not changed at all, wherein the editing efficiency is 0; 33 strains of the target site T2 have peak staggering or sequence change phenomena, the editing efficiency is 76.7%, PCR products of the target site T1 and T2 sequences of the broccoli BoMYB101 gene shown in figure 3 are directly sequenced to form a peak pattern, in figure 3, the target site T1 is not changed, and the target site T2 has a heavy peak or base change.

To further analyze the details of the editing of the target site gene sequence, a sequence fragment of a T2 target site amplified by a primer pair BoMYB101-F2+ BoMYB101-R2 of a #15 individual strain, #19 individual strain, #21 individual strain and #41 individual strain was inserted into pClone007 Simple Vector (Beijing Opticalke New technology Co., Ltd.). And transforming the connecting product into escherichia coli, coating the escherichia coli on a plate with 50 mg/L Amp for overnight culture at 37 ℃, selecting not less than 5 positive clones per single plant for sequencing analysis, and counting the base mutation type and frequency of the target gene. Among them, 7 monoclonals randomly selected from the #15 individual were sequenced: 1 base insertion occurs in 6 monoclonals, and deletion of a longer fragment (53 bases) occurs in 1 monoclone, and the editing frequency is 100%; the #19 single strain has 7 monoclonals which are subjected to deletion of 2 bases, and 1 monoclone which is not mutated; the 10 monoclonals detected by the #21 individual plant are all deletions of 10 basic groups, the editing frequency is 100 percent, and the plant is homozygous mutation; #41 individual strains randomly selected 9 monoclones for detection, 3 monoclones were subjected to single base insertion, 6 monoclones were subjected to 62 base deletion, the editing frequency was 100%, and the statistical chart of the single sequencing analysis of the target site T2 of the broccoli BoMYB101 gene is shown in FIG. 4.

The results show that the broccoli gene fixed-point editing method based on CRISPR/Cas9 provided by the invention can efficiently edit broccoli genes, even a homozygous mutant plant can be obtained in the T0 generation, and great help is provided for character observation or gene function research.

In summary, the CRISPR/Cas 9-based broccoli gene site-directed editing method provided by the invention is characterized in that CRISPR/Cas9 technology is used for performing site-directed knockout editing on broccoli BoMYB101 gene, 2 sgRNA target sites are designed according to broccoli BoMYB101 gene sequence, a knockout expression vector of the BoMYB101 gene is constructed, agrobacterium-mediated transformation of broccoli hypocotyl is performed, and molecular detection and screening of mutant plants are performed, so that high-efficiency broccoli gene editing plants are obtained, and application support is provided for character improvement and gene function research of disease resistance, cold resistance, breeding and the like of broccoli plants.

In addition, the CRISPR/Cas9 technology is used for carrying out base complementary pairing on a sgRNA sequence with a specific guide and a target sequence, guiding the Cas9 protein to be combined to the target sequence, carrying out DNA cutting to generate breakage, activating a non-homologous end connection or homologous recombination repair mechanism of a cell, and realizing mutation such as base deletion, insertion, substitution and the like. The CRISPR/Cas9 technology only modifies a target site sequence, an expression vector sequence in a T0 generation plant can be separated through progeny selfing to obtain a non-transgenic plant without any exogenous DNA sequence, and the mutant material is essentially indistinguishable from a mutant material obtained by a traditional breeding method, so that a new opportunity is provided for realizing an accurate, efficient, time-saving, labor-saving and safe agricultural breeding technology revolution, and a new technology is provided for broccoli breeding.

While the invention has been described in detail in the foregoing by way of general description, and specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. This invention is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It will be understood that the invention is not limited to the precise arrangements described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof.

The sequence at the 1 st to 8 th sites in the sequence table 1 is a NotI restriction endonuclease recognition site;

the sequence of 537-556 th site in the sequence table 1 is a type II endonuclease BsaI recognition site;

the sequence at 815-th and 822-th sites in the sequence table 1 is an SbfI restriction enzyme recognition site;

the sequence of the 710 th and 731 th sites in the sequence table 1 is a type II endonuclease BbsI recognition site;

the sequence at 10826-10831 in the sequence table 2 is a SacI restriction endonuclease recognition site;

the sequence at 10847-10852 in the sequence table 2 is XbaI restriction endonuclease recognition site;

the sequence from 1 st to 8 th in the sequence table 3 is an AscI restriction endonuclease recognition site;

the sequence of 5336-position 5343 in the sequence table 3 is NotI restriction endonuclease recognition site;

the sequence at the 189-208 th site in the sequence table 4 is a target site T1 sequence;

the sequence at position 1168-1187 in the sequence table 4 is the target site T2 sequence.

Sequence listing

<110> Olympic species of Zhejiang America, Ltd

<120> broccoli gene site-directed editing method based on CRISPR/Cas9

<130> 2021.06.23

<150> 2021109424786

<151> 2021-08-17

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 822

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gcggccgcct gataatataa aatctgttac ttctaaaact atacactatt tattctattt 60

tttgaatgaa agtatcttcg taaacacaca aaatattttg tgacgttgca attatacata 120

cacacaatat ctgcctcttc atactgacat tactgtgttc cctttcgtga ggtacgggcc 180

gagagagaac acagggtacg caccgattat gttcttatgc ccgcacaggg tacggaccgg 240

tcacctagta gtaactaaaa gctgatttac aaaaccatga atttttaaat gaatcacctt 300

ttgaataata ttaacgggcc agagagaaat cggagagagg cctgttagtg taataaacga 360

gcccattaat atgttgtaaa tgaagtgaaa cagtcccaca tcggttgtgt aagaagaaga 420

aactgtgttt atatagcgat ggagtgacga aggtgattga acaaagcacc agtggtctag 480

tggtagaata gtaccctgcc acggtacaga cccgggttcg attcccggct ggtgcaagag 540

accctgcagg gtctctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 600

atcaacttga aaaagtggca ccgagtcggt gcaacaaagc accagtggtc tagtggtaga 660

atagtaccct gccacggtac agacccgggt tcgattcccg gctggtgcag ggtcttcgtt 720

acagaagacc tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa 780

cttgaaaaag tggcaccgag tcggtgcttt ttttcctgca gg 822

<210> 2

<211> 11633

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

catggtagat ctgagggtaa atttctagtt tttctccttc attttcttgg ttaggaccct 60

tttctctttt tatttttttg agctttgatc tttctttaaa ctgatctatt ttttaattga 120

ttggttatgg tgtaaatatt acatagcttt aactgataat ctgattactt tatttcgtgt 180

gtctatgatg atgatgatag ttacagaacc gacgactcgt ccgtcctgta gaaaccccaa 240

cccgtgaaat caaaaaactc gacggcctgt gggcattcag tctggatcgc gaaaactgtg 300

gaattgatca gcgttggtgg gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag 360

gcagttttaa cgatcagttc gccgatgcag atattcgtaa ttatgcgggc aacgtctggt 420

atcagcgcga agtctttata ccgaaaggtt gggcaggcca gcgtatcgtg ctgcgtttcg 480

atgcggtcac tcattacggc aaagtgtggg tcaataatca ggaagtgatg gagcatcagg 540

gcggctatac gccatttgaa gccgatgtca cgccgtatgt tattgccggg aaaagtgtac 600

gtatcaccgt ttgtgtgaac aacgaactga actggcagac tatcccgccg ggaatggtga 660

ttaccgacga aaacggcaag aaaaagcagt cttacttcca tgatttcttt aactatgccg 720

gaatccatcg cagcgtaatg ctctacacca cgccgaacac ctgggtggac gatatcaccg 780

tggtgacgca tgtcgcgcaa gactgtaacc acgcgtctgt tgactggcag gtggtggcca 840

atggtgatgt cagcgttgaa ctgcgtgatg cggatcaaca ggtggttgca actggacaag 900

gcactagcgg gactttgcaa gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc 960

tctatgaact cgaagtcaca gccaaaagcc agacagagtc tgatatctac ccgcttcgcg 1020

tcggcatccg gtcagtggca gtgaagggcc aacagttcct gattaaccac aaaccgttct 1080

actttactgg ctttggtcgt catgaagatg cggacttacg tggcaaagga ttcgataacg 1140

tgctgatggt gcacgaccac gcattaatgg actggattgg ggccaactcc taccgtacct 1200

cgcattaccc ttacgctgaa gagatgctcg actgggcaga tgaacatggc atcgtggtga 1260

ttgatgaaac tgctgctgtc ggctttcagc tgtctttagg cattggtttc gaagcgggca 1320

acaagccgaa agaactgtac agcgaagagg cagtcaacgg ggaaactcag caagcgcact 1380

tacaggcgat taaagagctg atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga 1440

gtattgccaa cgaaccggat acccgtccgc aaggtgcacg ggaatatttc gcgccactgg 1500

cggaagcaac gcgtaaactc gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct 1560

gcgacgctca caccgatacc atcagcgatc tctttgatgt gctgtgcctg aaccgttatt 1620

acggatggta tgtccaaagc ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac 1680

ttctggcctg gcaggagaaa ctgcatcagc cgattatcat caccgaatac ggcgtggata 1740

cgttagccgg gctgcactca atgtacaccg acatgtggag tgaagagtat cagtgtgcat 1800

ggctggatat gtatcaccgc gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat 1860

ggaatttcgc cgattttgcg acctcgcaag gcatattgcg cgttggcggt aacaagaaag 1920

ggatcttcac tcgcgaccgc aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga 1980

ctggcatgaa cttcggtgaa aaaccgcagc agggaggcaa acaagctagc caccaccacc 2040

accaccacgt gtgaattaca ggtgaccagc tcgaatttcc ccgatcgttc aaacatttgg 2100

caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt 2160

ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga 2220

tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata 2280

tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa 2340

ttaaactatc agtgtttgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt 2400

attagaataa cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg 2460

tgcatgccaa ccacagggtt cccctcggga tcaaagtact ttgatccaac ccctccgctg 2520

ctatagtgca gtcggcttct gacgttcagt gcagccgtct tctgaaaacg acatgtcgca 2580

caagtcctaa gttacgcgac aggctgccgc cctgcccttt tcctggcgtt ttcttgtcgc 2640

gtgttttagt cgcataaagt agaatacttg cgactagaac cggagacatt acgccatgaa 2700

caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc agcaccgacg accaggactt 2760

gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc aagctgtttt ccgagaagat 2820

caccggcacc aggcgcgacc gcccggagct ggccaggatg cttgaccacc tacgccctgg 2880

cgacgttgtg acagtgacca ggctagaccg cctggcccgc agcacccgcg acctactgga 2940

cattgccgag cgcatccagg aggccggcgc gggcctgcgt agcctggcag agccgtgggc 3000

cgacaccacc acgccggccg gccgcatggt gttgaccgtg ttcgccggca ttgccgagtt 3060

cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc gaggccgcca aggcccgagg 3120

cgtgaagttt ggcccccgcc ctaccctcac cccggcacag atcgcgcacg cccgcgagct 3180

gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca ctgcttggcg tgcatcgctc 3240

gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg cccaccgagg ccaggcggcg 3300

cggtgccttc cgtgaggacg cattgaccga ggccgacgcc ctggcggccg ccgagaatga 3360

acgccaagag gaacaagcat gaaaccgcac caggacggcc aggacgaacc gtttttcatt 3420

accgaagaga tcgaggcgga gatgatcgcg gccgggtacg tgttcgagcc gcccgcgcac 3480

gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt ctgatgccaa gctggcggcc 3540

tggccggcca gcttggccgc tgaagaaacc gagcgccgcc gtctaaaaag gtgatgtgta 3600

tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata tgatgcgatg agtaaataaa 3660

caaatacgca aggggaacgc atgaaggtta tcgctgtact taaccagaaa ggcgggtcag 3720

gcaagacgac catcgcaacc catctagccc gcgccctgca actcgccggg gccgatgttc 3780

tgttagtcga ttccgatccc cagggcagtg cccgcgattg ggcggccgtg cgggaagatc 3840

aaccgctaac cgttgtcggc atcgaccgcc cgacgattga ccgcgacgtg aaggccatcg 3900

gccggcgcga cttcgtagtg atcgacggag cgccccaggc ggcggacttg gctgtgtccg 3960

cgatcaaggc agccgacttc gtgctgattc cggtgcagcc aagcccttac gacatatggg 4020

ccaccgccga cctggtggag ctggttaagc agcgcattga ggtcacggat ggaaggctac 4080

aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg catcggcggt gaggttgccg 4140

aggcgctggc cgggtacgag ctgcccattc ttgagtcccg tatcacgcag cgcgtgagct 4200

acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc agaacccgag ggcgacgctg 4260

cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa actcatttga gttaatgagg 4320

taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc ggccgtccga gcgcacgcag 4380

cagcaaggct gcaacgttgg ccagcctggc agacacgcca gccatgaagc gggtcaactt 4440

tcagttgccg gcggaggatc acaccaagct gaagatgtac gcggtacgcc aaggcaagac 4500

cattaccgag ctgctatctg aatacatcgc gcagctacca gagtaaatga gcaaatgaat 4560

aaatgagtag atgaatttta gcggctaaag gaggcggcat ggaaaatcaa gaacaaccag 4620

gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg cggttggcca ggcgtaagcg 4680

gctgggttgt ctgccggccc tgcaatggca ctggaacccc caagcccgag gaatcggcgt 4740

gacggtcgca aaccatccgg cccggtacaa atcggcgcgg cgctgggtga tgacctggtg 4800

gagaagttga aggccgcgca ggccgcccag cggcaacgca tcgaggcaga agcacgcccc 4860

ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag aatcccggca accgccggca 4920

gccggtgcgc cgtcgattag gaagccgccc aagggcgacg agcaaccaga ttttttcgtt 4980

ccgatgctct atgacgtggg cacccgcgat agtcgcagca tcatggacgt ggccgttttc 5040

cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc gctacgagct tccagacggg 5100

cacgtagagg tttccgcagg gccggccggc atggccagtg tgtgggatta cgacctggta 5160

ctgatggcgg tttcccatct aaccgaatcc atgaaccgat accgggaagg gaagggagac 5220

aagcccggcc gcgtgttccg tccacacgtt gcggacgtac tcaagttctg ccggcgagcc 5280

gatggcggaa agcagaaaga cgacctggta gaaacctgca ttcggttaaa caccacgcac 5340

gttgccatgc agcgtacgaa gaaggccaag aacggccgcc tggtgacggt atccgagggt 5400

gaagccttga ttagccgcta caagatcgta aagagcgaaa ccgggcggcc ggagtacatc 5460

gagatcgagc tagctgattg gatgtaccgc gagatcacag aaggcaagaa cccggacgtg 5520

ctgacggttc accccgatta ctttttgatc gatcccggca tcggccgttt tctctaccgc 5580

ctggcacgcc gcgccgcagg caaggcagaa gccagatggt tgttcaagac gatctacgaa 5640

cgcagtggca gcgccggaga gttcaagaag ttctgtttca ccgtgcgcaa gctgatcggg 5700

tcaaatgacc tgccggagta cgatttgaag gaggaggcgg ggcaggctgg cccgatccta 5760

gtcatgcgct accgcaacct gatcgagggc gaagcatccg ccggttccta atgtacggag 5820

cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc gaaaaggtct ctttcctgtg 5880

gatagcacgt acattgggaa cccaaagccg tacattggga accggaaccc gtacattggg 5940

aacccaaagc cgtacattgg gaaccggtca cacatgtaag tgactgatat aaaagagaaa 6000

aaaggcgatt tttccgccta aaactcttta aaacttatta aaactcttaa aacccgcctg 6060

gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc tgcaaaaagc gcctaccctt 6120

cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc ctatcgcggc cgctggccgc 6180

tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc gcggacaagc cgcgccgtcg 6240

ccactcgacc gccggcgccc acatcaaggc accctgcctc gcgcgtttcg gtgatgacgg 6300

tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 6360

cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc 6420

catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag 6480

cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga 6540

aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 6600

cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 6660

ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 6720

aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 6780

cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 6840

cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 6900

gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 6960

tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 7020

cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 7080

ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 7140

gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 7200

gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 7260

accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 7320

ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 7380

tcacgttaag ggattttggt catgcattct aggtactaaa acaattcatc cagtaaaata 7440

taatatttta ttttctccca atcaggcttg atccccagta agtcaaaaaa tagctcgaca 7500

tactgttctt ccccgatatc ctccctgatc gaccggacgc agaaggcaat gtcataccac 7560

ttgtccgccc tgccgcttct cccaagatca ataaagccac ttactttgcc atctttcaca 7620

aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa gttcctcttc gggcttttcc 7680

gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg gagtgtcttc ttcccagttt 7740

tcgcaatcca catcggccag atcgttattc agtaagtaat ccaattcggc taagcggctg 7800

tctaagctat tcgtataggg acaatccgat atgtcgatgg agtgaaagag cctgatgcac 7860

tccgcataca gctcgataat cttttcaggg ctttgttcat cttcatactc ttccgagcaa 7920

aggacgccat cggcctcact catgagcaga ttgctccagc catcatgccg ttcaaagtgc 7980

aggacctttg gaacaggcag ctttccttcc agccatagca tcatgtcctt ttcccgttcc 8040

acatcatagg tggtcccttt ataccggctg tccgtcattt ttaaatatag gttttcattt 8100

tctcccacca gcttatatac cttagcagga gacattcctt ccgtatcttt tacgcagcgg 8160

tatttttcga tcagtttttt caattccggt gatattctca ttttagccat ttattatttc 8220

cttcctcttt tctacagtat ttaaagatac cccaagaagc taattataac aagacgaact 8280

ccaattcact gttccttgca ttctaaaacc ttaaatacca gaaaacagct ttttcaaagt 8340

tgttttcaaa gttggcgtat aacatagtat cgacggagcc gattttgaaa ccgcggtgat 8400

cacaggcagc aacgctctgt catcgttaca atcaacatgc taccctccgc gagatcatcc 8460

gtgtttcaaa cccggcagct tagttgccgt tcttccgaat agcatcggta acatgagcaa 8520

agtctgccgc cttacaacgg ctctcccgct gacgccgtcc cggactgatg ggctgcctgt 8580

atcgagtggt gattttgtgc cgagctgccg gtcggggagc tgttggctgg ctggtggcag 8640

gatatattgt ggtgtaaaca aattgacgct tagacaactt aataacacat tgcggacgtt 8700

tttaatgtac tgaattaacg ccgaattaat tcgggggatc tggattttag tactggattt 8760

tggttttagg aattagaaat tttattgata gaagtatttt acaaatacaa atacatacta 8820

agggtttctt atatgctcaa cacatgagcg aaaccctata ggaaccctaa ttcccttatc 8880

tgggaactac tcacacatta ttatggagaa actcgagctt gtcgatcgac tctagctaga 8940

ggatcgatcc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc 9000

gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc 9060

gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc 9120

cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt 9180

cggcaagcag gcatcgccat gtgtcacgac gagatcctcg ccgtcgggca tgcgcgcctt 9240

gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg 9300

atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg 9360

gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat 9420

ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc 9480

caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac 9540

gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tggagttcat tcagggcacc 9600

ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc 9660

ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca 9720

agcggccgga gaacctgcgt gcaatccatc ttgttcaatc cccatggtcg atcgacagat 9780

ctgcgaaagc tcgagagaga tagatttgta gagagagact ggtgatttca gcgtgtcctc 9840

tccaaatgaa atgaacttcc ttatatagag gaaggtcttg cgaaggatag tgggattgtg 9900

cgtcatccct tacgtcagtg gagatatcac atcaatccac ttgctttgaa gacgtggttg 9960

gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg ggaccactgt 10020

cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat ttgtaggtgc 10080

caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga 10140

ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga 10200

ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttatcacatc 10260

aatccacttg ctttgaagac gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg 10320

gtgggggtcc atctttggga ccactgtcgg cagaggcatc ttgaacgata gcctttcctt 10380

tatcgcaatg atggcatttg taggtgccac cttccttttc tactgtcctt ttgatgaagt 10440

gacagatagc tgggcaatgg aatccgagga ggtttcccga tattaccctt tgttgaaaag 10500

tctcaatagc cctttggtct tctgagactg tatctttgat attcttggag tagacgagag 10560

tgtcgtgctc caccatgttg gcaagctgct ctagccaata cgcaaaccgc ctctccccgc 10620

gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 10680

tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt 10740

tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 10800

cagctatgac catgattacg aattcgagct cggtacccgg ggatcctcta gagtcgacct 10860

gcaggcatgc aagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 10920

gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 10980

aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgctaga 11040

gcagcttgag cttggatcag attgtcgttt cccgccttca gtttagcttc atggagtcaa 11100

agattcaaat agaggaccta acagaactcg ccgtaaagac tggcgaacag ttcatacaga 11160

gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacacac 11220

ttgtctactc caaaaatatc aaagatacag tctcagaaga ccaaagggca attgagactt 11280

ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct atctgtcact 11340

ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag 11400

gaaaggccat cgttgaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 11460

cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 11520

gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt 11580

cctctatata aggaagttca tttcatttgg agagaacacg ggggactctt gac 11633

<210> 3

<211> 5343

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggcgcgcctt accggtcaac atgtggagca cgacacactt gtctactcca aaaatatcaa 60

agatacagtc tcagaagacc aaagggcaat tgagactttt caacaaaggg taatatccgg 120

aaacctcctc ggattccatt gcccagctat ctgtcacttt attgtgaaga tagtggaaaa 180

ggaaggtggc tcctacaaat gccatcattg cgataaagga aaggccatcg ttgaagatgc 240

ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg tggaaaaaga 300

agacgttcca accacgtctt caaagcaagt ggattgatgt gataacatgg tggagcacga 360

cacacttgtc tactccaaaa atatcaaaga tacagtctca gaagaccaaa gggcaattga 420

gacttttcaa caaagggtaa tatccggaaa cctcctcgga ttccattgcc cagctatctg 480

tcactttatt gtgaagatag tggaaaagga aggtggctcc tacaaatgcc atcattgcga 540

taaaggaaag gccatcgttg aagatgcctc tgccgacagt ggtcccaaag atggaccccc 600

acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga 660

ttgatgtgat atctccactg acgtaaggga tgacgcacaa tcccactatc cttcgcaaga 720

cccttcctct atataaggaa gttcatttca tttggagagg acgtcgagag ttctcaacac 780

aacatataca aaacaaacga atctcaagca atcaagcatt ctacttctat tgcagcaatt 840

taaatcattt cttttaaagc aaaagcaatt ttctgaaaat tttcaccatt tacgaacgat 900

agccatggct cctaagaaga agcggaaggt tggtattcac ggggtgcctg cggctgacaa 960

gaagtactcc atcggcctcg acatcggcac caacagcgtc ggctgggcgg tgatcaccga 1020

cgagtacaag gtcccgtcca agaagttcaa ggtcctgggc aacaccgacc gccactccat 1080

caagaagaac ctcatcggcg ccctcctctt cgactccggc gagacggcgg aggcgacccg 1140

cctcaagcgc accgcccgcc gccgctacac ccgccgcaag aaccgcatct gctacctcca 1200

ggagatcttc tccaacgaga tggcgaaggt cgacgactcc ttcttccacc gcctcgagga 1260

gtccttcctc gtggaggagg acaagaagca cgagcgccac cccatcttcg gcaacatcgt 1320

cgacgaggtc gcctaccacg agaagtaccc cactatctac caccttcgta agaagcttgt 1380

tgactctact gataaggctg atcttcgtct catctacctt gctctcgctc acatgatcaa 1440

gttccgtggt cacttcctta tcgagggtga ccttaaccct gataactccg acgtggacaa 1500

gctcttcatc cagctcgtcc agacctacaa ccagctcttc gaggagaacc ctatcaacgc 1560

ttccggtgtc gacgctaagg cgatcctttc cgctaggctc tccaagtcca ggcgtctcga 1620

gaacctcatc gcccagctcc ctggtgagaa gaagaacggt cttttcggta acctcatcgc 1680

tctctccctc ggtctgaccc ctaacttcaa gtccaacttc gacctcgctg aggacgctaa 1740

gcttcagctc tccaaggata cctacgacga tgatctcgac aacctcctcg ctcagattgg 1800

agatcagtac gctgatctct tccttgctgc taagaacctc tccgatgcta tcctcctttc 1860

ggatatcctt agggttaaca ctgagatcac taaggctcct ctttctgctt ccatgatcaa 1920

gcgctacgac gagcaccacc aggacctcac cctcctcaag gctcttgttc gtcagcagct 1980

ccccgagaag tacaaggaga tcttcttcga ccagtccaag aacggctacg ccggttacat 2040

tgacggtgga gctagccagg aggagttcta caagttcatc aagccaatcc ttgagaagat 2100

ggatggtact gaggagcttc tcgttaagct taaccgtgag gacctcctta ggaagcagag 2160

gactttcgat aacggctcta tccctcacca gatccacctt ggtgagcttc acgccatcct 2220

tcgtaggcag gaggacttct accctttcct caaggacaac cgtgagaaga tcgagaagat 2280

ccttactttc cgtattcctt actacgttgg tcctcttgct cgtggtaact cccgtttcgc 2340

ttggatgact aggaagtccg aggagactat caccccttgg aacttcgagg aggttgttga 2400

caagggtgct tccgcccagt ccttcatcga gcgcatgacc aacttcgaca agaacctccc 2460

caacgagaag gtcctcccca agcactccct cctctacgag tacttcacgg tctacaacga 2520

gctcaccaag gtcaagtacg tcaccgaggg tatgcgcaag cctgccttcc tctccggcga 2580

gcagaagaag gctatcgttg acctcctctt caagaccaac cgcaaggtca ccgtcaagca 2640

gctcaaggag gactacttca agaagatcga gtgcttcgac tccgtcgaga tcagcggcgt 2700

tgaggaccgt ttcaacgctt ctctcggtac ctaccacgat ctcctcaaga tcatcaagga 2760

caaggacttc ctcgacaacg aggagaacga ggacatcctc gaggacatcg tcctcactct 2820

tactctcttc gaggataggg agatgatcga ggagaggctc aagacttacg ctcatctctt 2880

cgatgacaag gttatgaagc agctcaagcg tcgccgttac accggttggg gtaggctctc 2940

ccgcaagctc atcaacggta tcagggataa gcagagcggc aagactatcc tcgacttcct 3000

caagtctgat ggtttcgcta acaggaactt catgcagctc atccacgatg actctcttac 3060

cttcaaggag gatattcaga aggctcaggt gtccggtcag ggcgactctc tccacgagca 3120

cattgctaac cttgctggtt cccctgctat caagaagggc atccttcaga ctgttaaggt 3180

tgtcgatgag cttgtcaagg ttatgggtcg tcacaagcct gagaacatcg tcatcgagat 3240

ggctcgtgag aaccagacta cccagaaggg tcagaagaac tcgagggagc gcatgaagag 3300

gattgaggag ggtatcaagg agcttggttc tcagatcctt aaggagcacc ctgtcgagaa 3360

cacccagctc cagaacgaga agctctacct ctactacctc cagaacggta gggatatgta 3420

cgttgaccag gagctcgaca tcaacaggct ttctgactac gacgtcgacc acattgttcc 3480

tcagtctttc cttaaggatg actccatcga caacaaggtc ctcacgaggt ccgacaagaa 3540

caggggtaag tcggacaacg tcccttccga ggaggttgtc aagaagatga agaactactg 3600

gaggcagctt ctcaacgcta agctcattac ccagaggaag ttcgacaacc tcacgaaggc 3660

tgagaggggt ggcctttccg agcttgacaa ggctggtttc atcaagaggc agcttgttga 3720

gacgaggcag attaccaagc acgttgctca gatcctcgat tctaggatga acaccaagta 3780

cgacgagaac gacaagctca tccgcgaggt caaggtgatc accctcaagt ccaagctcgt 3840

ctccgacttc cgcaaggact tccagttcta caaggtccgc gagatcaaca actaccacca 3900

cgctcacgat gcttacctta acgctgtcgt tggtaccgct cttatcaaga agtaccctaa 3960

gcttgagtcc gagttcgtct acggtgacta caaggtctac gacgttcgta agatgatcgc 4020

caagtccgag caggagatcg gcaaggccac cgccaagtac ttcttctact ccaacatcat 4080

gaacttcttc aagaccgaga tcaccctcgc caacggcgag atccgcaagc gccctcttat 4140

cgagacgaac ggtgagactg gtgagatcgt ttgggacaag ggtcgcgact tcgctactgt 4200

tcgcaaggtc ctttctatgc ctcaggttaa catcgtcaag aagaccgagg tccagaccgg 4260

tggcttctcc aaggagtcta tccttccaaa gagaaactcg gacaagctca tcgctaggaa 4320

gaaggattgg gaccctaaga agtacggtgg tttcgactcc cctactgtcg cctactccgt 4380

cctcgtggtc gccaaggtgg agaagggtaa gtcgaagaag ctcaagtccg tcaaggagct 4440

cctcggcatc accatcatgg agcgctcctc cttcgagaag aacccgatcg acttcctcga 4500

ggccaagggc tacaaggagg tcaagaagga cctcatcatc aagctcccca agtactctct 4560

tttcgagctc gagaacggtc gtaagaggat gctggcttcc gctggtgagc tccagaaggg 4620

taacgagctt gctcttcctt ccaagtacgt gaacttcctc tacctcgcct cccactacga 4680

gaagctcaag ggttcccctg aggataacga gcagaagcag ctcttcgtgg agcagcacaa 4740

gcactacctc gacgagatca tcgagcagat ctccgagttc tccaagcgcg tcatcctcgc 4800

tgacgctaac ctcgacaagg tcctctccgc ctacaacaag caccgcgaca agcccatccg 4860

cgagcaggcc gagaacatca tccacctctt cacgctcacg aacctcggcg cccctgctgc 4920

tttcaagtac ttcgacacca ccatcgacag gaagcgttac acgtccacca aggaggttct 4980

cgacgctact ctcatccacc agtccatcac cggtctttac gagactcgta tcgacctttc 5040

ccagcttggt ggtgataagc gtcctgctgc caccaaaaag gccggacagg ctaagaaaaa 5100

gaagtaggat ccacctgatc tagagtccgc aaaaatcacc agtctctctc tacaaatcta 5160

tctctctcta tttttctcca gaataatgtg tgagtagttc ccagataagg gaattagggt 5220

tcttataggg tttcgctcat gtgttgagca tataagaaac ccttagtatg tatttgtatt 5280

tgtaaaatac ttctatcaat aaaatttcta attcctaaaa ccaaaatcca gtgacgcggc 5340

cgc 5343

<210> 4

<211> 1914

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggacggtg gtgaagagac gtcgacggcg agagggctga agaaaggtcc atggacgacg 60

acagaggatg cgatcttgac ggagtacgtg aggaaacgcg gtgaaggtaa ctggaacgcg 120

gtgcagaaga actcaggctt gctccggtgt gggaaaagct gccgtctacg gtgggcgaac 180

catctccggc caaatctaag gacaggatcc tttagtcctg atgaagagaa gatcatcatc 240

gagcttcacg ctaagttggg taacaaatgg gctcgtatgg cttctcaggt ttattttctc 300

ataaacaata ttttctcatt taaaaaatca cttattattc tccttatttg ttacttcaat 360

tctagaatta cagagcttta aatttttata tgcaattaaa attttctagt taaacacata 420

aattgcatgt ttcagtgtgt tttgaatcta ccgtctcttt ggtttggcat gattaatctt 480

gcatctcttc tattgctttt ttttatcctt ttagttgtaa aggttttttt tctattgtag 540

ttacctggaa gaactgacaa cgaaatcaaa aactattgga acacgaggat aaagagaaga 600

caacgagctg gtttgccttt attccctcac gagattcagc atctagggat tggtaatgat 660

gatgagtttg agattagatg ctttcagttc ccaaaccaag accatcctaa tcaccaaaat 720

atgattcaat acactaattc ctctaatact tcaccatcct cgtcttcatt ctcttcatca 780

tcttctcaac ctccaaaaga gctgtgttta gatcccttaa tctctactaa tcccggcctt 840

aatcagatcc ccaatatgtt ctctccttac aacaatagct ttgagaatga caataaccag 900

tttgttttct ctcttccttt ttcctcatcc tcatcgtcga atgagttatg taatcccgac 960

caacttcttg aactcatgtc agagaatttg gacacaaacg ttatcagtaa gaaagacatt 1020

gacgctacga gtcttattag agatcatgag acgataccga gttatttctc tttaggacta 1080

gacactaccg tcctagagct tccttcaaac caaacaccga ctcaatcgtg cacttccaat 1140

attatgcttt acaataatgt ccttcttgat tcacctgcgg ggaacagtgg attacttgac 1200

gccctcttgg aggaatctcg agccttgtct cgcggcggaa tcttcaagga cgctagggtt 1260

tcttcgagtg gtctatgtga ggatcaagac aagagagtga agatgaaatt tgagaatcgg 1320

ttgatagatc atctaagctc ttctcatcaa tcatcatttg gtaagaagcg ttatatgaat 1380

tcaaaccatt ttcacttata cattattttt tgctataacc attaatttat atttaataga 1440

aggcctagta gagatgattt gatccaaaaa aaagccaagt agatatgttt aatcaaagtg 1500

aaaccgaatc tagtttggat cttcggtaat atcgaataca tatttgattt ttactaatca 1560

tgtatttcat gttaatttgt gtttaataca tattaatctt aaaatgaaca gaatcaaact 1620

ctaatcttta cgagaagcac aatgacgcaa cgatgttgaa ggcaacagtg gatgatgatg 1680

acgacgacat gttgaagagc cttctcaaca gtttcccttc aaccacaccg ttacctgatt 1740

ggtaccagac gacagagatc ctaaaaggag gcctcaccga gcggaatatt gataggacac 1800

catcagggta ataacagggt ggagccacac aagccaccac cttcttccaa tgtagatcct 1860

atggcctcct tagggtcatc atcctactgg ggaaacatac ctagaatctg ctag 1914

Claims

1. A method for editing broccoli genes in a fixed point manner based on CRISPR/Cas9, which is characterized by comprising the following steps:

the complementary primer pair corresponding to target site sgT2 includes:

using restriction endonucleasesNotI andSbfi, mixingThe sgRNA expression frame plasmid containing the DNA fragments of the two target sites is connected to the pCAMBIA2301 plasmid containing the linker DNA fragment and the Cas9 expression frameNotI andSbfi, obtaining an expression vector plasmid edited by a BoMYB101 gene between enzyme cutting sites;

2. The method according to claim 1, wherein the step of ligating each target site sequence fragment into a sgRNA expression cassette, respectively, to construct a sgRNA expression cassette plasmid containing two target site DNA fragments comprises:

preparing a sgT1 target site assembly and connection reaction system according to the volume ratio of the linearized sgRNA expression frame plasmid after Bsa I digestion to the sgT1 target site DNA fragment, the T4 DNA ligase Buffer and the T4 DNA ligase of 15:2:2:1, and mixing the sgT1 target site DNA fragment and the DNA fragment of the sgT1 target siteBsaI, connecting the linearized sgRNA expression frame plasmid subjected to enzyme digestion at 4 ℃ overnight under the action of T4 DNA ligase Buffer and T4 DNA ligase, then transforming a connecting product into an escherichia coli DH5 alpha competent cell, coating the competent cell on an LB solid culture medium with 50 mu g/mL kanamycin, culturing at 37 ℃ overnight, screening positive clones, and extracting to obtain a sgRNA expression frame plasmid containing a sgT1 target site DNA fragment;

according to the ratio of 10 XNEB Buffer 2.1 to sg of DNA fragment containing sgT1 target siteRNA expression cassette plasmid, endonucleaseBbsⅠ、ddH₂Preparing a sgRNA expression frame enzyme digestion reaction system corresponding to sgT2 target sites with the volume ratio of O being 3:4:1:22, and carrying out endonuclease treatment on the sgRNA expression frame plasmid containing the DNA fragment with the sgT1 target sites by using type II endonuclease sitesBbsI, enzyme digestion, carrying out agarose gel electrophoresis on the enzyme digestion product, cutting the gel and recycling to obtain the productBbsI, digesting a linearized sgRNA expression frame plasmid;

according toBbsI, preparing a sgT2 target site assembly and connection reaction system by carrying out enzyme digestion on a linearized sgRNA expression cassette plasmid, an sgT2 target site DNA fragment, a T4 DNA ligase Buffer and a T4 DNA ligase in a volume ratio of 15:2:2:1, and carrying out enzyme digestion on the sgRNA expression cassette plasmid and the DNA fragment of the target site sgT2BbsI after enzyme digestion, linearized sgRNA expression frame plasmids are connected at 4 ℃ overnight under the action of T4 DNA ligase Buffer and T4 DNA ligase, then a connection product is transformed into escherichia coli DH5 alpha competent cells, the escherichia coli DH5 alpha competent cells are coated on an LB solid culture medium with 50 mu g/mL kanamycin and cultured at 37 ℃ overnight, positive clones are screened, and sgRNA expression frame plasmids containing two target site DNA fragments are extracted.

3. The method according to claim 1, wherein the linker DNA fragment is ligated to the expression vector pCAMBIA2301 plasmidSacI andXbathe method comprises the following steps of constructing pCAMBIA2301 plasmid containing linker DNA fragment between enzyme cutting sites, wherein the pCAMBIA2301 plasmid comprises the following steps:

4. The method according to claim 1, wherein the restriction enzyme is usedNotI andAsci, connecting Cas9 expression frame to pCAMBIA2301 plasmid containing linker DNA fragmentNotI andAscthe method comprises the following steps of I, obtaining pCAMBIA2301 plasmid containing linker DNA fragment and Cas9 expression frame between enzyme cutting sites, wherein the pCAMBIA2301 plasmid comprises the following steps:

according to the method, 10 XNEB Buffer is mixed with pCAMBIA2301 plasmid containing linker DNA fragment and restriction enzymeNotI, restriction enzymeAscⅠ、ddH₂Preparing a second pCAMBIA2301 linear enzyme digestion reaction system with the volume ratio of O to 5:5:1:1:38, and utilizing restriction enzymeNotI andAsccarrying out double enzyme digestion on the pCAMBIA2301 plasmid containing the linker DNA fragment and the Cas9 expression frame, carrying out agarose gel electrophoresis on the enzyme digestion product, cutting gel, and recovering the linearized pCAMBIA2301 plasmid containing the linker DNA fragment and the linearized Cas9 expression frame fragment;

5. The method according to claim 1, wherein the restriction enzyme is usedNotI andSbfi, subjecting the DNA piece containing the two target sitesThe sgRNA expression frame plasmid of the segment is connected to the pCAMBIA2301 plasmid containing the linker DNA fragment and the Cas9 expression frameNotI andSbfthe method comprises the following steps of I, obtaining an expression vector plasmid edited by a BoMYB101 gene between enzyme cutting sites, wherein the steps comprise:

6. The method according to claim 1, wherein the efficiency of the expression vector plasmid editing the BoMYB101 gene is 75-80%.