Construction method and application of CRISPR/Cas 9-mediated plant polygene editing vector

文档序号：163986 发布日期：2021-10-29 浏览：37次中文

阅读说明：本技术 一种CRISPR/Cas9介导的植物多基因编辑载体的构建方法和应用 (Construction method and application of CRISPR/Cas 9-mediated plant polygene editing vector ) 是由张辉汪冲罗鹏宇洪政张亚军于 2021-07-08 设计创作，主要内容包括：本发明公开了一种构建简便、高效的植物多基因编辑载体方法,其包括pMK-(1-12)中间载体及终载体pMMK-Cas9。载体构建的方法包括以下步骤：将多个sgRNA通过多顺反子tRNA-gRNA方式连接到pMK(1-12)中间载体上,每个中间载体上可以连接1-8个sgRNAs,得到pMK(1-12)-PTG载体；将多个pMK(1-12)-PTG载体上的U6/U3表达盒连接到pMMK-Cas9载体上,pMMK-Cas9可以连接2个到7个pMK(1-12)-PTG载体上的U6/U3表达盒,得到pMMK-PTG载体,根据不同的靶位点和小载体组合,可达到2-56个靶位点的多基因编辑。整个流程只需要一次PCR反应和两次“金门”连接体系即可完成,流程简单快速。本发明还以该载体敲除水稻LEA基因家族25个成员为例,验证该载体多基因编辑的高效性。(The invention discloses a method for constructing a simple and efficient plant polygene editing vector, which comprises a pMK- (1-12) intermediate vector and a final vector pMMK-Cas 9. The method for constructing the vector comprises the following steps: connecting a plurality of sgRNAs to pMK (1-12) intermediate vectors by a polycistronic tRNA-gRNA mode, wherein 1-8 sgRNAs can be connected to each intermediate vector to obtain a pMK (1-12) -PTG vector; the U6/U3 expression cassettes on a plurality of pMK (1-12) -PTG vectors are connected to a pMMK-Cas9 vector, the pMMK-Cas9 can be connected with U6/U3 expression cassettes on 2 to 7 pMK (1-12) -PTG vectors to obtain the pMMK-PTG vectors, and multi-gene editing of 2 to 56 target sites can be achieved according to different target sites and small vector combinations. The whole process can be completed only by one PCR reaction and two gold gate connection systems, and the process is simple and quick. The invention also takes the example that the carrier knocks out 25 members of the LEA gene family of rice as an example, and verifies the high efficiency of the multi-gene editing of the carrier.)

1. A construction method of a CRISPR/Cas9 mediated plant polygene editing vector is characterized by comprising the following steps:

(1) connecting a plurality of sgRNAs to pMK- (1-12) intermediate vectors by a polycistronic tRNA-gRNA mode, wherein 1 to a plurality of sgRNAs can be connected to each intermediate vector to obtain a pMK (1-12) -PTG vector;

(2) a plurality of U6/U3 expression cassettes on pMK (1-12) -PTG vectors are connected to a pMMK-Cas9 vector, and a pMMK-Cas9 expression cassette of U6/U3 on 2 to 7 pMK (1-12) -PTG vectors are connected to obtain the pMMK-PTG vectors.

2. The construction method according to claim 1, wherein the pMK (1-12) is a set of 12 intermediate vectors for CRISPR/Cas 9-mediated gene editing, wherein each of pMK1, pMK10 and pMK11 comprises OsU6 expression cassettes, and OsU6 expression cassettes have the nucleotide sequence shown in SEQ ID No. 1; pMK2, pMK3, pMK8 and pMK9 respectively contain a OsU3 expression cassette, and the nucleotide sequence of OsU3 expression cassette is shown as SEQ ID NO. 2; pMK4 and pMK5 respectively contain a OsU6a expression cassette, and the nucleotide sequence of the OsU6a expression cassette is shown as SEQ ID NO. 3; pMK6, pMK7 and pMK12 respectively contain a OsU6b expression cassette, and OsU6b nucleotide sequence is shown as SEQ ID NO. 4.

3. The construction method according to claim 1, characterized in that in step (1), a plurality of sgRNAs are constructed on a pMK (1-12) intermediate vector, a target sequence is firstly designed on line through a CRISPR-GE website according to a gene targeting site, primers are synthesized,

gRNA[x]-F(SEQ ID NO.5)：

5‘-TAGGTCTCCN₉N₁₀N₁₁N₁₂N₁₃N₁₄N₁₅N₁₆N₁₇N₁₈N₁₉N₂₀GTTTTAGAGCTAGA A-3’

gRNA[x]-R(SEQ ID NO.6)：

5’-CGGGTCTCAN₁₂N₁₁N₁₀N₉N₈N₇N₆N₅N₄N₃N₂N₁TGCACCAGCCGGG-3‘；

wherein N is₁-N₂₀Respectively represent the nucleotides from position 1 to position 20 of the 20bp target sequence.

4. The construction method according to claim 1, characterized in that, in the step (1), amplification is carried out simultaneously using amplification primers PTG pMK (1-12) F and PTG CRISPR R according to the difference of each minivector ligated to pMK- (1-12), the primer sequence of PTG pMK1/10/11F is shown in SEQ ID NO. 8; the primer sequence of PTG pMK2/3/8/9F is shown as SEQ ID NO. 9; the primer sequence of PTG pMK 4/5F is shown as SEQ ID NO. 10; the primer sequence of PTG pMK6/7/12F is shown as SEQ ID NO. 11; PTG CRISPR R is shown in SEQ ID NO. 12;

PTG pMK1/10/11F(SEQ ID NO.8)：

GTACGGGTCTCATGTGGAACAAAGCACCAGTGGTCTA

PTG pMK2/3/8/9F(SEQ ID NO.9)：

GTACGGGTCTCATGGCAACAAAGCACCAGTGGTCTA

PTG pMK 4/5F(SEQ ID NO.10)：

GTACGGGTCTCATGCCGAACAAAGCACCAGTGGTCTA

PTG pMK6/7/12F(SEQ ID NO.11)：

GTACGGGTCTCATGTTGAACAAAGCACCAGTGGTCTA

PTG CRISPR R(SEQ ID NO.12)：

GACTAGGTCTCCAAACAAAAAAAAAAGCACCGACTCG

simultaneously obtaining a tRNA-gRNA unit and amplifying to obtain an adapter fragment linking the tRNA-gRNA unit and a small vector, and connecting the tRNA-gRNA unit to a pMK (1-12) intermediate vector by a multi-fragment connection method to obtain a pMK (1-12) -PTG vector containing polycistronic tRNA-sgRNA; the multi-fragment connection method is to use IIS type restriction endonuclease to perform enzyme digestion and connected 'gold gate' cloning, and simultaneously connect a plurality of tRNA-gRNAs to pMK (1-12) intermediate vectors, wherein the IIS type restriction endonuclease is BsaI, AarI, BbsI, BsmBI or BsmBI.

5. The method of claim 4, wherein in step (1), the intermediate pMK (1-12) vector carries a type IIS restriction endonuclease cut site behind the U6/3 promoter, and the polycistronic tRNA-gRNA fragment is inserted between the two type IIS restriction endonuclease cut sites.

6. The construction method according to claim 1, wherein the step (2) links the U6/3 expression cassette on a plurality of pMK (1-12) -PTG vectors to the pMMK-Cas9 vector, and the order of the linkage of the pMK (1-12) intermediate vectors is:

when 2U 6/U3 are connected, pMK1+ pMK2 is connected to pMMK-Cas 9;

when 3U 6/U3 are connected, pMK1+ pMK3+ pMK4 is connected to pMMK-Cas 9;

when 4U 6/U3 are connected, pMK1+ pMK3+ pMK5+ pMK6 are connected to pMMK-Cas 9;

when 5U 6/U3 are connected, pMK1+ pMK3+ pMK5+ pMK7+ pMK8 are connected to pMMK-Cas 9;

when 6U 6/U3 are connected, pMK1+ pMK3+ pMK5+ pMK7+ pMK9+ pMK10 are connected to pMMK-Cas 9;

when 7U 6/U3 were ligated, pMK1+ pMK3+ pMK5+ pMK7+ pMK9+ pMK11+ pMK12 were ligated to pMMK-Cas 9.

7. The construction method according to claim 6, wherein the multiple pMK (1-12) intermediate vector ligation pMMK-Cas9 method uses the "gold gate" clone digested and ligated with type IIS restriction enzymes including BsaI, AarI, BbsI, BsmAI or BsmBI above multiple pMK (1-12) to simultaneously ligate U6/3 expression cassettes to pMMK-Cas 9; the U6/3 expression cassette above multiple pMKs (1-12) was ligated simultaneously between two type II restriction enzymes of pMMK-Cas 9.

8. A tool vector or a kit of tool vectors constructed according to the method of any one of claims 1 to 7.

9. Use of the tool vector of claim 8 in a CRISPR/Cas 9-mediated plant polygene editing system.

10. Use according to claim 8, wherein the plant is a dicotyledonous plant or a monocotyledonous plant.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a plant efficient and rapid polygene editing vector, and a construction method and application thereof.

Background

The CRISPR/Cas9 system as a third-generation gene editing technology has the characteristics of simple and convenient construction and high labeling efficiency, and is widely used for gene editing research. CRISP/Cas9 primarily includes two core elements: the expression cassette comprises a Cas expression cassette and a sgRNA expression cassette, wherein the Cas expression cassette consists of strong promoters (Ubi, 35S and the like) such as RNA polymerase II and terminators such as NOS; the sgRNA expression cassette is terminated by 6 or more consecutive polynucleotides T driven by an RNA polymerase III promoter (U6, U3, etc.). In research, multiple genes are often required to be simultaneously subjected to gene editing, when multiple gene editing is carried out in animals, a multiple gene editing material can be obtained only by co-transfecting plasmids of multiple sgRNA expression cassettes, and in plants, a Cas9 expression cassette, a sgRNA expression cassette and a screening marker are generally required to be assembled into a vector together, and multiple gene fixed-point editing is realized through stable genetic transformation mediated by agrobacterium.

At present, the most common plant polygene editing systems are mainly divided into two types: one is a multi-component transcription unit system (MCTU), namely, a plurality of sgRNA expression cassettes are assembled on a vector, the construction of the system generally comprises the steps of sequentially constructing a plurality of sgRNA expression cassettes on the vector through a plurality of different enzyme digestion connections, the construction process is time-consuming and labor-consuming, the number of engineered plant endogenous U6 and U3 promoters is limited, the efficiency of multigene editing can be influenced by excessively repeated promoters, and generally, the number of target sites of the MCTU system is not more than 8; another type is called double-component transcription unit (TCTU) system, in which sgRNA expression cassettes are formed by concatenating multiple sgrnas into one transcription unit through RNA self-cleavage elements, such as ribozymes, tRNA precursors, and Csy4, and releasing multiple sgrnas by RNA cleavage. Construction of such systems utilizes the "gold" cloning approach to join multiple DNA fragments together simultaneously by non-palindromic cohesive ends generated by type IIS restriction enzymes such as BsaI, Esp 3I, BbsI, AarI. Due to the limitation of the "gold gate" cloning method, construction becomes more difficult as the number of DNA fragments increases, generally, sgRNAs connected with one transcription unit does not exceed 8, and due to the influence of promoter activity, the transcription level of the sgRNAs far away from a promoter is reduced as the length of the transcription unit increases, so that the editing efficiency is influenced.

Disclosure of Invention

Based on the defects of complex construction process and limited number of editing sites of the multi-gene editing, the invention provides an efficient and rapid multi-gene editing vector for plants and a construction method thereof, which can realize simultaneous editing of 2-56 sites of a plant genome. The multi-gene editing vector combines the characteristics of MCTU and TCTU, and the construction method is simple and quick. Meanwhile, the multi-gene editing vector provided by the invention is used for targeting 25 members of the rich protein LEA family in the late stage of rice embryo, and the mutant plants edited at 23 loci are successfully obtained, which shows that the system has high editing efficiency on a plurality of loci.

The first purpose of the invention is to provide a construction method of a CRISPR/Cas9 mediated plant polygene editing vector, which is characterized by comprising the following steps:

Further, the pMK (1-12) is a set of 12 intermediate vectors for CRISPR/Cas 9-mediated gene editing, wherein each of pMK1, pMK10 and pMK11 contains one OsU6 expression cassette, and the nucleotide sequence of OsU6 expression cassette is shown as SEQ ID No. 1; pMK2, pMK3, pMK8 and pMK9 respectively contain a OsU3 expression cassette, and the nucleotide sequence of OsU3 expression cassette is shown as SEQ ID NO. 2; pMK4 and pMK5 respectively contain a OsU6a expression cassette, and the nucleotide sequence of the OsU6a expression cassette is shown as SEQ ID NO. 3; pMK6, pMK7 and pMK12 respectively contain a OsU6b expression cassette, and OsU6b nucleotide sequence is shown as SEQ ID NO. 4.

Further, sgRNA was constructed onto pMK (1-12) intermediate vector, comprising the following steps:

(A) according to a gene target site sequence, designing a target sequence on line through a CRISPR-GE website, synthesizing a primer, and amplifying tRNA-gRNA units with different target sequences by using pGTR (Xie et al ProcNatlAcadsiUSA, 2015,112: 3570-;

the characteristics of the primer sequences synthesized according to the target sequences are as follows:

gRNA[x]-F(SEQ ID NO.5)：

gRNA[x]-R(SEQ ID NO.6)：

the amplification mode is shown in fig. 1, the primers amplify tRNA-gRNA units with different target sequences by taking pGTR as a template, and the obtained tRNA-gRNA unit sequences are shown as follows:

TAGGTCTCC ₉ ₁₀ ₁₁ ₁₂ ₁₃ ₁₄ ₁₅ ₁₆ ₁₇ ₁₈ ₁₉ ₂₀NNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCA ₁ ₂ ₃ ₄ ₅ ₆ ₇ ₈ ₉ ₁₀ ₁₁ ₁₂NNNNNNNNNNNNTGAGACCCG。(SEQ ID NO.7)

said N is₁-N₂₀Respectively represent the nucleotides from position 1 to position 20 of the 20bp target sequence.

(B) Designing an amplification primer with a type II restriction enzyme cutting site: PTG pMK (1-12) F and PTG CRISPR R, which are subjected to amplification reaction simultaneously with the step (1), for linking tRNA-gRNA tandem elements to corresponding pMK (1-12) minivectors;

the type II restriction enzyme comprises (BsaI, AarI, BbsI, BsmAI or BsmBI);

preferably, the type II restriction enzyme cutting site is BsaI;

the amplification primers in the step (2) are as follows:

PTG pMK1/10/11F(SEQ ID NO.8)：

GTACGGGTCTCATGTGGAACAAAGCACCAGTGGTCTA

PTG pMK2/3/8/9F(SEQ ID NO.9)：

GTACGGGTCTCATGGCAACAAAGCACCAGTGGTCTA

PTG pMK 4/5F(SEQ ID NO.10)：

GTACGGGTCTCATGCCGAACAAAGCACCAGTGGTCTA

PTG pMK6/7/12F(SEQ ID NO.11)：

GTACGGGTCTCATGTTGAACAAAGCACCAGTGGTCTA

PTG CRISPR R(SEQ ID NO.12)：

GACTAGGTCTCCAAACAAAAAAAAAAGCACCGACTCG；

the PTG pMK (1-12) F and PTG CRISPR R primers carry a linker with a reverse complementary sequence to the corresponding pMK (1-12) intermediate vector cleavage site;

(C) and (3) connecting the tRNA-gRNA units and the joints obtained by amplification in the steps (1) and (2) to corresponding pMK (1-12) intermediate vectors by using an IIS type endonuclease enzyme digestion and connection 'gold gate' cloning method to obtain the pMK (1-12) -PTG vectors containing the polycistronic tRNA-sgRNA.

Preferably, each U6 or U3 expression cassette transcribes only up to 8 transcripts of the sgRNA to prevent decreased transcription levels.

Further, the type II restriction enzymes used in the multi-fragment ligation method include (BsaI, AarI, BbsI, BsmAI, or BsmBI).

Preferably, BsaI is used as the type II restriction enzyme.

(D) The obtained pMK (1-12) -PTG vector was subjected to colony PCR identification by universal primers M13F (SEQ ID NO.13) and M13R (SEQ ID NO.14), the number of inserted sgRNAs in positive clones was confirmed by amplification band size, and the type of inserted sgRNAs was further identified by Sanger sequencing. The colony PCR band size was 504bp +163 × n, where n is the number of sgrnas inserted.

Further, step (2) connects the U6/3 expression cassette on multiple pMK (1-12) -PTG vectors to the pMMK-Cas9 vector, and the connection sequence of the pMK (1-12) intermediate vectors is as follows:

when 2U 6/U3 are connected, pMK1+ pMK2 is connected to pMMK-Cas 9;

when 3U 6/U3 are connected, pMK1+ pMK3+ pMK4 is connected to pMMK-Cas 9;

when 4U 6/U3 are connected, pMK1+ pMK3+ pMK5+ pMK6 are connected to pMMK-Cas 9;

when 5U 6/U3 are connected, pMK1+ pMK3+ pMK5+ pMK7+ pMK8 are connected to pMMK-Cas 9;

when 6U 6/U3 are connected, pMK1+ pMK3+ pMK5+ pMK7+ pMK9+ pMK10 are connected to pMMK-Cas 9;

when 7U 6/U3 were ligated, pMK1+ pMK3+ pMK5+ pMK7+ pMK9+ pMK11+ pMK12 were ligated to pMMK-Cas 9.

As shown in FIG. 2, is a schematic connection diagram of the U6/3 expression cassette connected to the pMMK-Cas9 vector on the above-mentioned multiple pMK (1-12) -PTG vectors.

Further, the multiple pMK (1-12) intermediate vector ligation pMMK-Cas9 method uses type II endonuclease to cut and ligate a "gold gate" clone, and the U6/3 expression cassettes on the multiple pMK (1-12) intermediate vectors obtained in step (1) are ligated on the pMMK-Cas9 vector;

the type II restriction enzymes include (BsaI, AarI, BbsI, BsmAI or BsmBI); preferably, the type II restriction enzyme cleavage site used is AarI.

Further, the obtained plasmids were verified by Sanger sequencing.

The invention provides a vector for tandem expression of a plurality of sgRNAs obtained by the construction method.

The invention also provides a tool vector or a tool set vector obtained by the construction method.

The invention also provides a kit containing the primer and a tool carrier or a complete set of tool carriers.

The invention also provides application of the tool vector or the tool vector set in a CRISPR/Cas 9-mediated plant polygene editing system. The plant is a dicotyledonous plant and/or a monocotyledonous plant. Further preferably, the plant is rice.

The core of the invention is that 2-56 sgRNAs started by different promoters are efficiently and rapidly constructed by using common vectors and universal sequences, and simultaneously, different target gene segments are targeted, so that efficient and specific polygene editing is realized; meanwhile, the construction method is quick and flexible, different numbers of sgRNAs can be constructed according to needs, and the method has a wide application prospect.

Drawings

FIG. 1 is a schematic sequence diagram of tRNA-gRNA units.

In FIG. 2, A is a schematic diagram of the pMK (1-12) intermediate vector and the pMMK vector, and B is a schematic diagram of the connection sequence of the pMK (1-12) intermediate vector.

FIG. 3A is a schematic representation of the OsLEA24 knock-out intermediary vector pMK (1-12) -PTG assembly strategy; b is a schematic diagram of the assembly strategy of connecting the OsLEA24 knock-out intermediate vector to pMMK-Cas 9.

A in FIG. 4 is a schematic diagram of primers used for OsLEA24 knock-out identification; b is a map schematic diagram of the OsLEA24 knock-out vector; c is a schematic diagram of the identification of the OsLEA24 knock-positive clone.

FIG. 5A is a schematic diagram showing the gene editing efficiency and mutation type at each site of the LEA gene family of rice; b is a schematic diagram of specific mutation conditions of rice LEA1, LEA2, LEA4, LEA5, LEA6 and LEA 7.

Detailed Description

In order to facilitate the understanding of the technical scheme of the invention, the multi-gene editing vector is used for targeting 25 members of the LEA family of the late abundant proteins of rice embryos and successfully obtaining mutant plants with 23 edited loci by combining specific embodiments, and equipment and reagents used in each embodiment and test example can be obtained from commercial approaches without special description. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Example 1

The embodiment provides a method for quickly constructing multiple sgrnas into a pMK (1-12) vector, which includes the following steps:

(1) obtaining sequence information of OsLEA1, OsLEA2, OsLEA4, OsLEA5, OsLEA6, OsLEA7, OsLEA9, OsLEA10, OsLEA11, OsLEA13, OsLEA15, OsLEA17, OsLEA18, OsLEA19, OsLEA20, OsLEA21, OsLEA22, OsLEA23, OsLEA24, OsLEA27/28, OsLEA30, OsLEA31, OsLEA33 and OsLEA34 genes. And designing a target site of the target gene according to the database CRISPR-GE (http:// skl.scau.edu.cn /).

The target site is designed to avoid off-target of the core region (9-20) (2) GC content is 40-70% (3) and cannot contain GGTCTC sequence (4) to avoid designed linker duplication. The target sites, primer names and sequences were designed as shown in Table 1 below.

(2) The sequence containing the target site, tRNA and guide RNA was cloned by amplification with gold medal mix DNA polymerase (Beijing Ongchou Biotechnology Co., Ltd.) using pGTR (Xie et al, Proc Natl Acad Sci USA,2015,112:3570-3575) plasmid as a template and primers designed. The nomenclature of the individual bands of interest and the amplification procedure are detailed in tables 3 and 4 below.

After the amplification and amplification of the target band is finished, gel electrophoresis detection is carried out, the residual sample after the detection of the correct band size directly passes through a column recovery kit (Tiangen Biochemical technology Co., Ltd.) to recover and purify the target fragment, a plurality of DNA fragments are connected by a gold gate assembly method according to a certain sequence, the DNA fragments are connected into a pMK intermediate vector by enzyme digestion-connection cycling reaction of II type restriction enzyme BsaI (NEB company) and T4 DNA ligase (NEB company), and the connection reaction system is shown in the detailed table 5.

(3) 10uL of the reaction mixture was transformed into E.coli competent DH5a (Shanghai Weidi Biotechnology Co., Ltd.) and screened using a carbenicillin resistant plate. After resistant colonies are picked, PCR is used for positive clone detection, the sequences of detection primers are shown in Table 1, and the identified PCR reaction procedures are detailed in Table 6.

(4) After the PCR amplification is finished, performing gel electrophoresis detection, wherein the size of a PCR amplification fragment of a positive colony needs to consider the number of target points, and the size of the amplification fragment with 4 sgRNAs is about 1100 bp.

Carrying out plasmid extraction on the positive clone bacterial liquid with correct sequencing result, sending the positive clone bacterial liquid to a company for sequencing, and detecting the amplified fragment

The correct fragment sequence was as follows:

pMK1-PTG

pMK3-PTG

pMK5-PTG

pMK7-PTG

pMK9-PTG

pMK10-PTG

in the tandem fragment sequences shown in SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, underlined parts are target sequences, promoter sequences OsU6/U3/U6a/U6b are italicized, a gRNA framework sequence is thickened, and tRNA sequences are arranged between the gRNA framework sequence and the target sequences.

TABLE 1 OsLEA24 tRNA-gRNA unit amplification primers

Primer name	Oligonucleotide sequence (5 '-3')
		M13F	TGTAAAACGACGGCCAGT
LEA7R	GAAGTTAGCGAGCATGTCGT
		LEA6F	ATCGGCAGGAGCGGGACGAT
LEA17R	ATGGCGTCGAGGCAGGACAG
		LEA15NF	GCGACGCCATTGTGTCGAGC
LEA22R	AGACGTCGTAGTACGCGCTG
		LEA21F	AGCTAACTAGTGTTTGGCAA
UBI-N-R	ATCTCTAGAGAGGGGCACGA
		PTG pMK1/10/11F	GTACGGGTCTCATGTGGAACAAAGCACCAGTGGTCTA
PTG pMK2/3/8/9F	GTACGGGTCTCATGGCAACAAAGCACCAGTGGTCTA
		PTG PMK4/5F	GTACGGGTCTCATGCCGAACAAAGCACCAGTGGTCTA
PTG pMK6/7/12F	GTACGGGTCTCATGTTGAACAAAGCACCAGTGGTCTA
		PTG CRISPR R	GACTAGGTCTCCAAACAAAAAAAAAAGCACCGACTCG

TABLE 2 OsLEA24 genotype identification primers

TABLE 3 destination fragment names

	Part1	Part2	Part3	Part4	Part5
						pMK1-PTG	pMK1F-LEA1R	LEA1F-LEA2R	LEA2F-LEA4R	LEA4F-LEA5R	LEA5F-CRISPR R
pMK3-PTG	pMK3F-LEA6R	LEA6F-LEA7R	LEA7F-LEA9R	LEA9F-LEA10R	LEA10F-CRISPR R
						pMK5-PTG	pMK5F-LEA11R	LEA11F-LEA13R	LEA13F-LEA15R	LEA15F-LEA18R	LEA18F-CRISPR R
pMK7-PTG	pMK7F-LEA19R	LEA19F-LEA20R	LEA20F-LEA21R	LEA21F-LEA22R	LEA22F-CRISPR R
						pMK9-PTG	pMK3F-LEA23R	LEA23F-LEA24R	LEA24F-LEA27R	LEA27F-LEA28R	LEA28F-CRISPR R
pMK10-PTG	pMK1F-LEA30R	LEA30F-LEA31R	LEA31F-LEA33R	LEA33F-LEA34R	LEA34F-CRISPR R

TABLE 4 PCR reaction procedure

TABLE 5 pMK intermediate vector gold Gate Assembly reaction

TABLE 6 pMK-PTG Positive clone identification PCR reaction program

Example 2

This example will provide a method for constructing a multigene editing vector for connecting OsU6/3 expression cassettes on multiple pMK-PTG intermediate vectors to pMMK-Cas9, comprising the following steps:

pMK1-PTG, pMK3-PTG, pMK5-PTG, pMK7-PTG, pMK9-PTG and pMK10-PTG intermediate vectors obtained in example 1 were ligated in a gold-gated assembly method in this order, and OsU6/3 expression cassettes on the intermediate vectors were ligated into a pMMK-Cas9 gene editing vector by a restriction enzyme AarI type II (Thermofisher) and a DNA ligase T4 (NEB), and the ligation reaction system is described in Table 7.

10uL of the reaction mixture was taken to transform into E.coli competent Dh5a (Shanghai Weidi Biotech Co., Ltd.), and was screened using a kanamycin-resistant plate with a concentration of 100 ng/mL. After resistant colonies are picked, PCR is used for positive clone detection, the sequences of detection primers are shown in Table 1, and the identified PCR reaction procedures are detailed in Table 8.

After the PCR amplification is finished, carrying out gel electrophoresis detection, wherein the size of the PCR amplification fragment of the positive colony needs to consider the distance of the amplified fragment on the carrier, and the sizes of the amplification fragments of M13F-LEA7R, LEA6F-LEA17R, LEA15F-LEA22R and LEA21F-UBI-N-R are 1781bp, 1844bp, 2114bp and 2665bp respectively. The colony PCR and pMMK-PTG vector construction are schematically shown in FIG. 3 and FIG. 4.

pMMK-PTG

in the tandem fragment sequence shown in SEQ ID NO.21, the underlined parts are the target sequences, the promoter sequence is OsU6/U3/U6a/U6b in italics, the bold is the gRNA framework sequence, and a tRNA sequence is arranged between the gRNA framework sequence and the target sequence.

TABLE 7 pMMK-Cas9 editing vector gold gate assembly reaction

TABLE 8 pMMK-PTG Positive clone identification PCR reaction program

Example 3 transformation of Rice calli with pMMK-PTG Multi-Gene editing vector

The multigene editing vector pMMK-PTG obtained in example 2 was transformed into Agrobacterium (EHA105) by Agrobacterium-mediated transformation method, followed by infection of rice calli. The transformed variety is Xiushui 134. Detailed transformation procedures and various media formulation references used: komari T.efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the buildings of the T-DNA [ J ] Plant Journal for Cell & Molecular Biology,2010,6(2): 271-282).

Example 4 identification of transgenic plants

Extracting genome DNA of a regenerated plant of the transgenic rice by using a CTAB method, carrying out positive detection on the transgenic plant through a sgRNA sequence on an amplification carrier, wherein detection primers are M13F and LEA7R, the primer sequences refer to the table 1, and an inventor team detects 16 plants in a batch, wherein 13 plants are positive, and the positive rate is 81.25%.

For the transgenic rice plant which is detected to be positive by PCR, a detection primer which spans the front and back of the target site by 150 and 250bp is further designed aiming at the sequence near each target site. The specific nomenclature and sequence of the detection primers designed for 24 target genes in the present invention are shown in Table 9.

TABLE 9 pMMK-Cas9 genotype identifying primers

Example 5 analysis of the compiled results of Positive transgenic plants

The inventor group selected 11 strains (#1, #4, #5, #7, #8, #9, #12, #13, #14, #15, #16) from 13 positive transgenic seedlings for amplification and sequencing analysis of the target gene, and the editing efficiency and mutation types of these 11 strains are shown in FIG. 5A.

Of the 25 identified target sites, 19 of the remaining 24 target sites had a mutation rate of 100%, 1 had a mutation rate of 91%, 2 had a mutation rate of 81.8%, 1 had a mutation rate of 18.18% and one site was 0, except that LEA17 was the reference genome and the skilful 134 was different. The editing efficiency of the system as a whole reaches 100% at most sites, and 2 sites with low efficiency are probably related to the situation of the genome.

Further analysis as shown in FIG. 5B, found that most of the mutations at each site were consistent, e.g., the main mutation type of the mutation of LEA4 was-1 homozygous mutation, and the main mutation type of LEA 5+ A homozygous mutation. Most of the site mutation types are homozygous or biallelic, and only the main mutation types of the 5 sites LEA2, LEA7, LEA11, LEA30 and LEA33 are heterozygous mutations.

The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Shanghai university of Master

<120> construction method and application of CRISPR/Cas9 mediated plant polygene editing vector

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 349

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggatcatgaa ccaacggcct ggctgtattt ggtggttgtg tagggagatg gggagaagaa 60

aagcccgatt ctcttcgctg tgatgggctg gatgcatgcg ggggagcggg aggcccaagt 120

acgtgcacgg tgagcggccc acagggcgag tgtgagcgcg agaggcggga ggaacagttt 180

agtaccacat tgcccagcta actcgaacgc gaccaactta taaacccgcg cgctgtcgct 240

tgtgtggaga ccatctagac ggtctctgtt ttagagctag aaatagcaag ttaaaataag 300

gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttt 349

<210> 2

<211> 484

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caggaatctt taaacatacg aacagatcac ttaaagttct tctgaagcaa cttaaagtta 60

tcaggcatgc atggatcttg gaggaatcag atgtgcagtc agggaccata gcacaagaca 120

ggcgtcttct actggtgcta ccagcaaatg ctggaagccg ggaacactgg gtacgttgga 180

aaccacgtga tgtgaagaag taagataaac tgtaggagaa aagcatttcg tagtgggcca 240

tgaagccttt caggacatgt attgcagtat gggccggccc attacgcaat tggacgacaa 300

caaagactag tattagtacc acctcggcta tccacataga tcaaagctga tttaaaagag 360

ttgtgcagat gatccgtggc agagaccatc tagacggtct ctgttttaga gctagaaata 420

gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 480

tttt 484

<210> 3

<211> 546

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttttttcctg tagttttccc acaaccattt tttaccatcc gaatgatagg ataggaaaaa 60

tatccaagtg aacagtattc ctataaaatt cccgtaaaaa gcctgcaatc cgaatgagcc 120

ctgaagtctg aactagccgg tcaactatac aggctatcga gatgccatac acgagacggt 180

agtaggaact aggaagacga tggttgattc gtcaggcgaa atcgtcgtcc tgcagtcgca 240

tctatgggcc tggacggaat aggggaaaaa attggccgga taggagggaa aggcccaggt 300

gcttacgtgc gaggtaggcc tgggctctca gcgcttcgat tcgttggcac cggggtagga 360

tgcaatagag agcaacgttt agtaccacct cgcttagcta aactggactg ccttatatgc 420

gcgggtgctg gcttggctgc cgggagacca tctagacggt ctctgtttta gagctagaaa 480

tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 540

tttttt 546

<210> 4

<211> 439

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgcaagaacg aactaagccg gacaaaaaaa aaaaaggagc acatatacaa accggtttta 60

ttcatgaatg gtcacgatgg atgatggggc tcagacttga gctacgaggc cgcaggcgag 120

agaagcctag tgtgctctct gcttgtttgg gccgtaacgg aggatacggc cgacgagcgt 180

gtactaccgc gcgggatgcc gctgggcgct gcgggggccg ttggatgggg atcggtgggt 240

cgcgggagcg ttgaggggag acaggtttag taccacctcg cctaccgaac aatgaagaac 300

ccaccttata accccgcgcg ctgccgcttg tgttgggaga ccatctagac ggtctctgtt 360

ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc 420

accgagtcgg tgctttttt 439

<210> 5

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

taggtctccn nnnnnnnnnn ngttttagag ctagaa 36

<210> 6

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgggtctcan nnnnnnnnnn ntgcaccagc cggg 34

<210> 7

<211> 195

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

taggtctccn nnnnnnnnnn ngttttagag ctagaaatag caagttaaaa taaggctagt 60

ccgttatcaa cttgaaaaag tggcaccgag tcggtgcaac aaagcaccag tggtctagtg 120

gtagaatagt accctgccac ggtacagacc cgggttcgat tcccggctgg tgcannnnnn 180

nnnnnntgag acccg 195

<210> 8

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtacgggtct catgtggaac aaagcaccag tggtcta 37

<210> 9

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gtacgggtct catggcaaca aagcaccagt ggtcta 36

<210> 10

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gtacgggtct catgccgaac aaagcaccag tggtcta 37

<210> 11

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gtacgggtct catgttgaac aaagcaccag tggtcta 37

<210> 12

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gactaggtct ccaaacaaaa aaaaaagcac cgactcg 37

<210> 13

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgtaaaacga cggccagt 18

<210> 14

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

caggaaacag ctatgacc 18

<210> 15

<211> 1108

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggatcatgaa ccaacggcct ggctgtattt ggtggttgtg tagggagatg gggagaagaa 60

aagcccgatt ctcttcgctg tgatgggctg gatgcatgcg ggggagcggg aggcccaagt 120

acgtgcacgg tgagcggccc acagggcgag tgtgagcgcg agaggcggga ggaacagttt 180

agtaccacat tgcccagcta actcgaacgc gaccaactta taaacccgcg cgctgtcgct 240

tgtgtgaaca aagcaccagt ggtctagtgg tagaatagta ccctgccacg gtacagaccc 300

gggttcgatt cccggctggt gcaaaggtgc aggacatggt gaggttttag agctagaaat 360

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca 420

acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga cccgggttcg 480

attcccggct ggtgcagggg aaggtgagca aggccagttt tagagctaga aatagcaagt 540

taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcaacaaagc 600

accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt tcgattcccg 660

gctggtgcac gcgatgcagt cgaccaaggg ttttagagct agaaatagca agttaaaata 720

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcaacaa agcaccagtg 780

gtctagtggt agaatagtac cctgccacgg tacagacccg ggttcgattc ccggctggtg 840

cagcaagaaa caatggcgca gcgttttaga gctagaaata gcaagttaaa ataaggctag 900

tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttttttgt tttagagcta 960

gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg 1020

gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgtagcgcgt 1080

gcgccaattc tgcagacaaa tggccaat 1108

<210> 16

<211> 1242

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

caggaatctt taaacatacg aacagatcac ttaaagttct tctgaagcaa cttaaagtta 60

tcaggcatgc atggatcttg gaggaatcag atgtgcagtc agggaccata gcacaagaca 120

ggcgtcttct actggtgcta ccagcaaatg ctggaagccg ggaacactgg gtacgttgga 180

aaccacgtga tgtgaagaag taagataaac tgtaggagaa aagcatttcg tagtgggcca 240

tgaagccttt caggacatgt attgcagtat gggccggccc attacgcaat tggacgacaa 300

caaagactag tattagtacc acctcggcta tccacataga tcaaagctga tttaaaagag 360

ttgtgcagat gatccgtggc acaaagcacc agtggtctag tggtagaata gtaccctgcc 420

acggtacaga cccgggttcg attcccggct ggtgcaatcg gcaggagcgg gacgatgttt 480

tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca 540

ccgagtcggt gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac 600

agacccgggt tcgattcccg gctggtgcaa cgacatgctc gctaacttcg ttttagagct 660

agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc 720

ggtgcaacaa agcaccagtg gtctagtggt agaatagtac cctgccacgg tacagacccg 780

ggttcgattc ccggctggtg cattgcagga ggggttactc gggttttaga gctagaaata 840

gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgcaa 900

caaagcacca gtggtctagt ggtagaatag taccctgcca cggtacagac ccgggttcga 960

ttcccggctg gtgcagagcg agagcagttg tccgggtttt agagctagaa atagcaagtt 1020

aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttttt 1080

tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag 1140

tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta 1200

gtccgtagcg cgtgcgccaa ttctgcagac aaatggcctg ag 1242

<210> 17

<211> 1305

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ttttttcctg tagttttccc acaaccattt tttaccatcc gaatgatagg ataggaaaaa 60

tatccaagtg aacagtattc ctataaaatt cccgtaaaaa gcctgcaatc cgaatgagcc 120

ctgaagtctg aactagccgg tcaactatac aggctatcga gatgccatac acgagacggt 180

agtaggaact aggaagacga tggttgattc gtcaggcgaa atcgtcgtcc tgcagtcgca 240

tctatgggcc tggacggaat aggggaaaaa attggccgga taggagggaa aggcccaggt 300

gcttacgtgc gaggtaggcc tgggctctca gcgcttcgat tcgttggcac cggggtagga 360

tgcaatagag agcaacgttt agtaccacct cgcttagcta aactggactg ccttatatgc 420

gcgggtgctg gcttggctgc cgaacaaagc accagtggtc tagtggtaga atagtaccct 480

gccacggtac agacccgggt tcgattcccg gctggtgcac aaaggggaga gagaaccgtg 540

ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg 600

gcaccgagtc ggtgcaacaa agcaccagtg gtctagtggt agaatagtac cctgccacgg 660

tacagacccg ggttcgattc ccggctggtg caagcgagag ccctcctctc gtgttttaga 720

gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga 780

gtcggtgcaa caaagcacca gtggtctagt ggtagaatag taccctgcca cggtacagac 840

ccgggttcga ttcccggctg gtgcagcgac gccattgtgt cgagcgtttt agagctagaa 900

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 960

caacaaagca ccagtggtct agtggtagaa tagtaccctg ccacggtaca gacccgggtt 1020

cgattcccgg ctggtgcact gtcctgcctc gacgccatgt tttagagcta gaaatagcaa 1080

gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 1140

ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 1200

aagtggcacc gagtcggtgc ttttttgttt tagagctaga aatagcaagt taaaataagg 1260

ctagtccgta gcgcgtgcgc caattctgca gacaaatggc ctctc 1305

<210> 18

<211> 1195

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tgcaagaacg aactaagccg gacaaaaaaa aaaaggagca catatacaaa ccggttttat 60

tcatgaatgg tcacgatgga tgatggggct cagacttgag ctacgaggcc gcaggcgaga 120

gaagcctagt gtgctctctg cttgtttggg ccgtaacgga ggatacggcc gacgagcgtg 180

tactaccgcg cgggatgccg ctgggcgctg cgggggccgt tggatgggga tcggtgggtc 240

gcgggagcgt tgaggggaga caggtttagt accacctcgc ctaccgaaca atgaagaacc 300

caccttataa ccccgcgcgc tgccgcttgt gttgaacaaa gcaccagtgg tctagtggta 360

gaatagtacc ctgccacggt acagacccgg gttcgattcc cggctggtgc ataacaccac 420

caccgccgtg agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa 480

cttgaaaaag tggcaccgag tcggtgcaca aagcaccagt ggtctagtgg tagaatagta 540

ccctgccacg gtacagaccc gggttcgatt cccggctggt gcagatgggg gcgagcaagg 600

acagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 660

agtggcaccg agtcggtgca acaaagcacc agtggtctag tggtagaata gtaccctgcc 720

acggtacaga cccgggttcg attcccggct ggtgcaagca cgtacgtacg catcgagttt 780

tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca 840

ccgagtcggt gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac 900

agacccgggt tcgattcccg gctggtgcaa gctaactagt gtttggcaag ttttagagct 960

agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc 1020

ggtgcttttt tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 1080

tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag 1140

ttaaaataag gctagtccgt agcgcgtgcg ccaattctgc agacaaatgg cccac 1195

<210> 19

<211> 1242

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

caggaatctt taaacatacg aacagatcac ttaaagttct tctgaagcaa cttaaagtta 60

tcaggcatgc atggatcttg gaggaatcag atgtgcagtc agggaccata gcacaagaca 120

ggcgtcttct actggtgcta ccagcaaatg ctggaagccg ggaacactgg gtacgttgga 180

aaccacgtga tgtgaagaag taagataaac tgtaggagaa aagcatttcg tagtgggcca 240

tgaagccttt caggacatgt attgcagtat gggccggccc attacgcaat tggacgacaa 300

caaagactag tattagtacc acctcggcta tccacataga tcaaagctga tttaaaagag 360

ttgtgcagat gatccgtggc acaaagcacc agtggtctag tggtagaata gtaccctgcc 420

acggtacaga cccgggttcg attcccggct ggtgcacagc gcgtactacg acgtctgttt 480

tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca 540

ccgagtcggt gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac 600

agacccgggt tcgattcccg gctggtgcag aggaacacgg agagccaccg ttttagagct 660

agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc 720

ggtgcaacaa agcaccagtg gtctagtggt agaatagtac cctgccacgg tacagacccg 780

ggttcgattc ccggctggtg catgtgctcg cctccgccca tggttttaga gctagaaata 840

gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgcaa 900

caaagcacca gtggtctagt ggtagaatag taccctgcca cggtacagac ccgggttcga 960

ttcccggctg gtgcatacca ggggcagcac ggctagtttt agagctagaa atagcaagtt 1020

aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttttt 1080

tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag 1140

tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta 1200

gtccgtagcg cgtgcgccaa ttctgcagac aaatggcctg tc 1242

<210> 20

<211> 1108

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ggatcatgaa ccaacggcct ggctgtattt ggtggttgtg tagggagatg gggagaagaa 60

aagcccgatt ctcttcgctg tgatgggctg gatgcatgcg ggggagcggg aggcccaagt 120

acgtgcacgg tgagcggccc acagggcgag tgtgagcgcg agaggcggga ggaacagttt 180

agtaccacat tgcccagcta actcgaacgc gaccaactta taaacccgcg cgctgtcgct 240

tgtgtgaaca aagcaccagt ggtctagtgg tagaatagta ccctgccacg gtacagaccc 300

gggttcgatt cccggctggt gcagtgtact aggacgatga gccgttttag agctagaaat 360

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca 420

acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga cccgggttcg 480

attcccggct ggtgcaaaca cgtcgccgta catcacgttt tagagctaga aatagcaagt 540

taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcaacaaagc 600

accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt tcgattcccg 660

gctggtgcaa gctggtcctc cattctctgg ttttagagct agaaatagca agttaaaata 720

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcaacaa agcaccagtg 780

gtctagtggt agaatagtac cctgccacgg tacagacccg ggttcgattc ccggctggtg 840

cagatgttga cgccatgctc aggttttaga gctagaaata gcaagttaaa ataaggctag 900

tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttttttgt tttagagcta 960

gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg 1020

gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgtagcgcgt 1080

gcgccaattc tgcagacaaa tggccccc 1108

<210> 21

<211> 7200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21