Rice genetic transformation vector and application thereof in fixed-point insertion of DNA (Deoxyribose Nucleic Acid) fragment

Abstract

The invention discloses a rice genetic transformation vector and application thereof in fixed-point insertion of DNA fragments, and belongs to the technical field of plant genetic engineering. Comprising an rPE14 vector with a nucleotide sequence as shown in SEQ ID NO.1 and a pENTR4: gRNA41 vector connected with a pegRNA sequence. On the premise of no donor template, the DNA sequence of the fusion tag can be subjected to fixed-point insertion in front of the OsMAPK13 gene terminator of the rice, so that efficient in-situ labeling (three policy agrobacterium tumefaciens-mediated genetic transformation editing efficiencies are 66.67%, 30.68% and 29.55% respectively) is realized, the in-situ labeling process of the endogenous gene of the rice is greatly accelerated, and the in-situ labeling method has the advantages that the in-situ labeling efficiency of the endogenous gene of the rice is greatly improved. And research related to gene functions and proteins is further promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and in particular relates to a rice genetic transformation vector and its application in site-specific insertion of DNA fragments. Background Art

[0002] In recent years, with the rapid development of genome sequencing technology, the research on genes and their transcripts has been greatly promoted. However, the characterization of protein dynamics, including protein levels, subcellular localization, post-translational modifications, and interactions with macromolecules (such as proteins, DNA, RNA, carbohydrates and lipids) and small molecules (such as metabolites and ligands) is more challenging. Immunoassay technology is widely used in the research process of the above aspects. Immunoassay technology first requires the target protein to be used as an antigen and injected into the animal, and then the antibody is separated from the immune serum, and specific detection is performed based on the immune reaction between the antigen and the antibody. The biggest drawback of this method is that a corresponding antibody must be prepared for each replacement of a target protein, which is cumbersome and expensive. Therefore, it is very difficult to systematically produce reliable antibodies to specifically identify a single plant protein, but with the use of fusion tags, protein immunoassays have become universal and convenient. Currently, the commonly used methods for protein fusion tags include transposon method, biochemical synthesis method and transgenic method, among which the transposon method and biochemical synthesis method have the advantages of rapid realization, but may affect the protein structure and function. The transgenic method is to fuse a tag to the gene open reading frame (ORF) and transform it into the corresponding functional mutant plant. However, due to the random insertion of transgenes, such exogenous tagged proteins may not fully reproduce the properties of endogenous proteins even if the transgene is expressed under the promoter of the endogenous gene.

[0003] A preferred solution is to insert a DNA sequence encoding a fluorescent protein tag, FLAG or HA affinity tag into the gene of interest in vivo. With the rapid development of CRISPR technology, it has been widely used in many aspects such as animals, plants, human cells and microorganisms, making it possible. However, the fragment insertion technology based on the CRISPR system is restricted by factors such as editing efficiency and exogenous donor template delivery, which limits its large-scale application. Therefore, it is of great significance to develop an efficient DNA fragment site-specific insertion technology that does not require an exogenous donor template. Summary of the invention

[0004] The purpose of the present invention is to provide a rice genetic transformation vector and its application in site-specific insertion of DNA fragments to solve the problems existing in the above-mentioned prior art, accelerate the process of in situ labeling of rice endogenous genes, and further promote research related to gene function and protein.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a genetic transformation vector, which comprises an rPE14 vector having a nucleotide sequence as shown in SEQ ID NO.1.

[0007] Furthermore, the genetic transformation vector also includes a pENTR4:gRNA41 vector connected to a pegRNA sequence, wherein the nucleotide sequence of the pENTR4:gRNA41 vector is shown in SEQ ID NO.13.

[0008] Further, the pegRNA sequence includes any one of the following ac:

[0009] a. The sequence shown in SEQ ID NO.16;

[0010] b. The sequence shown in SEQ ID NO.17;

[0011] c. The sequence shown in SEQ ID NO.18.

[0012] The present invention also provides an artificial gene editing system for site-specific insertion of plant DNA fragments, and is further applied to in situ labeling of plant endogenous genes, including the genetic transformation vector.

[0013] Furthermore, the plant is rice.

[0014] The present invention also provides a method for achieving site-specific insertion of plant DNA fragments, comprising the steps of transforming the gene editing artificial system through Agrobacterium infection, and obtaining a series of regenerated plants through tissue culture.

[0015] The present invention also provides an application of the genetic transformation vector or the artificial gene editing system in site-specific insertion of plant DNA fragments.

[0016] The present invention discloses the following technical effects:

[0017] The gene editing artificial system of the present invention can realize the site-specific insertion of the fusion tag DNA sequence into the OsMAPK13 gene of rice in the absence of a donor template, thereby realizing in situ gene labeling (the genetic transformation editing efficiencies of the three strategies mediated by Agrobacterium are 66.67%, 30.68%, and 29.55%, respectively). This invention will greatly accelerate the process of in situ labeling of endogenous genes in rice and further promote research related to gene function and protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 There are two OsMAPK13 gene target sites;

[0020] Figure 2 The site-specific insertion repair modes of the DNA fragments corresponding to the three pegRNAs;

[0021] Figure 3 It is a comparison of the three pegRNA repair situations, where A is the in situ labeling of some mutants using the three strategies, and B is a bar graph of the editing efficiency of Agrobacterium-mediated genetic transformation using the three strategies. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Example 1, vector construction:

[0028] First, the above modules were assembled in the order of BamH I-kozak-bpNLS-SpCas9-linker-bpNLS-linker-M-MLV(ΔRNaseH)-bpNLS-c-myc NLS-Bcu I, and the nucleotide sequence encoding the corresponding protein was codon-optimized according to the preference of rice codons. The obtained nucleotide sequence was commissioned to Beijing Qingke Biotechnology Co., Ltd. for artificial synthesis. Afterwards, the Cas9 in pUbi-Cas9 (Large chromosomal deletions and heritable small genetic changes induced by CRISPR / Cas9 in rice. [J]. Nucleic Acids Research, 2014.) was replaced with the above synthetic sequence by BamH I and Bcu I restriction enzyme ligation, and the obtained vector was named rPE14 (SEQ ID NO.1).

[0029] GTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAA

[0030] TCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGCTAGAGCAGCTTGCCAACATGGTGG

[0031] AGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTA

[0032] TTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTG

[0033] TCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAA

[0034] AGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCA

[0035] CGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGT

[0036] GATAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAA

[0037] GACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCAT

[0038] TGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATG

[0039] CCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGA

[0040] TGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGC

[0041] AAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCA

[0042] AGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTC

[0043] TCTCTACAAATCTATCTCTCTCGAGCTTTCGCAGATCCCGGGGGGCAATGAGATATGAAAAAGC

[0044] CTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGAC

[0045] CTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGA

[0046] TATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTT

[0047] TGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTTAGCGAGAGCCTGAC

[0048] CTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCC

[0049] CGCTGTTCTACAACCGGTCGCGGAGGCTATGGATGCGATCGCTGCGGCCGATCTTAGCCAGAC

[0050] GAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCAT

[0051] ATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGC

[0052] GTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGC

[0053] ACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGG

[0054] TCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCT

[0055] GGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAG

[0056] CTTGCAGGATCGCCACGACTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGA

[0057] GCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCC

[0058] GATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACC

[0059] GATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGC

[0060] AAAGAAATAGAGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGTTTCTCCATAATAA

[0061] TGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCA

[0062] TATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCT

[0063] AAAACCAAAATCCAGTACTAAAATCCAGATCCCCCGAATTAATTCGGCGTTAATTCAGTACATT

[0064] AAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACACCACAATATATCCT

[0065] GCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAG

[0066] GCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCGTTGTAAGGCGGCAGACTTTGCTC

[0067] ATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGATGATCT

[0068] CGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCTGTGATCACCGCGGTT

[0069] TCAAAATCGGCTCCGTCGATACTATGTTTATACGCCAACTTTGAAAAACACTTTGAAAAAGCTGT

[0070] TTTCTGGTATTTAAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTTATAATTA

[0071] GCTTCTTGGGGTATCTTTAAAATACTGTAGAAAGAGGAAGGAAATAATAATGGCTAAAAATGA

[0072] GAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGA

[0073] ATGTCTCCTGCTAAGGTATAATAAGCTGGTGGGGAAAATGAAAACCTATATTTAAAAAATGACGG

[0074] ACAGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGG

[0075] AAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTG

[0076] CTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGAATGAAACAAAGCCCTGA

[0077] AAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTCCATCGACATATCGGATTGT

[0078] CCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGG

[0079] CCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATG

[0080] ATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTCCCACGGCGACCTGGGAGAC

[0081] AGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGG

[0082] GCGGACAAGTGGTATGACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAA

[0083] CAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATATT

[0084] ATATTTTACTGGATGAATTGTTTTAGTACCTAGAATGCATGACCAAAATCCCTTAACGTGAGTTT

[0085] TCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTC

[0086] TGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGG

[0087] ATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATA

[0088] CTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATA

[0089] CCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGG

[0090] TTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG

[0091] CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTAT

[0092] GAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGT

[0093] CGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG

[0094] TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC

[0095] TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA

[0096] CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTG

[0097] ATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGA

[0098] GCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTC

[0099] TCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGAC

[0100] TGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCT

[0101] GCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTT

[0102] TTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGT

[0103] GGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAG

[0104] CGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAA

[0105] GGGTAGGCGCTTTTTGCAGCTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGC

[0106] GGGTTTTAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTT

[0107] ATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGGT

[0108] TCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAAACAGACCTTT

[0109] TCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGTACATTAGGAACCGGCG

[0110] GATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGCCAGCCTGCCCCGCC

[0111] TCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTGAAACAG

[0112] AACTTCTTGAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTT

[0113] CTGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATC

[0114] AAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGCGGTAC

[0115] ATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCTCTTTACGATCTTGTA

[0116] GCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGCCGTTCTTGGCCTTCTTCGT

[0117] ACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGGTTTCTACCAGGTCGTCTTTCTG

[0118] CTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGC

[0119] GGCCGGGCTTGTCTCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGC

[0120] CATCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCTAC

[0121] GTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGACAGAC

[0122] GGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCACGTCATAGAGCATCGGAA

[0123] CGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTCCTAATCGACGGCGCACCGGCTGCCG

[0124] GCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCACGATTCACCGGGGCGTG

[0125] CTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCAT

[0126] CACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCG

[0127] GGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGGCCA

[0128] ACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGTTCTTGATTTTCC

[0129] ATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTCATTTGCTCATTTACTCTGGTAG

[0130] CTGCGCGATGTATTCAGATAGCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTC

[0131] AGCTTGGTGTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCC

[0132] AGGCTGGCCAACGTTGCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTT

[0133] GTGCTTTTGCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCC

[0134] AGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGC

[0135] GGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCAAGAATGGGCAGCTCGT

[0136] ACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCGTGCCTTTGATCGCCCGCGACACG

[0137] ACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGG

[0138] TCGGCGGTGGCCCATATGTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCC

[0139] TTGATCGCGGACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCG

[0140] CCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCG

[0141] GTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACA

[0142] GAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCT

[0143] GACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTATTT

[0144] ACTCATCGCATCATATACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACC

[0145] TTTTTAGACGGCGGCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTG

[0146] GCATCAGACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTC

[0147] GAACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTC

[0148] CTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGG

[0149] CGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTC

[0150] ACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGC

[0151] CGCCTCTTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGG

[0152] GGTGAGGGTAGGGCGGGGGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCC

[0153] GGGTGCGGTCGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACC

[0154] ATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCC

[0155] GGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGCCAGGCGGTCTA

[0156] GCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGG

[0157] CGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAG

[0158] TTCGGCCCGTTGGTTGGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCC

[0159] AGCGGCGGCGCTCTTGTTCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATG

[0160] CGACTAAAACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAA

[0161] CTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAAGCCGACT

[0162] GCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGGGGAACCCTGTGGTTGG

[0163] CATGCACATACAAATGGACGAACGGATAAACCTTTTCACGCCCTTTTAAATATCCGTTATTCTAA

[0164] TAAACGCTCTTTTCTCTTAGGTTTACCCGCCAATATATCCTGTCAAACACTGATAGTTTAAACTG

[0165] AAGGCGGGAAACGACAATCTGATCCAAGCTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCG

[0166] CAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGG

[0167] GATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAA

[0168] CGACGGCCAGTGCCAAGCTAGAGACGGGGATCACAAGTTTGTACAAAAAAGCTGAACGAGA

[0169] AACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATA

[0170] CTGTAAAACACAACATATCCAGTCATATTGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTT

[0171] ATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCGATCCGGCTTACTAAAAGCCAG

[0172] ATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCG

[0173] AAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGC

[0174] TATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAA

[0175] TGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAG

[0176] GTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGGCTGGTGAAATGCA

[0177] GTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGAT

[0178] ATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGAT

[0179] AAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACC

[0180] ACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGC

[0181] GAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTA

[0182] TACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGCATTATGTAG

[0183] TCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGC

[0184] TTTCTTGTACAAAGTGGTGAAGCTTGCATGCCTGCAGGCAGCGTGACCCGGTCGTGCCCCTCT

[0185] CTAGAGATAATGAGCATTGCATGTCTAAGTTATAAAAAATTACCACATATTTTTTTTGTCACACT

[0186] TGTTTGAAGTGCAGTTTATCTATCTTTATACATATATTTAAACTTTACTCTACGAATAATATAATCT

[0187] ATAGTACTACAATAATATCAGTGTTTTAGAGAATCATATAAATGAACAGTTAGACATGGTCTAAA

[0188] GGACAATTGAGTATTTTGACAACAGGACTCTACAGTTTTATCTTTTTAGTGTGCATGTGTTCTCC

[0189] TTTTTTTTTGCAAATAGCTTCACCTATATAATACTTCATCCATTTTATTAGTACATCCATTTAGGGT

[0190] TTAGGGTTAATGGTTTTTATAGACTAATTTTTTTAGTACATCTATTTTATTCTATTTTAGCCTCTAA

[0191] ATTAAGAAAACTAAAACTCTATTTTAGTTTTTTTATTTAATAATTTAGATATAAAATAGAATAAAA

[0192] TAAAGTGACTAAAAATTAAACAAATACCCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTT

[0193] CTTGTTTCGAGTAGATAATGCCAGCCTGTTAAACGCCGTCGACGAGTCTAACGGACACCAACC

[0194] AGCGAACCAGCAGCGTCGCGTCGGGCCAAGCGAAGCAGACGGCACGGCATCTCTGTCGCTGC

[0195] CTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGTCGGCATCCAGAA

[0196] ATTGCGTGGCGGAGCGGCAGACGTGAGCCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGG

[0197] CACGGCAGCTACGGGGGATTCCTTTCCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCCGTAA

[0198] TAAATAGACACCCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACACAC

[0199] ACAACCAGATCTCCCCCAAATCCACCCGTCGGCACCTCCGCTTCAAGGTACGCCGCTCGTCCT

[0200] CCCCCCCCCCCCCTCTCTACCTTCTCTAGATCGGCGTTCCGGTCCATGGTTAGGGCCCGGTAGT

[0201] TCTACTTCTGTTCATGTTTGTGTTAGATCCGTGTTTGTGTTAGATCCGTGCTGCTAGCGTTCGTA

[0202] CACGGATGCGACCTGTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGTTTCTCTTTGGGG

[0203] AATCCTGGGATGGCTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTTGTTTCGTTG

[0204] CATAGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTCGGGTCATC

[0205] TTTTCATGCTTTTTTTTTGTCTTGGTTGTGATGATGTGGTGTGGTTGGGCGGTCGTTCATTCGTT

[0206] CTAGATCGGAGTAGAATACTGTTTCAAACTACCTGGTGTATTTATTAATTTTGGAACTGTATGTG

[0207] TGTGTCATACATCTTCATAGTTACGAGTTTAAGATGGATGGAAATATCGATCTAGGATAGGTATA

[0208] CATGTTGATGTGGGTTTTACTGATGCATATACATGATGGCATATGCAGCATCTATTCATATGCTCT

[0209] AACCTTGAGTACCTATCTATTATAATAAACAAGTATGTTTTATAATTATTTTGATCTTGATATACTT

[0210] GGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCATACGCTATTTATTT

[0211] GCTTGGTACTGTTTCTTTTGTCGATGCTCACCCTGTTGTTTGGTGTTACTTCTGCAGGTCGACTC

[0212] TAGAGGATCTGGAATTCCCGGGTACCggatccgccaccATGGCCAAAAGGACGGCGGACGGGAGCG

[0213] AATTTGAGTCACCTAAGAAAAAGCGGAAGGTGGACAAAAAATATAGCATTGGTCTTGACATTG

[0214] GCACTAACTCTGTGGGGTGGGCTGTTATTACCGACGAGTACAAAGTTCCTTCGAAGAAATTCA

[0215] AGGTCTTGGGCAACACGGATAGGCACTCAATTAAGAAAAACTTGATTGGCGCTCTTCTTTTTG

[0216] ACTCGGGCGAGACAGCGGAGGCGACTCGCCTGAAGAGGACTGCTAGGAGGAGATATACCAG

[0217] GCGGAAGAATAGAATTTGTTATCTGCAGGAGATTTTCTCTAACGAAATGGCAAAAGTCGATGAT

[0218] AGCTTCTTCCATCGCCTGGAGGAGTCGTTTTTGGTCGAGGAAGATAAAAAGCATGAGAGGCA

[0219] CCCTATATTTGGCAACATTGTTGATGAGGTGGCGTACCATGAGAAATATCCGACAATTTACCAC

[0220] CTTAGGAAAAAACTCGTTGACAGTACTGATAAAGCGGATCTTCGGCTGATATACTTGGCTCTGG

[0221] CGCATATGATCAAATTCCGCGGCCACTTTTTGATCGAAGGTGACTTGAACCCGGATAATTCGGA

[0222] TGTGGACAAATTGTTTATCCAGCTGGTGCAAACTTACAACCAACTCTTTGAGGAGAACCCCAT

[0223] AAACGCCTCTGGCGTTGACGCGAAGGCCATACTTTCAGCTAGGCTCTCCAAATCACGCAAATT

[0224] GGAGAACCTCATAGCGCAATTGCCTGGTGAAAAAAAAAATGGTCTGTTTGGTAATTTGATTGC

[0225] ACTCAGTCTCGGGCTTACACCCAATTTCAAATCCAATTTTGACTTGGCCGAGGACGCTAAACT

[0226] TCAGCTGTCGAAAGATACGTATGATGACGATCTCGACAATCTCCTGGCCCAGATAGGCGACCA

[0227] GTATGCTGATTTGTTCTTGGCTGCGAAGAACCTGAGTGACGCAATTCTGCTTTCCGATATCCTC

[0228] CGCGTGAATACGGAAATAACAAAGGCCCCCCTGTCAGCTTCAATGATAAAGAGGTATGACGAG

[0229] CATCATCAAGACTTGACACTTCTTAAAGCTCTCGTTAGACAACAATTGCCGGAGAAATACAAG

[0230] GAGATTTTTTTCGACCAGTCCAAGAATGGATACGCAGGTTACATTGACGGCGGTGCGAGTCAG

[0231] GAGGAGTTCTATAAATTCATAAAGCCCATCTTGGAGAAAATGGATGGTACTGAGGAGCTTCTG

[0232] GTGAAACTCAAGCGCGAGGACCTTCTCAGGAAACAGAGAACTTTCGATAATGGCTCCATCCC

[0233] ACATCAGATCCATCTCGGAGAACTGCACGCTATCCTCCGCAGGCAGGAGGACTTTTATCCCTTC

[0234] CTTAAAGACAATAGAGAAAAAATCGAAAAAATCCTTACTTTCCGGATACCGTATTATGTCGGGC

[0235] CCTTGGCGCGCGGTAACAGCAGGTTCGCCTGGATGACCCGCAAAAGTGAGGAGACTATAACC

[0236] CCGTGGAACTTTGAGGAGGTTGTCGACAAAGGCGCGTCCGCACAATCCTTTATAGAGAGGATG

[0237] ACGAATTTTGACAAAAACTTGCCCAACGAGAAAGTCCTCCCTAAACACTCATTGCTGTATGAA

[0238] TACTTCACAGTTTATAACGAATTGACAAAAGTTAAATACGTTACAGAAGGCATGAGAAAACCT

[0239] GCCTTCCTGTCAGGCGAGCAGAAAAAGGCCATCGTTGATCTGCTGTTCAAAACTAACCGCAA

[0240] AGTCACTGTGAAGCAATTGAAAGAAGACTATTTCAAAAAGATAGAATGCTTCGATAGTGTTGA

[0241] AATCTCAGGAGTTGAGGACAGATTCAATGCGAGTTTGGGAACGTATCACGATCTGCTGAAGAT

[0242] TATAAAGGATAAGGATTTCCTTGACAACGAAGAGAACGAAGACATCCTGGAGGACATTGTTCT

[0243] GACTCTGACATTGTTTGAGGACAGAGAAATGATTGAGGAAAGGCTCCAAGACGTATGCACACC

[0244] TCTTCGACGATAAAGTTATGAAGCAACTGAAGCGGCGGAGATATACCGGCTGGGGCCGCCTGT

[0245] CTAGAAAGCTGATCAACGGTATCAGGGATAAGCAATCGGGGAAAAACTATTCTGGATTTCCTCA

[0246] AATCCGACGGTTTTGCTAATCGCATTTCATGCAACTTATTCACGATGACTCTCTTACATTCAAA

[0247] GAAGATATCCAAAAGCCAGGTTTCCGGGCAGGGGATTCGTTGCATGAGCATATTGCCAAT

[0248] CTCGCAGGTAGCCCCGCGATCAAGAAAGGCATATTGCAGACCGTTAAAGTTGTGGACGAACTC

[0249] GTGAAGGTTATGGGCAGGCACAAACCGGAGAATATAGTGATAGAAAATGGCTAGAGAAACCA

[0250] GACCACACAGAAGGGGCAAAAGAATTCTAGGGAGCGCATGAAACGGATCGAGGAAGGTATC

[0251] AAGGAACTCGGTTCCCAGATTCTTAAAGAGCACCCTGTTGAAAACACTCAGCTTCAAAATGA

[0252] AAAGCTTTATTTGTATTATTTGCAGAACGGGCGCGATATGTACGTCGACCAGGAGCTCGACATT

[0253] AACCGCCTCTCTGACTATGATGTTGATcacATCGTGCCGCAGAGCTTTTTGAAGGATGATAGCAT

[0254] TGACAACAAAGTTCTGACGCGGAGTGACAAAAATAGGGGCAAGTCTGATAATGTTCCCTCGG

[0255] AAGAAGTGGTCAAAAAGATGAAAAATTACTGGAGACAGCTTCTCAACGCTAAGCTGATCACC

[0256] CAAAGGAAGTTTGATAATTTGACCAAGGCGGAACGGGGAGGTCTCTCCGAGCTCGACAAAGC

[0257] AGGGTTTATCAAACGCCAGCTGGTCGAGACGAGGCAAATAACTAAGCATGTGGCACAGATCCT

[0258] GGACAGTCGGATGAATACGAAATATGACGAGAACGACAAGCTGATTAGGGAAGTTAAAGTCAT

[0259] AACCTTGAAATCGAAACTCGTTTCTGACTTCCGCAAGGATTTTCAATTCTACAAGGTTCGGGA

[0260] GATAAACAACTACCATCATGCACACGACGCCTATTTGAATGCCGTTGTGGGAACTGCTCTCATT

[0261] AAGAAATATCCGAAACTGGAATCCGAGTTCGTTTATGGGGACTACAAGGTGTACGATGTTCGC

[0262] AAGATGATTGCTAAATCGGAACAGGAAATTGGGAAAGCAACGGCTAAGTACTTCTTCTACAGC

[0263] AATATTATGAATTTCTTTAAAACGGAGATTACTCTCGCCAATGGAGAGATCCGGAAAAGACCCC

[0264] TTATAGAGACCAACGGTGAAACAGGTGAGATTGTTTGGGATAAAGGAAGAGATTTTGCGACG

[0265] GTCAGAAAGGTCTTGTCGATGCCCCAGGTCAACATCGTCAAAAAAACAGAAGTTCAAACGGG

[0266] CGGGTTCTCAAAGGAGTCTATACTTCCTAAACGGAACTCCGACAAACTCATTGCTCGCAAAAA

[0267] AGATTGGGACCCCAAAAAATATGGGGGCTTCGACTCGCCAACCGTTGCATATTCAGTGCTCGT

[0268] GGTCGCTAAAGTTGAGAAAGGTAAGTCCAAGAAACTCAAATCTGTCAAAGAGCTGTTGGGCA

[0269] TCACAATTATGGAACGGAGTAGCTTCGAGAAAAATCCTATTGACTTCCTGGAGGCGAAAGGCT

[0270] ACAAGGAGGTGAAAAAAGATCTGATCATCAAGCTGCCCAAATACTCCCTCTTTGAATTGGAAA

[0271] ATGGTCGCAAAAGGATGCTCGCCAGCGCCGGCGAGCTCCAGAAGGGAAATGAGTTGGCCTTG

[0272] CCGAGTAAATATGTCAATTTCCTCTACCTCGCATCGCACTACGAGAAACTGAAGGGATCGCCG

[0273] GAGGATAACGAGCAAAAACAACTGTTCGTTGAACAACATAAACATTACCTGGATGAAATTATC

[0274] GAACAGATCTCTGAATTTAGCAAAAGGGTTATCCTTGCAGACGCCAATCTGGATAAGGTTCTTT

[0275] CGGCGTACAACAAACATAGGGATAAACCAATCCGCAGAACAGGCAGAAAATATCATACACTTGT

[0276] TCACTCTTACCAATCTGGGTGCTCCCGCAGCGTTTAAGTACTTTGACACAACCATCGATCGCAA

[0277] GAGGTACACAAGTACCAAGGAAGTTCTTGACGCCACTCTTATTCATCAATCGATCACAGGTCT

[0278] CTATGAAACCCGGATAGACCTTTCTCAGCTTGGAGGGTGATTCAGGGGGGTCCAGCGGTGGGTAG

[0279] CAAGAGGACTGCGGACGGGAGCGAATTTGAGTCACCCAAAAAGAAAGAAGGTGTCAGGC

[0280] GGAAGTAGTGGCGATCTACCCTGAATATCGAAGATGAATACCGGCTGCATGAGACATCGAAA

[0281] GAACCGGATGTTTCTCTCGGGTCGACATGGCTGTCGGATTTTCCGCAAGCTTGGGGCCGAGACC

[0282] GGCGGTATGGGTCTCGCTGTCAGGCAGGCACCCTTGATCATACCCTTGAAGGCCACATCGACA

[0283] CCGTCTCAATAAAACAGTACCCCCATGAGCCAGGAAGCACGGCTCGGAATAAAGCCACATATT

[0284] CAGCGGCTTTTGGATCAGGGATTCTCGTTCCTTGCCAATCTCCTTGGAACACACCTCTCCTGC

[0285] CGGTTAAGAAGCCGGGCACTAACGATTACAGACCCGTTCAAGATCTCAGAGAAGTGAACAAG

[0286] AGAGTCGAAGACATCCACCCAACTGTTCCTAATCCCTACAATTTGCTCTCGGGATTGCCACCGT

[0287] CACATCAATGGTATACTGTTCTTGACCTTAAAGATGCATTCTTCTGTTTGAGATTGCACCCTACT

[0288] TCTCAACCGCTTTTTGCATTTGAGTGGCGCGATCCAGAGATGGGTATATCAGGGCAACTCACTT

[0289] GGACAAGACTGCCACAAGGGTTTAAAAACTCCCCTACTCTGTTTAACGAAGCCTTGCATAGAG

[0290] ACCTGGCCGATTTCAGGATACAGCACCCTGATCTGATCCTCCTGCAGTACGTCGACGACCTGC

[0291] TGTTGGCTGCTACATCAGAACTCGATTGCCAACAAGGTACTAGAGCTCTTCTCCAGACGCTTG

[0292] GAAATCTGGGGTATCGCGCGAGCGCTAAAAAGGCCCAGATATGTCAAAAACAAGTTAAGTACC

[0293] TGGGGTATTTGCTCAAGGAAGGGCAAAGGTGGTTGACGGAAGCGAGGAAGGAAACGGTGAT

[0294] GGGCCAGCCAACACCTAAGACACCGCGGCAGCTGAGGGAATTCCTCGGGAAGGCTGGTTTTT

[0295] GTCGGCTGTTCATCCCTGGTTTCGCGGAAATGGCGGCACCGCTGTATCCTCTGACGAAACCGG

[0296] GGACCCTGTTCAATTGGGGACCAGATCAACAGAAAGCGTACCAGGAAATCAAGCAAGCGCTG

[0297] TTGACCGCTCCGGCTCTTGGTCTCCCCGACCTCACTAAACCTTTCGAGCTCTTTGTCGACGAA

[0298] AAGCAAGGGTATGCGAAAGGTGTTCTTACACAAAAACTTGGACCGTGGCGCAGACCTGTTGC

[0299] CTACTTGAGTAAAAAGCTTGACCCCGTTGCGGCCGGGTGGCCACCATGCCTGCGCATGGTGGCCGCCATAGCCGTCCTCACTAAGGATGCCGGTAAGCTGACGATGGGTCAGCCCTTGGTCATACTCGCTCCCCATGCCGTGGAGGCACTCGTCAAACAACCCCCCGATCGCTGGCTGAGCAATGCAAGGATGACGCACTACCAAGCACTTCTCCTGGACACCGACCGCGTCCAATTTGGCCCGGTTGTCGCGCTTAACCCGGCGACTCTTTTGCCTCTTCCTGAAGAGGGTTTGCAGCATAACTGCCTGGACATCCTCGCTGAAGCTCATGGGACCCGCCCGGACTTGACAGATCAGCCTCTCGGCGGCAGCAAACGGACGGCCGATGGGAGCGAATTTGAATCACCTAAGAAGAAAAGGAAAGTCGGTTCGGGCCCCGCGGCCAAGAGAGTCAAACTTGACTGAactagtGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGAAATTAATTC

[0300] Among them, the nucleotide sequences of the series of modules BamH I-kozak-bpNLS-SpCas9-linker-bpNLS-linker-M-MLV(ΔRNase H)-bpNLS-c-myc NLS-Bcu I are as follows:

[0301] BamH I (SEQ ID NO.2): ggatcc.

[0302] Kozak (SEQ ID NO.3): gccaccATGGCC

[0303] bpNLS1 (SEQ ID NO.4): AAAAAGGACGGCGGACGGGAGCGAATTTGAGTCACCTAAGAAAAAGCGGAAGGTG

[0304] SpCas9 (SEQ ID NO.5): GACAAAAAATATAGCATTGGTCTTGACATTGGCACTAACTCTGTGGGGTGGGCTGTTATTACCGACGAGTACAAAGTTCCTTCGAAGAAATTCAAGGTCTTGGGCAACACGGATAGGCACTCAATTAAGAAAAACTTGATTGGCGCTCTTCTTTTTGACTCGGGCGAGACAGCGGAGGCGACTCGCCTGAAGAGGACTGCTAGGAGGAGATATACCAGGCGGAAGAATAGAATTTGTTATCTGCAGGAGATTTTCTCTAACGAAATGGCAAAAGTCGATGATAGCTTCTTCCATCGCCTGGAGGAGTCGTTTTTGGTCGAGGAAGATAAAAAGCATGAGAGGCACCCTATATTTGGCAACATTGTTGATGAGGTGGCGTACCATGAGAAATATCCGACAATTTACCACCTTAGGAAAAAACTCGTTGACAGTACTGATAAAGCGGATCTTCGGCTGATATACTTGGCTCTGGCGCATATGATCAAATTCCGCGGCCACTTTTTGATCGAAGGTGACTTGAACCCGGATAATTCGGATGTGGACAAATTGTTTATCCAGCTGGTGCAAACTTACAACCAACTCTTTGAGGAGAACCCCATAAACGCC

[0305] TCTGGCGTTGACGCGAAGGCCATACTTTCAGCTAGGCTCTCCAAATCACGCAAAT

[0306] TGGAGAACCTCATAGCGCAATTGCCTGGTGAAAAAAAAAATGGTCTGTTTGGTAA

[0307] TTTGATTGCACTCAGTCTCGGGCTTACACCCAATTTCAAATCCAATTTTGACTTGG

[0308] CCGAGGACGCTAAACTTCAGCTGTCGAAAGATACGTATGATGACGATCTCGACAA

[0309] TCTCCTGGCCCAGATAGGCGACCAGTATGCTGATTTGTTCTTGGCTGCGAAGAAC

[0310] CTGAGTGACGCAATTCTGCTTTCCGATATCCTCCGCGTGAATACGGAAATAACAAA

[0311] GGCCCCCCTGTCAGCTTCAATGATAAAGAGGTATGACGAGCATCATCAAGACTTG

[0312] ACACTTCTTAAAGCTCTCGTTAGACAACAATTGCCGGAGAAATACAAGGAGATTT

[0313] TTTTCGACCAGTCCAAGAATGGATACGCAGGTTACATTGACGGCGGTGCGAGTCA

[0314] GGAGGAGTTCTATAAATTCATAAAGCCCATCTTGGAGAAAATGGATGGTACTGAG

[0315] GAGCTTCTGGTGAAACTCAAGCGCGAGGACCTTCTCAGGAAACAGAGAACTTTC

[0316] GATAATGGCTCCATCCCACATCAGATCCATCTCGGAGAACTGCACGCTATCCTCCG

[0317] CAGGCAGGAGGACTTTTATCCCTTCCTTAAAAGACAATAGAGAAAAAATCGAAAAA

[0318] ATCCTTACTTTCCGGATACCGTATTATGTCGGGCCCTTGGCGCGCGGTAACAGCAG

[0319] GTTCGCCTGGATGACCCGCAAAAGTGAGGAGACTATAACCCCGTGGAACTTTGA

[0320] GGAGGTTGTCGACAAAGGCGCGTCCGCCAATCCTTTATATAGAGGATGACGAAT

[0321] TTTGACAAAACTTGCCCAACGAGAAAGTCCTCCCTAAACACTCATTGCTGTATG

[0322] AATACTTCACAGTTTATAACGAATTGACAAAGTTAAATACGTTACAGAAGGCATG

[0323] AGAAAACCTGCCTTCCTGTCAGGCGAGCAGAAAAAGGCCATCGTTGATCTGCTG

[0324] TTCAAAACTAACCGCAAAGTCACTGTGAAGCAATTGAAAGAAGACTATTTCAAA

[0325] AAGATAGAATGCTTCGATAGTGTTGAAATCTCAGGAGTTGAGGACAGATTCAATG

[0326] CGAGTTTGGGAACGTATCACGATCTGCTGAAGATTATAAAGGATAAGGATTTCCTT

[0327] GACAACGAAGAGAACGAAGACATCCTGGAGGACATTGTTCTGACTCTGACATTG

[0328] TTTGAGGACAGAGAAATGATTGAGGAAAGGCTCCAAAGACGTATGCACACCTCTTC

[0329] GACGATAAAGTTATGAAGCAACTGAAGCGGCGGAGATATACCGGCTGGGGCCGC

[0330] CTGTCTAGAAAGCTGATCAACGGTATCAGGGATAAGCAATCGGGGAAAACTATTC

[0331] TGGATTTCCTCAAATCCGACGGTTTTGCTAATCGCAATTTCATGCAACTTATTCACG

[0332] ATGACTCTCTTACATTCAAAGAAGATATCCAAAAAGCCCAGGTTTCCGGGCAGGG

[0333] GGATTCGTTGCATGAGCATATTGCCAATCTCGCAGGTAGCCCCGCGATCAAGAAA

[0334] GGCATATTGCAGACCGTTAAAGTTGTGGACGAACTCGTGAAGGTTATGGGCAGGC

[0335] ACAAACCGGAGAATATAGTGATAGAAATGGCTAGAGAAAACCAGACCACACAGA

[0336] AGGGGCAAAAGAATTCTAGGGAGCGCATGAAACGGATCGAGGAAGGTATCAAGG

[0337] AACTCGGTTCCCAGATTCTTAAAGAGCACCCTGTTGAAAACACTCAGCTTCAAAA

[0338] TGAAAAGCTTTATTTGTATTATTTGCAGAACGGGCGCGATATGTACGTCGACCAGG

[0339] AGCTCGACATTAACCGCCTCTCTGACTATGATGTTGATcacATCGTGCCGCAGAGCT

[0340] TTTTGAAGGATGATAGCATTGACAACAAAGTTCTGACGCGGAGTGACAAAAATAG

[0341] GGGCAAGTCTGATAATGTTCCCTCGGAAGAAGTGGTCAAAAAGATGAAAAATTAC

[0342] TGGAGACAGCTTCTCAACGCTAAGCTGATCACCCAAAGGAAGTTTGATAATTTGA

[0343] CCAAGGCGGAACGGGGAGGTCTCTCCGAGCTCGACAAAGCAGGGTTTTATCAAAC

[0344] GCCAGCTGGTCGAGACGAGGCAAATAACTAAGCATGTGGCACAGATCCTGGACA

[0345] GTCGGATGAATACGAAATATGACGAGAACGACAAGCTGATTAGGGAAGTTAAAGT

[0346] CATAACCTTGAAATCGAAACTCGTTTCTGACTTCCGCAAGGATTTTCAATTCTACA

[0347] AGGTTCGGGAGATAAACAACTACCATCATGCACACGACGCCTATTTGAATGCCGT

[0348] TGTGGGAACTGCTCTCATTAAGAAATATCCGAAACTGGAATCCGAGTTCGTTTATG

[0349] GGGACTACAAGGTGTACGATGTTCGCAAGATGATTGCTAAATCGGAACAGGAAAT

[0350] TGGGAAAGCAACGGCTAAGTACTTCTTCTACAGCAATATTATGAATTTCTTTAAAA

[0351] CGGAGATTACTCTCGCCAATGGAGAGATCCGGAAAAGACCCCTTATAGAGACCAA

[0352] CGGTGAAACAGGTGAGATTGTTTGGGATAAAGGAAGAGATTTTGCGACGGTCAG

[0353] AAAGGTCTTGTCGATGCCCCAGGTCAACATCGTCAAAAAAACAGAAGTTCAAAC

[0354] GGGCGGGTTCTCAAAGGAGTCTATACTTCCTAAACGGAACTCCGACAAACTCATT

[0355] GCTCGCAAAAAAGATTGGGACCCCAAAAAATATGGGGGCTTCGACTCGCCAACC

[0356] GTTGCATATTCAGTGCTCGTGGTCGCTAAAGTTGAGAAAGGTAAGTCCAAGAAAC

[0357] TCAAATCTGTCAAAGAGCTGTTGGGCATCACAATTATGGAACGGAGTAGCTTCGA

[0358] GAAAAATCCTATTGACTTCCTGGAGGCGAAAGGCTACAAGGAGGTGAAAAAAGA

[0359] TCTGATCATCAAGCTGCCCAAATACTCCCTCTTTGAATTGGAAAATGGTCGCAAAA

[0360] GGATGCTCGCCAGCGCCGGCGAGCTCCAGAAGGGAAATGAGTTGGCCTTGCCGA

[0361] GTAAATATGTCAATTTCCTCTACCTCGCATCGCACTACGAGAAACTGAAGGGATCG

[0362] CCGGAGGATAACGAGCAAAAACAACTGTTCGTTGAACAACATAAACATTACCTGG

[0363] ATGAAATTATCGAACAGATCTCTGAATTTAGCAAAAGGGTTATCCTTGCAGACGCC

[0364] AATCTGGATAAGGTTCTTTCGGCGTACAACAAACATAGGGATAAACCAATCCGCGAACAGGCAGAAAATATCATACACTTGTTCACTCTTACCAATCTGGGTGCTCCCGCAGCGTTTAAGTACTTTGACACAACCATCGATCGCAAGAGGTACACAAGTACCAAGGAAGTTCTTGACGCCACTCTTATTCATCAATCGATCACAGGTCTCTATGAAACCCGGATAGACCTTTCTCAGCTTGGAGGTGAT

[0365] Linker1(SEQ ID NO.6):TCAGGGGGGTCCAGCGGTGGTAGC

[0366] bpNLS2(SEQ ID NO.7):AAGAGGACTGCGGACGGGAGCGAATTTGAGTCACCCAAAAAGAAAAGAAAGGTG

[0367] Linker2(SEQ ID NO.8):TCAGGCGGAAGTAGTGGCGGATCT

[0368] M-MLV(ΔRNase H)(SEQ ID NO.9):

[0369]

[0370] bpNLS3(SEQ ID NO.10):AAACGGACGGCCGATGGGAGCGAATTTGAATCACCTAAGAAGAAAAGGAAAGTCc-myc NLS(SEQ ID NO.11):CCCGCGGCCAAGAGAGTCAAACTTGAC

[0371] Bcu I (SEQ ID NO.12): actagt

[0372] The expression and stability of pegRNA were optimized, and the nucleotide sequence encoding the corresponding protein was codon-optimized according to the preference of rice codons. The obtained nucleotide sequence gRNA41 was commissioned to Beijing Qingke Biotechnology Co., Ltd. for artificial synthesis. Afterwards, it was assembled with pENTR4 by BamH I and EcoR I restriction enzyme ligation to obtain a vector named pENTR4:gRNA41 (SEQ ID NO.13). The Bsa I restriction site was used to clone the pegRNA sequence of the target gene.

[0373] pENTR4:gRNA41 (SEQ ID NO.13)

[0374] CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCTAGCATGGATCTCGGGGACGTCTAACTACTAAGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGGAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGTGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAACTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTCCTGTTAGTTAGTTACTTAAGCTCGGGCCCCAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAATTGATAAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCAGGCTCCACCATGGGAACCAATTCAGTCGACTGGATCCATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACAT

[0375] GGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAA

[0376] GACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCG

[0377] GATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGT

[0378] GGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCT

[0379] CTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAA

[0380] AAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATTGGCAGA

[0381] CATACTGTCCCACAAATGAAGATGGAATCTGTAAAAGAAAACGCGTGAAATAATG

[0382] CGTCTGACAAAGGTTAGGTCGGCTGCCTTTAATCAATACCAAAGTGGTCCCTACC

[0383] ACGATGGAAAAACTGTGCAGTCGGTTTGGCTTTTTCTGACGAACAAATAAGATTC

[0384] GTGGCCGACAGGTGGGGGTCCACCATGTGAAGGCATCTTCAGACTCCAATAATGG

[0385] AGCAATGACGTAAGGGCTTACGAAATAAGTAAGGGTAGTTTGGGAAATGTCCACT

[0386] CACCCGTCAGTCTATAAATACTTAGCCCCTCCCTCATTGTTAAGGGAGCAAAATCT

[0387] CAGAGAGATAGTCCTAGAGAGAGAAAGAGAGCAAGTAGCCTAGAAGTAGTCAAG

[0388] GCGGCGAAGTATTCAGGCACGTGGCCAGGAAGAAGAAAAGCCAAGACGACGAA

[0389] AACAGGTAAGAGCTAAGCATCTAGAAAGTTGAAAACAATCTTCAAAAGTCCCAC

[0390] ATCGCTTAGATAAGAAAACGAAGCTGAGTTTATATACAGCTAGAGTCGAAGTAGT

[0391] GATTGAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAG

[0392] ACCCGGGTTCGATTCCCGGCTGGTGCAAGAGACCAAAGGAGGTCTCAGGCCGGC

[0393] ATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAA

[0394] TGGGACTTTTTTTTGATATCTCCGGGGCTAATTGAATATGAAGATGAAGATGAAATA

[0395] TTTGGTGTGTCAAATAAAAAGCTGGTGTGCTTAAGTTTGTGTTTTTTTCTTGGCTT

[0396] GTTGTGTTATGAATTTGTGGCTTTTTCTAATATTAAATGAATGTAAGATCTCATTATA

[0397] ATGAATAAACAAATGTTTCTATAATCCATTGTGAATGTTTTGTTGGATCTCTTCTGC

[0398] AGCATATAACTACTGTATGTGCTATGGTATGGACTATGGAATATGATTAAAGATAAG

[0399] GAATTCGCGGCCGCACTCGAGATATCTAGACCCAGCTTTCTTGTACAAAGTTGGCA

[0400] TTATAAGAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAA

[0401] AATAAAATCATTATTTGCCATCCAGCTGCAGCTCTGGCCCGTGTCTCAAAATCTCT

[0402] GATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTT

[0403] ACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAG

[0404] GCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGA

[0405] TAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGC

[0406] CAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGA

[0407] GATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATT

[0408] TTATCCGTACTCCTGGTGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACA

[0409] GCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCT

[0410] GGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACA

[0411] GCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTT

[0412] GATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGA

[0413] AAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGAT

[0414] TTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTT

[0415] GGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCC

[0416] TCGGTGAGTTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATA

[0417] ATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAG

[0418] AATTGGTTAATTGGTTGTAACATTATTCAGATTGGGCCCCGTTCCACTGAGCGTCA

[0419] GACCCCGTAGAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTCTGCGCGTAAT

[0420] CTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGAT

[0421] CAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC

[0422] CAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA

[0423] GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGG

[0424] CGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCG

[0425] CAGCGGTCGGGCTGAACGGGGGGTTCGTCACACAGCCCAGCTTGGAGCGAAC

[0426] GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTT

[0427] CCCGAAGGGAGAAAGCGGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGG

[0428] AGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

[0429] GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC

[0430] GGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT.

[0431] Example 2: Design and cloning of recognition sequences for OsMAPK13 gene.

[0432] The OsMAPK13 genome sequence was obtained from the MSU / TIGR rice genome database (http: / / rice.plantbiology.msu.edu / ), and then two suitable target sites were selected near the stop codon (the sequence near the OsMAPK13 gene terminator is shown in SEQ ID NO.19) ( Figure 1 As shown in Figure 1), OsMAPK13 gRNA1: 5'-UGUGACCAGGAUGCUCUCCU-3' (SEQ ID NO. 14); OsMAPK13 gRNA2: 3'-CGCAAGUCGGCCGGUACUAG-5' (SEQ ID NO. 15). Figure 2 As shown in the figure, three pegRNAs were designed for OsMAPK13, of which pegRNA1 and 2 target gRNA1; pegRNA3 is a double target, and the corresponding spacer sequence in the pegRNA is marked with a horizontal line:

[0433] pegRNA1 (SEQ ID NO.16):

[0434] CGATTCCCGGCTGGTGCA TGTGACCAGGATGCTCTCCTGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTACTGATGCCCTAGGCTATTTGTCATCGTCGTCTTTATAATCGGAGAGCATCCTAAATTGAATTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAAGGCCGGCATGGTCCCAGC

[0435] pegRNA2(SEQ ID NO.17):

[0436] CGATTCCCGGCTGGTGCA TGTGACCAGGATGCTCTCCT GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCATTACTATTTGTCATCGTCGTCTTTATAATCGGAGAGCATCCTAAATTGAATTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAAGGCCGGCATGGTCCCAGC

[0437] pegRNA3(SEQ ID NO.18):

[0438] CGATTCCCGGCTGGTGCA TGTGACCAGGATGCTCTCCT GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCATTACTATTTGTCATCGTCGTCTTTATAATCGGAGAGCATCCTAAATTGAATTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAAGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACATCGAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCA GATCATGGC CGGCTGAACGCGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCATTACTAGATTATAAAGACGACGATGACAAATAGTAATGATTCAGCCGGAGCTTTACTTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAAGGCCGGCATGGTCCCAGC

[0439] Sequence near the terminator of OsMAPK13 gene (SEQ ID NO.19):

[0440] TCTATTGCCCGAACTATGATAAGTCCCCCAAGATCACAAGAAAAGGGCAAGAATCGTGCATCTGCTTATCCAAATGGCATAATCAATCTAAACTCCAATCCTAAGATCTATTTAAAGAGTGCTAGTATCAGTGCTTCAACGTGCATCATTAGAGGGAATAAAGGCCCGAAGGTATTTGCCTACTGCCAGTATGATGACAGTGCTTAAATTTTAGCCTGACACTTGGTAAATCTTATGAGTAAATTATCAGTGCATTTTTTCCCTCGTATCTTTGTTTTTGTGGGTCTGACTAAGTTTTACTCTGTTTCACTTTGCCTTTTTTTTTTTCACTCTGAAGG

[0441] AGAATGGTATCTCTGAGGACATGGAAGAGGTTGTCTATGAGTTGTCAGATAATGTG

[0442] ACCAGGATGCTCTCCTAGGGCATCAGTAACCAGCGTTCAGCCGGCCATGATCTTG

[0443] ATGCAATTCCAAATTCCAATAACTGTTTATACCATCAGTATCATGGATCCTTGCCTAT

[0444] ATCTATGAGCTGATACTCTAGGCTCTTAGCTTATATCACCTATTGATCATCTTCCAAA

[0445] AGCATCAAGCTTGGAGAAATGAGTTGTCTTTGTCAATTCGAGGCCAAATTTGGCA

[0446] ACTCGGCTTGTTGTACATGCATTTTCCTCATAGCCTGGCAGTGTGTGATGACTCTATCTGCCATATGAACAATCTGGCACTTTTTACCTTAGTACCATATTACCGTTTCAGAA.

[0447] The above three pegRNAs were commissioned to Beijing Qingke Biotechnology Co., Ltd. for artificial synthesis. Afterwards, MH-F1 (nucleotide sequence SEQ ID NO.20 is 5'-3': CGATTCCCGGCTGGTGCA) and R1 (nucleotide sequence SEQ ID NO.21 is 5'-3': GCTGGGACCATGCCGGCC) were used, pegRNA1 was used as a template, high-fidelity polymerase was used and PCR amplification was performed according to the recommended procedure in the instruction manual, the recovered band was infused with the pENTR4:gRNA41Bsa I digestion product, and the obtained vector was named pENTR4:gRNA41-pegRNA1, and the rPE14 vector and the pENTR4:gRNA41-pegRNA1 vector connected with the pegRNA sequence were constructed into the final rice genetic transformation vector through the LR reaction of Gateway.

[0448] The construction process of pENTR4:gRNA41-pegRNA2,3 is the same as 1.

[0449] Example 3: Transformation of japonica rice variety Kitaake using the rPE14 system.

[0450] The rice genetic transformation vectors of the above three different repair strategies were transformed into Agrobacterium EHA105 (Agrobacterium EHA105 was purchased from Beijing Bomade Gene Technology Co., Ltd.) by electroporation, and rice Agrobacterium-mediated genetic transformation was performed according to the method disclosed by Hiei et al. (Hiei Y., Ohta S., Komari T., and Kumashiro T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6: 271–282.) to obtain T0 generation rice plants.

[0451] Example 4: Identification of in situ labeling of the OsMAPK13 gene in T0 generation transgenic rice.

[0452] The genomic DNA of resistant calli and transgenic plants was extracted by CTAB method.

[0453] According to the OsMAPK13 genome sequence, two pairs of primers were designed for second-generation sequencing and first-generation sequencing respectively:

[0454] OsMAPK13-HF1 (SEQ ID NO. 22): ggagtgagtacggtgtgcGGTTGTCTATGAGTTGTCAG

[0455] OsMAPK13-HR1 (SEQ ID NO. 23): gagttggatgctggatggTATCAGCTCATAGATATAGGCAAG (next generation sequencing, 157 bp, PE150)

[0456] OsMAPK13-F1 (SEQ ID NO.24): CTCTGAAGGAGAATGGTATC

[0457] OsMAPK13-R1 (SEQ ID NO. 25): ATGGCAGATAGAGTCATCAC (first generation sequencing, 355 bp)

[0458] The results of second-generation and first-generation sequencing were analyzed. Figure 3 As shown, the three strategies of Agrobacterium-mediated genetic transformation editing efficiency were 66.67%, 30.68% and 29.55%, respectively, of which the precision editing efficiency was 66.67%, 1.14% and 9.09%, respectively. The above results show that all three strategies can achieve site-specific insertion of DNA fragments, thereby achieving in situ gene labeling, and the strategy using microhomology sequence repair is the most efficient and more precise.

[0459] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the scope of protection determined by the claims of the present invention. In addition, the examples described are only one application of the present invention, so the use of the relevant repair mode in the present invention for site-directed insertion of other DNA fragments (not limited to the DNA sequence of the fusion tag) should also fall within the scope of protection determined by the claims of the present invention.

Claims

1. A genetic transformation vector, characterized in that: The genetic transformation vector includes the rPE14 vector whose nucleotide sequence is shown as SEQ ID NO.

1.

2. The genetic transformation vector according to claim 1, characterized in that The genetic transformation vector also includes a pENTR4:gRNA41 vector connected to a pegRNA sequence, wherein the nucleotide sequence of the pENTR4:gRNA41 vector is shown in SEQ ID NO.

13.

3. The genetic transformation vector according to claim 2, characterized in that The pegRNA sequence includes any one of the following ac: a. The sequence shown in SEQ ID NO.16; b. The sequence shown in SEQ ID NO.17; c. The sequence shown in SEQ ID NO.

18.

4. A gene editing artificial system for site-specific insertion of plant DNA fragments, characterized in that: The invention comprises the genetic transformation vector according to any one of claims 1 to 3.

5. The gene editing artificial system according to claim 4, characterized in that: The plant is rice.

6. A method for achieving site-specific insertion of plant DNA fragments, characterized in that: The method comprises the steps of transforming the gene-editing artificial system described in any one of claims 4 to 5 by Agrobacterium infection, and obtaining a series of regenerated plants by tissue culture.

7. Use of the genetic transformation vector described in any one of claims 1 to 3 or the gene editing artificial system described in any one of claims 4 to 5 in the site-specific insertion of plant DNA fragments.

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