Set of base editing artificial system for paddy

An artificial system and base editing technology, applied in the field of base editing artificial systems, can solve the problem that the efficiency is only one thousandth to two percent, and achieve the effect of high base editing efficiency

Active Publication Date: 2019-07-30
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, researchers have mainly used traditional methods such as homologous recombination mechanisms to obtain gene function gain mutants, while in most higher organisms including rice, non-homologous end-joining repair The dominant position in homologous recombination custom mutants is only 1 / 1000 to 2%

Method used

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  • Set of base editing artificial system for paddy
  • Set of base editing artificial system for paddy
  • Set of base editing artificial system for paddy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1.1 Construction of pUbi:rBE14 and pUbi:rBE17

[0053] First, artificially synthesize the fragment of SEQ ID No.2, and then clone it into the cloning vector pUC57 (the vector is commercially available) to obtain pUC57-2. The amino acid sequence encoded by SEQ ID No.2 is shown in SEQ ID No.1. The segments of SEQ ID No.10 were artificially synthesized, and then cloned into the cloning vector pUC57 to obtain pUC57-10. The amino acid sequence encoded by SEQ ID No.10 is shown in SEQ ID No.11.

[0054] Then, clone SEQ ID No.6 (the ubiquitin promoter UbiP of maize), SEQ ID No.2, and SEQ ID No.8 (Nos terminator) into the pCAMBIA 1300 vector according to the direction from 5' to 3', and named is pUbi:rBE14. Cloning SEQ ID No.6 (maize ubiquitin promoter UbiP), SEQ ID No.10, and SEQ ID No.8 (Nos terminator) into the pCAMBIA 1300 vector according to the direction from 5' to 3', named pUbi: rBE17.

[0055] 1.2 Construction of pENTR4: sgRNA

[0056] According to the direction f...

Embodiment 2

[0058] Base editing of OsWRKY45 and OsSERK2 using pUbi:rBE14

[0059] 2.1 Design and cloning of recognition sequences for OsWRKY45 and OsSERK2 genes

[0060] The transcript and genome sequences of each gene were obtained from the MSU / TIGR rice genome database (http: / / rice.plantbiology.msu.edu / ). Principles for designing target sequences: 1) Determine whether the editing site contains nucleotide base A or T, and whether the amino acid changes caused by the mutation of the corresponding base A to G or the corresponding base T to C are in line with expectations; When the nucleotide sequence of T is used as the target nucleotide sequence, its reverse complementary sequence is used for base editing; 2) Search for the PAM motif sequence in the 3' end direction, so that the mutant base A or T is in the target nucleus Positions 4 to 7 in the direction of the 5' end of the nucleotide sequence. Wherein, the PAM motifs recognized by the pUbi:rBE14 system are NGG, NAG, GAGN and AAGN, et...

Embodiment 3

[0081] Base editing of OsMPK6 using pUbi:rBE14 and pUbi:rBE17, respectively

[0082] The PAM motifs recognized by the pUbi:rBE14 system and the pUbi:rBE17 system are NGG, NAG, GAGN and AAGN, etc., and the N is A or G or C or T.

[0083] According to the target nucleotide sequence primer design principle in embodiment 2, the target nucleotide sequence of OsMPK6 is designed to contain the primer that matches with the BsaI restriction site end connection: gOsMPK6-F1 (SEQ ID No.22) and gOsMPK6-R1 ( SEQ ID No. 23).

[0084] After synthesizing the primers, use T4 polynucleotide kinase to phosphorylate the primers, anneal to form double strands, clone gOsMPK6-F1 / R1 into the BsaI restriction site of the pENTR4:sgRNA vector, and confirm that the inserted fragment is completely correct by sequencing . Wherein, the forward primer represents the target nucleotide sequence inserted into the BsaI site on the pENTR4:sgRNA carrier, as shown in SEQIDNo.24. Wherein, the 2nd, 4th and 7th posi...

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Abstract

The invention relates to a set of base editing artificial system for paddy. The system comprises an I adjusting element and an II adjusting element. The I adjusting element can encode a nucleotide sequence of an amino acid sequence I, wherein the amino acid sequence I comprises an amino acid sequence shown in SEQ ID No.1. The II adjusting element comprises an (II-1) nucleotidesequence and an (II-2) nucleotide sequence from the end 5' to the end 3' in sequence. The (II-1) nucleotide sequence comprises a target nucleotide sequence, the (II-2) nucleotide sequencecomprises an sgRNA nucleotide sequence derived from streptococcus pyogenes, and the (II-1) nucleotide sequence and the (II-2) nucleotide sequence are in transcription fusion.

Description

technical field [0001] This application relates to a base editing artificial system for rice. Background technique [0002] Rice (Oryza sativa L.) is one of the world's major food crops, feeding nearly half of the world's population, including almost the entire population of East and Southeast Asia. China is the country with the highest total rice output in the world, accounting for about 30% of the global total. In the production process, the three major diseases of rice, mainly rice blast, rice smut and sheath blight, seriously restrict the growth and development of rice, resulting in a decrease in rice yield and quality, threatening global food security. Therefore, it is a major issue for the sustainable development of human society to increase the yield, improve the quality of rice, and increase the research on the disease resistance and stress resistance of rice plants to ensure the stable supply of food. As a model plant of monocotyledonous plants, rice's research te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/113
CPCC12N15/113C12N15/8213C12N2310/10
Inventor 周焕斌严芳旷永洁
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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