Artificial carrier system with site-specific mutagenesiss and site-specific mutagenesis method

A technology of artificial systems and regulatory elements, applied in botany equipment and methods, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc., can solve the problem of low editing efficiency of GC and AC at target sites

Active Publication Date: 2017-09-19
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, preliminary results found that existing single-base replacement technologies, such as the rBE3 system, are based on the m...

Method used

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  • Artificial carrier system with site-specific mutagenesiss and site-specific mutagenesis method

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Embodiment 1

[0047] 1. Construction of vectors

[0048] Through the routine operation of DNA cloning, the constitutive promoter Ubi-p (SEQ ID No.17), SEQ ID No.8, and Nos terminator (SEQ ID No.20) of maize were cloned in order from 5' to 3' onto the pUbi-ccdB vector, named pUbi:rBE5, and used for transgenic rice plant research.

[0049] The OsU6-p promoter (SEQ ID No.18), two BsaI restriction sites (SEQ ID No.14), sgRNA sequence (SEQ ID No.13), (T)8 terminator (SEQ ID No. 21), japonica rice U6 snRNA promoter (SEQ ID No.19), two BtgZI restriction sites (SEQ ID No.15), sgRNA sequence (SEQ ID No.13), (T)8 terminator (SEQ ID No. .21) Cloning into the multiple cloning site of the pENTR4 vector in the order from 5' to 3', named pENTR4:sgRNA. The two BtgZI or two BsaI restriction sites can be used to clone the target sequence of the specific gene in the following example 2 respectively.

[0050] 2. Design and cloning of recognition sequence for Pi-d2 gene.

[0051] Transcript sequences and ge...

Embodiment 2

[0067] Through the routine operation of DNA cloning, the constitutive promoter Ubi-p (SEQ ID No.17), SEQ ID No.28 and Nos terminator (SEQ ID No.20) of maize were cloned in order from 5' to 3' onto the pUbi-ccdB vector, named pUbi:rBE3. Linearize pENTR4:gPi-d2 with AatII enzyme digestion, and then transfer the targeting sequence transcription element into pUbi:rBE3 through Gateway reaction, and obtain the final vector pUbi:rBE3-gPi-d2, which is used for transgenic rice plant research . Its genome target site sequence for Pi-d2 is the same as that in Example 1, which is 5'-TTATTATTGTCATTATGCTC-3' (SEQ ID No. 24). The nucleotide sequence shown in SEQ ID No.28 encodes the amino acid sequence shown in SEQ ID No.29.

[0068] Other operations are the same as in Example 1.

[0069] The edited plants that obtained Pi-d2 were not screened in the transgenic plant population.

[0070] The present application obtained transgenic rice containing rBE3 (pUbi:rBE3-gPi-d2) and rRE5 (pUbi:rB...

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Abstract

The application relates to a set of artificial systm applied for site-specific alternation of rice genome basic group and a site-specific mutagenesis method. The artificial system comprises a I adjusting element and a II adjusting element; the I adjusting element comprises a nucleotide sequence capable of encoding an amino acid sequence I; the amino acid sequence I is selected from one of SEQ ID Nos. 1-6; the II adjusting element comprises a II-1 nucleotide sequence and a II-2 nucleotide sequence from 5' terminal to 3' terminal; the II-1 nucleotide sequence comprises a target nucleotide sequence; the II-2 nucleotide sequence comprises sgRNA nucleotide sequence originated from Streptococcus pyogenes; the II nucleotide sequence is transcribed and fused with the II-2 nucleotide sequence, and its product can guide the protein encoded by the I adjusting element to a target site to be mutated in the target biological genome, and induce and mutate C at the target site to be one of T, A and G, or G is mutated to be one of A, T and C.

Description

technical field [0001] The invention relates to a set of artificial system and method for site-directed mutation of plant genome bases, in particular to an artificial system for single-base substitution in rice genome. Background technique [0002] Rice (Oryza sativa L.) is one of the main food crops in the world, and nearly half of the world's population, including almost the entire population of East and Southeast Asia, feeds on rice. In China, the sown area of ​​rice accounts for 1 / 4 of the national grain crops, while the output accounts for more than half. Increasing yield, improving rice quality, and increasing rice plant resistance to disease and stress to ensure a stable supply of food are major issues for the sustainable development of human society. Rice is also a model system of monocotyledonous plants, and its research techniques, methods, theories and achievements have an important guiding role for other gramineous plants, such as wheat, corn, and sorghum. [0...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/8205C12N15/8241
Inventor 周焕斌严芳
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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