TALE nuclease simplified framework construction for plant gene fixed-site shearing

A nuclease and plant technology, applied in plant genetic improvement, plant products, genetic engineering, etc.

Pending Publication Date: 2019-11-19
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this technology is rarely used in plants, and there is

Method used

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  • TALE nuclease simplified framework construction for plant gene fixed-site shearing
  • TALE nuclease simplified framework construction for plant gene fixed-site shearing
  • TALE nuclease simplified framework construction for plant gene fixed-site shearing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Chemical Synthesis of the Streamlined Backbone Coding Sequences for Targeted Editing of Plant Gene TALENs

[0019] The simplified framework of TALENs was synthesized by gene synthesis method (Nucleic Acids Research, 2004, 32, e98). The designed primers are:

[0020] SplantTALENs-1 ATGGACCCAATCCGTTCTCGTACTCCATCTCCTGCACGTGAACTGCTGCCTGGTCCACAA (shown in SEQ ID NO.2)

[0021] SplantTALENs-2 GTGCACCACCACGATCAGCAGTAGGCTGGACCCTGTCAGGTTGTGGACCAGGCAGCAGTT (shown in SEQ ID NO.3)

[0022] SplantTALENs-3 TGCTGATCGTGGTGGTGCACCACCTGCTGGTGGTCCACTGGACGGTCTTCCTGCTCGTCG (shown in SEQ ID NO.4)

[0023] SplantTALENs-4 TGGTGCAGGTGGAGATGGCAGCCGAGTCCGAGACATAGTACGACGAGCAGGAAGACCGTC (shown in SEQ ID NO.5)

[0024] SplantTALENs-5 TGCCATCTCCACCTGCACCATCTCCTGCATTCTCTGCTGGTTCCTTTCTCCGACCTGCTGC (shown in SEQ ID NO.6)

[0025] SplantTALENs-6 TCAAGAAGGGAGGTGTCAAGAAGGGATGGATCGAACTGACGCAGCAGGTCGGAGAAGGAA (shown in SEQ ID NO.7)

[0026] SplantTALENs-7 CTTGACACCCTCCCTTCTTGACTCGATGCCTGCTGTT...

Embodiment 2

[0052] Example 2: Chemical Construction of Plant Expression Elements for Simplified Skeletons of Plant Gene TALENs

[0053] The simplified skeleton of plant TALENs The promoter of SplantTALENs is CaMV35S+Omega [Omega is the translation enhancer from TMV virus], the terminator is Nos, and the N-terminal and C-terminal sequences required by TALEN are added to the N-terminal of the FokI cleavage domain ( Including the N-terminal 163 amino acid sequence and the C-terminal 63aa skeleton), a small fragment of DNA sequence with BamHI and SacI restriction endonuclease sites at both ends was introduced between the N-terminal and C-terminal sequences for insertion The above constructed target site binding functional modules. Both ends of SplantTALENs have EcoRI and HindIII cutting sites, which facilitate the cloning and identification of plant expression units

[0054] The CaMV35S+Omega and Nos terminators were amplified by PCR from a conventional vector, and then the promoters, the re...

Embodiment 3

[0062] Example 3: The streamlined backbone of plant gene TALENs is used for plant gene splicing

[0063]Phytoene desaturase (PDS) is a key enzyme in the carotenoid synthesis pathway. The inactivation of PDS gene function will lead to the ineffective synthesis of carotenoids, and the chlorophyll will be destroyed under light conditions, making Tissue that should be green turns white, a phenomenon known as photobleaching, which provides a visually apparent phenotypic change for loss of gene function.

[0064] In order to verify the application effect of TALENs streamlined framework in plant gene splicing. In this study, 15 bases of the target site were selected according to the genomic DNA sequence of the rice OsPDS1 gene and the Arabidopsis AtPDS1 gene, and the uniqueness of the target site in the rice genome was guaranteed by Blast analysis with the rice genome. According to the recognition of base T by NG, the recognition of base C by HD, the recognition of base A by NI, the...

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PUM

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Abstract

The invention discloses a TALE nuclease simplified framework sequence for plant gene shearing. The terminal N of the framework contains 136 amino acids, the terminal C contains 63 amino acids, repetition modules of TALEs for recognizing specific DNA sequences are constructed in the middle of the framework, Fok1 type-II endonucleases are fused at the terminal C, tiny fragments of two different endonucleases are inserted in the terminal N and the terminal C, and through the two endonucleases, the repetition modules of the TALEs can be randomly inserted to construct TALE nucleases capable of shearing the specific DNA sequences. The TALE nucleases are simplified to construct the phytoene dehydrogenase shear nucleases, and after plants are transformed, whitened seedlings are obtained.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and specifically constructs a simplified framework of TALENs suitable for plant expression, that is, the N-terminal contains 136 amino acids, and the C-terminal contains 63 amino acids. It is fused with FokI type II endonuclease to construct a TALE nuclease capable of cutting specific DNA sequences. Background technique [0002] The genome-directed editing technologies emerging in recent years include the following artificial nucleases: zinc finger nucleases (ZFNs), TALE nucleases (TALENs), and the CRISPR / Cas system. These artificial nucleases can generate DNA double-strand breaks at DNA target sites, and their site-directed editing of the genome is achieved by controlling the DNA repair pathway. At present, the main applied technologies are CRISPR / Cas system and TALENs technology. [0003] In 2009, Moscou et al. (Moscou, Science, 2009, 326(5959): 1501) from the Department of Plant Pathology,...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/53A01H5/00A01H6/46A01H6/20
CPCC12N15/8218C12N15/825C12N9/001C12Y103/99029
Inventor 彭日荷姚泉洪田永生高建杰许晶付晓燕李振军韩红娟王波王丽娟张福建黄悠楠张文慧
Owner SHANGHAI ACAD OF AGRI SCI
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