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A pair of talens for efficient editing of rice waxy gene, its recognition of targeting sites and its application

A technology for identifying components in rice, applied in the fields of biotechnology and genetic engineering, can solve problems such as low efficiency, limited gene knockout, and undetectable genetic modification

Active Publication Date: 2019-07-26
深圳华大基因农业控股有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has been applied to a variety of organisms, many targets still cannot be detected genetically modified or have low efficiency due to factors such as chromosomal structural inhibition of selected recognition sites, DNA modification or invalid expression, or partial TALENs folding. Gene knockout at the individual level is very limited

Method used

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  • A pair of talens for efficient editing of rice waxy gene, its recognition of targeting sites and its application
  • A pair of talens for efficient editing of rice waxy gene, its recognition of targeting sites and its application
  • A pair of talens for efficient editing of rice waxy gene, its recognition of targeting sites and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0201] Example 1 TALENs target sequence design

[0202] 1. Download the rice (Oryza sativa) WAXY gene from NCBI, and design 5 pairs of TALENs combinations. The WAXY gene sequence is shown in Appendix 1.

[0203] 2. TALENs recognition sites are designed according to the following general principles:

[0204] (1) The 0th base is T, that is, the base before the first in the recognition sequence is the 0th, which is T;

[0205] (2) The length of the spacer sequence (Spacer) between the two recognition sequences is between 15-30;

[0206] (3) The length of the recognition sequence is between 15-24.

[0207] 3. The designed TALENs target sequence recognition sites and spacer regions are as follows. The underline is the TALEN recognition binding region, and the middle is the spacer region.

[0208]

Embodiment 2

[0209] Embodiment 2 TALENs construction

[0210]According to the classic Golden Gate method (Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research, 2011, Vol. 39, No. 12 e82 doi: 10.1093 / nar / gkr218 ) for TALE assembly, the four modules of NI, NG, HD, and NN recognize the four bases of A, T, C, and G, respectively. The combined modules were constructed on the background plasmid pTAL3 vector (purchased from Addgene, USA), and the sequence was verified to be correct by sequencing. pTAL3 vector such as figure 1 shown.

Embodiment 3

[0211] Example 3 TALENs expression vector construction

[0212] The TALENs coding region in the correctly sequenced pTAL3 vector was connected to the transformed plant expression vector pCMBIA1301 using KpnI and SpeI, BglII and BstEII respectively (pCMBIA1301 was purchased from the China Plasmid Vector Strain and Cell Line Gene Collection Center, and pCMBIA1301 was transformed as follows: ubi+ The MCS+nos expression cassette (sequence SEQ ID NO.: 30) was added to the pCMBIA1301 vector using HindIII and EcoRI restriction enzymes). Finally, the expression vector contains a screening gene—hygromycin gene, and a pair of TALENs are placed under the CaMV 35S (cauliflower mosaic virus) promoter and Ubi (maize ubiquitin 1) promoter, such as image 3 shown. For plant expression vector plasmid information, see figure 2 .

[0213] The ubi+MCS+nos expression cassette sequence is shown in SEQ ID NO.:30.

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Abstract

The invention provides a pair of TALENs for efficiently editing a rice WAXY gene, and an identification targeting site and an application thereof, and concretely provides a transcription activator-like effect nuclease (TALEN) gene for efficiently targeting the rice endogenous WAXY gene, and a method using a recombinant plasmid containing the gene to carry out oriented targeting. The TALENs contain a pair of TALE proteins and DNA endonuclease catalytic subunits respectively fused with the TALE proteins, and can respectively identify and cut two adjacent sites of the rice endogenous WAXY gene. Synthesis of a nucleotide sequence for encoding the TALENs and construction of a vector including the nucleotide sequence are realized on the basis of designing the amino acid sequence of the TALEs, and the cell targeting efficiency is greatly improved through plasmid transfection of cells by the TALENs.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering. Specifically, the invention relates to a pair of TALENs for efficiently editing the rice WAXY gene, its recognition targeting site and its application. Background technique [0002] The cultivation of rice in the world can be traced back to 10,000 years ago, has a long history, and rice feeds 50% of the world's population at the same time. With the improvement of social living standards and the continuous improvement of people's living standards, people's requirements for rice have gradually shifted from quantity to quality, so the quality of rice has attracted more and more attention. Rice quality mainly consists of milling quality, appearance quality, cooking and eating quality and nutritional quality. Among them, the quality of cooking and eating is the indicator that consumers are most concerned about. The eating quality of rice is largely related to the relative content...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/11C12N9/22C12N15/55C12N5/04A01H5/00A01H6/46
Inventor 王磊许奇武原辉刘远红
Owner 深圳华大基因农业控股有限公司
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