Novel reaction system for rapidly constructing plant gene site-directed knockout carrier

A site-directed knockout and reaction system technology, applied in the field of molecular biology, can solve the problems that are unfavorable for rapid and high-throughput gene site-directed knockout vector construction, limit vector throughput, and high construction costs, so as to reduce experimental losses and reduce the use of and the effect of reducing the cost of material resources

Inactive Publication Date: 2017-10-17
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process is not only relatively cumbersome and time-consuming, but also depends on some imported kits and enzymes, and the construction cost is high
The most important thing is that such a two-step construction system greatly limits the throughput of vector construction, which is not conducive to the construction of rapid and high-throughput gene-directed knockout vectors

Method used

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  • Novel reaction system for rapidly constructing plant gene site-directed knockout carrier
  • Novel reaction system for rapidly constructing plant gene site-directed knockout carrier
  • Novel reaction system for rapidly constructing plant gene site-directed knockout carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 Construction method of the vector based on CRISPR-Cas system

[0048] 1) Use the pOsU3-gRNA plasmid as a template, use sgRNA recombination primers (Table 1) to amplify the OsU3-sgRNA sequence, and homologously integrate it into the pCAMBIA 1305.1 linear vector after double digestion with EcoRI and KpnⅠ

[0049] ( figure 1 ). The ligation product was transformed into DH5a, and positive clones were selected for sequencing. A new plasmid vector containing the correct sequence of OsU3-sgRNA was obtained by sequencing, named pCAMBIA 1305.1-sgRNA;

[0050] 2) Using the pJIT163-2NLSCas9 plasmid as a template, the genomic sequence encoding Cas9 was amplified using Cas9 recombination primers (Table 1), and integrated into the pCAMBIA 1305.1-SgRNA vector digested with Xba I and Hind III by homologous recombination. The ligation product was transformed into DH5a, and positive clones were selected for sequencing. A new plasmid containing the correct sequence of the...

Embodiment 2

[0054] Example 2 The reaction system capable of rapidly constructing rice gene-directed knockout vectors

[0055] ① Appropriate amount of 100ng of 1305CRISPR plasmid DNA prepared in 1; ② 10X buffer 1 μL of restriction endonuclease AarⅠ; ③ 0.2 μL of 50X Oligo of restriction endonuclease AarⅠ; ④ 1 μL of double-stranded DNA of target site; ⑤ Restriction endonuclease AarⅠ0. 2 μL; ⑥T4 DNA ligase 0.1 μL; ⑦ATP 1 μL, add water to a total volume of 10 μL.

Embodiment 3

[0056] Example 3 The method for rapidly constructing rice gene-directed knockout vector

[0057] The reaction system described in Example 2 was placed at 37° C. for 5 minutes and 16° C. for 5 minutes. After 5-10 cycles, the reaction product was directly used to convert DH5a. After the colonies grown on the plate were cultured overnight at 37°C, 1-3 colonies were randomly selected and sent to the company for sequencing confirmation. Any plasmid containing the target gene sequence at the cloning site is the correct plasmid that has been constructed.

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Abstract

The invention discloses a novel reaction system for rapidly constructing a plant gene site-directed knockout carrier. The system comprises 1) based on a CRISPR-Cas system carrier, the carrier comprises the DNA molecules having the following characteristics: an agrobacterium infection method is used for carrier conversion to the plant, a carrier capable of simultaneously realizing driving of SgRNA by an OsU3 promoter for transcription and driving of Cas9 protein by an Ubiquitin promoter for expression; 2) restriction enzyme AarI; 3) buffer and Oligo required by a reaction of the restriction enzyme AarI; 4) target gene site specific DNA double chain; and 5) T4DNA ligase and ATP required by an enzyme reaction. The system only requires 5-10 reaction cycles for rapidly and efficiently completing a construction process of plasmid, greatly simplify the operation flow, and satisfies the requirement of rapid construction with high flux for plant gene site-directed knockout plasmid.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a novel reaction system capable of rapidly constructing plant gene-directed knockout vectors. Background technique [0002] The CRISPR-Cas system is an acquired immune system of bacteria. When bacteria are infected by phages, they can obtain DNA fragments of phages and integrate them into the genome. When this type of phage invades bacteria again, through the specific recognition of the invading phage nucleic acid, the CAS protein is used to cut, so that this type of phage is degraded, thereby forming immunity. [0003] The full name of CRISPR is Clustered regularly interspaced short palindromic repeats, that is, clustered, regularly spaced, short palindromic repeats. The unusual stretch of DNA was first discovered by Osaka University in Japan while studying a bacterial gene encoding alkaline phosphatase. These fragments are composed of simple repeats, and there are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/22A01H5/00
CPCC12N9/22C12N15/8213
Inventor 万建民陈赛华江玲王益华王迪郑天慧李景芳赵志刚刘裕强
Owner NANJING AGRICULTURAL UNIVERSITY
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