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Method for editing cotton genes

A genome editing, base technology, applied in the field of genetic engineering

Active Publication Date: 2018-06-26
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there have been many reports about the application of CRISPR / Cas9 system in different organisms, but cotton, as an important fiber crop, has not been reported so far.

Method used

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  • Method for editing cotton genes
  • Method for editing cotton genes
  • Method for editing cotton genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Cloning and optimization of pGhU6-7 promoter and pGhU6-9 promoter

[0050] 1. Blast of the pGhU6 promoter

[0051] Primers were designed using Primer5 software by homologous alignment with the Arabidopsis U6-26 snRNA gene.

[0052] 2. Cloning of the promoter

[0053] Using the genomic DNA of upland cotton YZ1 (Jin et al.2006) as a template, use the following PCR system and program to amplify to obtain two pGhU6, and finally use pGEM-T Easy Vector Systems for cloning and sequencing verification, and separate the promoters with the correct sequence Named as pGhU6-7 promoter, pGhU6-9 promoter. The primer sequences for amplifying the pGhU6-7 promoter are respectively pGhU6-7F (5'-TTAATCTGATGCTCCACCTGCTTTTGAT-3') and pGhU6-7R (5'-CATCTGATGCTTCTTTCGCTTATATAAACG-3'), and the primer sequences for amplifying the pGhU6-9 promoter are respectively pGhU6-9F (5'-TGATTGCTAGAATTTGCAGTAGGCTT-3') and pGhU6-9R (5'-CATCTGATGCTTCTTTCGCTTATAAACG-3'). The PCR system is shown i...

Embodiment 2

[0060] Example 2: Construction of pRGEB32-GhU6.7-NPTII and pRGEB32-GhU6.9-NPTII vectors

[0061] 1. Connection of pRGEB32 with pGhU6-7I and pGhU6-9I

[0062] Firstly, the pRGEB32 vector was digested, and the digestion system was shown in Table 3, placed at 37°C for 5 hours, and then the digested product was purified using a gel recovery kit (purchased from Wuhan Huaxin Sunshine Biotechnology Co., Ltd.).

[0063] Table 3 50μL enzyme digestion system

[0064]

[0065] Amplify from plasmids with the correct sequences of pGhU6-7I and pGhU6-9I, introduce BSAⅠ and gRNA sequences by overlapping extension PCR, and then use a gel extraction kit to purify the PCR products, digest the PRGEB32 vector with pGhU6-7I, pGhU6 The -9I fragment was ligated by ClonExpress II One Step Cloning Kit (Vazyme C112-02), transformed into Escherichia coli competent, and positive clones were picked for sequencing, and the sequenced correct plasmids were named pRGEB32-GhU6.7, pRGEB32-GhU6. 9.

[0066]...

Embodiment 3

[0077] Example 3: Construction of pRGEB32-GhU6.7-NPTⅡ-sgRNA and pRGEB32-GhU6.9-NPTⅡ-sgRNA vectors

[0078] 1. sgRNA design of GhCLA gene

[0079] Four sgRNAs were designed in the exon region of Gh_A10G2292 gene of upland cotton 1-deoxyxylulose-5-phosphate synthase (Cloroplasto alterados, CLA), named sgRNA1, sgRNA2, sgRNA3, sgRNA4 respectively.

[0080] Table 7GhCLA gene sgRNA statistical table

[0081]

[0082] 2. sgRNA is connected to pRGEB32-GhU6.7-NPTⅡ, pRGEB32-GhU6.9-NPTⅡ vector

[0083]The sequence inserted into the vector is the repeat sequence of tRNA and gRNA, and the repeat sequence is spliced ​​by overlapping extension PCR (Urban et al.1997). The target is added with a primer adapter, and then connected to the vector by a one-step cloning method. Using gRNA2 and gRNA4 as combination 1, and gRNA1 and gRNA3 as combination 2, they were ligated with pRGEB32-GhU6.7-NPTⅡ and pRGEB32-GhU6.9-NPTⅡ vectors respectively. The successfully constructed vectors were named 7NC1...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for editing cotton genes. A CRISPR-Cas9 system for cotton genome editing and a construction method thereof are disclosed. According to the method, two upland cotton U6 promoters pGhU6-7 and pGhU6-9 are obtained by cloning, and Bsa I restriction site is mutated. Two vectors pRGEB32-GhU6.7-NPTII and pRGEB32-GhU6.9-NPT II of the CRISPR-Cas9 vector with editing capability in cotton are constructed using an optimized promoter. A cotton endogenous gene GhCLA is selected as a target gene, and four sgRNAs targeting the gene are designed to guide the Cas9 protein to cleave at the specific site of GhCLA. The whitening phenotype, enzymatic map and sequencing of cotton showed that a CRISPR / Cas9 gene editing system driven by pGhU6-7I and pGhU6-9I has editing capability in cotton.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for editing the genome of upland cotton. The invention relates to constructing two high-efficiency transformation vectors of upland cotton, and using the two vectors to edit the upland cotton genome. Background technique [0002] Gene mutation is one of the common methods to study gene function, from EMS mutation, radiation mutagenesis, T-DNA insertion, transposon insertion and other random mutation methods, to zinc finger nuclease (ZFN) and transcription activator-like effector nucleic acid Enzyme (TALEN) target gene site-directed modification technology is a breakthrough in the field of biotechnology. CRISPR refers to a short palindromic repeat sequence with clustered regular intervals. After the target sequence is cut through the CRISPR system, the individual organism will start its own repair mechanism, including non-homologous recombination repair and h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66C12N15/113
CPCC12N9/1247C12N15/66C12N15/8205C12Y207/07006
Inventor 金双侠张献龙张军王鹏程涂礼莉梁思佳孙琳
Owner HUAZHONG AGRI UNIV
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