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Method of constructing cotton mutant library with high through throughput based on CRISPR/Cas9 system

A high-throughput, mutant technology for genetic engineering

Inactive Publication Date: 2019-03-26
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, in plants, the high-throughput application of CRISPR / Cas9 has only been reported in rice, and cotton, as an important fiber crop, has not been reported so far

Method used

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  • Method of constructing cotton mutant library with high through throughput based on CRISPR/Cas9 system
  • Method of constructing cotton mutant library with high through throughput based on CRISPR/Cas9 system
  • Method of constructing cotton mutant library with high through throughput based on CRISPR/Cas9 system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Cloning and optimization of tRNA and CcdB gene

[0044] 1. Cloning of tRNA

[0045] The tRNA was amplified using the reported vector PGTR4 as a template. The PCR system and PCR conditions are shown in Table 1-2. PCR primers are described below.

[0046] Table 1 tRNA cloning PCR system

[0047] components

Volume (μL)

double distilled water

16.1

10×taq Buffer

2

dNTP

0.3

S primer

0.2

As primer

0.2

Taq

0.2

template-PGTR4

1

[0048] Table 2 PCR conditions for tRNA clones

[0049]

temperature

time

pre-denatured

95℃

4min

transsexual

95℃

30s

annealing

58℃

30s

extend

72℃

10s

cycle

28

last extension

72℃

5min

save

4℃

∞

[0050] The primer sequences for amplifying tRNA were tRNA-S (5'-AAGAAGCATCAGATGGGCAAACAAAGCACCAGTGGT-3') and tRNA-AS (5'-CTGCATCGGTCTCCTGCACCAGCCGGG-...

Embodiment 2

[0063] Embodiment 2: Construction of Htkt (High-throughput-kan-tRNA) carrier

[0064] 1. Connection of tRNA-CcdB and pRGEB32-GhU6.7-NPTⅡ

[0065] Firstly, the pRGEB32-GhU6.7-NPTⅡ vector was digested, and the enzyme digestion system was shown in Table 7, and placed at 37°C for 5 hours, and then the enzyme digestion product was digested using a gel recovery kit (purchased from Wuhan Huaxin Sunshine Biotechnology Co., Ltd.) purification.

[0066] Table 7 50μL enzyme digestion system

[0067]

[0068]

[0069] The digested pRGEB32-GhU6.7-NPTII vector and the tRNA-CcdB fragment were ligated with the ClonExpress II OneStep Cloning Kit (Vazyme C112-02). The ligation system was shown in Table 8. The ligated product was transformed into Escherichia coli competent. Pick positive clones for sequencing, and name the plasmid with correct sequencing as Htkt (High-throughput-kan-tRNA) (the sequence of this vector is shown in SEQ ID NO: 5).

[0070] Table 8 5μL connection system

[...

Embodiment 3

[0072] Embodiment 3: Construction of Htkt-sgRNA carrier

[0073] 1. sgRNA design of upland cotton genes

[0074] For 280 upland cotton genes, about three sgRNAs were designed in the exon region of each gene (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR), a total of 844 sgRNAs were designed, respectively Named sgRNA1, sgRNA2...sgRNA844, synthesized by GenScript Biotechnology Co., Ltd.

[0075] 2. Mixture of 844 sgRNAs

[0076] Mix 50 tubes of sgRNA with 10 μL each into one tube. Name the mixed sgRNA from the 1st to 50th tube as H1, and name the mixed sgRNA from the 51st to 100th tube as H2, and so on, and mix the sgRNA from the 801st to 844th tube Named H17. There are 17 tubes of sgRNA in total.

[0077] 3. Overlap extension PCR amplification of sgRNA double strands

[0078] Overlap extension PCR amplification of sgRNA double-stranded primers are Htkt-S1 (5'-TTCCCGGCTGGTGCA-3') and Htkt-AS1 (5'-ATTTCTAGCTCTAAAAC-3'). The PCR reaction conditions are shown in Table 9, an...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method of constructing a cotton mutant library with high through throughput based on a CRISPR / Cas9 system. A converting vector of upland cotton is constructed, and an application of high throughput edition in a genome of upland cotton by using the vector is carried out. TRNA and CcdB gene fragments are cloned by means of primers with joints and the gene fragments are connected to a Prgeb32-gHu6.7-NBTII converting vector edited by the genome of upland cotton to form a vector Htkt which canedits the genome of upland cotton with high throughout. By means of the characteristic of the high throughput vector, upland cotton drone fragments can be designed in batches and are mixed to construct the vector to obtain the cotton mutant library finally. A high throughput sequencing result verifies that the sgRNA coverage rate of the high throughput vector reaches up to 97%.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a method for constructing a cotton mutant library based on a high-throughput CRISPR / Cas9 system to construct a high-throughput transformation vector for upland cotton, which can be used to perform high-throughput transformation in the upland cotton genome edited application. 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 it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/82C12N15/66A01H5/00A01H6/60
CPCC12N15/8218C12N15/902
Inventor 郭小平徐孝兰张军
Owner HUAZHONG AGRI UNIV