Establishment and application of plant multi-gene knockout vector

A technology for knocking out vectors and multiple genes, which can be used in the introduction of foreign genetic material using vectors, recombinant DNA technology, etc., which can solve the problems of time-consuming, labor-intensive, and inability to obtain multiple mutants.

Active Publication Date: 2015-12-02
CHINA NAT RICE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has the following problems: 1. There may not be the required single mutant; 2. If there is a close linkage bet

Method used

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  • Establishment and application of plant multi-gene knockout vector
  • Establishment and application of plant multi-gene knockout vector
  • Establishment and application of plant multi-gene knockout vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1, construction of binary expression vector pC1300-Cas9

[0043] With primer pair 35S-F:5'-AAA AAA GTACCCCTACTCCAAAAATG-3'( labeled as KpnI, Marked as BamHI restriction site) and 35S-R:5'-ACA AGCTTGGGCTGTCCTCTCC-3'( Marked as the BglII restriction site) PCR amplification of plasmid pJIT163-2NLSCas9 (provided by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences), KODFX DNA polymerase (purchased from Toyobo Biotechnology Co., Ltd.) PCR amplification of the target band, reaction conditions as follows:

[0044]

[0045] Reaction program: denaturation at 94°C for 2 minutes; then denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, extension at 68°C for 40 seconds, and 31 cycles of amplification; finally extension at 68°C for 5 minutes.

[0046] A constitutive 2×CaMV35S promoter fragment of about 740bp was obtained, which was double-digested with KpnI and BglII and then ligated into the KpnI and BamHI rec...

Embodiment 2

[0047] Embodiment 2, the construction of intermediate carrier SK-gRNA

[0048] Use primer pair gRNA-F:5'-TTG GA CTCGAG GATTATGTGGAAAAAAAAGCACC-3'( Marked as KpnI restriction site, Marked as BamHI restriction site, ____ is marked as XbaI restriction site, _ is marked as XhoI restriction site) and gRNA-R:5'-CCGCGGTG GCTAGC CGATTAAGGAATCT-3'( Marked as the NotI restriction site, Marked as BglII restriction site, _ marked as NheI restriction site, ____ marked as SalI restriction site) PCR amplification plasmid pU3-gRNA (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences); KODFX DNA polymerase ( Purchased from Toyobo Biotechnology Co., Ltd.) PCR amplification target band, the reaction conditions are as follows:

[0049]

[0050] Reaction program: denaturation at 94°C for 2 minutes; then denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, extension at 68°C for 30 seconds, and 31 cycles of amplification; finally exten...

Embodiment 3

[0055] Example 3, Selection of target sequence in monocot rice and construction of a single gRNA intermediate vector

[0056] In the present invention, four genes of OsPDS, Os02g23823, OsMPK2 and Os05g02070 are simultaneously knocked out as an example, but not limited thereto.

[0057] 1. Selection of target sequence and primer design

[0058] Find on the genome sequence of the target gene NNNNNNNNNNNNNNNN↓NNN The sequence is the target sequence (single underscore indicates the target sequence, double underscore indicates the PAM sequence, ↓ indicates the proposed cutting mutation site of CAS9 protein, at the first 3 bases of PAM.) Design two complementary DNA sequences: in the forward direction Add GGCA before the sequence and AAAC before the reverse complementary sequence. The specific structure is as image 3 shown.

[0059] details as follows:

[0060] Target gene: OsPDS, gene accession number: LOC_Os03g08570.1, target sequence: TTGGTCTTTGCTCCTG↓CAG (The double u...

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Abstract

The invention discloses establishment and application of a plant multi-gene knockout vector. A method of the establishment of the plant multi-gene knockout vector comprises the following steps of (1) establishing a binary expression vector pC1300-Cas9; (2) establishing an intermediate vector SK-9RNA; (3) establishing single objective gRNA; and (4) serially connected a plurality of pieces of gRNA and establishing a final binary expression vector. A plurality of gRNA sequences are connected to the binary expression vector of an existing Cas9 sequence by an isocaudamer connecting method, and multiple plant mutants can be obtained by an agrobacterium tumefaciens infected transgenic technology. By an isocaudamer connecting establishment strategy, pieces of gRNA can be combined, and multiple plant mutants can be obtained by transgenosis once, so that an efficient and convenient multiple plant mutants preparing method is established.

Description

technical field [0001] The invention belongs to the technical field of carrier construction using genetic engineering methods in biotechnology. Background technique [0002] Multiple mutants are of great significance in the study of basic plant functions and crop breeding. The traditional method of obtaining multiple mutants is to complete by crossing each other and segregating offspring between single mutants. However, this method has the following problems: 1. There may not be the required single mutant; 2. If there is a close linkage between several genes, it is almost impossible to obtain multiple mutants; 3. The whole process is time-consuming and labor-intensive. [0003] In recent years, the modified Type II CRISPR-Cas9 system has successfully achieved targeted genome modification in multiple species, proving that CRISPR-Cas9 is a simple, efficient and widely used tool for editing genomes. The system mainly uses endonuclease Cas9 and positioned guide RNA (gRNA) mole...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/82
Inventor 王克剑王春沈兰付亚萍严长杰
Owner CHINA NAT RICE RES INST
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