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Plasmid vector and method for building plant population by using plasmid vector

A plasmid vector, plant technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, and introduction of foreign genetic material using vectors, which can solve the problems of low coverage of whole genome coding genes, complicated operation and high cost

Active Publication Date: 2018-05-15
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mutant populations constructed by physical and chemical mutagenesis and insertional mutagenesis have problems such as complex operations, low mutation rate, long time-consuming screening and isolation, and high cost.
For example, for physical and chemical mutagenesis of rice mutant populations, tedious hybridization, gene map cloning and other processes are involved in the later stage; for insertion mutant populations, the target gene is easier to clone, but the coverage rate of the whole genome coding gene is not high
In particular, they all involve a large-scale seed production process in the later stage, which consumes a lot of manpower and material resources.

Method used

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  • Plasmid vector and method for building plant population by using plasmid vector
  • Plasmid vector and method for building plant population by using plasmid vector
  • Plasmid vector and method for building plant population by using plasmid vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Embodiment 1: Construction of recombinant plasmid

[0084] The technical route for constructing the carrier is as follows:

[0085] 1. Construction of pUbi-ccdB-Cas9 recombinant plasmid

[0086] Hind III digestion was performed on the binary vector pH3 owned by our laboratory, and the 8.8kb vector backbone was recovered, which was named pH4 after self-ligation with T4DNA Ligase (Takara). The elements included in pH4 are: CaMV35S promoter, hygromycin gene, NOS terminator, pVS1RepA, pVS1 origin of replication. Use BsaI and Hind III to perform double enzyme digestion on pH4, and recover a 5.5kb large fragment, which includes the main elements: CaMV35S promoter, hygromycin gene, and NOS terminator. At the same time, using pH4 as a template, H3-F (SEQ ID No.8, introducing a restriction endonuclease BsaI site) and H3-R (SEQ ID No.9) as primers, using the high-fidelity enzyme I- 5 TM 2 × High-Fidelity Master Mix (purchased from Cloning (Beijing) Biotechnology Co., Ltd.) a...

Embodiment 2

[0092] Example 2: Using pHZLib2 system to construct a plasmid library of rice endogenous MPK gene family and rice transformation

[0093] 1. Construction of pHZLib2-OsMPKs plasmid library

[0094] Synthetic oligonucleotide sequence OsMPK1-oligo-1 (SEQ ID No.16: CCCGCGCGCTGTCGCTTGTGTG TCATCCAGTACAACATCTT GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG, the underline is the target nucleotide sequence of OsMPK1), OsMPK2-oligo-1 (SEQ ID No.17: CCCGCGCGCTGTCGCTTGTGTG ATGGCCATCACGGTGGCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG, the underline is the target nucleotide sequence of OsMPK2), OsMPK3-oligo-1 (SEQ ID No.18: CCCGCGCGCTGTCGCTTGTGT GAAGTATTACTACTCGATG GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG, the underline is the target nucleotide sequence of OsMPK3), OsMPK4-oligo-1 (SEQ ID No.19: CCCGCGCGCTGTCGCTTGTGTG CTAATGGCATGGGAAACCA GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG, the underline is the target nucleotide sequence of OsMPK4), OsMPK5-oligo-1 (SEQ ID No.20: CCCGCGCGCTGTCGCTTGTGTG TCAGGCC GACGATGACGCA GTTTTAG...

Embodiment 3

[0106] Example 3: Detection of OsMPKs rice mutant populations

[0107] 1) Extraction of genomic DNA

[0108] About 0.1 g of the leaves of the T0 generation plants were cut and ground with a grinder after quick freezing in liquid nitrogen; 600 μl of 2×CTAB DNA extract (containing 1 / 1000 β-mercaptoethanol) was added, shaken and mixed, and then lysed at 65°C for 45 minutes. Add 500 μl of chloroform and shake vigorously to form an emulsion, and centrifuge at 14000 rpm for 10 min. After centrifugation, transfer the supernatant to a 1.5ml centrifuge tube, add an equal volume of isopropanol, invert and mix well, centrifuge at 14000rpm for 10min, discard the supernatant, add 700μl 70% ethanol to wash the white precipitate, centrifuge at 14000rpm for 5min, discard the supernatant Place the centrifuge tubes in a fume hood to air dry for 10 min. Add 30 μl ddH 2 O dissolves DNA. The DNA solution was stored at -20°C for later use.

[0109] 2) PCR amplification and sequencing detection...

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Abstract

The invention relates to a plasmid vector and a method for building a plant population by using the plasmid vector. The plasmid vector contains, at a replication origin, a gene expression box for expressing Cas9 protein, a first promoter, nucleotide sequences containing suicide gene sequences, a gRNA scaffold element and a termination signal, and at least one first enzyme cutting site located on the nucleotide sequence 5' end containing the suicide gene sequence and at least one second enzyme cutting site located at the nucleotide sequence 3' end containing the suicide gene sequence; furthermore, no first enzyme cutting site and no second enzyme cutting site exist in other positions of the plasmid vector. The first promoter is located at the upstream of the termination signal; the nucleotide sequences containing suicide gene sequences and the gRNA scaffold element are all located between the first promoter and the termination signal.

Description

technical field [0001] The application relates to a plasmid vector and a method for establishing a plant population using it. Background technique [0002] CRISPR / Cas9 genome editing technology has become an important means of current plant gene function and application research. It can realize the gene targeting technology of artificially modifying the genetic material at a specific site in the genome of an organism, and the genetic information changes caused by it can be stably transmitted and functionally presented between generations. The principle of genome-specific editing is to use artificial nucleases to cut at the target site of the biological genome to generate a DNA double-strand break (DSB), thereby activating the cell's DSB repair mechanism to achieve the goal. Generally, this technology can realize the mutation of one site, or the simultaneous screening of a few site mutations. [0003] Although CRISPR / Cas9 technology has been used to create large-scale mutan...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N9/22A01H5/00A01H6/46
CPCC12N9/22C12N15/8213
Inventor 周焕斌旷永洁严芳
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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