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A kind of plasmid carrier and the method for using it to establish plant colony

A plasmid vector and nucleotide sequence technology, applied in the field of plasmid vectors and the establishment of plant populations by using them, can solve the problems of low coverage of whole genome coding genes, long time for screening and separation, low mutation rate, etc., so as to reduce false positives. The effect of plant probability, reducing field workload and increasing positive rate

Active Publication Date: 2021-09-28
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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  • A kind of plasmid carrier and the method for using it to establish plant colony
  • A kind of plasmid carrier and the method for using it to establish plant colony
  • A kind of plasmid carrier and the method for using it to establish plant colony

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 TC ATCCAGTACAACATCTT 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 GTTT...

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 present application relates to a plasmid vector and a method for establishing a plant mutant population on a large scale using the plasmid vector. The plasmid vector comprises at the origin of replication, a gene expression cassette for expressing the Cas9 protein, a first promoter, a nucleotide sequence comprising a suicide gene sequence, a gRNA scaffold element and a termination signal, and a nucleoside located at the said suicide gene sequence At least one first restriction site at the 5' end of the acid sequence and at least one second restriction site at the 3' end of the nucleotide sequence comprising the suicide gene sequence; and no other position in the plasmid vector The first restriction site and the second restriction site; wherein, the first promoter is located upstream of the termination signal, and the nucleotide sequence comprising the suicide gene sequence and the gRNA scaffold Elements and are located between said first promoter and said 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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Patent Type & Authority Patents(China)
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