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Method of developing corn dwarfing material via gene editing

A gene editing, corn technology, applied in the fields of biotechnology and genetic breeding, can solve the problems of reduced yield, lodging, and incomplete application of corn breeding

Pending Publication Date: 2019-08-16
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The improvement of maize production in the 20th century mainly relied on increasing the planting density of maize per unit area (Duvick et al., 2010). However, if the density is too high, there will be the risk of lodging, which may reduce the yield, so it will reduce to a certain extent. Maize Plant Height Helps Increase Maize Yield
Many maize dwarf mutants have been identified over the past few decades but have not been fully utilized in maize breeding

Method used

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  • Method of developing corn dwarfing material via gene editing
  • Method of developing corn dwarfing material via gene editing
  • Method of developing corn dwarfing material via gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Design of editing sites based on CRISPR / Cas9

[0029] Design target sites through CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR2 / ) website, maize target genes ZmGA20ox3, ZmGA20ox5 and related information from the database MaizeGDB (https: / / www.maizegdb.org / ) download. Upload the exon sequence of the target gene to the CRISPR-P website, select the reference genome as the maize genome, and obtain potential editing sites near 5'-NGG. Two target sites are designed for each gene and located on the first exon of the gene, and the two target sites (located in different expression cassettes) are connected in series to the same gene editing vector. ZmGA20ox3 target site sequence is 5’-GGAGCCATTCCTGTGGCCGC-3’ and 5’-CTGTCCTTCGGCTTCCACGA-3’, ZmGA20ox5 target site sequence is 5’-AGATCCCCGCGCCATTCCTG-3’ and 5’-CTGTCGTTCGGCTACCACGA-3’,

Embodiment 2

[0030] Example 2 Construction of Gene Editing Vectors

[0031] Primers were designed according to the selected editing sites and the restriction sites at the multiple cloning sites of vectors pCBC-MT1T2 and pBUE411. The primer information is shown in Table 1:

[0032] Table 1 Amplification Primers

[0033]

[0034]

[0035] Use two rounds of PCR to amplify the target fragment. The first round of PCR reaction uses the first pair of primers MT1-F0 / MT2-R0 to amplify the pCBC-MT1T2 plasmid as a template, and the second round of PCR reaction uses the second pair of primers MT1-BsF / MT2-BsR uses the first-round amplification product as a template to amplify, connects the two target sites in series, and obtains the target fragment.

[0036] The first round reaction system is:

[0037]

[0038] The second round reaction system is:

[0039]

[0040] The two-round PCR amplification reaction program was: 94°C for 2min; 98°C for 10s, 58°C for 30s, 68°C for 1min, 35 cycles; ...

Embodiment 3

[0050] Example 3 Gene Editing Vector Transformed into Agrobacterium LBA4404

[0051] The gene ZmGA20ox3 and ZmGA20ox5 editing vectors constructed in Example 2 were respectively transformed into Agrobacterium LBA4404, and the specific steps were as follows:

[0052] (1) Take 5 μL of the plasmid and add it to 200 μL of Agrobacterium competent cells;

[0053] (2) Ice bath for 30 minutes;

[0054] (3) Take it out from the ice, and quickly place it in liquid nitrogen for quick freezing for 5 minutes;

[0055] (4) Take it out from the liquid nitrogen and incubate in a water bath at 37°C for 5 minutes;

[0056] (5) Take it out and put it on ice for 5 minutes;

[0057] (6) Add 800 μL of blank YEB medium, and recover on a shaker at 28°C at 200 rpm for 4-5 hours;

[0058] (7) 4000rpm normal temperature centrifugation for 5min;

[0059] (8) Discard part of the supernatant, and suspend the precipitate with the remaining about 200 μL supernatant, spread it on a YEB solid culture plate...

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Abstract

The invention provides a method of developing a corn dwarfing material via gene editing. The method comprises designing, for target gene ZmGA20ox3 / ZmGA20ox5 of corn, a sgRNA based on CRISPR / Cas9, connecting a fragment having sgRNA-coding DNA to a Cas-carrying carrier, and converting the corn with the constructed carrier to achieve site-directed mutagenesis of the gene ZmGA20ox3 / ZmGA20ox5 so as toobtain a transgenic corn plant with corresponding gene function deficiency. The biological functionality of the corn gene ZmGA20ox3 / ZmGA20ox5 is disclosed herein for the first time; the corn gene ZmGA20ox3 / ZmGA20ox5 is edited through CRISPR / Cas9 technology; a mutant material with no transgenic gene insert fragment is acquired through further screening; the corn dwarfing material has important breeding value.

Description

technical field [0001] The invention relates to the fields of biotechnology and genetic breeding, in particular to a method for creating dwarf corn materials using gene editing technology. Background technique [0002] The improvement of maize production in the 20th century mainly relied on increasing the planting density of maize per unit area (Duvick et al., 2010). However, if the density is too high, there will be the risk of lodging, which may reduce the yield, so it will reduce to a certain extent. Maize plant height helps to increase maize yield. At the end of the 1960s, the world-famous "Green Revolution" set off a frenzy that semi-dwarf plants could increase yields. High-yield varieties of semi-dwarf rice and wheat were successfully created during the "Green Revolution". Many maize dwarf mutants have been identified over the past few decades but have not been fully utilized in maize breeding. [0003] Gibberellins (GAs), a natural tetracyclic diterpene carboxylic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8261
Inventor 刘允军张姣姣王国英
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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