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Method for increasing length of wheat grains by using gene editing to knock out grain development related protein TaGSR1

A technology related to protein and wheat grains, applied in genetic engineering, plant genetic improvement, chemical instruments and methods, etc., to achieve broad market application prospects, important breeding application value, and the effect of increasing wheat yield

Active Publication Date: 2021-11-30
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after searching, it was found that the coding sequence of the wheat TaGSR1 gene and its encoded grain development-related protein TaGSR1, as well as the method of using gene editing to knock out the grain development-related protein TaGSR1 to increase the length of wheat grains, have not been reported.

Method used

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  • Method for increasing length of wheat grains by using gene editing to knock out grain development related protein TaGSR1
  • Method for increasing length of wheat grains by using gene editing to knock out grain development related protein TaGSR1
  • Method for increasing length of wheat grains by using gene editing to knock out grain development related protein TaGSR1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of expression vector

[0038] 1. Design of sgRNA targeting TaGSR1

[0039] In order to design an sgRNA capable of editing the coding region of the TaGSR1 gene, the sequence of the TaGSR1 gene was first amplified in the wheat variety Shixin 828. The primers used were TaGSR1-F and TaGSR1-R, and the PCR reaction system was: KOD-FXNEO buffer : 25 μL, dNTP (2mM): 10 μL, TaGSR1-F (10 μM): 1.5 μL, TaGSR1-F (10 μM): 1.5 μL, Shixin 828gDNA (50ng / μL): 1 μL, KOD-FX NEO: 1 μL, ddH 2 O make up 50 μL. The PCR reaction program was: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 12 sec; annealing at 58°C for 20 sec, extension at 68°C for 25 sec, and 35 cycles of reaction; renaturation at 68°C for 5 min.

[0040] PCR results such as figure 1 As shown, lane 1 is the Transgen 2K plus marker, and lane 2 is the target band of TaGSR1.

[0041] The PCR product was sent to Qingdao Qingke Zixi Biotechnology Co., Ltd. for sequencing. The sequence of the T...

Embodiment 2

[0050] Example 2 Obtaining and identification of transgenic offspring

[0051] 1. TaGSR1 transgenic offspring obtained

[0052] The recombinant binary expression vector pBUE411-TaGSR1 constructed in Experimental Example 1 was transformed into Agrobacterium EHA105 competent cells, specifically: the recombinant binary expression vector pBUE411-TaGSR1 was added to Agrobacterium EHA105 competent cells in an ice bath for 5 minutes, and then quickly frozen in liquid nitrogen 5min, heat shock reaction at 37°C for 5min, then ice-bathed for 5min, added LB without anti-antibody, placed in a shaking table at 28°C for 2h, and then applied to LB (containing rifampicin, streptomycin and kanamycin) with an applicator ) plate, cultured upside down at 28°C until clones grow out. Pick a single clone and inoculate it in the LB medium containing the corresponding resistance, shake the bacteria at 28°C, 160rpm, and cultivate for 24 hours.

[0053] The JW1 wheat seeds about 15 days after pollinat...

Embodiment 3

[0061] Example 3 Phenotypic identification of wheat offspring with TaGSR1 gene knockout

[0062] In order to obtain homozygous knockout lines with three homologous copies of TaGSR1.2A, TaGSR1.2B and TaGSR1, restriction amplified polymorphic sequence (Caps) markers were developed for the editing site, and the selfing of gene editing mutants Progeny were identified until a homozygous mutant for TaGSR1.2A, TaGSR1.2B and TaGSR1 simultaneous knockout was obtained.

[0063] TaGSR1.2A, Caps-2A, TaGSR1.2B and TaGSR1 genes were amplified by PCR. The PCR reaction system is: KOD-FX NEO buffer: 10 μL, dNTP (2mM): 4 μL, Caps-2A-F (or Caps-2B-F, or Caps-2D-F) (10 μM): 0.6 μL, Caps-2A -R (or Caps-2B-R, or Caps-2D-R) (10μM): 0.6μL, gene knockout strain gDNA (about 20ng / μL): 1μL, KOD-FX NEO: 0.4μL, ddH 2 O make up 20 μL. The PCR reaction program was: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 12 sec; annealing at 58°C for 20 sec, extension at 68°C for 45 sec, and 35 cycle...

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Abstract

The invention discloses a method for increasing the length of wheat grains by using gene editing to knock out grain development related protein TaGSR1. The method comprises the following steps of: designing single-stranded guide RNA of a targeted TaGSR1 gene, and constructing a plant binary expression vector pBUE411-TaGSR1 for knocking out the TaGSR1 gene in wheat; and transforming the plant binary expression vector into common wheat through agrobacterium to obtain transgenic wheat with the TaGSR1 gene knocked out, and increasing the length of the wheat grains, wherein the nucleotide sequence of the TaGSR1 gene is shown as SEQ ID NO. 4. Experiments prove that the transgenic wheat with the TaGSR1 gene knocked out increases the length of the wheat grains and the thousand seed weight. According to the technical scheme, a feasible method is provided for realizing rapid breeding of wheat and increasing the yield of wheat by using a gene engineering technology, and the method has important breeding application value and wide market application prospect.

Description

technical field [0001] The invention relates to a method for increasing the length of wheat grains, in particular to a method for increasing the length of wheat grains by knocking out the grain development-related protein TaGSR1 by gene editing; it belongs to the technical field of plant genetic engineering and molecular breeding. Background technique [0002] Wheat is the second largest food crop in the world, providing 20% ​​carbohydrates and 23% protein for humans. Human food security is faced with problems such as the reduction of arable land, global warming, continuous population growth, and increased environmental pollution. How to increase wheat production under limited resource conditions is of great significance to food security. [0003] Brassinolide (BR), as the sixth plant hormone in plants, plays a very important role in the processes of cell elongation and division, leaf senescence, plant morphogenesis, and plant stress resistance. Due to the extensive role of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/10A01H6/46
CPCC07K14/415C12N15/8261C12N15/8216
Inventor 樊敏吕金洋白明义李根英韩超
Owner SHANDONG UNIV