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System for acquiring dwarf rice by modifying OsHPH gene through CRISPR-Cas and application of system

A rice and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of decreased resistance to diseases and insect pests of varieties, single genetic background of rice varieties, and high plant height

Active Publication Date: 2018-06-01
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

However, since the 1960s, there are not many excellent dwarf sources available for breeding. Excessive use of limited dwarf sources may lead to a single genetic background of rice varieties, resulting in a decline in the resistance of varieties to diseases and insect pests
In particular, with the breakthrough and development of hybrid breeding technology, the full use of heterosis has also brought about the problem of high plant height

Method used

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  • System for acquiring dwarf rice by modifying OsHPH gene through CRISPR-Cas and application of system
  • System for acquiring dwarf rice by modifying OsHPH gene through CRISPR-Cas and application of system
  • System for acquiring dwarf rice by modifying OsHPH gene through CRISPR-Cas and application of system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, the vector construction of CRISPR / Cas9 of the coding gene OsHPH of rice control plant height-related protein OsHPH

[0054] 1. Acquisition of OsHPH gene

[0055] Using the genomic DNA of rice Kitaake (Oryza sativa var. Kitaake) as a template, PCR amplification was performed with the following primers primer1 and primer2 to obtain the target gene. The underlined part is the linker for In-Fusion enzyme ligation.

[0056] Primer1: 5'- ATCCTCTAGAGTCGAC ATGGTGGCCGAGCACCCCACGC-3';

[0057] primer2: 5'- ATCCTCTAGAGTCGAC TCAGCTGGCCGCCTCGAC-3'.

[0058] After recovering and purifying the PCR product, it was connected to the B-zero (purchased from Beijing Quanshijin Company) sequencing vector, transformed into DH5α competent cells, and the positive clones were selected for sequencing.

[0059] Sequencing results showed that the length of the amplified PCR product was 1.1 Kb, the sequence was the nucleotide sequence shown in SEQ ID No.3, and it was named OsHPH g...

Embodiment 2

[0068] Example 2, Cultivation of OsHPH gene edited transgenic plant Kitaake and its identification

[0069] 1. Breeding transgenic plants with edited OsHPH gene

[0070] CRISPR / Cas9-gRNA1 was mediated by Agrobacterium tumefaciens EHA105 to transform Kitaake japonica rice, the specific method is as follows:

[0071] 1. The recombinant vector CRISPR / Cas9-gRNA1 obtained in Example 1 was introduced into Agrobacterium tumefaciens EHA105 by heat shock method to obtain recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector CRISPR / Cas9-gRNA1. The recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector CRISPR / Cas9-gRNA1 was cultured at 28°C for 16 hours, and the cells were collected. The bacterium was diluted with N6 liquid medium (Sigma, catalog number C1416) containing 100 μM acetosyringone to obtain the diluted bacterial solution, and the OD of the diluted bacterial solution was 600 ≈0.5.

[0072] 2. Mix and infect the mature embryog...

Embodiment 3

[0080] Example 3. Breeding transgenic plants Nipponbare (Nip), Zhongzuo 1022 (1022), Zhongzuo 9017 (9017), Dongjing (DJ), Zhonghua 11 (ZH11), Dianjingyou (DJY) with edited OsHPH gene

[0081] Using the same method as in Example 2, the transgenic plants Nipponbare (Nip), Zhongzuo 1022 (1022), Zhongzuo 9017 (9017), Dongjing (DJ), Zhonghua 11 (ZH11), Yunnan For the results of Japonica Excellent (DJY), see Figure 2-8 As shown, the phenotype of reduced plant height can also be achieved in these varieties, which also fully demonstrates that OsHPH is a gene that regulates plant height in rice.

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Abstract

The invention provides a rice plant height related gene OsHPH, and an application to regulating the height of a rice plant. The gene is edited through a CRISPR-cas system to reduce the height of the rice plant at different degrees, and a T0-generation is subjected to segregation of posterity to breed a plant having no transgenic ingredients. The plant can be produced and applied as a crossing parent, and the undesirable phenomenon that a crossbred F1 is too high to cause lodging in the agricultural production is solved.

Description

technical field [0001] The present invention belongs to the related field of crop molecular breeding. Specifically, the present invention relates to the coding editing and application of plant genes using CRISPR / Cas9 technology, in particular to genes related to plant height development and their coding genes and applications. Background technique [0002] Rice (Oryza sativa L.) is one of the most important food crops in my country and even in the world. The increase of its yield is of great strategic significance to solve the future global food problems. Plant height is one of the most important agronomic traits in rice. The worldwide "Green Revolution" is marked by crop dwarf breeding. However, since the 1960s, there are not many excellent dwarf sources available for breeding. Excessive use of limited dwarf sources may lead to a single genetic background of rice varieties, resulting in a decline in the resistance of varieties to diseases and insect pests. In particular, ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29C12N15/113A01H5/00A01H6/46
CPCC07K14/415C12N15/113C12N15/8213C12N15/8261C12N2310/10C12N2810/10
Inventor 隋毅孙尧阴涛吴传银张皓珊程子祥
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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