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Method for obtaining rice changed in tillering by utilizing CRISPR-Cas to modify an OsTAC1 gene

A rice and gene technology, applied in the field of genetic engineering, can solve problems such as mistargeting, affecting the specific knockout of target genes, etc.

Active Publication Date: 2018-05-04
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

Therefore, whether the sgRNA can specifically and accurately target the target gene is a prerequisite for whether CRISPR / Cas9 can specifically knock out the target gene. Whether it is off-target or wrongly targeted, it will affect the specificity of CRISPR / Cas9 to the target gene knockout

Method used

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  • Method for obtaining rice changed in tillering by utilizing CRISPR-Cas to modify an OsTAC1 gene
  • Method for obtaining rice changed in tillering by utilizing CRISPR-Cas to modify an OsTAC1 gene
  • Method for obtaining rice changed in tillering by utilizing CRISPR-Cas to modify an OsTAC1 gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The construction of embodiment 1 expression vector

[0026] According to the backbone sequence of the pCas9 vector cut by AarI, add the linker sequence AGATGATCCGTGGCA...N before and after the 20bp obtained 20 …GTTTTAGAGCTATGC was used as the F primer, and the obtained F primer was reverse-complemented to obtain the R primer, which was sent to the company for synthesis (sequence is as follows).

[0027] AGATGATCCGTGGCA TGCACCATCAATGAGAACAA GTTTTAGAGCTATGC sgRNA-exon2-F

[0028] GCATAGCTCTAAAAC TTGTTTCCATTGATGGTGCA TGCCACGGATCATCT sgRNA-exon2-R

[0029] AGATGATCCGTGGCA ATACTTGCAATTGGCACGCT GTTTTAGAGCTATGC sgRNA-exon3-1-F

[0030] GCATAGCTCTAAAAC AGCGTGCCAATTGCAAGTAT TGCCACGGATCATCT sgRNA-exon3-1-R

[0031] AGATGATCCGTGGCA CGAAAATCGTCATTGTTGCT GTTTTAGAGCTATGC sgRNA-exon3-1-F

[0032] GCATAGCTCTAAAAC AGCAACAATGACGATTTTCG TGCCACGGATCATCT sgRNA-exon3-1-R

[0033] AGATGATCCGTGGCA TGTAAAATAAGTAGGTCATG GTTTTAGAGCTATGC sgRNA-exon4-F

[0034] GCATAGCTCTAAAAC ...

Embodiment 2

[0039] Example 2 Agrobacterium-mediated transformation

[0040] Take EH-tac1-sgRNA-exon3-1 as an example. Using Agrobacterium-mediated transformation of the constructed EH-tac1-sgRNA-exon3-1 into an indica rice variety 93-11 (Oryza sativa L.93-11, Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Germplasm Bank) The specific method is as follows:

[0041] 1) Cultivate the EH-tac1-sgRNA-exon3-1 bacterial solution at 28°C for 16 hours, collect the bacterial cells, and dilute it into N6 liquid medium containing 100 μmol / L (purchased by Sigma, C1416) to a concentration of OD600≈0.8, Obtain the bacterial liquid;

[0042] 2) Mix and infect the 93-11 mature embryo coleoptile tissue that has been cultivated for one month with the above-mentioned bacterial liquid for 30 minutes, blot the bacterial liquid with filter paper, and then transfer it to a co-cultivation medium (N6 solid co-cultivation medium, purchased by Sigma Company) , co-cultured at 24°C for ...

Embodiment 3

[0048] Example 3 Molecular Identification of Transgenic Rice

[0049] The DNA of the four T0-transformed tac1-sgRNA-exon3-1 rice plants obtained above was extracted as a template for PCR molecular detection, and the T0-transformed pCas9 rice was used as a control. The specific method is: use primer3-1-F: ACCAGGTGTTCAATTGGCTG and primer3-1-R: CCCCAGCAACAATGACGATT as primer pairs for PCR amplification, PCR reaction system: 10×PCRBuffer for KOD-plus-Neo 5ul, 2mM dNTPs 5ul, 25mM MgSO 4 3ul, DNA (200ng / ul) 1ul, primer3-1-F (10pmol / ul) 1.5ul, primer3-1-R (10pmol / ul) 1.5ul, KOD-plus-Neo (1U / ul) 1ul, ddH 2 O 32ul, total volume 50ul. Amplification reaction system: 94°C for 2min; 98°C for 10sec, 58°C for 30sec, 68°C for 30min, 40 cycles, and the PCR product was sent to the company for sequencing verification. The results showed that the four T0 transfected tac1-sgRNA-exon3-1 plants were all edited positive plants.

[0050] According to the method described above, the number of trans...

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Abstract

The invention provides a method for cultivating a transgenic plant of which a tillering angle is reduced, i.e., edits a protein for regulating and controlling the tillering angle of rice, which is from oryza rice (Oryza sativa var.93-11) and has a name of OsTAC1. Particularly, the coding gene is subjected to knock-out of the OsTAC1 by utilizing a CRISPR / Cas9 gene editing technology so as to obtainthe transgenic plant. Compared to a tillering angle of a contrast wild type plant, the tillering angle of the transgenic plant is reduced. In addition, the invention illustrates a relationship between function weakening type mutation and function loss type mutation and the tillering angle of the plant. SgRNA provided by the invention has high editing efficiency and has wide application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to editing the OsTAC1 gene by using the CRISPR / Cas9 system to obtain rice varieties with reduced tiller angles. Background technique [0002] Rice (Oryza sativa L.) is one of the most important food crops in my country and even in the world. With the continuous increase of population, the increase of its yield is of great strategic significance to solve the global food problems in the future. [0003] Plant type is one of the important agronomic traits affecting rice population yield, and increasing rice planting density is an effective way to increase rice yield. The tiller angle of rice refers to the angle between the lateral tiller and the main stem. As one of the important factors affecting the ideal plant shape of rice, it determines the planting density per unit area and crop yield. The ideal tiller angle can not only avoid plant diseases induced by too high field humidity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/46
CPCC12N15/113C12N15/8213C12N15/8261C12N2310/10
Inventor 隋毅阴涛孙尧吴传银程子祥张皓珊
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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