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Corn transcription factor ZmbHLH167 and application thereof

A transcription factor, corn technology, applied in the fields of application, genetic engineering, angiosperms/flowering plants, etc., can solve the problem of unobserved mutant phenotypes, etc.

Active Publication Date: 2017-10-20
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Subsequently, it was reported in the literature that RNA interference technology was used to silence the ortholog of ZOU in maize (that is, ZmbHLH167). overt mutant phenotype

Method used

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  • Corn transcription factor ZmbHLH167 and application thereof
  • Corn transcription factor ZmbHLH167 and application thereof
  • Corn transcription factor ZmbHLH167 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: The CRISPR-Cas9 transgenic vector of ZmbHLH167 was constructed and used for transgenic transformation.

[0030] The CRISPR-Cas9 vector suitable for maize that has been constructed in our laboratory was selected as the vector for Agrobacterium transformation of maize immature embryos. Digest the vector with Pst I, and use the synthetic ZmbHLH167 The specific sequence connection guide RNA sequence is inserted into the vector together with the corn U6 promoter and terminator ( figure 1 ), after the sequencing is correct, transform into EHA105 Agrobacterium strain by electric shock. Select the immature embryos of the PBPA corn line 8-12 days after pollination, about 1.5mm in size, as the recipient material, and carry out the transformation of immature embryos. The specific process:

[0031] 1. Agrobacterium infection for 10 minutes-co-cultivation at 20°C for 3 days.

[0032] 2. Recovery culture at 28°C for 7 days-screening culture (bialaphos 1.5 mg / l) at 28°...

Embodiment 2

[0038] Embodiment two: ZmbHLH167 Detection of the expression of ZmbHLH167 in the transgenic No. 4 and No. 6 event frameshift mutation materials

[0039] 1. Take 8-10 immature seeds.

[0040] 2. Extract the total protein of ZmbHLH167 mutant material and wild-type material with frameshift mutation respectively. Use liquid nitrogen to grind to powder grade, put into EP tube, add IP lysate, and lyse on ice for 20min.

[0041] 3. Centrifuge at the highest speed and remove the supernatant. Take 4 μl of protein from each of the two samples, add 1 μl of 5×SDS protein loading buffer mixed with 1M DTT, denature at 99°C for 10 minutes, and immediately insert the protein sample on ice.

[0042] 4. SDS-PAGE electrophoresis, the stacking gel is 5%, and after half an hour of 80V electrophoresis, the separating gel is 12.5%, and the electrophoresis time is about 2 hours.

[0043] 5. 200mA transfer film for 1h. Block with 5% milk in TBST for 1 h at room temperature.

[0044] 6. Dilute i...

Embodiment 3

[0050] Embodiment three: ZmbHLH167 Phenotype Observation of CRISPR-Cas9 Event 4 Mutant Kernel

[0051] Observed under natural light and on a light box, respectively ZmbHLH167 The phenotype of the grain of the transgenic frameshift mutant material ( Figure 4 ). Observation ZmbHLH167 Compared with the wild-type grains on the same ear, the grains of transgenic frameshift mutant materials were significantly smaller. Observed on the light box ZmbHLH167 Compared with the wild-type grains, the grains of transgenic frameshift mutant materials showed a significantly lower light transmittance, showing a powdery endosperm phenotype.

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Abstract

The invention relates to a corn transcription factor ZmbHLH167 and an application of the corn transcription factor ZmbHLH167. A base sequence of the gene is shown as SEQ ID NO (sequence identifier number): 1. A corn immature embryo is transformed by taking a gene segment SEQ ID NO: 2 of the sequential coding protein ZmbHLH167 as guide RNA (ribonucleic acid) by use of a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 technology, and a plant of a gene deletion mutant is obtained. Compared with a wild seed, a corn seed of the genetically modified mutant is smaller significantly, but a germination rate is not influenced. Biochemical analysis shows that a starch content of the corn seed of the genetically modified mutant is decreased obviously, a content of protein and total oil and fat is significantly increased, and a genetic resource is provided for creating high quality corn.

Description

technical field [0001] The invention relates to a maize transcription factor ZmbHLH167 and its application. technical background [0002] corn( Zea mays ) is one of the most important food crops in the world, but also important livestock feed and important industrial raw materials. With the continuous increase of the world population, improving the yield and quality of corn is of great significance to human beings. Corn endosperm is the main nutrient storage place for corn kernels. The endosperm contains a large amount of starch (accounting for about 80% of the dry weight of the endosperm) and protein (accounting for about 10% of the dry weight of the endosperm). The germ of corn contains a lot of oil. [0003] Researchers hope to improve the yield and quality of corn by studying a series of corn kernel mutants. For example, opaque2 The mutant increases the content of lysine, an essential amino acid for humans, by nearly 2 times. Studies have shown that the improvement...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/82C12N9/22A01H5/10
CPCC07K14/415C12N9/22C12N15/8213C12N15/8245C12N15/8247C12N15/8251C12N15/8261
Inventor 宋任涛冯帆祁巍巍朱晨光
Owner SHANGHAI UNIV
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