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Zea mays transcription factor ZmbZIP22 and application thereof

A technology of transcription factor and corn, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of high background

Active Publication Date: 2017-10-27
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But its disadvantage is that the background is higher

Method used

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  • Zea mays transcription factor ZmbZIP22 and application thereof
  • Zea mays transcription factor ZmbZIP22 and application thereof
  • Zea mays transcription factor ZmbZIP22 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Using yeast one-hybrid assay to screen potential transcription factors of 27kDa γ-gliadin

[0040] Firstly, the SMART method was adopted to obtain the homogeneous cDNA fragments of corn kernels. single hybrid vector pGADT7-Rec2 Connection transformations were obtained with approximately 1.0 x 10 6 clone. In the construction of the bait plasmid, the 27kDa γ-prolamin gene promoter was used as the bait.

[0041] Results: The constructed library plasmid and bait plasmid were transformed into yeast Y187 by PEG / LiAc method, and 10 6a yeast transformant. Through the screening of DDO and TDO, a total of 27 suspected positive clones were obtained. A bZIP-type transcription factor gene was screened out of these cloned plasmids, sequenced and transformed into yeast ZmbZIP22 , see figure 1 .

Embodiment 2

[0042] Example 2: Verification of ZmbZIP22 binding to 27kDa γ-gliadin gene promoter in vivo

[0043] 1. Take wild-type immature corn kernels 15 days after pollination, and carry out vacuum cross-linking with 1% formaldehyde.

[0044] 2. Extract the nucleus from the cross-linked grains, and break the chromatin into fragments of about 300 bp by ultrasound.

[0045] 3. Add rabbit pre-immune serum and incubate at 4°C for 1 hour to pre-purify the chromatin.

[0046] 4. Add 40 μl GE Protein A agarose beads and incubate at 4°C for 1 hour.

[0047] 5. Centrifuge the incubated liquid at 800g for 2min, divide the supernatant into two equal parts, add the self-made ZmbZIP22 antibody and the same amount of IgG respectively, and incubate overnight at 4°C.

[0048] 6. Add 20 μl GE Protein A agarose beads and incubate at 4°C for 90 minutes.

[0049] 7. Centrifuge and wash the column with IP buffer for 5 times in total.

[0050] 8. Elute with elution buffer. Add Proteinase K to digest th...

Embodiment 3

[0053] Example 3: Transcriptional activation of the 27kDa γ-gliadin gene promoter by ZmbZIP22

[0054] The transcriptional activation of the 27kDa γ-gliadin gene promoter by ZmbZIP22 was detected using a dual fluorescent transcriptional activation system. The schematic diagram of the strategy is shown in image 3 .

[0055] 1. pass Hind III and Bam H I The target gene promoter was linked into the reporter vector pGreen-0800 fused with the luciferase gene by the enzyme point, and the transcription factor ZmbZIP22 open reading frame was linked into the effector vector driven by the 35S promoter.

[0056] 2. The vector was transformed into Agrobacterium strain GV3101 by heat shock method.

[0057] 3. Infect the tobacco leaf cells with Agrobacterium to transiently express the proteins carried by the reporter carrier and the effector carrier.

[0058] 4. After 48h, the infected leaf protein was extracted.

[0059] 5. Use a full-wavelength fluorescence detector (TECAN) to det...

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Abstract

The invention relates to an application of a Zea mays grain transcription factor in the aspect of regulation and control of alcohal-soluble proteins. The factor comprises a base sequence shown in SEQ ID NO: 1. The protein ZmbZIP22 encoded by the sequence can be directly bonded to a 27kDa gamma-gliadin promoter and activate the 27kDa gamma-gliadin promoter. Gene-deficient mutant plants are obtained through carrying out Zea mays immature-embryo conversion by taking a gene fragment, shown in SEQ ID NO: 2, of ZmbZIP22 as a guide RNA by using a CRISPR-Cas9 technology. Compared with wild type grains, transgenic mutants have Zea mays grains with irregular and relative-thin protein body shells, the content of alcohal-soluble proteins in mature grains is remarkably lowered, the content of essential amino acids such as lysine, which are deficient to the conventional Zea mays, in the mature grains is remarkably increased, and thus, the nutritional quality of Zea mays is improved.

Description

technical field [0001] The invention relates to a maize transcription factor ZmbZIP22 and its application. technical background [0002] corn( Zea mays ) is one of the most productive grass crops in the world. In addition to being used as grain, corn is also an important source of feed for poultry and livestock, and is also an important industrial raw material. So corn has played an important role in the progress of society. With the continuous improvement of people's living standards, higher requirements are placed on the quality of the food they ingest. Therefore, the improvement of corn quality has become an important research topic. The main components of corn kernels include starch, protein and oil, among which protein determines the nutritional value of corn kernels, so the protein quality of corn is an important research trait. [0003] Glamin is the main storage protein in corn grain, accounting for more than 60% of the total protein. However, the content of ess...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8251C12N15/8253C12N15/8254
Inventor 宋任涛李朝斌祁巍巍朱晨光
Owner SHANGHAI UNIV
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