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Application of ZmWRKY70 protein and coding gene thereof in plant drought resistance

A technology of transgenic plants and plants, applied in applications, plant peptides, plant products, etc.

Active Publication Date: 2022-06-21
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are a large number of transcription factors in plants. Transcription factors regulate their expression by binding to specific sequences of target genes, and play an important role in integrating external environmental signals and regulating plant growth and development. However, transcription factors play an important role in plant response to biotic and abiotic stresses, such as drought. , The specific mechanism of cold resistance still needs to be further explored

Method used

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  • Application of ZmWRKY70 protein and coding gene thereof in plant drought resistance
  • Application of ZmWRKY70 protein and coding gene thereof in plant drought resistance
  • Application of ZmWRKY70 protein and coding gene thereof in plant drought resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1, the acquisition of WRKY70 gene and the construction and detection of CRISPR / Cas9 gene editing vector

[0051] 1. Acquisition of WRKY70 gene

[0052] In order to study the molecular mechanism of WRKY family proteins in plant drought resistance, the WRKY70 gene was directionally mutated from the B73 genome of maize (Zea mays L.) by using CRISPR / Cas9 technology.

[0053] The maize WRKY70 gene consists of 5524 bases (sequence 1), the reading frame of the T01 transcript is sequence 3, and the encoded protein is sequence 2, named WRKY70 protein. The transcript consists of 3 exons, the 584th to 2136th base of the reading frame, the 2755th to 2895th base, the 3978th ​​to 4924th base, and the rest are introns sequence. The gene is derived from B73 corn. Since the same DNA segment sequence of corn can produce different transcripts and translate different proteins, the different transcripts produced by the DNA segment sequence and the translated proteins with differen...

Embodiment 2

[0079] Example 2, Construction and Identification of WRKY70 Gene CRISPR-Cas9 Plants

[0080] 1. Preparation of recombinant bacteria

[0081] The CRISPR / Cas9 gene editing vector pBCXUN-WRKY70 CRISPR-Cas9 mutant plasmid constructed in Example 1 was transformed into competent Agrobacterium strain EHA105 by heat shock method, and a positive clone was identified by colony PCR, which was named EHA105 / pBCXUN-WRKY70 CRISPR -Cas9.

[0082] 2. Construction of WRKY70 gene CRISPR-Cas9 plants

[0083] Inoculate a single colony of Agrobacterium EHA105 / pBCXUN-WRKY70 CRISPR-Cas9 correctly identified in 2-3 mL of liquid medium containing 100 μg / mL kanamycin and 50 μg / mL rifampicin, culture with shaking at 28°C overnight, and in the second Transfer to a large amount of liquid medium containing antibiotics for shaking culture, collect the bacteria after several transfers, and resuspend to OD 600 Between 0.8-1.0.

[0084] The recombinant strain EHA105 / pBCXUN-WRKY70 CRISPR-Cas9 was transferred...

Embodiment 3

[0094] Example 3, WRKY70 Gene CRISPR-Cas9 Mutant Maize Phenotype Detection under Drought Treatment

[0095]1. Sow T2 generation CRISPR-Cas9 mutant corn seeds and wild-type corn B73 seeds in small pots with nutrient soil, covering 50cm 2 After the soil is filled with water, the remaining water in the tray is poured out, and seedlings emerge after 7 days of cultivation to obtain T2 generation CRISPR-Cas9 mutant corn seedlings and wild-type corn seedlings;

[0096] 2. Transplant the well-growing T2 generation CRISPR-Cas9 mutant corn seedlings and wild-type corn B73 seedlings into new pots filled with nutrient soil, 3 plants per pot, water normally and cultivate for 7 days. Three replicates were set up, with 6 pots for each replicate.

[0097] 3. After completing step 2, continue without watering for 14 days (drought treatment), and resume watering for 7 days again.

[0098] After 7 days of resuming watering, the difference in phenotype is obvious, the results are as follows f...

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Abstract

The invention discloses application of a ZmWRKY70 protein and a coding gene thereof in plant drought resistance. The DNA molecule provided by the invention is a WRKY70 gene and is as follows: (b1) a DNA molecule with a coding region as shown in a sequence 1 in a sequence table; (b2) a DNA molecule with a coding region as shown in a sequence 3 in the sequence table; after the WRKY70 gene provided by the invention is mutated by a CRISPR-Cas9 gene editing technology, the growth of a control plant which is not mutated under a drought treatment condition is obviously superior to that of a mutant plant, which shows that the drought resistance of the plant can be obviously reduced by gene mutation. In the embodiment of the invention, the drought-sensitive plant is obtained by adopting a CRISPR-Cas9 gene editing technology, compared with a traditional breeding mode, the time is short, the purposiveness is strong, a gene resource is provided for cultivating and improving a new variety of the drought-resistant plant, and a theoretical basis is provided for illuminating a molecular mechanism of a WRKY transcription factor in plant drought stress signal response.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of a ZmWRKY70 protein and its coding gene in plant drought resistance. Background technique [0002] Corn is one of the three major food crops in the world. It has a wide range of planting areas, but its distribution in various regions is not uniform. Different regions have different environments, different climatic conditions, and different precipitation. Among them, drought is an important factor affecting corn production. Drought at the corn seedling stage will inhibit the growth rate of corn, resulting in a severe shortening of the development period. Drought will also inhibit the plant height of corn, causing leaf wilting, thereby reducing photosynthesis. Drought at the corn filling stage will lead to underfull kernels, which will lead to corn yield. reduce. More than half of my country's corn is planted on the dry land that relies on natural precipi...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N15/84A01H5/00A01H6/46
CPCC07K14/415C12N15/8273C12N15/8218
Inventor 巩志忠王瑜蒋杉
Owner CHINA AGRI UNIV
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