Application of Maize Casein Kinase 2ck2α2 and its Encoding Gene Based on High Temperature Stress Response
A protein kinase and gene encoding technology, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve the problems of high temperature resistance genetic resources and lack of germplasm resources in maize.
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Embodiment 1
[0023] Example 1: Casein kinase 2 in response to high temperature in corn α Identification of subunits
[0024] 1. Identification of casein kinase 2 affected by high temperature in maize by qRT-PCR α subunit
[0025] To determine ZmCK2α1 、 α2 、 α3 、 Whether α4 is affected by high temperature, the three-leaf stage seedlings of the maize hybrid Zhengdan 958 were treated with high temperature stress for 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h and 10 h respectively, and then the third The total RNA of the two leaves was analyzed by qRT-PCR. The results showed that ZmCK2α2 and ZmCK2α3 were significantly induced by high temperature stress, and the expression of ZmCK2α2 increased most significantly. and ZmCK2α4 expression had little effect (see figure 1 ).
[0026] 2. ZmCK 2 α 2 Analysis of cis-acting elements in the promoter region of genes
[0027] For further analysis ZmCK2α2 Whether it responds to stress, the cis-acting elements of its promoter were analyzed. First fou...
Embodiment 2
[0029] Embodiment 2: ZmCK 2 α 2. High temperature resistance function test of genes
[0030] Malondialdehyde (MDA) content is the embodiment of the degree of peroxidation of plant cell membranes. The high content of malondialdehyde indicates that the degree of peroxidation of plant cell membranes is high and the cell membrane is seriously damaged. while H 2 O 2 The accumulation of content can directly or indirectly oxidize biological macromolecules such as nucleic acids and proteins in cells, and damage the cell membrane, thereby accelerating the aging and disintegration of cells. Catalase (CAT) can scavenge H 2 O 2 , is one of the important enzymatic defense systems in plants. for sure ZmCK2α2 Whether it is involved in the resistance of maize to high temperature, using the maize protoplast transient transformation system, the overexpression vector OE -ZmCK 2 α 2 and ZmCK 2 α The double-stranded RNA of 2 and distilled water were transformed into maize leaf protop...
Embodiment 3
[0032] Example 3: Based on ZmCK 2 α Characterization of high temperature resistance function of 2 transgenic plants
[0033] To further explore ZmCK 2 α 2 The function under high temperature stress was obtained by maize genetic transformation technology. ZmCK 2 α 2 Overexpression homozygous lines. First, by homologous recombination ZmCK 2 α 2 Recombination into the binary expression vector pFGC5941 containing the 35S promoter, then transform the plasmid with the correct sequencing result into Agrobacterium GV3101, and finally use the Agrobacterium-mediated in situ transformation of maize mature embryos to transform pFGC5941- ZmCK2α2 The vector is transformed into maize, and T is obtained after herbicide screening 0 Generation positive plants, and then selfed to T 2 generation and obtained the ear of the transgenic plant.
[0034] 1. Detection of transgenic lines by qRT-PCR ZmCK 2 α 2 expression
[0035] To identify overexpressed transgenes in ZmCK 2 α Wh...
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