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Application of corn CIPK42 protein and coding gene of corn CIPK42 protein in regulation and control of salt stress tolerance of plants

A technology for encoding genes and transgenic plants, applied in the field of plant genetic engineering, can solve the problems of long cycle, difficult to determine gene function, low transformation efficiency, etc., and achieve the effect of improving salt tolerance and growth performance, and improving salt tolerance.

Active Publication Date: 2020-05-19
新疆农业科学院核技术生物技术研究所(新疆维吾尔自治区生物技术研究中心)
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  • Description
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  • Application Information

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Problems solved by technology

Some functional genes of crops can improve their stress resistance in Arabidopsis, but after transforming crops, their stress resistance has not been significantly improved
In addition, due to the problems of long transformation period and low transformation efficiency in maize transformation, there are still relatively few functional genes in maize that can improve its stress resistance.
A large number of studies have shown that the CIPK gene family selectively regulates the regulatory pathways of plants under various stresses, which also makes it difficult to determine the function of the genes

Method used

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  • Application of corn CIPK42 protein and coding gene of corn CIPK42 protein in regulation and control of salt stress tolerance of plants
  • Application of corn CIPK42 protein and coding gene of corn CIPK42 protein in regulation and control of salt stress tolerance of plants
  • Application of corn CIPK42 protein and coding gene of corn CIPK42 protein in regulation and control of salt stress tolerance of plants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 Contains the construction of the expression vector of CIPK42 protein kinase gene

[0053] 1. Based on the sequence of the known target gene, primers were designed to amplify the ZmCIPK42 gene sequence from the cDNA of the maize B73 inbred line, and the target fragment was recovered with an agarose gel kit.

[0054] 2. Ligate the recovered fragments with the pGEM-T vector, then transform Escherichia coli DH5α competent cells, obtain positive single clones, send them for sequencing, after sequence comparison, obtain single clones consistent with the original sequence, shake the bacteria and extract the single clones The plasmid was cloned and named pGEM-ZmCIPK42.

[0055] 3. Using pGEM-ZmCIPK42 as the entry vector and pCAMBIA3301 plasmid as the expression vector, the recombinant vector pCAMBIA3301-ZmCIPK42 was obtained through recombination reaction.

[0056] Schematic diagram of expression vector construction see figure 1 .

Embodiment 2

[0057] Embodiment 2 Transformation and screening of Arabidopsis containing CIPK42 protein kinase gene

[0058] 1. The ZmCIPK42 overexpression vector constructed in Example 1 was transformed into Agrobacterium Gv3101.

[0059] 2. Transformation of wild type Arabidopsis thaliana by floral dipping method.

[0060] 3. Convert the T obtained after conversion 0 The generation seeds were vernalized for 3 days, and then directly sowed in nutrient soil for growth. After about two weeks of normal growth, spray T with 0.5‰ of PPT (phosphinothricin) 0 Transformed plants were screened from Arabidopsis thaliana.

[0061] 4. Since the transformation overexpression vector pCAMBIA3301 used has the Bar gene against PPT (phosphinothricin), it can be transferred into the plant along with the target gene during transformation, so most of the untransformed plants die after spraying PPT (phosphinothricin) , while the transformed plants were still able to grow normally. The transformed plants wer...

Embodiment 3

[0064] Embodiment 3 Containing the transformed maize of CIPK42 protein kinase gene and screening

[0065] 1. Preparation of plant receptor material: 10-13 days after the pollination of the corn inbred line B104, take the corn young ears and peel off the bract leaves on the ultra-clean workbench, and take out the immature embryos. Put the immature embryos with the scutellum up, inoculate them on 1 / 2 MS medium, inoculate 20-40 immature embryos in each dish, and culture them in the dark at 28°C for 2-3 weeks, then the callus can be induced.

[0066] 2. Preservation method of the bacterial strain: pick up the single colony of the constructed ZmCIPK42 Agrobacterium LB4404 (the ZmCIPK42 overexpression vector constructed in Example 1 is transferred into the Agrobacterium LB4404 to obtain ZmCIPK42 Agrobacterium LB4404) with an inoculation loop and streaked on the On the YEP solid medium with corresponding antibiotics, pick a single colony of Agrobacterium after 2 days of subculture, a...

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Abstract

The invention relates to the technical field of plant genetic engineering, and particularly discloses an application of a corn CIPK42 protein and a coding gene of the corn CIPK42 protein in regulationand control of salt stress tolerance of plants. The corn ZmCIPK42 gene is found to be able to positively regulate and control the salt tolerance of the plants; and the salt tolerance of the plants can be effectively improved by increasing of the expression quantity of the ZmCIPK42 gene. According to the invention, transgenic corn and arabidopsis thaliana plants with ZmCIPK42 overexpression are constructed; and compared with a non-transgenic wild type, the transgenic corn and arabidopsis thaliana plants are significantly improved in salt tolerance and growth. Discovery of the salt-tolerant function of the ZmCIPK42 gene provides a novel gene target and resource for cultivation of salt-tolerant plant varieties, is of great significance to research of a salt-tolerant molecular mechanism of the plants, and lays a certain theoretical foundation for research of a salt stress response mechanism of the plants and a molecular mechanism of resisting adverse environments.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the application of a corn CIPK42 protein and its coding gene in regulating plant tolerance to salt stress. Background technique [0002] To adapt to environmental changes during growth, plants encounter a series of biotic and abiotic stress factors, such as drought, salinity and pathogen infection. Most of these environmental stresses are related to calcium signaling. Calcium ions (Ca 2+ ) is a second messenger in cells, involved in almost all signal transduction of abiotic and biotic stress. Ca 2+ Dependent signaling pathways are involved in many aspects of plant development and environmental response. Studies have shown that upon stimulation by environmental factors, cell-encoded transporters sense these signals temporally and spatially and pass them on to downstream genes. Plant cells possess an array of calcium sensor proteins that sense Ca 2+ And tran...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/82C12N15/11A01H5/00
CPCC12N9/1205C12N15/8273C12N15/8261
Inventor 陈勋基黄全生李晓荣李娟郝晓燕陈果李建平常晓春高升旗足木热木·吐尔逊
Owner 新疆农业科学院核技术生物技术研究所(新疆维吾尔自治区生物技术研究中心)
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