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Application of CKX2 gene and CKX3 gene for improving cold resistance capability of plants

A gene and plant technology, applied in the field of application of genes CKX2 and CKX3 in improving plant cold resistance, can solve the problems that have not been reported and applied, and achieve the effect of good application value

Active Publication Date: 2018-04-06
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the CKX gene has become an important target for molecular genetic improvement of crops, its role in the process of plant response to low temperature stress has not been reported and applied.

Method used

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  • Application of CKX2 gene and CKX3 gene for improving cold resistance capability of plants
  • Application of CKX2 gene and CKX3 gene for improving cold resistance capability of plants
  • Application of CKX2 gene and CKX3 gene for improving cold resistance capability of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of CKX2 or CKX3 gene overexpression vector

[0037]In order to understand the effect of cytokinin content on plant frost resistance, we targeted the oxidases CKX2 and CKX3, which can participate in the regulation of cytokinin metabolism in plants in response to drought and high-salt stress. The CKX2 and CKX3 genes were cloned from the Arabidopsis thaliana genome. According to the sequence analysis of the coding regions, primers 1-4 were designed to amplify the coding regions of the genes CKX2 and CKX3, and respectively connected to the overexpression vector pBINHygTx.

[0038] Primer 1: CKX2-F:5'-gtcgacGTTGTAATTCTCTTTCTTCAAAAA-3' (SEQ ID NO: 5)

[0039] Primer 2: CKX2-R:5'-ggtaccTCAAAAGATGTCTTGCCCTGG-3' (SEQ ID NO: 6)

[0040] Primer 3: CKX3-F: 5'-gtcgacAAGAATCAAGCTATTCATAA-3' (SEQ ID NO: 7) Primer 4: CKX3-R: 5'-ggtaccCTAACTCGAGTTTATTTTTT-3' (SEQ ID NO: 8)

[0041] The specific method for constructing 35S: CKX2 / 3 is: amplify the 5' and 3' of th...

Embodiment 2

[0042] Example 2 Construction and detection of CKX2 or CKX3 gene overexpression plants

[0043] The pBINHygTx vectors containing CKX2 or CKX3 gene described in Example 1 were respectively transformed into Agrobacterium GV3101 strains, and then respectively transformed into wild-type Arabidopsis plants to obtain Arabidopsis transgenic seedlings. The specific method is: inoculate the Agrobacterium containing the target vector in 100mL LB three-antibody liquid culture medium (kanamycin 50 μg / mL, rifampicin 50 μg / mL, gentamicin 50 μg / mL), and shake culture at 28°C Overnight, stay OD 600 The value is 1.0-2.0, with 5000rpm, centrifuge at room temperature for 10min, and collect the thalline; use 200mL transformation solution (1 / 2MS, 5% sucrose, 40μL Silwet L-77) to suspend the thallus; soak the Arabidopsis inflorescence in Agrobacterium In the transformation solution for 1min, put on a fresh-keeping bag to moisturize and place in a dark place to keep the temperature low. The next da...

Embodiment 3

[0050] Example 3 Phenotype Analysis of Low Temperature Tolerance in Overexpressed CKX2 or CKX3 Gene Plants

[0051] Cytokines play a vital role in regulating plant growth and development and responding to environmental factors. Previous literatures reported that the process of cold acclimation can effectively improve the frost resistance of plants. The internal mechanism is that a period of adaptation to low temperatures above zero can activate the rapid increase in the expression of plant cold response genes, accumulate a large amount of osmotic substances and protective proteins in the cells, and make plants better adapt to the subsequent subzero temperatures. In this embodiment, the cold acclimatization condition is set to grow at 4° C. for 4 days under normal light.

[0052] First, the seedlings of the transgenic plants overexpressing CKX2 and CKX3 were grown for 2 weeks, and then the culture plates were subjected to low temperature treatment, the treatment time was gradi...

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Abstract

The invention discloses an application of CKX2 gene and CKX3 gene for improving cold resistance capability of plants. The CKX2 gene and CKX3 gene are sourced from Col ecotype arabidopsis thaliana, wherein the nucleic acid sequences of cDNAs of the plant are represented as the SEQ ID No.1 and SEQ ID No.2. The invention provides a gene resource for culturing a novel species of cold resistant plant.The application has excellent potential application value and lays a theoretical foundation for researching mechanism of stress responding signal of plants and molecular mechanisms of tolerance against adverse environments.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to the application of genes CKX2 and CKX3 in improving cold resistance of plants. Background technique [0002] Under natural and agricultural production conditions, plants are constantly subjected to various biotic and abiotic stresses. The scientific and technological means that people have mastered can effectively overcome some adverse environmental factors such as soil nutrient deficiency, hypoxia, pathogenic bacteria infection and weed competition. In recent years, with global climate anomalies, the adverse effects of extreme temperatures on plants have become increasingly prominent. In 2008, southern my country experienced a historically rare low-temperature freezing disaster, and the continuous freezing weather had a huge impact on agricultural production. The disaster-affected area across the country reached 15,000 hm 2 , The direct economic loss ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/82A01H5/00A01H6/20
CPCC12N9/0026C12N15/8273C12Y105/99012
Inventor 施怡婷侯凌燕杨淑华
Owner CHINA AGRI UNIV
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