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, it

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

Example Embodiment

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

[0037] In order to understand the effect of cytokinin content on the ability of plants to resist freezing, we focused on the oxidases CKX2 and CKX3, which are involved in the regulation of the cytokinin metabolism pathway 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 region, primers 1 to 4 were designed to amplify the coding regions of genes CKX2 and CKX3, and respectively connect them to the overexpression vector pBINHygTx.

[0038] Primer 1: CKX2-F: 5'-gtcgacGTTGTAATTCTCTTCTTCAAAAA-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...

Example Embodiment

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

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

Example Embodiment

[0050] Example 3 Analysis of low temperature tolerance related phenotypes of plants overexpressing CKX2 or CKX3 genes

[0051] Cell division hormone plays a vital role in regulating plant growth and development and responding to environmental factors. Previous literature reports that the process of cold acclimation can effectively improve the frost resistance of plants. The internal mechanism is that a period of adaptation to above zero temperature can activate the rapid increase in plant cold response gene expression, accumulate a large amount of osmotic substances and protective proteins in the cell, so that the plant can better adapt to the subsequent freezing temperature. In this embodiment, the cold acclimation conditions are set to grow for 4 days at 4°C under normal light.

[0052] The seedlings of the transgenic plants overexpressing CKX2 and CKX3 were first grown for 2 weeks, and then the culture plate was treated with a low temperature, the treatment time was -1°C to -6°...

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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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