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Grape polyphosphoinositide kinase gene VvIPK2 and application thereof in improving cold resistant ability of plants

A technology of grape inositol polyphosphate and transgenic plants, which is applied in the field of genetic engineering and can solve problems such as low temperature stress of VvIPK2 that has not been seen yet

Inactive Publication Date: 2011-08-24
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on whether VvIPK2 is involved in the response of grapes to low temperature stress.

Method used

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  • Grape polyphosphoinositide kinase gene VvIPK2 and application thereof in improving cold resistant ability of plants
  • Grape polyphosphoinositide kinase gene VvIPK2 and application thereof in improving cold resistant ability of plants
  • Grape polyphosphoinositide kinase gene VvIPK2 and application thereof in improving cold resistant ability of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Determination of the impact of low temperature on the relative expression of VvIPK2 in 'F-242' leaves

[0034] 1. Clone the VvIPK2 gene

[0035] Using the method of homologous cloning, the Arabidopsis polyphosphoinositide kinase gene AtIPK2β(AT5G61760) CDS was used for WU-BLAST sequence alignment, and the homologous sequence of the polyphosphoinositide kinase gene in grape was retrieved, and the DNAstar software package analyzed its openness. Read frame (open reading frame, ORF), and use it as a template to design specific primers

[0036] VvIPK2-CDS-sense: 5'-ATGCTTAAGGTCCCGGATCATC-3' (as shown in SEQ ID No: 3);

[0037] VvIPK2-CDS-antisense: 5'-GCGTTATTCAGTGGTACCATTCTCC-3', (as shown in SEQ ID No: 4) was synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0038] The total RNA of grapes was extracted by CTAB method, RNA LA PCR of Dalian Bao Biological Company TM Kit (AMV) Ver.1.1 kit reverse transcription PCR. According to the requirements of the ...

Embodiment 2

[0064] Example 2. Determination of the effect of IPK2 inhibitor LY-294002 on SOD enzyme activity, Cu / Zn SOD gene expression, MDA content and relative permeability of cell membrane in 'F-242' leaves under low temperature conditions

[0065] The present invention uses SOD enzyme activity, MDA content and relative permeability of cell membrane as grape cold-resistant physiological index respectively, and the mensuration of SOD enzyme activity and MDA content is with reference to the method of " plant physiology experiment " (Zhu Guanglian etc., 1990), and cell membrane relative permeability The conductivity of the exudate was measured with a DDS-II conductivity meter. Each experiment was repeated 3 times. The control and treatment were the mean values ​​and standard errors of the three treatments. The significant difference was analyzed by Duncan's new multiple range method.

[0066] Spray H on the robust 3-4 week-old 'F-242' test-tube plantlets. 2 o 2 (0.1%), ascorbic acid (asc...

Embodiment 3

[0082] Embodiment 3, measure low temperature stress to the H in 'F-242' blade 2 o 2 The effect of content

[0083] grape leaf H 2 o 2 The determination method of the content refers to the methods of Zhang Xiaoli et al. (2009) and Xia Jinchan et al. (2005), and the absorbance value at 240nm wavelength is measured by an ultraviolet spectrophotometer. Each experiment was repeated 3 times.

[0084] h 2 o 2 It is an important molecule involved in the response of plants to stress. Depend on Image 6 It is known that low temperature treatment at 4°C can cause H in leaves of ‘F-242’ 2 o 2 Burst occurred at 9h (p2 o 2 Participated in the process of 'F-242' responding to low temperature stress.

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Abstract

The invention provides a grape polyphosphoinositide kinase gene VvIPK2 with a sequence of SEQ ID NO:1 in a sequence table and also provides grape polyphosphoinositide kinase with an amino acid sequence of SEQ ID NO:2. In the invention, the polyphosphoinositide kinase gene VvIPK2 of a test tube seedling of a wine grape rootstock variety 'F-242' is cloned, and tests prove that grape blades VvIPK2 participate in a grape cold resisting process and the gene VvIPK2 is transferred to arabidopsis, therefore, the invention provides a theoretical basis for improving the grape resistance by using a transgenic technology. According to the invention, H2O2 is proved to be positioned on the upstream of the VvIPK2 to play a role; and the grape polyphosphoinositide kinase gene VvIPK2 has important guiding significance in researching influence of two signal elements on gene expression with cold regulation, change of transcriptome and change of protein level by means of molecular biology, information biology and proteomics, and deeply researching a plant-influenced low-temperature forced signal transduction mechanism.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a grape polyphosphate inositol kinase gene VvIPK2 and its application. Background technique [0002] Low temperature is one of the main environmental stress factors encountered in the process of plant growth, and the loss of various crops due to low temperature and cold damage is as high as hundreds of billions of dollars every year. Grape is one of the important economic crops. It is a perennial deciduous fruit tree sensitive to low temperature. The grapes planted in northern my country cause huge economic losses every year due to low temperature and cold damage and late spring cold. The stress of plants to low temperature is a complex process, including the stages of low temperature signal perception, signal transduction and transcriptional regulation. Plants can sense low temperature through changes in plasma membrane fluidity, calcium ion-permeable channels on the plasma m...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/63A01H5/00
Inventor 刘新侯丽霞赵方贵车永梅李希东
Owner QINGDAO AGRI UNIV
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