Glutathione peroxidase GhGPX8 of cotton and application thereof

A glutathione peroxidase, cotton technology, applied in the field of plant genetic engineering, can solve problems such as complex action process

Active Publication Date: 2017-07-21
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in general, because there are many types of GPXs genes and their corresponding proteins, and the action process is relat

Method used

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  • Glutathione peroxidase GhGPX8 of cotton and application thereof
  • Glutathione peroxidase GhGPX8 of cotton and application thereof
  • Glutathione peroxidase GhGPX8 of cotton and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Present embodiment mainly introduces to cotton glutathione peroxidase GGPX8 The cloning of the gene and the analysis process of its base sequence are as follows.

[0046] According to the sequencing results of the upland cotton TM-1 transcriptome, specific primers were designed, and PCR technology was used to clone the full-length sequence of the target gene GhGPX8. The primer sequences were designed as follows:

[0047] 8-F: 5'-CCCGGTACCATGGCTTCTCAATCTTCTA-3',

[0048] 8-R: 5'-CCCGGATCCAGCCAGCAGTTTCTTAA-3';

[0049] Using the kit for extracting cotton DNA and RNA, extract the DNA and RNA of upland cotton respectively, reverse transcribe the RNA into cDNA, and use the DNA and cDNA of upland cotton as templates to carry out GGPX8 Amplification of the sequence;

[0050] The PCR reaction conditions were: 95°C, 5 min; 95°C, 40 s, 55°C, 40 s, 72°C, 30 s, 32 cycles; 72°C, 5 min.

[0051] Recover the PCR amplification product (use the PCR recovery kit), and perform sequenc...

Embodiment 2

[0059] Preliminary results showed that under various stress treatments, the cotton plant GGPX8 The expression levels of genes showed up-regulation, especially when treated with high temperature, jasmonic acid or salicylic acid, GGPX8 The expression of the gene was obviously induced, that is, the gene may be involved in the anti-oxidation regulation of cotton in response to high temperature and plant hormones. In order to further determine the cotton response mode and response situation in the face of different treatments, the inventor conducted a further experiment by using real-time quantitative PCR technology, and the relevant experiments are briefly introduced as follows.

[0060] Cotton samples:

[0061] Cotton is grown in quartz sand containing Hoagland's nutrient solution to the stage of two leaves and one heart (natural growth or after adversity treatment), and the materials of the four tissues of root, stem, true leaf and cotyledon are respectively taken;

[0062] Wh...

Embodiment 3

[0100] On the basis of the result analysis of the foregoing embodiment 2, the inventor thinks that it is necessary to GGPX8 The location of the gene in the cell was further analyzed, thus constructing the GGPX8 Overexpression of the cDNA sequence (SEQ ID NO.2) p1300GFP- GGPX8 The specific process of recombinant vector is briefly introduced as follows.

[0101] First, design a restriction enzyme Kpn I and Bam The primer sequence of H I restriction site is as follows:

[0102] 1300GFP-GPX8-F: 5'-CCCGGTACCATGGCTTCTCAATCTTCTA-3',

[0103] 1300GFP-GPX8-R: 5'-CCCGGATCCAGCCAGCAGTTTCTTAA-3';

[0104] Then, using the cDNA sample prepared in Example 2 as a template, carry out PCR amplification, and reclaim the amplified product;

[0105] The 3rd, to recovered PCR amplification product and p1300GFP The carrier adopts Bam H I and Kpn I performed double enzyme digestion, and the enzyme digestion system was referred to the Takara manual (Takara, Japan); 1.0% agarose gel el...

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Abstract

The invention specifically relates to a glutathione peroxidase GhGPX8 gene with antioxidation effect in cotton and application of the same to gene engineering, belonging to the field of plant gene engineering. The gene includes 1491 bp bases and has a base sequence as shown in SEQ ID No. 1. A coding sequence of the gene includes 501 bp bases; a protein coded by the coding sequence is glutathione peroxidase GhGPX8 of cotton and contains 166 amino acids. According to the invention, a vector p1300GFP-GhGPX8 and a vector pYES2-GhGPX8 are constructed to prove the GhGPX8 is located in the cytoplasm of mesophyll cells and indicate the glutathione peroxidase GhGPX8 is capable of removing active oxygen. Overexpression of the GhGPX8 gene is applicable to cultivation of antioxidative cotton varieties and improvement of the adversity stress resistance of cotton.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a glutathione peroxidase GhGPX8 gene in cotton with antioxidant effect and its specific application in genetic engineering. Background technique [0002] During the whole growth period, plants will be subjected to various biotic and abiotic stress environments, and various stress factors will eventually lead to the accumulation of excessive active oxygen in plants, thereby affecting the growth and development of plants. Existing studies believe that reactive oxygen species are a by-product of plant metabolism and play an important role in cell signal transmission and redox balance. Low concentrations of reactive oxygen species can act as signal molecules in plants, while high concentrations of reactive oxygen species can be toxic to plant cells and oxidize intracellular components such as DNA, protein, and plasma membranes, thereby affecting plant cells. grow...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/08C12N15/82A01H5/00
CPCC12N9/0065C12N15/8271C12Y111/01009
Inventor 苗雨晨李坤陈明杨李海鹏郭敬功宋纯鹏
Owner HENAN UNIVERSITY
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