Cotton glutathione peroxidase ghgpx8 and its application
A technology of glutathione peroxidase and cotton, which is applied in the field of plant genetic engineering and can solve problems such as complex action processes
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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-GhGPX8 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 ele...
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