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Development of wide stress protein family gene specific molecular marker and application thereof

A protein family and molecular marker technology, applied in the field of biological genetic engineering

Inactive Publication Date: 2009-10-28
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very few reports on the development of specific primers, chromosome positioning, etc., especially in such studies on crops.

Method used

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  • Development of wide stress protein family gene specific molecular marker and application thereof
  • Development of wide stress protein family gene specific molecular marker and application thereof
  • Development of wide stress protein family gene specific molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] 1. Gene sequence:

[0071] Firstly, using the nucleic acid sequences of the hv.3739 extensive stress protein genes of the barley varieties Steptoe and Morex obtained by the inventor's clone, the results of comparing the nucleic acid sequences with the software DNAMAN 5.11 are as attached figure 1 As shown, the sequence comparison diagram was obtained.

[0072] 2. Genomic DNA extraction:

[0073] Genomic DNA of barley cultivars Morex and Steptoe and their DH populations were extracted by SDS method.

[0074] The steps are:

[0075] (1) Take a small amount of fresh young leaves, grind them into fine powder with liquid nitrogen, put them into a 2.0ml centrifuge tube, add SDS extract (100mM Tris-Cl pH5.8, 100mMNaCl, 2% SDS) preheated to 65°C 1ml mixed;

[0076] (2) 65 ℃ water bath for 50 minutes, during which gently shake to mix. After cooling to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently until the supernatant becomes milky, ...

Embodiment 2

[0109] 1. Gene sequence:

[0110] Firstly, using the nucleic acid sequences of the hv.7364 extensive stress protein genes of the barley varieties Steptoe and Morex obtained by the inventor's clone, the results of comparing the nucleic acid sequences through the software DNAMAN 5.11 are shown in the appendix figure 2 As shown, the sequence comparison diagram was obtained.

[0111] 2, with the second step of embodiment 1.

[0112] 3. Design primers based on the differences in known related sequences:

[0113] Compare the partial DNA sequences of Morex and Steptoe of the hv.3739 gene by DNAMAN5.11 software, and then use the DNA sequence of the hv.7364 gene of Morex to insert the base sequence GGCCGGGAA region more than the sequence of Steptoe, and design primers:

[0114] F: 5'-CTCTGGCAGACATGGCAGC-3';

[0115] R: 5'-AGAGATAACTGAGGAAAACACACTG-3'.

[0116] 4. PCR amplification:

[0117] Using the DNA of Steptoe and Morex as a template, the primers designed in step 3 were used...

Embodiment 3

[0138] 1. Gene sequence:

[0139] Firstly, using the nucleic acid sequences of the hv.23267 extensive stress protein genes of the barley varieties Steptoe and Morex obtained by the inventor's clone, the results of comparing the nucleic acid sequences through the software DNAMAN 5.11 are shown in the appendix image 3 As shown, the sequence comparison diagram was obtained.

[0140] 2, with the second step of embodiment 1.

[0141] 3. Design primers based on the differences in known related sequences:

[0142] Compare the partial DNA sequences of Morex and Steptoe of the hv.23267 gene by DNAMAN5.11 software, and then use the base sequence AACT region missing from the DNA sequence of the hv.23267 gene of Morex than the sequence of Steptoe to design primers:

[0143] F: 5'-CTTAACCAACTAACCAAGGAGAT-3';

[0144] R: 5'-CATGAGTACAGTAGATGGTTGGG-3'.

[0145] 4. PCR amplification:

[0146] Using the DNA of Steptoe and Morex as a template, the primers designed in step 3 were used for ...

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Abstract

The invention is based on partial DNA sequences of USP genes of Steptoe and Morex, which are two varieties of hordeum vulgare. DNA MAN 5.11 software is used for comparing the gene sequences of the two materials to find base mutation, insertion and deletion domains; the different domains are used for designing a primer so as to cause only one of Steptoe and Morex to be capable of amplifying a target strip; PCR amplification is utilized to detect the condition of the target strip amplified by a DH population material to be detected and data is sorted out according to the requirement of Mapmaker 3.0 software. The Mapmaker 3.0 software is used for positioning the USP genes on a chromosome; a hordeum vulgare DH population resistance QTL map and a real-time fluorescent quantization PCR technology are utilized to analyze the relation between the USP genes and resistance; and the genes are led into crops by a transgenic technology or a chromosome engineering technology, thus effectively enhancing the external pressure resisting capability of living beings and remarkably improving the resisting capability of the crops.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to the development and application of a gene-specific molecular marker utilizing a broad stress protein family. Background technique [0002] The Universal Stress Protein (USP) gene family is a class of small cytoplasmic bacterial proteins. USP genes were first identified in Escherichia coli and are widespread in archaea and bacteria. When the cells are under most stress conditions (nutrient deficiency, heat, osmotic stress and ultraviolet damage, etc.), the expression of USP will be enhanced. In individuals with strong resistance ability, the expression level is higher than that in sensitive individuals. It also has a significant effect in cells that have stopped growing. Phosphorylation of tryptophan and threonine proteins, mainly through tyrosine phosphorylation of proteins, plays a very important role in the cell growth arrest phase. However, its p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/82
Inventor 魏育明李伟滔周练祁鹏飞郑有良
Owner SICHUAN AGRI UNIV
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