Unlock instant, AI-driven research and patent intelligence for your innovation.

A section of DNA molecule, recombination expression vector containing the DNA molecule and use thereof

A DNA molecule and expression vector technology, applied in the field of DNA molecules, can solve problems such as difficulty in obtaining stable, high-expression proteins

Inactive Publication Date: 2009-09-16
CAPITAL NORMAL UNIVERSITY
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the bottleneck factor that restricts the wide application of this method is: compared with general eukaryotic genes, due to the complexity of the structure of the low-molecular-weight glutenin subunit gene, it is difficult to obtain stable and high expression levels using existing in vitro expression vectors. protein, but the protein with low expression level is not enough to identify its function and further study its molecular structure by powder mixing experiment

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A section of DNA molecule, recombination expression vector containing the DNA molecule and use thereof
  • A section of DNA molecule, recombination expression vector containing the DNA molecule and use thereof
  • A section of DNA molecule, recombination expression vector containing the DNA molecule and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the construction of recombinant expression vector

[0035] 1. Synthesis of DNA molecules

[0036] A DNA molecule was artificially synthesized, the deoxyribonucleotide sequence of which was shown as sequence 2 in the sequence listing (that is, the 5079-5184 positions from the 5' end of the sequence 3 in the sequence listing). The 1st-6th position from the 5' end of sequence 2 in the sequence listing is the NdeI restriction site, and the 7th-63rd is the DNA molecule that promotes the transcription and translation of the target gene (its deoxyribonucleotide sequence is as sequence in the sequence listing 1), the 64th-81st position is the histidine coding sequence, the 82-99th position is the thrombin cleavage site, the 100th position is a base added to eliminate the frameshift mutation, and the 101-106th position It is the NcoI restriction site. The schematic diagram of the structure of the synthesized DNA molecule is shown in figure 1 shown. Among them, ...

Embodiment 2

[0045] Example 2, Expression and Identification of LMW-GS Gene AmLMW-m1

[0046] Using the gene AmLMW-m1 cloned from Goatwort caudate PI254863 as material, the effectiveness of the recombinant expression vector EP-1 obtained in the above-mentioned Example 1 was initially verified, and at the same time, the gene AmMW-m1 synthesized in step 1 in the above-mentioned Example 1 was used as the material. The plasmid pET-30a of the DNA molecule served as a control.

[0047] Genomic DNA of A. caudates PI254863 was extracted and used as a template to amplify AmLMW-m1 gene (the accession number of AmLMW-m1 gene in NCBI gene bank is EU329425) by PCR. Design primers to remove the signal peptide, and add EcoR I and Xho I restriction sites to the 5' ends of the Forward and Reverse primers, respectively, so that the amplified fragment can be inserted into the correct reading frame. The primer sequences are as follows:

[0048] Forward: 5'-ACA GAATTC ATGGAGACTAGCTGC-3',

[0049] Reverse: ...

Embodiment 3

[0056] Example 3, Expression and identification of LMW-GS genes ZyLMW-m1 and ZyLMW-m2

[0057] Using the genes ZyLMW-m1 and ZyLMW-m2 from bread wheat Zhongyou 9507 as materials, the effectiveness of the recombinant expression vector EP-1 obtained in the above-mentioned Example 1 was further verified, and at the same time, it did not contain the steps in the above-mentioned Example 1 1 DNA molecule synthesized from plasmid pET-30a was used as a control.

[0058] The genomic DNA of You 9507 in bread wheat was extracted and used as a template to carry out the ZyLMW-m1 gene and ZyLMW-m2 gene (the accession numbers of ZyLMW-m1 gene and ZyLMW-m2 gene in NCBI Gene Bank are EU329426 and EU329427, respectively). PCR amplification. Design primers to remove the signal peptide, and add EcoR I and Xho I restriction sites to the 5' ends of the Forward and Reverse primers, respectively, so that the amplified fragment can be inserted into the correct reading frame. The primer sequences are a...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a section of DNA molecule. The DNA molecule is any DNA molecule of the following 1) to 3): 1) DNA molecule composed of deoxyribonucleotide sequence expressed by sequence 1 of the sequence table; 2) DNA molecule that can be hybridized with the DNA molecule limited by the 1) and can promote target gene to transcribe and translate in strict conditions; 3) DNA molecule that has more than 90% of homology with the DNA sequence limited by the 1) and can promote target gene to transcribe and translate. The recombination expression vector constructed by the DNA molecule is not only applied to the expression of low molecular weight glutelin subunit gene but also further applied to prokaryotic expression of special gene that cannot be expressed by conventional carrier and has similar structure with low molecular weight glutelin subunit, and is also applied to prokaryotic expression of other ordinary genes. The invention can provide supports for structure and function identification of low molecular weight glutelin subunit, and has important realistic meaning for quality improving of wheat.

Description

technical field [0001] The invention relates to a segment of DNA molecule, a recombinant expression vector containing the DNA molecule and its application. Background technique [0002] Wheat low molecular weight glutenin accounts for 40% of the endosperm storage protein content and 60-70% of the gluten content. It is the most abundant protein component in the endosperm. It acts as a "skeleton" in the dough and imparts As well as the viscoelasticity required for other specialized foods, it plays an important role in the quality of wheat. Due to the high heterogeneity and complexity in the composition of wheat low molecular weight glutenin, it is difficult to separate and analyze, and the biological identification technology is not yet mature, which seriously restricts people's research on low molecular weight glutenin. [0003] So far, at least 80 low-molecular-weight glutenin genes with complete coding frames have been cloned from wheat and its relatives. Since subunit ide...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N5/10C12N1/00C12P21/02
Inventor 晏月明李小辉王轲王顺利张淼怡
Owner CAPITAL NORMAL UNIVERSITY