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OYE 3 gene deriving from saccharomyces cerevisiae and preparation method and application thereof

A Saccharomyces cerevisiae and gene technology, applied in the field of crop genetics and breeding, can solve problems such as hindering the self-purification process of water bodies

Active Publication Date: 2012-07-04
上海金丰裕米业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If this waste water is discharged directly into the water body, it will hinder the self-purification process of the water body

Method used

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  • OYE 3 gene deriving from saccharomyces cerevisiae and preparation method and application thereof
  • OYE 3 gene deriving from saccharomyces cerevisiae and preparation method and application thereof
  • OYE 3 gene deriving from saccharomyces cerevisiae and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Synthesis of Saccharomyces cerevisiae OYE3 gene by gene synthesis

[0025] According to the plant preference code, the gene encoding Saccharomyces cerevisiae OYE3 was re-synthesized, and the synthetic primers of the OYE3 gene were as follows:

[0026] OYE3-1:

[0027] GAA, ATGCCATTTGTAAAAGGTTTTGAGCCGATCTCCCTAAGAGACACAAACCTT

[0028] OYE3-2:

[0029] GATGTGCAAGCTGAGTGTTACCAATCTTAATTGGTTCAAAAAAGGTTTGTGTCTCTTAGGG

[0030] OYE3-3:

[0031] TAACACTCAGCTTGCACATCGTGCGGTTATGCCCCCATTGACCAGAATGAGGGCCACTCA

[0032] OYE3-4:

[0033] ATACACACAGCAGCCCACTCCTTATTTGGAATATTTCCGGGGTGAGTGGCCCTCATTCTGGT

[0034] OYE3-5:

[0035] AGGAGTGGGCTGCTGTGTATTATGGTCAGCGTGCTCAAAGACCTGGTACCATGATCATCA

[0036] OYE3-6:

[0037] TCATAGCCGCCGGCTTGAGGGGAAATAAACGTACCTTCCGTGATGATCATGGTACCAGGT

[0038] OYE3-7:

[0039] CCTCAAGCCGGCGGCTATGACAACGCCCCTGGGATTTGGTCTGATGAGCAGGTCGCTGAG

[0040] OYE3-8:

[0041] ACGACTGACAATCATGGATGGCTAAAAAGATATTCTTCCACTCAGCGACCTGCTCATCAG

[0042] OYE3-9:

[...

Embodiment 2

[0090] Embodiment 2: Saccharomyces cerevisiae OYE3 enzyme gene expression vector construction

[0091] Respectively use BamHI and SacI to double-enzyme digest the PCR products, recover the DNA fragments with 1% agarose gel, connect the recovered OYE3 enzyme gene fragments to the pYPx245 plasmid containing double 35S promoters by T4DNA ligase, enzyme digestion identification and sequence analysis The recombinant plasmid AH128 containing Saccharomyces cerevisiae OYE3 enzyme gene was determined. The expression vector also contains a GUS reporter gene and a kanamycin resistance marker gene with an intron, the vector such as figure 1 shown.

Embodiment 3

[0092] Embodiment 3: Agrobacterium cultivation

[0093] The recombinant plasmid was introduced into Agrobacterium by electric shock method. Pick Agrobacterium tumefaciens LBA4404 single bacterium to 25mL YEB medium (50mg / L rifampicin) for overnight culture, take 5mL bacterial liquid transfer to 100mL YEB medium (50mg / L rifampicin), cultivate to OD600=0.7 -0.8, place the bacterial solution on ice for 10 minutes, centrifuge at 5000 rpm for 10 minutes, and collect the bacterial cells at 4°C, add 100 mL of sterile double distilled water to wash twice. Add 4mL of 10% glycerol to suspend the bacteria and transfer to a 50ml centrifuge tube. Centrifuge at 5500rpm for 10min at 4°C. Collect the bacteria, add 500 μL of 10% glycerol to suspend the bacteria, and transfer to a 1.5ml centrifuge tube. Take 70 μL of the Agrobacterium competent cells prepared above, add 1 μL of the recombinant plasmid AH128, mix well with a yellow pipette tip with the head removed, and transfer to a 0.1 cm e...

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Abstract

The invention discloses an OYE 3 gene deriving from saccharomyces cerevisiae and a preparation method and application thereof. A nucleotide sequence of the OYE 3 gene is SEQ ID NO 1, and a coded protein sequence of the OYE 3 gene is SEQ ID NO 2. The OYE 3 gene is prepared through a gene synthesis method and transformed into arabidopsis, tobacco, tomatoes and rice, germination percentage of seeds of obtained transgenic plants is improved by more than 2 times compared with non-transgenic plants, and root length of the transgenic plants is increased by more than 45% compared with the non-transgenic plants if the obtained transgenic plants are on a flat plate containing trinitrotoluene (TNT). The gene can promote plants to take part in degrading of the TNT, capacity of the transgenic plants for degrading the TNT is improved, and beneficial help for plants to recover TNT pollution is provided.

Description

technical field [0001] The invention belongs to the field of crop genetics and breeding, and in particular relates to an OYE3 gene derived from Saccharomyces cerevisiae and its preparation method and application. Background technique [0002] 2,4,6-Trinitrotoluene (TNT) is an energetic material widely used in military and civil blasting industries. Because TNT is toxic, carcinogenic and mutagenic, it is very harmful to the environment. It is mainly manifested in: human inhalation, mild cases can cause liver disease (toxic hepatitis), aplastic anemia and cataracts, severe cases can cause death When the content of TNT in the water reaches 1mg / L, the fish will die; TNT enters the soil and will interact with several main compounds in the soil to increase the concentration of organic matter in the soil. Therefore, it will cause far-reaching harm to the environment. Foreign countries began to study microbial degradation of TNT in the 1940s. After years of continuous exploration a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/39C07H21/04C12N15/82B09C1/00
Inventor 朱波姚泉洪彭日荷熊爱生薛永付晓燕田永生赵伟金晓芬
Owner 上海金丰裕米业有限公司