Lycium chinense Miller GGPS1 gene for improving stress resistance of plant and recombinant vector comprising the gene

A technology of recombinant vectors and genes, applied in the direction of plant gene improvement, introduction of foreign genetic material using vectors, plant products, etc.

Inactive Publication Date: 2014-04-30
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] With the continuous discovery of the medicinal value and health care function of lycopene and carotenoids, the demand for the types and yields of lycopene and carotenoids will also increase. However, lycopene carotenoids Difficult to synthesize chemically

Method used

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  • Lycium chinense Miller GGPS1 gene for improving stress resistance of plant and recombinant vector comprising the gene
  • Lycium chinense Miller GGPS1 gene for improving stress resistance of plant and recombinant vector comprising the gene
  • Lycium chinense Miller GGPS1 gene for improving stress resistance of plant and recombinant vector comprising the gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Cloning of geranylgeranyl pyrophosphate synthase gene LmGGPS1 from Lycium barbarum

[0050] GGPS1 3' end cloning:

[0051] Using Trizol reagent, extract total RNA from 100 mg of fresh Lycium barbarum leaves, and design upstream primers according to the Unigene sequence of the transcriptome. The upstream primer of the geranyl geranyl pyrophosphate synthase gene LmGGPS1 in Lycium barbarum is LmGGPS1-F1:5'TAGGTGGCGGGAACGAAGAT- 3', using the 3' FULLRACE Core Set Ver.2.0 (TaKaRa, Japan) kit to amplify the complete gene sequence. Specific steps: ①Using totalRNA as a template, use 3'RACE Adapter primers for reverse transcription reaction to synthesize 1st Strand cDNA. The reaction system is as follows:

[0052] RNA 2μL. 3' RACE Adapter 1μL 5×M-MLV Buffer 2μL dNTP Mixture 1μL RNase Inhibitor 0.25 μL Reverse Transcriptase M- MLV 0.25 μL RNase Free ddH 2 o 3.5 μL

[0053] Reaction conditions: 42°C, 60min; 70°C, 1...

Embodiment 2

[0064] Construction process of cloning vector pMD19-T-LmGGPS1

[0065] The LmGGPS1 gene shown in the sequence listing is connected to the pMD19-T carrier, and the reaction system is as follows:

[0066] LmGGPS1 full-length recovery product 4μL pMD19-T vector 1μL Solution I 5μL

[0067] LmGGPS1 PCR fragment is the reclaimed in embodiment 1 GGPS1 full length product.

[0068] Reaction conditions: 16°C, 30min. The ligation product was transformed into competent E-Coli.TOP10, spread on LB agar plate medium containing 40ml 25mg / ml X-Gal, 16ml 50mg / ml IPTG, 100mg / L Amp and cultured to form a single colony. Select white colonies, colony PCR method confirms the length of the insert in the T vector, which is consistent with expectations, and sends the vector to Huada Gene Company for sequencing. We obtained the 1074bp base sequence of the gene, blasted it at NCBI, and compared it with solanum The family homology is high, indicating that the gene was s...

Embodiment 3

[0070] Construction process of Escherichia coli expression vector pET28a-LmGGPS1

[0071] The Escherichia coli containing the pMD19-T-LmGGPS1 plasmid obtained in the expanded culture experiment example 2 was extracted, the plasmid was double-digested with BamHI and SalI, and the pET28a empty vector was double-digested with BamHI and SalI, purified and ligated , to obtain the Escherichia coli expression vector pET28a-LmGGPS1, and connect the digested products of the two.

[0072] The connection system is as follows:

[0073] pET28a empty vector fragment 2μL pMD19-T-LmGGPS1 restriction fragment 5μL 5×T4 DNA ligase buffer 2μL T4 DNA ligase 1μL

[0074] The ligation product was transformed into competent Escherichia coli BL21. Spread on LB plates containing 100mg / L kana resistance and culture at 37°C. After 12 hours, a single colony was picked for colony PCR verification, and the colony PCR verification positive bacteria were shaken to extra...

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Abstract

The invention relates to a Lycium chinense Miller geranylgeranyl pyrophosphate synthetase (GGPS) gene and a recombinant vector comprising the gene, and specifically relates to cloning of GGPS gene (i) Lm ( / i) (i) GGPS1 ( / i) in Lycium chinense Miller. The total RNA is extracted from fresh Lycium chinense Miller leaves, LmGGPS1 in Lycium chinense Miller is cloned to obtain a complete gene sequence 1134bp; an Escherichia coli expression vector pET28a-LmGGPS1 and a binary plant expression vector pCAMBIA2300-LmGGPS1 are constructed; and the vectors are transformed into Agrobacterium C58 cells by an electroporation method; and the cells are used for tobacco transformation, so as to obtain transgenic tobacco. Through the tests, the transgenic tobacco gains greatly improved content of plant lycopene, and increased content of carotene. The invention can be used for the preparation of transgenic maize, soybean, rice, peanut, sunflower, potato, cotton, millet, barley, and flower and vegetable plants.

Description

technical field [0001] The present invention relates to a kind of Lycium barbarum ( Lycium chinense Miller ) Geranylgeranyl pyrophosphate synthase gene (Geranylgeranyl pyrophosphate synthase, GGPS ) and a recombinant vector including the gene, specifically the geranyl geranyl pyrophosphate synthase gene in Lycium barbarum LmGGPS1 clone. Background technique [0002] Geranyl Geranyl pyrophosphate (GGPP) is an important intermediate metabolite ubiquitous in the biological world. In plants, GGPP participates in the production of chlorophyll, carotenoids, gibberellins, plastoquinones, vitamin E, monoterpenes and benzoquinones, etc. The synthesis of products has an important impact on plant photosynthesis, growth and development, and product quality. The product was directly catalyzed by GGPS. GGPP is not only the most direct precursor of carotenoid biosynthesis, but also the precursor of gibberellin, chlorophyll and plastoquinone in plants. The synthesis of GGPP is the ra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/70C12N15/84C12N1/21C12N5/10A01H5/00
Inventor 季静王罡贾翠翠田小卫刁进进曹海燕
Owner TIANJIN UNIV
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