Medlar carotenoid synthase gene lycB and plasmid comprising the gene

A technology of carotene and synthesizing enzymes, applied in the field of genetic engineering, to achieve the effect of improving nutritional quality

Inactive Publication Date: 2006-12-27
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cloning of Lycium barbarum carotenoid synthase gene has not been reported yet

Method used

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  • Medlar carotenoid synthase gene lycB and plasmid comprising the gene
  • Medlar carotenoid synthase gene lycB and plasmid comprising the gene
  • Medlar carotenoid synthase gene lycB and plasmid comprising the gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Preparation of PCR template

[0019] Lycium barbarum cDNA library was provided by our laboratory, and λ phage DNA was extracted by liquid culture method.

[0020] Those skilled in the art can construct a wolfberry cDNA library in the laboratory. The construction steps of the library: (1) extraction of total RNA and purification of mRNA; (2) synthesis of cDNA double strands; (3) connection of cDNA and adapter with ligase;

[0021] (4) Removal of small fragments and redundant adapters; (5) Cloning of cDNA into vectors; (6) Packaging of phages; (7) Quality inspection of cDNA libraries.

[0022] In the process of cDNA library construction, mRNA isolation kits, cDNA synthesis kits, LambdaDNA packaging kits, vector λExCell, etc. were used, all of which have been commercialized.

Embodiment 2

[0024] PCR amplification in vitro

[0025] A pair of primers were designed and synthesized according to the conserved sequences of LycB genes in citrus, Arabidopsis, pepper, tomato, tobacco, daffodil, etc.: Primer 1 (5′ end primer SEQ ID No.3) 5′-GGA TCC ATG GAT ACT TTA GTG AAA ACTCCA-3'; Primer 2 (3' end primer SEQ ID No. 4) 5'-GC GGC CGC TTA TTC TTT GTC CCG CAATAA GTT-3'. In the 25μl reaction system, add 10×PCR Buffer (Mg 2+ 1SmM) 2.5μl, dNTP (each 2.5mmol / L) 2μl, primer 1 (20μM) and primer 2 (20μM) each 1μl, Ex Taq DNA polymerase (5U / μl) 0.5μl, template DNA 1.5ng. Reaction conditions: 94°C 3min→(94°C 1min→59°C 1min→72°C 2min) 35 →72°C for 10 minutes. The amplification reaction was carried out on a German UNOII Biometra DNA expander. Electrophoresis on 1% agarose gel was performed after the reaction.

Embodiment 3

[0027] Cloning and DNA sequence determination of amplified products

[0028] The PCR amplified products were recovered using Baobiology DNA Fragment Recovery Kit. The recovered product was ligated with the pGEM-T vector under the action of T 4 DNA ligase, and the ligated product was transformed into Escherichia coli JM109. Blue-white selection was performed on ampicillin-containing media. White colonies were randomly picked, and a small amount of plasmid DNA was extracted by alkaline lysis, and identified by PCR and double enzyme digestion (SacI, NcoI). Sequence determination was performed by Huada Gene Shanghai Dingan Biotechnology Co., Ltd. using a DNA fluorescence automatic sequence analyzer.

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PUM

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Abstract

This invention exposed a kind of synthase gene lycB of medlar carotenoids as well as the plasmid that includes this gene. The synthase gene lycB of medlar carotenoids has the nucleotide sequence indicated in the SEQ ID No. 1 sequence listing. The synthase gene lycB of medlar carotenoids in this invention can help to control the metabolism and accumulation of carotenoids in plant by the gene engineering approach, create the strains with higher carotenoids content, improve the nutrition of fruit and vegetable and realize the production of natual carotenoids in factory.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a wolfberry carotenoid synthetase gene lycB and a plasmid containing the gene. Background technique [0002] Plant carotenoids are synthesized via the isoprenoid pathway. GGPP (geranyl geranyl pyrophosphate) is the direct precursor of this pathway. Two GGPPs form phytoene under the action of PSY (phytoene synthase), and phytoene undergoes continuous desaturation. Hydrogen reacts to form streptosporine, lycopene. Lycopene is the branch point of further synthetic metabolism of carotenoids. Under the action of LycB (lycopene β-cyclase), lycopene is cyclized to form β-carotene. In LycB (lycopene β-cyclase) Under the joint action of LycE (lycopene ε-cyclase), α-carotene is formed, and lutein is further formed. With the development of molecular biology research methods, the genes encoding these enzymes have been isolated and identified from plants such as tom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/63
Inventor 季静王罡郑阳霞
Owner TIANJIN UNIV
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