Medlar carotenoid synthase gene PSY 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

At present, there is no report on the cloning of caroten

Method used

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

Examples

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) ligation of cDNA and adapters with ligase; (4) removal of small fragments and redundant adapters; (5) Cloning the cDNA into the vector; (6) packaging of the phage; (7) quality inspection of the cDNA library.

[0021] 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

[0023] PCR amplification in vitro

[0024] A pair of primers were designed according to the conserved sequences of PSY genes in plants such as tomato, pepper, Arabidopsis, and corn: primer 1 (forward SEQ ID No.3) GAA TTC ATG TCT ATT TGT ACG CTA TGG GTT GTT; primer 2 (reverse , SEQ ID No.4) GC GGC CGC TCA TGT TTG GGG TAT CAT AAA AGA, in 25μL reaction system, add 10×PCRBuffer (Mg 2+ 15mmol·L -1 )3.0μL, dNTP (each 2.5mmol·L -1 ) 2.5 μL, primer 1 (20 μmol L -1 ) and primer 2 (20μmol·L -1 ) each 1.5 μL, TaqDNA polymerase (5U·μL -1 ) 0.5 μL, template DNA 1.5ng. Reaction conditions: 94°C 3min→(94°C 1min→55°C 1min→72°C 2min) 35 →72°C for 10 minutes. The amplification reaction was carried out on a German UNOII Biometra DNA amplification instrument. Electrophoresis on 1% agarose gel was performed after the reaction. (See figure 1 )

Embodiment 3

[0026] Cloning and DNA sequence determination of amplified products

[0027] The PCR amplification products were recovered using the DNA Fragment Recovery Kit of Beijing Dingguo Biotechnology Development Center. The recovered product was ligated with the pCEM-T vector, 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 PSY of medlar carotenoids as well as the plasmid that includes this gene. The synthase gene PSY of medlar carotenoids has the nucleotide sequence indicated in the SEQ ID No. 1 sequence listing. The synthase genes PSY 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 PSY and a plasmid containing the gene. Background technique [0002] Carotenoids in plants are synthesized by isoprene compounds or terpenoids. There are five enzymes involved in the metabolic backbone, namely phytoene synthase (PSY), phytoene desaturase (PDS), ξ-carotene desaturase (ZDS), lycopene β - Cyclase (LycB) and Lycopene epsilon-cyclase (LycE). With the development of molecular biology research methods, genes encoding these enzymes have been isolated and identified from tomato, tobacco, pepper, Arabidopsis, corn and other plants. It is reported that the total carotene content in wolfberry is 2.9527mg·g -1 , its high content is rare in plants. At present, there is no report on the cloning of carotenoid synthase gene from Lycium barbarum at home and abroad. The main technical literatures are: Tao Jun, Zhan...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/63
Inventor 季静王罡郑阳霞
Owner TIANJIN UNIV
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