Carotenoid synthetic route Tagetes erecta violaxanthin de-epoxidase (TeVDE1) gene and application

A technology of carotene and cyclooxygenase, which is applied in the field of plant genetic engineering and can solve problems such as genes that have not been cloned and separated

Active Publication Date: 2016-09-28
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, marigold, which is an important plant source of lutein, has not yet cloned and isolated some important genes of its lutein synthesis pathway.

Method used

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  • Carotenoid synthetic route Tagetes erecta violaxanthin de-epoxidase (TeVDE1) gene and application
  • Carotenoid synthetic route Tagetes erecta violaxanthin de-epoxidase (TeVDE1) gene and application
  • Carotenoid synthetic route Tagetes erecta violaxanthin de-epoxidase (TeVDE1) gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: High-throughput transcriptome sequencing of marigold

[0014] Since the marigold genome has not been determined, in order to obtain the sequence of marigold functional gene transcripts, the tissue samples of the pigmented marigold cultivar Chrysanthemum King were used to extract 1 RNA each from the leaves, flower buds, and mature flowers of three individual plants, and conduct high-pass analysis. Quantitative transcriptome sequence determination and assembly annotation.

[0015] 1. Reagents

[0016] Plant RNA extraction reagent Trizol was purchased from Invitrogen Company, DNase I (Dnase I) was purchased from Takara Company, RNA Library Prep Kit was purchased from Beijing Biomike Biotechnology Co., Ltd., and other reagents were imported. packaged or domestically produced analytically pure products.

[0017] 2. Plant material

[0018] Pigment marigold (Tagetes erecta L.) cultivar Juwang was bred by Chifeng Xinhui Horticultural Company.

[0019] 3. Method ...

Embodiment 2

[0053] Example 2: Cloning of Tagetes TeVDE1 gene

[0054] According to the data assembly of the marigold violaxanthin decycloxygenase gene TeVDE1 according to Example 1, PCR-specific primers were designed using the primer design software Primer Premier 5.0, total RNA was extracted from marigold petals, and reverse transcription-PCR (reverse transcription- PCR, RT-PCR) to obtain the TeVDE1 gene.

[0055] 1. Reagents

[0056] Plant RNA extraction reagent Trizol was purchased from Invitrogen Company, DNase I (Dnase I) was purchased from Takara Company; reverse transcriptase (TransScript Reverse Transcriptase) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; high-fidelity DNA polymerase PrimeStar was purchased from TaKaRa Company; the cloning vector pEASY-Blunt SimpleCloning Vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the primers were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd., and the rest of the reagents were imported or do...

Embodiment 3

[0106] Embodiment 3: Determination of lutein content in marigold petals by HPLC method

[0107] The content of lutein in different tissues of the pigmented marigold cultivar Juwang was determined by high performance liquid chromatography (HPLC).

[0108] 1. Reagents

[0109] Lutein standard products were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and the rest of the reagents were imported subpackages or domestic analytically pure products.

[0110] 2. Method

[0111] Take samples of marigold leaves, flower buds and mature petals, grind them with liquid nitrogen, weigh 0.15g, add 1mL ethanol (containing 0.1% 2,6-di-tert-butyl-4-methylphenol), vortex for 15s, add 400mL 50 %KOH aqueous solution, vortex for 15s, in 60°C water bath for 60min, vortex every 15min. Extract 3 times with 3.3 mL of n-hexane, combine the extracts, and take 50 μL of the filtrate for loading. Chromatographic conditions: high performance liquid chromatography (WATERS, USA); column ...

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Abstract

The invention discloses a carotenoid synthetic route Tagetes erecta violaxanthin de-epoxidase (TeVDE1) gene. The TeVDE1 is characterized in that the nucleotide sequence of the TeVDE1 gene is shown as SEQ ID NO: 1 in a sequence table, the amino acid sequence of the TeVDE1 gene is shown as SEQ ID NO: 2 in the sequence table. The TeVDE1 gene is obtained from tagetes erecta petal tissue in a clone mode. Real-time quantification analysis and a xanthophyll content determination result show that the expression quantity of the TeVDE1 gene in Tagetes erecta flower buds is high, and the expression level of the TeVDE1 is related to the xanthophyll content. The SEQ ID NO: 1 gene has an effect on xanthophyll synthetic accumulation, and the TeVDE1 obtained from the pigment tagetes erecta in a clone mode can be used for increasing the pigment contents of xanthophylls and other carotenoid.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to the cloning of the violaxanthin deepoxygenase gene TeVDE1 of the carotenoid synthesis pathway from the pigment marigold, the nucleotide sequence and protein sequence of the gene, and the use of the gene in improving Applications in the accumulation of plant carotenoid synthesis. Background technique [0002] According to "Northern Gardening" (2007, No. 1, pp. 44-46), marigold (Tagetes erecta L.) is a 1-year herbaceous plant of the genus Marigold in the Asteraceae family. It is native to America, Mexico and other places. Colorful, strong plant adaptability and short production cycle, it has been widely introduced all over the world. A variety of secondary metabolites can be extracted from marigold flowers. The yellow pigments in petals are mainly lutein esters, lutein and other trace carotenoids. Marigold petals can be made into four forms of commod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02A01H5/00
CPCC12N9/0055C12N15/825C12Y110/99003
Inventor 黄胜雄曹徐绿牛向丽刘建刘莹刘永胜刘国庆田丽
Owner HEFEI UNIV OF TECH
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