Lilium spp. ANS (Anthocyanidin Synthase) gene and cloning method thereof

A lily and gene technology, applied in the field of lily ANS gene and its cloning, can solve the problem of less research on gene cloning and so on

Inactive Publication Date: 2014-02-12
BEIJING UNIV OF AGRI
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  • Abstract
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Problems solved by technology

At present, the research on oriental lily mainly focuses on cultivation technology, tissue culture, postharvest physiology, etc. There are few studies on gene cloning.

Method used

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  • Lilium spp. ANS (Anthocyanidin Synthase) gene and cloning method thereof
  • Lilium spp. ANS (Anthocyanidin Synthase) gene and cloning method thereof
  • Lilium spp. ANS (Anthocyanidin Synthase) gene and cloning method thereof

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Embodiment Construction

[0028] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings; it should be understood that the preferred embodiments are only for illustrating the present invention, rather than limiting the protection scope of the present invention.

[0029] The fully colored oriental lily 'Justina' buds were used as experimental materials, which were quickly frozen in liquid nitrogen and stored in a -80°C ultra-low temperature freezer for later use.

[0030] 1. Total RNA Extraction

[0031] Using Plant RNAzol reagent, the specific method is as follows:

[0032] 1) Put the petals in a mortar, add liquid nitrogen and grind quickly. After grinding sufficiently, add RNAzol at a ratio of 100mg tissue / mL RNAzol and immediately transfer to a centrifuge tube.

[0033] 2) Stand at room temperature for 10 minutes to fully lyse the petals.

[0034] 3) Centrifuge at 4°C and 12,000 rpm for 5 minutes, and pipette the supernata...

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Abstract

The invention discloses a lilium spp. ANS (Anthocyanidin Synthase) gene cloning method. The method comprises the following steps of: extracting and purifying lilium spp. petal RNAs (Ribonucleic Acids); synthesizing a first cDNA (complementary Deoxyribose Nucleic Acid) chain; performing a polymerase chain reaction (PCR) by applying a pair of primers for amplifying the first lilium spp. cDNA chain; performing 3'RACE (3'Rapid-Amplification of CDNA Ends) cloning on a PCR product by applying a 3'RACE primer; performing 5'RACE cloning on the PCR product by applying a 5'RACE primer; splicing an obtained conserved sequence, a 3'-end sequence and a 5'-end sequence by using Seqman software to obtain a cDNA full-length sequence with 1,356 bases of a gene A; and performing a PCR amplified reaction, rubber cutting recovery, connection and transformation, blue-white selection and a colony PCR.

Description

technical field [0001] The invention relates to a gene and its cloning method, especially the lily ANS gene and its cloning method. Background technique [0002] Flower color is one of the most important traits of flowers, which is mainly determined by three types of pigments: flavonoids, carotenoids and alkaloids. Flavonoids are the decisive pigment group for the formation of most flower colors. They exist in the petal epidermal cell fluid, among which anthocyanins belong to the red series, and the others belong to the yellow series; carotenoids are mainly yellow, orange or red, and they are widely present in Flowers, leaves, peels and roots of plants; alkaloids are involved in the formation of yellow, orange and red petals. There are many structural genes and regulatory genes involved in the synthesis of these three types of pigments, including multiple metabolic steps, and the mechanism of action is relatively complex. [0003] Traditional flower color breeding is time-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/10
Inventor 张克中崔金腾王瑜张睿鹂
Owner BEIJING UNIV OF AGRI
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