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Peanut SPL (squamosa promoter-binding protein-like) transcription factor gene, as well as encoded protein and application thereof

A technology of transcription factor and peanut, applied in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2014-08-20
山东省农业科学院高新技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SPL is a kind of transcription factor unique to plants, which is involved in a variety of metabolic pathways and physiological and biochemical processes, but there is no report on the ability of SPL to promote anthocyanin accumulation. This study is innovative and prospective

Method used

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  • Peanut SPL (squamosa promoter-binding protein-like) transcription factor gene, as well as encoded protein and application thereof
  • Peanut SPL (squamosa promoter-binding protein-like) transcription factor gene, as well as encoded protein and application thereof
  • Peanut SPL (squamosa promoter-binding protein-like) transcription factor gene, as well as encoded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: determination of peanut anthocyanin content

[0031] Referring to the methods of Mancinelli et al (1991) and Serrano et al (2012), the anthocyanin contents in the roots, stems, and leaves of the peanut purple mutant and Fenghua No. 1 were respectively determined. Specific steps are as follows:

[0032] (1) Weigh 50 mg of peanut material, place it in a 1.5 mL centrifuge tube, and grind it into powder with liquid nitrogen.

[0033] (2) Add 700 μl acidic methanol (CH 3 OH:HCl volume ratio is 99:1), extract overnight at 4°C. (Note: Three sets of biological repetitions were set up, and each set was measured 3 times.)

[0034] (3) Centrifuge at 12,000 rpm for 1 min at 4°C, take 600 μL of supernatant and place in a new centrifuge tube, then add 1 mL of chloroform, and then add 400 μl of distilled water.

[0035] (4) Centrifuge at 12000 rpm for 10 min at 4°C, and the supernatant is used for the determination of anthocyanin content.

[0036] (5) Use a spectrop...

Embodiment 2

[0043] Embodiment 2: peanut SPL2-1 gene cloning

[0044] The extraction method of peanut total RNA is as follows:

[0045] (1) Weigh 1g of the mixed material of Fenghua No. 1 stem and leaf, grind it thoroughly in liquid nitrogen, and then quickly transfer it to a pre-cooled 1.5mL Eppendof tube (about 200mg sample per tube).

[0046] (2) Preheat CTAB extract at 65°C (components: 2wt% cetyltrimethylammonium bromide (CTAB), 1.4mol / L NaCl, 20mmo / L ethylenediaminetetraacetic acid (EDTA, pH8.0 ), 100mmol / L Tris-HCl (pH8.0), 2wt% polyvinylpyrrolidone (pvp-40)).

[0047] (3) Add 600 μL of preheated CTAB extract, mix quickly, and incubate at 65°C for 5 minutes, and mix thoroughly for 3 to 5 times during this period.

[0048] (4) After cooling to room temperature, add an equal volume (600 μL) of water-saturated phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) mixture, and mix well.

[0049] (5) 4°C, 12000rpm, centrifuge for 15min.

[0050] (6) Transfer the supernatant to a new...

Embodiment 3

[0129] Embodiment 3: Real-time PCR quantitatively detects the expression level of SPL2-1

[0130] Preparation of cDNA template:

[0131] Using the peanut purple mutant unearthed for about two weeks and Fenghua No. 1 as materials, total RNA was extracted from roots, stems, and leaves by the above-mentioned CTAB method, and the qualified RNA samples were reverse-transcribed, and the reverse-transcribed products were Can be used as a template for Real-time PCR.

[0132] Real-time PCR reaction system and reaction procedure:

[0133] The fluorescent dye is FastStart Universal SYBR Green master (ROX) from Roche, and the reaction system is as follows:

[0134]

[0135] Mix the above reaction solution evenly, and then carry out PCR reaction.

[0136] Reaction program: 95°C for 10min; 95°C for 15s, 60°C for 1min, at which time fluorescence signal was collected, 40 cycles; 95°C for 15s, 60°C for 1min; 0.3°C / s to 95°C, 95°C for 15s, during this period continuous Collect fluorescen...

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Abstract

The invention relates to a peanut SPL (squamosa promoter-binding protein-like) transcription factor gene, as well as an encoded protein and application thereof. The nucleotide sequence of a peanut anthocyanin accumulation regulatory transcription factor AhSPL2-1 is as shown in SEQ (sequence) ID (identity) No. 1. The peanut AhSPL2-1 transcription factor provided by the invention plays an important role in the aspect of plant anthocyanin accumulation, and has potential application values in cultivation of high-anthocyanin crops.

Description

technical field [0001] The invention relates to a peanut SPL transcription factor gene, its encoded protein and its application, and belongs to the technical field of biotechnology. Background technique [0002] Peanut (Arachis hypogaea L.) is an important oil crop and economic crop in China. my country is the world's largest peanut producer and exporter. The development of peanut industry has important strategic significance for the development of my country's national economy and grain and oil security. [0003] Anthocyanin is a super antioxidant natural product, which has the functions of preventing cardiovascular disease, delaying cell aging, slowing down diabetes, improving vision and anti-cancer, so it is widely used in health food and cosmetics. At present, black foods such as black rice and black beans are favored by consumers because they are rich in anthocyanins, vitamins and various trace elements. Colored peanuts, also known as multicolored peanuts, can be divid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82C12N1/21C07K14/415A01H5/00
Inventor 王兴军李明夏晗李长生赵术珍侯蕾李爱芹赵传志
Owner 山东省农业科学院高新技术研究中心
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