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Plant-anthocyanin synthesis control gene and application thereof

An anthocyanin and plant technology, applied in the fields of vegetable breeding and molecular genetics, can solve the problems of time-consuming and laborious, destructive germplasm materials, delaying the breeding process, etc., and achieve the effects of high detection accuracy, low cost and simple operation

Active Publication Date: 2018-05-11
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the color of purple-heart Chinese cabbage leaf bulbs can only be displayed after the bulbs are mature, and the leaf bulbs need to be cut and observed plant by plant in the field. The operation is not only extremely destructive to the germplasm materials, but also time-consuming and labor-intensive. greatly delayed the breeding process

Method used

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  • Plant-anthocyanin synthesis control gene and application thereof
  • Plant-anthocyanin synthesis control gene and application thereof
  • Plant-anthocyanin synthesis control gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Extraction of Chinese cabbage genome total RNA

[0035] 1) Grind the material stored in ultra-low temperature with liquid nitrogen, add about 50 mg of the ground powder, add 1 mL of Bizol solution, mix vigorously, let stand on ice for 15 min; centrifuge at 12000×g for 15 min at 4°C, take the supernatant, add 200 μL of chloroform, Cover the tube cap, shake vigorously, and let stand on ice for 15 minutes; centrifuge at 12,000×g for 15 minutes at 4°C, carefully pipette the supernatant into another centrifuge tube, add an equal volume of pre-cooled (-20°C) isopropanol, up and down Mix by inverting several times, and precipitate at -20°C for more than 2 hours;

[0036] 2) Centrifuge at 10000×g for 10 min at 4°C. Discard the supernatant, add 1mL 75% ethanol (depleted enzyme-free ddH 2 O preparation, that is, containing 0.1% DEPC, obtained by sterilizing for 40 minutes) wash the precipitate twice; discard the supernatant, air-dry the RNA on the ultra-clean work...

Embodiment 2

[0039] Embodiment 2: Extraction of Chinese cabbage genome total DNA

[0040] 1) Take 0.2g fresh tender leaves of Chinese cabbage with main veins removed, stuff them into a 2mL centrifuge tube with steel balls (steel balls need to be cleaned with 75% alcohol by mass fraction), put them into liquid nitrogen for quick freezing, and use tissue grinding Grind into powder;

[0041] 2) Add 700 μl of CTAB extract (CTAB: 2%, Tris-HCl (pH=8.0): 100 mmol / L, EDTA: 20 mmol / L, NaCl: 1.4 mol / L) preheated at 65°C to the centrifuge tube, Then add 10 μL of β-mercaptoethanol and mix quickly;

[0042] 3) Then put the centrifuge tube into an oven at 65°C, shake it every 5-10 minutes, and incubate for 45 minutes;

[0043] 4) Take out the centrifuge tube, add an equal volume of a mixture of phenol: chloroform: isoamyl alcohol (25:24:1), shake well for 15 minutes, and centrifuge at 10000 r / min for 10 minutes at room temperature;

[0044] 5) Transfer the upper liquid phase (about 700 μl) to another...

Embodiment 3

[0050] Embodiment 3: PCR amplification and gene detection of Chinese cabbage purple gene

[0051] Using the total DNA in Example 2 as a template, the purple gene of purple heart Chinese cabbage, the gene of purple traits in the parent of Porphyra stalk and the allele in white leafy Chinese cabbage and the detection of the gene were respectively obtained.

[0052] 1) 20 μl PCR reaction system: 2 μl of 50ng / μl template DNA, 10 μl of 2×Taq Master Mix, 1 μl of 10 μM upstream and downstream primers, supplement the reaction system with sterile deionized water to 20 μl.

[0053] The primer sequences are:

[0054] Upstream primer: 5'-ATGGAGGGTTCGTCCCAAG-3'

[0055] Downstream primer: 5'-TCAAGTTCCAGTTTCTCTCATCC-3'

[0056] 2) The PCR amplification reaction is carried out on a PCR instrument

[0057] The temperature and reaction conditions were as follows: pre-denaturation at 94°C for 3 min; then denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for a t...

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Abstract

The invention discloses a plant-anthocyanin synthesis control gene and application thereof. The sequence of the disclosed gene is shown in the SEQ ID No.1. A 3,772-bp klenow-fragment insertion mutation of the gene exists in first intron of white heading brassica rapa, and the gDNA sequence of the mutation is shown in the SEQ ID No.5. According to the mutation in the intron in the gDNA nucleotide sequence of the gene, three specificity PCR primers of the Marker 1, the Marker 2 and the Marker 8 are designed, wherein the Marker 1 and the Marker 2 are co-dominant markers, and the Marker 8 is a maternal marker. The DNA of the target brassica rapa is subjected to PCR amplification and identified through agarose gel electrophoresis, drinamyl hybrid strains and homozygous strains in segregation population can be rapidly identified through the Marker 1 and the Marker 2, and the drinamyl hybrid strains and the homozygous strains in the segregation population can also be distinguished through theMarker 8 in cooperation with the heading characteristics.

Description

technical field [0001] The invention belongs to the fields of vegetable breeding and molecular genetics, and in particular relates to a gene for controlling purple traits of purple-heart Chinese cabbage, molecular markers and applications thereof. Background technique [0002] Chinese cabbage (Brassica rapa L.) belongs to Brassica Brassica Brassica subspecies. It is one of the main vegetable crops originated in my country and one of the largest vegetable crops in my country. It is closely related to people's daily life and health. closely related. With the improvement of people's living standards, in recent years, people's requirements for the quality of Chinese cabbage are increasing day by day, and the breeding of orange and purple Chinese cabbage has been paid more and more attention by scholars and breeders at home and abroad. [0003] Due to the rich content of anthocyanins, purple Chinese cabbage is not only beneficial to improve the stress resistance of cabbage crops,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/20C12Q1/6895C12N15/11
CPCC07K14/415C12N15/825C12Q1/6895C12Q2600/13
Inventor 张鲁刚何琼薛一花
Owner NORTHWEST A & F UNIV
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