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Saussurea involucrata chalcone synthetase (chs), its coded protein and cloning method

A gene and positive monoclonal technology, applied in biochemical equipment and methods, enzymes, enzymes, etc.

Inactive Publication Date: 2007-09-05
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Before the publication of this patent, no other publications or reports of the Saussurea chalcone synthase gene and its cDNA full-length sequence and the amino acid sequence of the encoded protein mentioned in this patent application have been found

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The establishment of embodiment 1 snow lotus cDNA library

[0039] The jellyfish snow lotus (Saussurea.medusa) 1-15 days old callus is based on the red line of jellyfish snow lotus used for the construction of the bank Obtained by induction in the laboratory, cultured and subcultured in MS medium supplemented with 2mg / L NAA and 0.5mg / LBA at 25±1°C, 16h light / 8h dark.

[0040]Weigh 1-14 days of red callus, 50 mg per day, a total of 700 mg mixed samples in a mortar, and use TRIZOL Reagent (purchased from GIBCO Company) to extract total RNA. Take 1-3 μl of total RNA sample, and synthesize the first-strand cDNA with the PolyT primer of the CDSIII restriction site in the SMART cDNA Library Construction Kit (Catlog#: K1051-1) (CLONTEC). Use LD PCR (long distance PCR) Amplify the cDNA first strand to obtain double stranded cDNA.

[0041] Proteinase K was added to digest and remove the Taq enzyme in the cDNA, then the cDNA was digested with Sfi I, and then the cDNA was size-s...

Embodiment 2

[0042] Example 2 Screening Saussurea chalcone synthase gene chs cDNA from the library

[0043] According to the conserved amino acid sequence of the chs gene published in GenBank, the upstream and downstream partial degenerate primers were designed and synthesized with the software DNAMAN4.0 (Lynnon Biosoft, USA):

[0044] CP 1 : 5′-AAT GCG ATG AAT GAA TGG GG-3′

[0045] CP 2 : 5′-AAG CAA CCG TGT TAC ATC AT-3′

[0046] Take 1 μL of each sub-library as a template, and use CP 1 、CP 2 As a primer, use 25 μL of the system to lyse the phage by heating at 95°C for 10 minutes, add the PCR amplification mixture, and perform TD-PCR amplification. Program: Denaturation at 94°C for 5 minutes, followed by 1 minute at 94°C, annealing at 65-50°C for 1 minute (each cycle annealing temperature is 0.5°C lower than that of the previous cycle), extension at 72°C for 1 minute, and 30 cycles; denaturation at 94°C for 1 minute, Anneal at 50°C for 1 min, extend at 72°C for 1 min, 15 cycles; ex...

Embodiment 3

[0048] Example 3PCR method for cloning Saussurea chalcone synthase gene chs full-length cDNA

[0049] According to the sequence of the chs cDNA published in GenBank, a primer was designed at the upstream of the open reading frame, that is, at the 5′ end, according to the conservation, and then a primer was designed from the middle of the sequencing sequence using the software DNAMAN4.0:

[0050] CP 3 : 5′-ATG GTG ACC GTC GAG GAA GTC CG-3′

[0051] CP 4 : 5′-GCA CTA CTA GTG GCG TTG-3′

[0052] A small amount of total RNA was extracted with TRIZOL Reagent, 2 μL of total RNA was taken, and the first-strand cDNA was synthesized under the action of M-MLV reverse transcriptase (purchased from Promega) in a 20 μL reaction mixture system, using this first-strand cDNA as a template, With the above CP 3 , CP 4 Perform PCR amplification for primers: denaturation at 94°C for 5 minutes, followed by 1 minute at 94°C, annealing at 55°C for 1 minute, extension at 72°C for 1 minute, and 3...

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PUM

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Abstract

A method for constructing Saussurea involucrate cDNA library, screening encoding gene of Saussurea involucrate by degenerate primer and cloning chs by specific primer, and its chs full-length cDNA sequence and encoded amino-acid polypeptide sequence are disclosed. The length of cDNA is 1,313bp and it encodes 389 amino-acid polypeptides, it can be used to construct positive-sense gene constructor, control plant or plant culture and improve flavone yield.

Description

technical field [0001] The invention belongs to the field of biotechnology. The invention discloses a method for cloning a chalcone synthase gene by screening Saussurea cDNA library and a PCR method, and provides a full-length cDNA nucleic acid sequence for cloning the chalcone synthase gene and its encoded protein sequence. Background technique [0002] Snow lotus, also known as snow lotus, belongs to the genus Asteraceae. It is a class of rare and precious medicinal plants commonly used by the people and belongs to perennial herb. It is generally distributed on high mountain flowstone beaches above 4,000 meters above sea level. As early as in "Northwest Regions" and "Xiangyuan Xiaozhi", there are records about snow lotus. It has the functions of expelling cold and dehumidification, promoting blood circulation and promoting menstruation, strengthening tendons and yang, anti-inflammation, analgesia, and contracting uterus. It is used by folks to treat rheumatoid arthritis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/52C12N9/00C12N15/63C12N1/21
Inventor 赵德修虞珍珍程丽琴徐亮胜金治平
Owner INST OF BOTANY CHINESE ACAD OF SCI
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