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A kind of primer based on hmgr 3'-UTR and rRNA ITS and its application

A-UTR, primer sequence technology, applied in the field of molecular biology, can solve problems such as death

Active Publication Date: 2020-12-11
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pericarpium bark contains the poisonous vaccine of willow willow, which has a strong cardiotonic effect but is also a toxic ingredient. Overdose can even cause death

Method used

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  • A kind of primer based on hmgr 3'-UTR and rRNA ITS and its application
  • A kind of primer based on hmgr 3'-UTR and rRNA ITS and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: The method for obtaining the primer sequence

[0069] 1. Obtaining homologous sequences and designing homologous primers

[0070] Through NCBI search, Acanthopanax (registration number: JQ905593), Arabidopsis thaliana (registration number: AY488113), Panax notoginseng (registration number: KP702301), ginseng (registration number: GU565098), American ginseng (registration number: FJ755158HMGR) were respectively obtained. ) of the HMGR sequence in NCBI using blast for homologous sequence analysis, and then using Clustal X software for multi-sequence homology alignment, and HMGR homologous PCR primers were designed according to the conserved region of the HMGR ORF region (HMGR primer sequence: upstream Fa :5 ， -agtaaattcaggggggagtcata-3 ， and downstream Fb:5 ， -caccattttcaactttaactggtg-3 ， ), the primer was verified to amplify DNA fragments in the subsequent PCR of the DNA samples of Wujiapi, indicating that the PCR primers were also conserved in Wujiapi. ...

Embodiment 2

[0097] Embodiment two: the application of primer sequence

[0098] The three pairs of primer sequences obtained in the present invention are used for the inspection of the bark of Wujia and Xiangjia. The test samples can be fresh products, dry products or processed products that do not damage DNA.

[0099] 1. Sample DNA extraction, the specific steps are as follows:

[0100] Ⅰ. Take a sample of 1.0~1.5g and place it in a mortar, add liquid nitrogen into the mortar to submerge the sample, and grind it quickly;

[0101] Ⅱ. Put the ground sample into a 2.0mL EP tube, add 800μL of CTAB extract solution (pH 8.0 65°C) to submerge the sample, shake gently to mix;

[0102] Ⅲ. Put the EP tube in a water bath at 65°C for 30min~60min, shake the EP tube gently every 10min to mix the solution, centrifuge at 4°C at 12000rpm for 10min;

[0103] Ⅳ. Take the supernatant into a new 2.0mL centrifuge tube, add an equal volume of phenol:chloroform solution (V / V=1:1), shake gently for 2 minutes t...

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Abstract

The invention provides three pairs of primers based on HMGR 3'-UTR and rRNA ITS and a primer acquisition method and application of the primers based on HMGR 3'-UTR and rRNA ITS. The three pairs of primers based on a high-specificity 3'-UTR and rRNA ITS sequence are high in sequence specificity and capable of precisely identifying cortex acanthopanacis and cortex periplocae samples. Downstream sequences are obtained by a RACE (rapid-amplification of cDNA ends) technology for amplification under the condition that whole genome sequences of cortex acanthopanacis and cortex periplocae are unknown, new sequence fragments are acquired by means of high-throughput sequencing, and a traditional Chinese medicine identifying function is realized. In application of the primer, the DNA instead of the RNA is extracted from the samples, so that requirements on the samples are low, and the samples can be fresh, dry or simply processed (according to processing methods low in DNA damage).

Description

technical field [0001] The patent of the present invention relates to the technical field of molecular biology, specifically a primer based on HMGR 3'-UTR and rRNAITS and its application. Background technique [0002] 3-Hydroxy-3-methylglutaryl CoA reductase (HMGR) catalyzes the generation of mevalonate from 3-hydroxy-3-methylglutaryl-CoA, and HMGR is the rate-limiting step in the biosynthesis of eleutheroside enzyme. Eleutheroside is the active ingredient of Wujiapi and Acanthopanax, and it is also the target compound for species identification and quality identification. The 3' untranslated region of mRNA (3'-UTR) is a nucleotide sequence extending from the stop codon at the end of the mRNA coding region to the front of the poly-A tail (Poly-A). The 3'-UTR of eukaryotes plays an important role in the regulation of gene expression. It can regulate the stability of mRNA, control its translation, and assist in the identification of codons. The 3'-UTR is highly specific amon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6895C12Q1/6869
Inventor 唐勋曾阳司怀军
Owner GANSU AGRI UNIV