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Molecular biological method for quickly identifying gender of scandent hop

A technique of molecular biology, Humulus chinensis, applied in the field of molecular biology

Inactive Publication Date: 2014-02-05
HENAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at present, there is no report about the method of AFLP-SCAR molecular markers to identify the sex of Humulus japonicus

Method used

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  • Molecular biological method for quickly identifying gender of scandent hop
  • Molecular biological method for quickly identifying gender of scandent hop
  • Molecular biological method for quickly identifying gender of scandent hop

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Screening of sex-specific AFLP molecular markers in Humulus japonicus

[0014] Specifically include the following steps:

[0015] (1) Genomic DNA extraction of Humulus chinensis: select the Humulus humulus that has bloomed and can be identified as male or female by flower structure. Put an appropriate amount of leaves into a mortar, add liquid nitrogen to quickly grind them into powder, and transfer them to a centrifuge tube; add CTAB extraction buffer preheated to 65°C, and incubate at 65°C for 60 minutes; add an equal volume of phenol: chloroform: iso Amyl alcohol (25:24:1, V / V / V), 10000×g, centrifuge at room temperature for 10 min; take the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1, V / V), mix Homogenize, 10000×g, centrifuge at room temperature for 10min; take the supernatant, add 2 times the volume of pre-cooled absolute ethanol, and let it stand at -20°C for 1h; pick out the flocculent DNA precipitate with a thin glass rod, and...

Embodiment 2

[0032] Example 2: Cloning and sequence analysis of Humulus japonicus male-specific AFLP molecular markers

[0033] The male-specific bands amplified by the primer combination (E-CTT / M-GAC) were recovered, cloned into the pMD-18T (Dalian Bao Biology) vector, ligated and transformed by standard methods, and screened by conventional PCR methods A clone containing the target fragment was obtained, and a 194bp male-specific fragment was obtained by sequencing, the sequence of which was shown in SEQ ID NO:1. The sequence was compared on Genebank, and no homologous sequence was found.

Embodiment 3

[0034] Example 3: Transformation of SCAR markers and identification of Humulus sex

[0035] According to the sequence of the male-specific AFLP molecular marker of Humulus japonicus, specific primers were designed to convert the sex-specific AFLP marker into a SCAR marker. The sequences of the primers are as follows:

[0036] SCARF: 5'-TTGAAGAAATCTCATCCTCAAAGA-3':

[0037] SCARR: 5'-GACCTTGGCCACAGGAATAG-3'.

[0038] That is, using the genomic DNA of the female Humulus and the male Humulus as templates, PCR amplification was performed with this primer, and the primer only amplified a 153bp fragment in the genome of the male Humulus, but no amplification occurred in the female Humulus. add fragments, such as figure 2 shown.

[0039] Among them, the PCR reaction system used is: 1 U of Taq DNA polymerase, 2 μL of dNTP, 100-200 ng of genomic DNA, which contains 0.1 μmol / L of male-specific upstream and downstream primers, and supplemented with double distilled water to a final v...

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Abstract

The invention relates to a molecular biological method for identifying gender of scandent hop, belonging to the technical field of the molecular biology. The specific implementing process is as follows: a designed specific primer sequence is utilized and a scandent hop individual genome DNA (deoxyribonucleic acid) is used as a template for PCR (Polymerase Chain Reaction) amplification, the reaction system is 25muL, the reaction compositions are as follows: 1U of TaqDNA polymerase, 2muL of dNTP (diethyl-nitrophenyl thiophosphate) and 100-200ng of genome DNA, the PCR amplification is performed on a PCR instrument, the electrophoretic separation is performed to the amplified product with 1.5% of sepharose gel, ethidium bromide is used for dying, the process is observed and recorded by photograph under a gel-imaging system, DL2000 marker is used as a molecular weight mark, the scandent hop with an objective stripe with size of 153bp is male, and the scandent hop without the stripe is female. The SCAR (sequence characterized amplified regions) primer is used for predicting the gender of the scandent hop, a simple and stable molecular marker is provided for identifying the gender of the scandent hop, and the molecular biological method has the important application value in the aspects of production and human health.

Description

technical field [0001] The invention relates to a molecular biology method for quickly identifying the sex of humulus humulus, belonging to the technical field of molecular biology. Background technique [0002] Humulus scandens L, also known as Lalayang and Lalateng, is a plant of the genus Humulus in the Cannabaceae subfamily (Cannaboideae), and is an annual weed widely distributed in my country. Humulus has unisexual flowers, dioecious plants, and the sex performance is stable. The flowering period is July-August, and the fruiting period is August-September. Humulus is a non-clinical commonly used Chinese herbal medicine. The whole herb can be used medicinally, especially its seeds contain humulone and lupulone, etc., which have the functions of clearing away heat and detoxification, diuresis, detumescence, antibacterial and anti-inflammatory, strengthening the stomach and inhibiting bacteria, and inhibiting viruses, etc., and can improve animal immunity, Prevent diseas...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6879C12Q1/6895C12Q2600/156
Inventor 高武军李书粉王连军张国俊邓传良卢龙斗
Owner HENAN NORMAL UNIV
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