Tetraena mongolica microsatellite locus, as well as amplification primer and application thereof

A technology of microsatellite sites and amplification primers, which is applied in the field of sequence and detection of Tetragalus, can solve the problems of molecular genetics research that needs to be deepened, and achieve obvious results

Inactive Publication Date: 2019-02-22
CHINA SHENHUA ENERGY CO LTD +2
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  • Summary
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AI Technical Summary

Problems solved by technology

A series of work has been carried out on the conservation biology of Tetragalus, but the research on its molecular genetics needs to be deepened urgently

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1: DNA extraction

[0066] Collect Sihe wood samples (collected from wild species in Wuhai City), use silica gel to dry, and use a plant DNA extraction kit (EasyPure Plant Genomic DNA Kit, Beijing Quanshijin Biological Co., Ltd.) to carry out DNA extraction to obtain Sihe Wood Genome DNA.

Embodiment 2

[0067] Example 2: Construction of Tetragalus microsatellite enrichment library

[0068] The extracted genomic DNA was used to construct a microsatellite enrichment library using the AFLP rapid separation method (Fast Isolation by AFLP of Sequences Containing repeats, FIASCO). The specific process was as follows: Take about 250 ng of genomic DNA, digest it with MseI (New England Biolabs), and simultaneously Ligated with the AFLP receptor linker of MseI (5'TAC TCA GGA CTC AT-3' / 5'-GAC GAT GAG TCC TGA G-3'). After diluting the digested product tenfold, use AFLP receptor-specific primers (5'-GAT GAG TCC TGA GTAAN-3', referred to as MseI-N, N in the sequence represents any nucleotide) for PCR amplification. After the amplified product was electrophoresed on 1% agarose gel for 30 minutes, a diffuse band larger than 200bp was produced.

[0069] Use probe (AC)12 (ACACACACACACACACACACACAC) or (AG)12 (AGAGAGAGAGAGAGAGAGAGAG) (Sangon Bioengineering (Shanghai) Co., Ltd.) to hybridize wit...

Embodiment 3

[0071] Example 3: Screening of positive clones

[0072] Pick white plump colonies and place them in Amp (0.6g / L)+LB liquid medium for expansion (37°C, 5h). A three-primer PCR method (Zane et al., 2002) was used to detect positive recombinants containing microsatellite fragments. In the experiment, the M13 vector universal primer (M13-47) (5'-CGC CAG GGT TTT CCC AGT CAC GAC-3') and the corresponding biotin-free probe (AC)12 / (AG)12 corresponding to the clone were used for reaction . If there are two or more bands in the product, it can be considered that the single clone contains the target sequence, otherwise, it is considered to have no target sequence. 130 positive recombinants with the target sequence were successfully detected by the three-primer method, and these recombinants were sequenced.

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Abstract

The invention belongs to the technical field of biology, and provides a tetraena mongolica microsatellite locus and a primer sequence thereof. The tetraena mongolica microsatellite locus has a sequence as shown in SEQ ID NO: 1-10. The tetraena mongolica microsatellite locus can be used in molecular ecological research, pedigree analysis, germplasm resources investigation, assistant breeding and individual recognition of tetraena mongolica, and provides basic materials to studying plant drought resisting mechanisms.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the sequence and detection of Tetragalus. Background technique [0002] The West Ordos area is a special area in the steppe desert area in the west of my country, where several rare and endangered plants such as Tetraena mongolica Maxim., Ammopiptanthus mongolicus (Maxim.) Cheng f., Potaninia mongolica Maxim., Helianthemum soongaricum Schrenk, Reanmuria trigyna Mxim., Tugarinovia mongolica Iljin, Prunus mongolica Maxim. and Haloxylon ammodendron (C.A.Mey.) Bunge) and others have attracted much attention. Among them, the unique and rare relict plant Tetragalus is an ancient relic plant of the genus Tetragalus in the family Tribulus, which is distributed in a narrow area in the desert area of ​​central Asia, mainly in the northwest of the Ordos Plateau and south of the Kubuqi Desert. , east of the Ulan Buhe Desert, the foothills of Table Mountain (Mount Alabazau) north of the Amalek Dese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 郭洋楠智颖飙李强王义张保卫赵利清
Owner CHINA SHENHUA ENERGY CO LTD
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