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Kelp gametophyte DNA solution preparation method and sex determination method

A gametophyte and kelp technology, applied in the field of marine biotechnology and seaweed molecular genetics, can solve the problems of negative impact of the environment and experimenters, cumbersome experimental steps, labor and financial resources, etc., to save sample storage space, reduce costs and errors The effect of low ratio

Active Publication Date: 2018-09-14
SHANDONG ORIENTAL OCEAN SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods need to prepare various reagents and buffers in the preparation stage, and the samples also need to be pretreated accordingly, and then they have to go through multi-step operations such as extraction, precipitation, and purification. The experimental steps are cumbersome, labor-intensive and financial; The reagents used are generally toxic and can negatively affect the laboratory environment and experimenters

Method used

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  • Kelp gametophyte DNA solution preparation method and sex determination method
  • Kelp gametophyte DNA solution preparation method and sex determination method
  • Kelp gametophyte DNA solution preparation method and sex determination method

Examples

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Effect test

Embodiment 1

[0041] Example 1: To verify the feasibility of the preparation method of Laminaria gametophyte DNA solution which can be used for PCR amplification.

[0042] 6 kelp species 901 kelp (901), Zaohoucheng kelp (ZHC), Benniu kelp (BN), Dongfang No. 6 kelp (Dong 6) provided by the Germplasm Preservation Laboratory of Seaweed Center of Shandong Oriental Ocean Technology Co., Ltd. , Korean kelp (HG), Lianza kelp (LZ), 3 gametophytes for each male and female, a total of 36 gametophytes (see Table 1 for the number of gametophytes). Utilize two methods to obtain the laminar gametophyte genomic DNA solution: one utilizes the plant genomic DNA extraction kit of Tiangen Company to extract 36 laminaria gametophyte DNAs; Laminaria gametocytes with a wet weight of about 15-30 mg were placed in a 1.5 mL sterilized centrifuge tube, and 500 μL of sterilized deionized water was added, thoroughly ground, boiled in water for 20 minutes, and allowed to settle at room temperature for 30 minutes) to ob...

Embodiment 2

[0045] Example 2: Verification of the correctness of double-primer PCR sex identification of Laminaria gametophytes.

[0046] 631301♀♂ mixed gametophyte clone provided by the germplasm preservation laboratory of Muping Branch of Seaweed Center of Shandong Oriental Ocean Technology Co., Ltd., S.ang 1301 ♀♂ mixed gamete clone, S.jap 1301♀♂ mixed gametophyte clone, 491302♀♂♂ mixed, 261102♀♂ mixed; 5 Laminaria female gametophytes and 5 Laminaria male gametophytes were randomly selected from the Laminaria gametophyte sample in Example 1, and a total of 15 clonal lines were tested (gametophyte numbering See Table 2). Take an appropriate amount of gametophyte clones in a 1.5 mL sterilized centrifuge tube, add 500 μL of sterilized deionized water, grind thoroughly, bathe in boiling water for 20 minutes, and settle at room temperature for 30 minutes to obtain a DNA solution of 15 Laminaria gametophyte clones. The first method is to use Laminaria gametophyte female-specific primer H...

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Abstract

The invention discloses a kelp gametophyte DNA solution preparation method and a sex determination method. The preparation method comprises the steps that kelp gametophyte is added with sterile deionized water, full grinding is conducted, after boiling water bath is conducted, room-temperature standing is conducted, supernatant is transferred into a sterile centrifuge tube, by means of a female specific primer HFM4 and a male specific primer Msj68-16-3, a round of double-primer PCR amplification is conducted, and the sex and confusing situation of multiple varieties or strains of kelp gametophyte are identified rapidly and accurately. The method of preparing a kelp gametophyte DNA solution easily and rapidly without using any molecular reagents is provided, a kelp female gametophyte specific marker is developed, the pertinence is high, the amplification efficiency is high, and the stability is good; by combining a kelp male gametophyte specific marker developed by a lab, the method ofidentifying the sex by means of the kelp gametophyte DNA solution is built, the sex of the kelp gametophyte can be detected, and whether or not the male and female mixed phenomenon occurs is determined.

Description

technical field [0001] The invention belongs to the field of marine biotechnology seaweed molecular genetics, and mainly relates to a method for preparing a kelp gametophyte DNA solution which can be used for PCR amplification, and a method for quickly identifying the sex of the kelp gametophyte. technical background [0002] kelp( Saccharina japonica ) belongs to Phaeophyta (Phaeophyta), Phaeophyceae (Phaeophyceae), Laminariales (Laminariales), Laminariaceae (Laminariaceae), Laminaria ( Saccharina ); is an important economic seaweed in China, which is widely used in medicine, chemical industry, food and other fields. In the 1970s, the separation and cloning technology of male and female gametophytes of Laminaria was established, which laid the foundation for the protection, development and utilization of Laminaria germplasm resources. [0003] Laminaria gametophyte cloning can maintain its excellent character and purity for a long time, which is the best preservation for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6879
CPCC12N15/1003C12Q1/6806C12Q1/6879
Inventor 刘延岭李言姜黎明彭捷王利芹赵聚萍武瑞娜李晓捷潘金华田萍萍
Owner SHANDONG ORIENTAL OCEAN SCI TECH
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