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pSP107 plasmid as well as application and construction method thereof

A plasmid, mfa1.2 technology, applied in the biological field

Active Publication Date: 2018-12-14
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When UET1 and UET2 or when UEMT3 and UEMT2 are co-cultured with two sexually compatible smut fungus, when two sexually compatible smut fungus are co-cultured, the colonies cultured in vitro change from yeast type to hyphae type, and the colony grows aerial Mycelia, mycelia can infect Zizania, make Zizania pregnant, but it is easy to produce gray Zizania

Method used

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  • pSP107 plasmid as well as application and construction method thereof
  • pSP107 plasmid as well as application and construction method thereof
  • pSP107 plasmid as well as application and construction method thereof

Examples

Experimental program
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Effect test

example 1

[0026] Example 1: Extraction of Genomic DNA in Ustilago smut UET1 Strain

[0027] 1. Heat the CTAB extract in a water bath at 65°C half an hour in advance;

[0028] 2. Take UET14 bacteria (thickness about 5mm) into a 2.0mL centrifuge tube with a pipette tip, add about 10 stainless steel balls (diameter 1mm), and mark; add 800ul preheated CTAB extract in a fume hood; shaker 70Hz , shake for 150s, break the bacteria;

[0029] 3. Water bath at 65°C for 1 hour;

[0030] 4. Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), and mix well;

[0031] 5. In the fume hood, let it stand at room temperature for 15 minutes; at room temperature, centrifuge at 10,000 rpm for 10 minutes; at this time, there is a layering phenomenon in the centrifuge tube, the supernatant contains nucleic acid, the middle is a protein layer, and the bottom is steel balls and impurities; in a 1.5mL centrifuge tube (steps 4 and 5 can be repeated until the intermediate protein layer is invisi...

example 2

[0037] Example 2: mfa1.2 gene cloning

[0038] Using the genomic DNA in the UET1 bacterial strain obtained in Example 1 as a template, according to the designed mfa1.2F and mfa1.2R primers, clone mfa1.2 Gene. The nucleotide sequence of mfa1.2F is shown in SEQ ID No.2, and the nucleotide sequence of mfa1.2R is shown in SEQ ID No.3.

[0039] PCR amplification system: (50 ul)

[0040] 10×PCR buffer (takara) 5ul

[0041] dNTP mix (takara) 4ul

[0042] mfa1.2F 1ul

[0043] mfa1.2R 1ul

[0044] LATaq (takara) 0.5ul

[0045] dd H2O 37.5ul

[0046] UET1UET1 DNA 1ul

[0047] The PCR reaction conditions are as follows:

[0048]

[0049] PCR products were detected by 1% agarose gel electrophoresis. After cutting the gel, the product was recovered with the Magen Gel DNA Mini Recovery Kit. Store in a -20°C refrigerator for later use.

example 3

[0050] Example 3: E1 gene cloning

[0051] Using the genomic DNA in the UET1 bacterial strain obtained in Example 1 as a template, carry out PCR according to the designed bE1F and bE1R primers, and clone E1 Gene. The nucleotide sequence of bE1F is shown in SEQ ID No.4, and the nucleotide sequence of bE1R is shown in SEQ ID No.5.

[0052] PCR amplification system: (50 ul)

[0053] 10×PCR buffer (takara) 5ul

[0054] dNTP mix (takara) 4ul

[0055] bE1F 1ul

[0056] bE1R 1ul

[0057] LATaq (takara) 0.5ul

[0058] dd H 2 O 37.5ul

[0059] UET1UET1 DNA 1 μl

[0060] The PCR reaction conditions are as follows:

[0061]

[0062] PCR products were detected by 1% agarose gel electrophoresis. After cutting the gel, the product was recovered with the Magen Gel DNA Mini Recovery Kit. Store in a -20°C refrigerator for later use.

[0063] mfa1.2 and E1 Gene electrophoresis amplification picture as figure 1 shown.

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Abstract

The invention discloses a pSP107 plasmid as well as application and a construction method thereof, and belongs to the field of biotechnology. The invention provides a novel plasmid, pSP107 plasmid, aswell as the application and the construction method of the pSP107 plasmid. The plasmid provided by the invention can be transformed into protoplasts of UET2 and UEMT2 to obtain a UeTSP strain and a UeMTSP strain; and the two strains can infect zizania latifolia and cause zizania latifolia to be pregnant, thereby providing a new idea for artificial zizania latifolia gestation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to pSP107 plasmid and its application and construction method. Background technique [0002] Ustilago smut ( Ustilago esculenta ) is a unique pathogenic fungus that infects Zizania plants ( Zizania latifolia ) After that, it can inhibit the heading and flowering of the plants and stimulate the expansion of the base of the stem to form edible wild rice stems. In East Asia and Southeast Asia, especially China, Zizania is a highly nutritious aquatic vegetable and an important medicinal material. However, in the field, there are often gray water bamboos full of teliospores and sesame water bamboos formed by a small amount of teliospores, which are easy to cause pneumonia after being eaten by mistake, which is not conducive to the development of the water bamboo industry. In view of the successful development of artificial breeding technology, how to control the formation of ...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/66C12N1/15C12R1/645
CPCC07K14/37C12N15/66C12N15/80
Inventor 张雅芬叶子弘于金梦夏文强俞晓平崔海峰
Owner CHINA JILIANG UNIV
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