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Ustilago esculenta haploid strain UeTSP and application thereof

A haploid and bacterial strain technology, applied in the fields of application, fungi, biochemical equipment and methods, etc., can solve problems such as the trouble of artificial fertilization

Active Publication Date: 2019-01-04
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the emergence of the above situation, unnecessary troubles have been brought to the artificial pregnancy

Method used

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  • Ustilago esculenta haploid strain UeTSP and application thereof
  • Ustilago esculenta haploid strain UeTSP and application thereof
  • Ustilago esculenta haploid strain UeTSP and application thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0024] Example 1: Extraction of Genomic DNA in Ustilago smut UET1 Strain

[0025] 1. Heat the CTAB extract in a water bath at 65°C half an hour in advance.

[0026] 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. In the fume hood, add 800ul preheated CTAB extraction solution. The vibrating instrument is 70Hz, vibrate for 150s, and break up the bacteria.

[0027] 3. Water bath at 65°C for 1 hour.

[0028] 4. Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), and mix well.

[0029] 5. In the fume hood, let stand at room temperature for 15 minutes. Centrifuge at 10000rpm for 10min at room temperature. At this time, stratification occurs in the centrifuge tube. The supernatant contains nucleic acid, the middle is a protein layer, and the bottom is steel balls and impurities. Carefully pipette the supernatant into a new 1.5mL centrifuge tube (steps 4 a...

example 2

[0035] Example 2: mfa1.2 gene cloning

[0036] 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.1, and the nucleotide sequence of mfa1.2R is shown in SEQ ID No.2.

[0037] PCR amplification system: (50 ul)

[0038] 10×PCR buffer (takara) 5ul

[0039] dNTP mix (takara) 4ul

[0040] mfa1.2F 1ul

[0041] mfa1.2R 1ul

[0042] LATaq (takara) 0.5ul

[0043] dd H2O 37.5ul

[0044] UET1 DNA 1ul

[0045] The PCR reaction conditions are as follows:

[0046]

[0047] 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

[0048] Example 3: E1 gene cloning

[0049] Taking the genomic DNA in the UET1UET1 bacterial strain obtained in Example 1 as a template, carry out PCR according to the bE1F and bE1R primers designed, and clone E1 Gene. The nucleotide sequence of bE1F is shown in SEQ ID No.3, and the nucleotide sequence of bE1R is shown in SEQ ID No.4.

[0050] PCR amplification system: (50 ul)

[0051] 10×PCR buffer (takara) 5ul

[0052]dNTP mix (takara) 4ul

[0053] bE1F 1ul

[0054] bE1R 1ul

[0055] LATaq (takara) 0.5ul

[0056] dd H 2 O 37.5ul

[0057] UET1UET1 DNA 1 μl

[0058] The PCR reaction conditions are as follows:

[0059]

[0060] 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.

[0061] mfa1.2 with E1 Gene electrophoresis amplification picture as image 3 shown. mfa1.2 The nucleotide sequence is shown in SEQ ...

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Abstract

The invention relates to an ustilago esculenta haploid strain UeTSP and application thereof and belongs to the field of a biotechnology. On one hand, the ustilago esculenta haploid strain UeTSP is provided; on the other hand, the application of the ustilago esculenta haploid strain UeTSP in impregnation is provided. The ustilago esculenta haploid strain UeTSP and the application thereof have the advantages that the ustilago esculenta haploid strain UeTSP can directly infect zizania aquatica plants to make zizania aquatica pregnant without producing teliospores, that is to say that an unfavorable grey angle expression type is not formed, and a new idea is provided for artificial impregnation of zizania aquatica.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a haploid strain of Ustilago smut UeTSP and an application thereof. 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 forma...

Claims

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Application Information

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IPC IPC(8): C12N1/14A01G22/00A01G7/06C12R1/645
CPCA01G7/06A01G22/00C12N1/145C12R2001/645
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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