Specific gene sequence of tilletia controversa kuhn, specific SCAR marker and PCR detection method

A technology of dwarf black powder fungus and wheat dwarf black ear, which is applied in the biological field, can solve problems such as failure to separate, and achieve the effects of high detection sensitivity and reliability, and high practical application value.

Inactive Publication Date: 2011-08-03
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2004, Gao Qiang (Gao Qiang. Molecular detection of Tilletia tritici smut [D]. Changsha, Hunan Agricultural University, 2004) used RAPD technology to find a line that could isolate Tilletia tritici strains and Tilletia tritici strains Different from the bands of other smut strains, but unfortunately no different bands were found between Tilletia dwarf and Tilletia erythroderma, and the two could not be separated
At present, there are no reports at home and abroad based on ISSR method to obtain specific primers for the detection of T. dwarf.

Method used

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  • Specific gene sequence of tilletia controversa kuhn, specific SCAR marker and PCR detection method
  • Specific gene sequence of tilletia controversa kuhn, specific SCAR marker and PCR detection method
  • Specific gene sequence of tilletia controversa kuhn, specific SCAR marker and PCR detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: screening the specific ISSR specific primers of T. tritici

[0048] 1.1 Extraction of T. tritici DNA

[0049] The improved CTAB / SDS method was used to extract the DNA of T. tritici.

[0050] (1) Sample pretreatment: Take one smut gall (about 10 mg), crush it and add it to a 2 ml sterilized screw centrifuge tube, add 25 mg diatomaceous earth, 250 mg sterilized glass beads with a diameter of 1 mm, and vortex Vortex to mix. Place at -20°C overnight.

[0051] (2) Water bath: Add 660 μl of preheated SDS / CTAB extraction buffer and 5 μl of proteinase K solution to the centrifuge tube, mix with a vortexer, and bathe in a constant temperature water bath at 65°C for 1 hour

[0052] (3) Grinding: Place the centrifuge tube in MP FastPrep-24, a rapid nucleic acid extractor, set at 6.5M / s, and process once every 10s.

[0053] (4) Extraction: take the supernatant, add an equal volume of chloroform / isoamyl alcohol mixed solution (24:1), and centrifuge at 15493×g for 10 ...

Embodiment 2

[0065] Example 2 Determination of Sequence of Specific Fragment of Tilletia tritici and Obtaining Specific SCAR Marker

[0066] According to the sequence sequencing results of the T. tritici specific fragments obtained in Example 1, the sequences were analyzed and compared with DNAMAN 5.2.2 software, and three pairs of TCK specialized SCAR primers TCKSF(1-3) / TCKSR (1-3). These primers can amplify bands of 372bp, 496bp and 419bp respectively in all TCK bacterial strains, but there is no amplification product such as Tilletia tritici and Tilletia tritici figure 2 , 5 , 7. Therefore, the above three fragments are specific SCAR markers of T. tritici.

[0067] The sequences of primers TCKSF(1-3) and TCKSR(1-3) are as follows:

[0068] TCKSF1 (5'-TGG TGG TCG GGAAAG ATT AGA-3')

[0069] TCKSR1 (5'-GGG ACG AAG GCA TCA AGA AG-3')

[0070] TCKSF2(5'-TTG CTG GCT CTT CGC CCT GA-3')

[0071] TCKSR2 (5'-TTG CCC GTC TTG CGG TTG AT-3')

[0072] TCKSF3 (5'-CACACACACACAGGAAGCA-3')

...

Embodiment 3

[0074] Example 3: Verification of Tilletia tritici-specific SCAR markers

[0075] The TCKSF(1-3) / TCKSR(1-3) specific SCAR marker primers TCKSF(1-3) / TCKSR(1-3) for T. tritici constructed in the present invention were synthesized by Shanghai Biological Engineering Company, and the total volume of the amplification reaction system for specific SCAR markers was 25 μl, wherein Contains 10×PCR-buffer 2.5μl, Mg 2+ 2μl (25mM), dNTPs 0.3μl (10mM), TCKSF (1-3) / TCKSR (1-3) each 1μl (10μM), Taq DNA polymerase 0.3μl (2.5U / μl), template DNA (wheat dwarf fishy black 1 μl (20ng / μl) of DNA extracted from powdery mildew, Tilletia tritici and Tilletia tritici, and 25 μl in sterilized double distilled water.

[0076] PCR amplification conditions were 94°C for 5min; 94°C for 30Sec, TCKSF1, 3 / TCKSR1, 3 55°C for 30Sec (TCKSF2 / TCKSR2 60°C for 30Sec), 72°C for 1min, 30 cycles; 72°C for 10min; 4°C forever. After PCR amplification, take 5 μl of the amplified product and add 1 μl of sample buffer to e...

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Abstract

The invention relates to a specific gene sequence of tilletia controversa kuhn, a specific SCAR marker and application thereof. For the specific gene sequence of the tilletia controversa kuhn, according to the nucleotide sequences of the specific fragments shown in Seq No.1, Seq No.2 and Seq No.3, the nucleotide sequences of the SCAR marker, which are shown in Seq No.4, Seq No.5 or Seq No.6 and are high in specificity and sensitivity, can be obtained, so that various primer selections are provided for the PCR technique to detect the wheat dwarf bunt. The detection sensitivity of the high-specificity primer TCKSF(1-2) / TCKSR(1-2) which is designed according to the sequences of the SCAR marker reaches 1ng / 25mu l, so that the tilletia controversa kuhn can be identified from various similar strains.

Description

Technical field: [0001] The invention belongs to the field of biological technology, and relates to a specific gene fragment, a specific SCAR marker and a PCR detection method of Tilletia tritici. technical background [0002] Tilletia controversa Kühn (TCK for short) caused by Tilletia controversa Kühn (TCK) is an important international quarantine disease, which has devastating effects on wheat production and is also one of the main species of foreign biological invasion research in my country. 1 (Wang Yuan. T. dwarf smut [M]. Selected Plant Quarantine Pests Entering China, edited by the Animal and Plant Quarantine Bureau of the People's Republic of China and the Plant Quarantine Laboratory of the Ministry of Agriculture, Beijing: China Agricultural Press, 1997, 95 ~98). In the popular years of wheat dwarf smut, the yield loss caused by it is generally 20-50%, and in severe cases, it can reach 75-90%, or even extinction. The bacteria are highly resistant to stress, and the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68C12R1/645
Inventor 高利陈万权刘太国
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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