Detection primers and kits and detection methods for the diversity of endophytic fungi in Zhongshan fir

A technology for detecting primers and endophytic fungi, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Detect the effect of simple steps

Active Publication Date: 2020-11-10
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a technical solution for the identification of endophytic fungi diversity of Zhongshan fir, to solve the problem in the prior art that there is no method for identifying the diversity of endophytic fungi in the leaves of Taxus genus, especially Zhongshan fir

Method used

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  • Detection primers and kits and detection methods for the diversity of endophytic fungi in Zhongshan fir
  • Detection primers and kits and detection methods for the diversity of endophytic fungi in Zhongshan fir
  • Detection primers and kits and detection methods for the diversity of endophytic fungi in Zhongshan fir

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] (1) Put 0.3g of Zhongshan fir leaves into a mortar pre-added with liquid nitrogen, grind to powder, transfer to a 2.0mL Eppendorf tube, add 800μL of CTAB, 2μL of β-mercaptoethanol, vortex and mix, place in 65 ℃ water bath for 30 minutes, shake and mix once every 10 minutes;

[0055] (2) After cooling at room temperature, add 800 μL of chloroform, mix by inverting, and emulsify at room temperature for 20-30 minutes;

[0056] (3) Centrifuge at 12000rpm for 10min at room temperature, transfer the supernatant to a new 1.5mL Eppendorf tube, add isopropanol 0.5 times the volume of the supernatant, invert and mix until a white flocculent precipitate appears, and place it at -20°C to precipitate 1h;

[0057] (4) Centrifuge at 12000 rpm for 1 min at room temperature, remove the supernatant, and add 1 mL of 75% ethanol to wash the DNA precipitate;

[0058] (5) Repeat (4) once;

[0059] (6) Aspirate the supernatant, and put it in the ultra-clean table to ventilate and dry for 4...

Embodiment 2

[0067] Using the extracted DNA as a template, use 10 μmol / L of Primer F1 / Primer R1 and Primer F2 / Primer R2 as primers, respectively, and use TransFast Taq DNA polymerase kit (Beijing Quanshijin Biotechnology Co., Ltd.) to perform PCR amplification , the specific PCR reaction system is as follows:

[0068]

[0069] The experimental results show that: when the total amount of the system is 20 μL, the amplification band is weak, and the concentration of the product recovered from the gel is low; when the total amount of the system is 25 μL and 50 μL, the concentration of the product recovered from the gel is equivalent due to the limitation of the amount of sample loaded. Therefore, it is preferred A total of 25 μL of the system was used for PCR amplification.

Embodiment 3

[0071] The PCR amplification program includes: pre-denaturation at 98°C for 3 min; denaturation at 95°C for 30 s, annealing at 54-60°C for 30 s, extension at 72°C for 15-60 s, and 25-40 cycles; final extension at 72°C for 7 min. The amplification procedures of different primer sets are different, so in order to optimize the amplification efficiency, the following test analysis was carried out respectively:

[0072] (1)Primer F1 / Primer R1

[0073]

[0074] The experimental results show that the amplification efficiency is lower when the annealing temperature is 54°C and 60°C, and the amplification efficiency is higher when the annealing temperature is 56°C and 58°C; the extension time is proportional to the length of the amplified fragment, and 56°C When the extension time is 45s and 60s, the amplification efficiency is the highest. Considering the time cost, the preferred extension time is 45s; the number of amplification cycles is positively correlated with the concentrati...

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Abstract

The invention, which belongs to the technical field of biology, relates to a detection primer, a kit and a detection method for endophytic fungi diversity of taxodium zhongshanshan. The detection primer comprises a first primer pair and a second primer pair. The sequences of the first primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2; and the sequences of the second primer pair are shown as SEQID NO.3 and SEQ ID NO.4. The detection primer provided by the invention is obtained by analyzing and designing the diversity of leaf fungi of the taxodium zhongshanshan and has high sensitivity and detection concentration. The detection method provided by the invention is a detection scheme with simple steps and low cost; and a theoretical reference can be provided for detecting the diversity ofendophytic fungi in different varieties of leaves in different regions of the taxodium zhongshanshan.

Description

technical field [0001] The invention relates to a detection primer, a kit and a detection method for endophytic fungus diversity of Zhongshan fir, belonging to the field of biotechnology. Background technique [0002] The genus Taxodium Rich. belongs to Taxodiaceae, and it is divided into 3 species, Taxodium distichum (Linn.) Rich.], Pool Cedar (T.ascendens Brongn) and Mexican Bald Cedar (T. mucronatum Tenore). Bald cypresses are all tall trees. Due to their deep roots, huge root system, and rapid base expansion, they are widely used in wetland afforestation, windbreak and soil stabilization, and gully filling in Europe and the United States. Hybrid Bald Cedar, also known as Taxodium ‘Zhongshanshan’, is a general term for excellent clones obtained by crossing between two species of Taxodium and Mexican Bald Cedar. The excellent Zhongshan fir variety combines the main advantages of Bald Cedar and Mexican Bald Cedar: fast growth, salt and alkali resistance, flood resistance,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 张凡陆小清殷云龙於朝广王传永周艳威陈红李云龙蔡小龙
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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