Method for detecting number of arbuscular mycorrhizal fungi in wheat rhizosphere soil based on real-time fluorescence quantification PCR

An arbuscular mycorrhizal fungi, real-time fluorescence quantitative technology, applied in the biological field, can solve the problems of arbuscular mycorrhizal fungi counting and other problems that have not yet been seen in qPCR technology, and achieve a simple and accurate identification method, high amplification efficiency, and high linearity.

Inactive Publication Date: 2016-10-12
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Method for detecting number of arbuscular mycorrhizal fungi in wheat rhizosphere soil based on real-time fluorescence quantification PCR
  • Method for detecting number of arbuscular mycorrhizal fungi in wheat rhizosphere soil based on real-time fluorescence quantification PCR
  • Method for detecting number of arbuscular mycorrhizal fungi in wheat rhizosphere soil based on real-time fluorescence quantification PCR

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Embodiment 1

[0038] (1) Construct the target plasmid

[0039] According to the 28s rDNA conservative sequence of ribosomal DNA of common fungi in the arbuscular mycorrhizal family and soil on the NCBI website, the BioEdit software was used for comparison and then the upstream and downstream primers for specifically amplifying arbuscular mycorrhizal specific fragments were designed such as SEQ ID No. 1 and SEQ ID No.2.

[0040] The applicant took samples from the Liuhe Transgenic Experiment Base of Jiangsu Academy of Agricultural Sciences to obtain transgenic wheat and its receptor rhizosphere soil, using UltraClean TM soil DNA Isolation Kit, extract total DNA from soil and dissolve total DNA in ddH 2 In O; use SEQ ID No.1 and SEQ ID No.2 as the upstream and downstream primers, and the total soil DNA as the template to amplify the target band:

[0041] The PCR reaction system is 25 μL, in which rTaq enzyme 0.125 μL, 10×PCR buffer 2.5 μL, dNTP (dATP, dTTP, dCTP, dGTP each 2.5mM) 2 μL, MgCl 2 (25...

Embodiment 2

[0058] Example 2 Sensitivity detection of fluorescence quantitative PCR detection system

[0059] For the standard plasmid (2.0×10 10 copies / µl) 10 times dilution, 1*10 8 copies / µL-1*10 3 copies / µL, use SEQ ID No.1 and SEQ ID No.2 for fluorescence quantitative amplification to detect the sensitivity of amplification. Refer to Example 1 for specific reaction system and reaction conditions.

[0060] Get the amplification curve as Figure 4 As shown, Figure 4 Among them, 1, 2, 3, 4, 5, 6, 7, 8 represent the standard plasmid stock solution in turn, 10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -5 ,10 -6 ,10 -7 Double dilution; perform agarose gel electrophoresis on the amplification results, such as Figure 5 As shown, Figure 5 Among them, M: DL 2000 maker, 1, 2, 3, 4, 5, 6, 7, 8 represent the standard plasmid stock solution, 10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -5 ,10 -6 ,10 -7 Fold dilution; by Figure 4 It can be seen that in the 10 -6 Before the double dilution, the amplification curve still showed an...

Embodiment 3

[0061] Example 3 Analysis of growth dynamics of arbuscular mycorrhizal fungi in rhizosphere soil during the main growth period of transgenic wheat

[0062] The genetically modified wheat and its recipients are planted in the Liuhe Transgenic Experimental Base of Jiangsu Academy of Agricultural Sciences. Each variety (line) has 4 replicates, and a random block design. The area of ​​each plot is 10 (length) × 6 (width) m 2 .

[0063] The wheat rhizosphere soil was collected by the diagonal five-point sampling method at the same place during the sowing, jointing, filling, and maturation periods of wheat growth, and DNA was extracted using the specific primers and amplification system provided in Example 1. Amplify and calculate according to the standard curve to obtain the bacterial content of the initial arbuscular mycorrhizal fungi in the soil sample.

[0064] The established Real-time qPCR was used to detect the number of arbuscular mycorrhizal fungi in the rhizosphere soil during th...

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Abstract

The invention provides a method for detecting the number of arbuscular mycorrhizal fungi in wheat rhizosphere soil based on real-time fluorescence quantification PCR. The method includes the steps that firstly, sample total DNA of wheat rhizosphere soil is extracted and serves as a template of PCR amplification; then, amplification is conducted with SEQ ID No.1 and SEQ ID No. 2 as primers, and specific fragments of 129 bp are obtained; after bond and conversion are conducted, qPCR amplification is conducted. By means of the method, arbuscular mycorrhiza is authenticated, absolute quantification can be conducted on the arbuscular mycorrhiza accurately, the copy number of the arbuscular mycorrhizal fungi in all periods of duration of transgenic wheat is obtained, and in the growth and development stages of wheat, the arbuscular mycorrhiza copy number tends to be gradually increased as a whole. The rapid, convenient and precise authentication method is built, a theoretical and test basis is provided for subsequent tests, great significance is provided for further evaluation of safety of transgenic crops, and compared with an existing method, the method is high in sensitivity, high in specificity, good in repeatability, convenient to operate and visual in result.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting the number of arbuscular mycorrhizal fungi in wheat rhizosphere soil based on real-time fluorescent quantitative PCR. Background technique [0002] Arbuscular mycorrhiza ( Arbuscular mycorrhiza , AM) Fungi are a class of plant symbiotic bacteria that have attracted much attention. They can form mutually beneficial symbiosis with the roots of most crops. AM fungi can not only stimulate the effective absorption of mineral elements such as phosphorus and nitrogen by plants, but also have an active role in enhancing plant disease resistance and stress resistance. [0003] In recent years, the large-scale planting of genetically modified crops has caused people to worry about environmental issues. Among them, the impact of genetically modified crops on the soil ecosystem (microbial species, population, quantity and biodiversity) is currently a hot research topic. Therefor...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06C12R1/645
CPCC12Q1/6851C12Q2531/113C12Q2545/113
Inventor 吴季荣仇剑波赵晶晶邢宇俊史建荣
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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