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Rhopalosiphum padi GAPDH reference gene partial sequence, cloning method and application

The invention relates to a technology of constricted tube aphid and cloning method, which is applied in the field of molecular biology to achieve the effects of improving detection efficiency, shortening detection time, and optimizing PCR amplification procedure.

Inactive Publication Date: 2015-06-10
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cloning of the GAPDH gene of A. graminearum and its application as an internal reference gene have not been reported at home and abroad.

Method used

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  • Rhopalosiphum padi GAPDH reference gene partial sequence, cloning method and application
  • Rhopalosiphum padi GAPDH reference gene partial sequence, cloning method and application
  • Rhopalosiphum padi GAPDH reference gene partial sequence, cloning method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Cloning of the GAPDH gene of Aphid graminearum

[0047] 1.1. Extraction of total RNA from Aphid graminearum:

[0048] TRIzol (Amion, USA) method was used to extract the total RNA of Aphid spp.

[0049] According to the different tools used for grinding samples (using imported products without RNase / DNase), the RNA extraction process is as follows: about 0.05-0.1g of fresh samples or frozen Aphid spp. are fully ground into powder in liquid nitrogen, Quickly transfer to a 1.5mL centrifuge tube frozen in liquid nitrogen, then add 1mL TRIzol, shake vigorously to mix, let stand on ice for 5min; 200μl chloroform, shake vigorously for 15s, and let stand on ice for 5min. Centrifuge at 12,000 rpm at 4°C for 10 min; transfer the supernatant to a new centrifuge tube, add 400 μL of chloroform supernatant, shake vigorously to mix, and let stand on ice for 5 min. Centrifuge at 12,000rpm at 4°C for 10min; transfer the supernatant to a new centrifuge tube, add isopropanol...

Embodiment 2

[0060] Example 2.Detection of GAPDH gene in two different stages of Aphid graminearum by specific RT-qPCR

[0061] 2.1. Sample preparation

[0062] The total RNA extracted from two different stages (adult winged aphids and adult apterous aphids) of A. graminearum was used as templates respectively, and the specific extraction method was described in 1.1 of Example 1 for details.

[0063] 2.2. qPCR amplification primer synthesis

[0064] qPCR amplification upstream primer (SEQ ID NO4): 5'-ACTACTGTTCACGCTACCACCG-3';

[0065] qPCR amplification downstream primer (SEQ ID NO5): 5'-GCTGCTTCCTTGACCTTACCTT-3'.

[0066] Synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. (PAGE purification method).

[0067] 2.3. Fluorescent quantitative PCR (RT-qPCR)

[0068] RT-qPCR was performed using a two-step method, run on a 7500 real time PCR machine (Applied Biosystems). The RT part uses the kit FastQuant RT Kit (with gDNase) from Beijing Tiangen (TIANGEN) Company, using the total RN...

Embodiment 3

[0072] Example 3. As an internal reference gene for detecting the relative expression of barley yellow dwarf virus (BYDV-PAV) CP gene in aphids

[0073] 3.1. Sample preparation

[0074] The avirulent and non-toxic aphids with and without wings fed on oat plants infected with BYDV-PAV, respectively, when the feeding time intervals were 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h. sampling. At each feeding time interval, 8 aphids of each wing type were taken together as a sample to extract RNA. The whole experiment was repeated 3 times. The RNA extraction methods of all samples are described in 1.1 in Example 1 for details.

[0075] 3.2. qPCR amplification primer synthesis of CP gene

[0076] qPCR amplification upstream primer (SEQ ID NO6): 5'-CGGGGCTGAGGTATTCGTAT-3';

[0077] qPCR amplification downstream primer (SEQ ID NO7): 5'-AGGACTTTGAGGCGGATTTG-3'.

[0078] Synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. (PAGE purification method).

[0079] 3.3. RT-qPCR ampl...

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Abstract

The invention relates to a rhopalosiphum padi GAPDH reference gene partial sequence, a cloning method and an application, belongs to the field of molecular biology, and particularly relates to a rhopalosiphum padi reference gene GAPDH with a nucleotide sequence represented in SEQ ID No.1. The obtained nucleotide sequence of the rhopalosiphum padi reference gene GAPDH (999bp) can be used as reference genes for research of rhopalosiphum padi function genes or gene expressions at different development stages as well as relative levels of expressions of viruses in the nucleotide sequence when being used as a virus transmission medium in future.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a partial sequence of a reference gene GAPDH gene of Aphid graminearum, a cloning method and its application as an internal reference gene in RT-qPCR. Background technique [0002] The cereal aphid (Rhopalosiphum padi L., RP) belongs to the Homoptera: Aphididae. This aphid is widely distributed in the world and is one of the important pests of grasses. In addition to being able to directly affect grain yield by absorbing plant nutrition, it mainly causes harm as a carrier of gramineous crops and weed virus diseases. Aphid graminearum transmits yellow dwarf virus in a persistent non-proliferative way, and is the dominant vector of many barley yellow dwarf viruses in the family Flaviviridae and the genus Flavivirus. [0003] At the same time as the sap of the phloem of the plants infected by yellow dwarf disease was sucked by the aphid, it also fed on the vi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/10C12Q1/68
Inventor 刘艳武科科王锡锋
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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