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Rhopalosiphum padi Linnaeus EF1-alpha reference gene partial sequence as well as cloning method and application thereof

The invention relates to a technology of constricting aphid and cloning method, which is applied in the field of molecular biology to achieve the effects of optimizing PCR amplification procedures, improving detection efficiency and improving reliability.

Inactive Publication Date: 2014-05-21
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

As a nuclear gene of aphids, the partial gene sequence of EF1-α has become the basis for the classification of genetic diversity in the population of A. graminearum, but it has not been reported as a housekeeping gene to study the expression changes of other genes.

Method used

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  • Rhopalosiphum padi Linnaeus EF1-alpha reference gene partial sequence as well as cloning method and application thereof
  • Rhopalosiphum padi Linnaeus EF1-alpha reference gene partial sequence as well as cloning method and application thereof
  • Rhopalosiphum padi Linnaeus EF1-alpha reference gene partial sequence as well as cloning method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Cloning of the EF1-α gene of Aphid graminearum

[0046] 1.1. Extraction of total RNA from Aphid graminearum:

[0047] Total RNA was extracted from Aphid graminis (Aphidless adult) by TRIzol (Amion, USA).

[0048] 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; Add 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, ...

Embodiment 2

[0060] Example 2.Detection of specific RT-qPCR of EF1-α gene in two different stages of Aphid graminearum

[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'-TAGACGCTATCCTACCCCCCA-3';

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

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

[0067] 2.3. 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 used the kit FastQuant RT Kit (with gDNase) from Beijing Tiangen (TIANGEN) Co., Ltd., using the total RNA extracted from the winged adu...

Embodiment 3

[0071] Example 3. As an internal reference gene for detecting barley yellow dwarf virus (BYDV-PAV) CP gene in poisonous aphids

[0072] 3.1. Sample preparation

[0073] The non-toxic aphids with and without winged adults fed on oat plants infected with BYDV-PAV, and when the feeding time intervals were 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h, respectively 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.

[0074] 3.2. qPCR amplification primer synthesis of CP gene

[0075] qPCR amplification upstream primer (SEQ ID NO6): 5'-CGGGGCTGAGGTATTCGTAT-3';

[0076] qPCR amplification downstream primer (SEQ ID NO7): 5'-AGGACTTTGAGGCGGATTTG-3'.

[0077] Synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. (PAGE purification method).

[0078] 3.3. RT-qPCR amplification

[0079] ...

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Abstract

The invention relates to a rhopalosiphum padi Linnaeus EF1-alpha reference gene partial sequence as well as a cloning method and application thereof, belongs to the field of molecular biology, and particularly relates to rhopalosiphum padi Linnaeus EF1-alpha, and the nucleotide sequence of the rhopalosiphum padi Linnaeus EF1-alpha is indicated as SEQ ID NO.1. The nucleotide sequence of the obtained rhopalosiphum padi Linnaeus EF1-alpha (837bp) can be used as a reference gene for studying the hopalosiphum padi Linnaeus EF1-alpha functional gene or the gene expression at different development periods as well as a reference gene for studying the relative level of the expression of virus in the body when being used as a virus transmission interbody.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a partial sequence of the reference gene EF1-α 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 ...

Claims

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Application Information

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