Method for detecting resistance of diamondback moth to bt insecticidal protein cry1ac based on abcc2 gene and its kit

A technology of insecticidal protein and detection method, applied in the biological field, can solve the problems of low sensitivity, long cycle, poor repeatability, etc., and achieve the effect of strong specificity, high sensitivity and accuracy

Inactive Publication Date: 2017-02-01
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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Problems solved by technology

[0006] Aiming at the problems of low sensitivity, long period, poor repeatability and high material requirements in the existing Plutella xylostella Bt insecticidal protein Cry1Ac resistance detection technology, the present invention intends to use specific fluorescent quantitative PCR primer screening and optimization of the re

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  • Method for detecting resistance of diamondback moth to bt insecticidal protein cry1ac based on abcc2 gene and its kit
  • Method for detecting resistance of diamondback moth to bt insecticidal protein cry1ac based on abcc2 gene and its kit
  • Method for detecting resistance of diamondback moth to bt insecticidal protein cry1ac based on abcc2 gene and its kit

Examples

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Example Embodiment

[0045] Example 1

[0046] The real-time fluorescent quantitative PCR technology was used to detect the midgut cDNA samples of DBM1Ac-S and SZ-R 4th instar larvae and the establishment of the detection kit and its use method.

[0047] 1. Taking the conserved fragment region of the ABCC2 gene in Plutella xylostella as the target sequence (SEQ ID NO.1), according to the principle of real-time PCR primer design, the specific fluorescent quantitative primer sequence is designed as follows:

[0048] Forward primer (qCC2-F): 5'-AGTCTTGGCACGCAAACGG-3' (SEQ ID NO. 2);

[0049] Reverse primer (qCC2-R): 5'-CGAACAGACGCATGAAGGACAT-3' (SEQ ID NO.3);

[0050] The specific primer amplified fragment sequence such as figure 1 As shown, its amplification specificity is as figure 2 (Among them, 1: PCR amplified fragment of the target gene ABCC2; 2: PCR amplified fragment of the internal reference gene L32; M: Marker I.) As shown in the figure, it can be seen that the primer has high amplification specifi...

Example Embodiment

[0085] Example 2

[0086] The availability of the kit and detection method in Example 1 was verified by the other three Bt resistant diamondback moth populations DBM1Ac-R, NIL-R and SH-R. The specific experimental process is shown in Example 1.

[0087] The final fluorescent quantitative PCR reaction test results are as follows Image 6 As shown in, A: Example 1 of the 4th instar larval midgut cDNA negative control sample of the diamondback moth population DBM1Ac-S sensitive to Bt insecticidal protein; B: Example 1 produced resistance to Bt insecticidal protein Cry1Ac Positive control sample of midgut cDNA from the 4th instar larvae of Plutella xylostella population SZ-R; C: Test sample for the midgut cDNA of 4th instar larvae of Plutella xylostella population SH-R which is resistant to Btk; D: To kill Bt The midgut cDNA sample of the 4th instar larvae of the diamondback moth DBM1Ac-R, which is resistant to protein Cry1Ac; E: the 4th instar larvae of the near-isogenic population NI...

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Abstract

The invention discloses a method and kit for detecting the resistance of plutella xylostella (L.) to an insecticidal protein Cry1Ac generated by Bt (bacillus thuringiensis) based on an ABCC2 (ATP-binding cassette C2) gene through real-time fluorescent quantitative PCR (polymerase chain reaction). The kit comprises the following target gene-specific primer sequences: SEQIDNO.2 of a forward primer and SEQIDNO.3 of a reverse primer. With the advantages of less manpower, small sample amount, large detection amount, high sensitivity, strong specificity, rapidness, simplicity, convenience, accuracy and reliability, the method can be used for effectively detecting the level of the resistance of plutella xylostella (L.) to the insecticidal protein Cry1Ac generated by Bt, is suitable for early rapid diagnosis of the resistance of plutella xylostella (L.) to the insecticidal protein Cry1Ac generated by Bt, can be also used for real-time monitoring, warning and forecasting of the resistance of plutella xylostella (L.) in the fields to the insecticidal protein Cry1Ac generated by Bt, and has broad application prospects.

Description

technical field [0001] The invention relates to the field of biology, in particular to a real-time fluorescent quantitative PCR detection method and a kit thereof for the resistance of diamondback moth to Bt insecticidal protein Cry1Ac. Background technique [0002] diamondback moth Plutella xylostella (L.), also known as diamondback moth, square moth, belongs to Lepidoptera (Lepidoptera) Plutellidae (Plutellidae), hosts as many as more than 40 species, but mainly harms Brassicaceae plants, is a worldwide harmful cross Important pests of cauliflower vegetables. In my country, since the 1970s, diamondback moth has gradually become the main pest of cruciferous vegetables in South China and the middle and lower reaches of the Yangtze River, and its distribution has continued to expand northward (Zhao et al., 1994). At present, the annual cost of controlling Plutella xylostella is as high as 4-5 billion US dollars worldwide (Furlong et al., 2013). The main reason why the diam...

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/124C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 张友军郭兆将康师朱勋吴青君王少丽徐宝云谢文
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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