Detection method and kit for the resistance of diamondback moth to bt insecticidal protein cry1ac based on abcc3 gene

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: 2017-06-09
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 reaction system and other steps , to establish a fast and accurate real-time fluorescent quantitative PCR molecular detection method and a fast, simple, accurate and efficient detection kit for detecting Bt resistance of Plutella xylostella, providing an effective tool for effective detection of Bt resistance in Plutella xylostella

Method used

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  • Detection method and kit for the resistance of diamondback moth to bt insecticidal protein cry1ac based on abcc3 gene
  • Detection method and kit for the resistance of diamondback moth to bt insecticidal protein cry1ac based on abcc3 gene
  • Detection method and kit for the resistance of diamondback moth to bt insecticidal protein cry1ac based on abcc3 gene

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Detection of midgut cDNA samples of 4th instar larvae of DBM1Ac-S and SZ-R by real-time fluorescent quantitative PCR technology and establishment of the detection kit and its application method.

[0048] 1. Taking the conserved fragment region of the midgut ABCC3 gene of Plutella xylostella xylostella as the target sequence (SEQ ID NO.1), according to the principle of real-time fluorescent quantitative PCR primer design, the specific fluorescent quantitative primer sequence was designed as follows:

[0049] Forward primer (qCC3-F): 5′-TCAACCGCTTCACCAAGGACAT-3′ (SEQ ID NO.2);

[0050] Reverse primer (qCC3-R): 5′-CGGCGTTCAGCACCAGGAT-3′ (SEQ ID NO.3);

[0051] The specific primer amplifies the fragment sequence as figure 1 As shown, its amplification specificity is as figure 2 (Among them, 1: the PCR amplified fragment of the target gene ABCC3; 2: the PCR amplified fragment of the internal reference gene L32; M: Marker I.) As shown in the figure, it can be seen from th...

Embodiment 2

[0087] The availability of the kit and detection method in Example 1 was verified by other three Bt-resistant diamondback moth populations DBM1Ac-R, NIL-R and SH-R. The specific experimental process is shown in Example 1.

[0088] The final fluorescent quantitative PCR reaction test results are as follows: Figure 6Shown in, wherein, A: negative control sample of midgut cDNA of the 4th instar larvae of the Plutella xylostella population DBM1Ac-S sensitive to Bt insecticidal protein in A: in embodiment 1; positive control sample of midgut cDNA of 4th instar larvae of Plutella xylostella population SZ-R; C: cDNA test sample of midgut of 4th instar larvae of Plutella xylostella population SH-R resistant to Btk; D: insecticidal to Bt The midgut cDNA of the 4th instar larvae of the Plutella xylostella population DBM1Ac-R resistant to the protein Cry1Ac to be tested; E: the 4th instar larvae of the near isogenic line population NIL-R of the Plutella xylostella resistant to the Bt in...

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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 ABCC3 (ATP-binding cassette C3) 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.), belonging to Lepidoptera (Lepidoptera) Plutellidae (Plutellidae), is an important vegetable pest that is harmful worldwide, with more than 40 kinds of hosts, and has become a major vegetable production worldwide. obstacle. Plutella xylostella mainly damages leaves with larvae throughout the growth period of cruciferous vegetables, which greatly reduces the yield and quality of vegetables. When serious occurrences occur in individual fields, the yield can be reduced by more than 90%, or even become extinct. The reason why the diamondback moth is so harmful is mainly because it has super adaptability to chemical pesticides. At present, it has produced varying degrees of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/124C12Q2600/158C12Q2531/113C12Q2561/113C12Q2545/101
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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