Method and kit for detecting acetylcholin esterase A201S mutation and drug resistance of tomato leaf miners
A technology of cholinesterase, moth acetyl, applied in the field of molecular biology
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0052] Example 1: Cloning of Tomato Acety Acoury Esterase Ace Estera AcE-1 Gene
[0053] 1. Senior sequence clone of the tomato sub-acetyl cholecence esterase ACE-1 gene mRNA coding area
[0054] (1) Total RNA extraction
[0055] The 3-year-old larvae 5 head was placed in a 1.5 ml centrifuge tube, and the liquid nitrogen treatment was used to ground it into a powder, then RNA was extracted using Trizol method, and stored at -80 ° C for standby.
[0056] (2) CDNA synthesis
[0057] According to the reverse transcription kit (Easyscript first-strand cDNA Synthesis Supermix), the reverse transcription synthesis cDNA, and stores to be stored in alternate.
[0058] (3) Design of amplification primers by mRNA coding area
[0059] On the basis of transcription group sequencing, two pairs of primers ACE-1-F1 and ACE-1-R1, ACE-1-F2 and Ace-1-R2 were designed with Oligo 7 software to amplify tomato submerged pulp Ace-1 gene mRNA. Code sequence, its sequence is:
[0060] ACE-1-F1: 5'-AtcgTGG...
Embodiment 2
[0078] Example 2: TaqMan-MGB detection method of acetylcholine esterase gene mutation A201S
[0079] 1.A201S mutation TAQMAN-MGB method detection and synthesis of specific primers and specific probes
[0080] On the basis of obtaining the a201s mutant site of the ACE-1 gene, the A201S mutation site of the ACE-1 gene is designed and synthesized for the A201S mutant site of the ACE-1 gene, and synthesized the A201S mutant site of the ACE-1 gene. The A201SF and A201SR are located in the region of the genomic structure, and PCR amplification can be performed in genomic DNA as a template. The amplification product is 88 bp, and the amplified special primer sequence is:
[0081] A201SF: 5'-TatttggaggtaAccccCaca-3 '
[0082] A201SR: 5'-acaatggagagacagcaaatgaag-3 '
[0083] For the case of the two alleles of the tomato sub-moth A201S mutant site, two specific probes for TAQMan-MGB detection were designed based on its sequence characteristics, respectively for two allele types. The A201SPR...
Embodiment 3
[0110] Example 3: TAQMAN-MGB method detection of organophosphorus resistance of the main intrusion population of tomatoes
[0111] (1) Collection of tomatoes and DNA extraction
[0112] In 2017-2019, my country Tomato Louisi Moth main invasion area Xinjiang (Boltara, Yili) and Yunnan (Yuxi, Linyi, Dehong) in the field population, a total of 240 tomato lubs Samples were used to detect resistant mutations of its organophosphorus resistance-related gene acetylcholine esterase. The genomic DNA to be identified was extracted to obtain a genomic DNA solution, which was stored at -20 ° C.
[0113] (2) TAQMAN-MGB method detection
[0114] The TAQMAN-MGB detection method established by Example 2 was used to detect the A201S site mutation of the tomato sub-moth population of tomato in the above region. In particular, PCR amplification is performed using amplified special primers A201SF and A201SR, and identification of A201S mutation genotypes using specific probes A201SPR and A201SPs. The ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More