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Low-temperature tolerance-related gene fad9 of apple snail, its encoded protein and its cloning method

A low temperature tolerance, apple snail technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problem of no relevant research reports on the function and role of FAD9 gene, and achieve high positive rate and strong credibility , highly reproducible effect

Active Publication Date: 2020-12-29
CHINA JILIANG UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although there have been many studies on the FAD9 gene, and a large number of cDNA sequences of FAD9 have also been cloned, the apple snail is a worldwide invasive alien organism, and there is no relevant research report on the function and role of the FAD9 gene in the apple snail.

Method used

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  • Low-temperature tolerance-related gene fad9 of apple snail, its encoded protein and its cloning method
  • Low-temperature tolerance-related gene fad9 of apple snail, its encoded protein and its cloning method
  • Low-temperature tolerance-related gene fad9 of apple snail, its encoded protein and its cloning method

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1. Extraction of total RNA from apple snail muscle tissue and synthesis of cDNA

[0039] (1) Take 10mg-50mg of fresh apple snail liver tissue, wash it with DEPC water for 3-5 times, and quickly store it in liquid nitrogen;

[0040] (2) Extraction of total RNA: Grind the liver tissue of the apple snail into fine powder under liquid nitrogen conditions, and then transfer it to a centrifuge tube containing 0.3-0.5 mL of cell lysate; extract the total RNA of the liver tissue;

[0041] (3) The cDNA of apple snail liver tissue was synthesized by reverse transcription and stored at -20°C for later use.

Embodiment 2

[0042] Embodiment 2. The design of the apple snail FAD9 gene degenerate primer

[0043] Search the amino acid sequence of FAD gene in NCBI database, select the amino acid sequence of FAD9 from different species, and then use CODEHOP program to design degenerate primers.

[0044] The main steps of using CODEHOP to design primers are as follows: save the amino acid sequences of the different species queried above in FASTA format, and submit the results to Blockmaker (http: / / blocks.fhcrc.org / blocks / make_blocks.html) to make conserved regions Then submit the obtained conserved region to the server for primer design. The main parameters of the design are: degeneracy (Degeneracy) 128; annealing temperature (Temperature) 60°C; genetic code (Genetic code): standard, try Select primers with low degeneracy, suitable Tm value and target fragment length and send them to the company for synthesis. The designed primer sequences are as follows:

[0045] Upstream primer UP1: 5'-TCGTATgaytay...

Embodiment 3

[0053] Example 3. Cloning of apple snail FAD9 gene

[0054] UP1 and UP2 were amplified by PCR with designed demerger primers to obtain a 705bp FAD9 intermediate fragment ( figure 1 ). The PCR reaction system is 25 μL: 10xrTaq buffer 2.5 μL, dNTPs (each 10 nmol / L) 0.5 μL, MgCl 2 (25mM) 1.5μL, cDNA 1μL, Taq enzyme 0.5μL, add ddH2O water to make up to 25μL. The PCR reaction program was: 95°C for 30s, 58°C for 45s, 72°C for 1min, 35 cycles; 72°C for 10°C extension.

[0055] Using the FAD9-3F, FAD9-3R primer pair, a 2100bp 3' end fragment was amplified.

[0056] Using the FAD9-5F, FAD9-5R primer pair, a 425bp 5' end fragment was amplified.

[0057] The above amplified fragments were spliced ​​using DNAman software to obtain the full-length sequence of the apple snail FAD9 gene with a full length of 2334bp. The nucleotide sequence and amino acid sequence of the full-length gene ORF are respectively provided by SEQ ID NO:1 and SEQ ID NO:2.

[0058] Based on the amino acid seque...

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Abstract

The invention discloses a pomacea canaliculata low-temperature tolerance related gene FAD9 as well as coded protein and cloning method thereof, and belongs to the field of biotechnology. The inventionalso discloses application of the FAD9 gene. The pomacea canaliculata low-temperature tolerance related gene FAD9 disclosed by the invention is rapid and effective with high repeatability, and is animportant implementation of the FAD9 gene family. The pomacea canaliculata is an important invasive species; the pomacea canaliculata low-temperature tolerance related gene FAD9 disclosed by the invention is helpful in developing a novel biological pesticide, and the low-temperature tolerance of organism is reduced by blocking the synthesis of unsaturated fatty acid in the organism so as to achieve an aim of controlling further northward invasion of pomacea canaliculata. The invention is of great significance to the study and measurement of biological and ecological novel insecticides of pomacea canaliculata and the resistance mechanism study on the low-temperature tolerance of pomacea canaliculata.

Description

technical field [0001] The invention relates to the field of freshwater mollusk biology, in particular to a gene caltractin related to low temperature tolerance of apple snails, a protein encoded by it and a cloning method thereof. Background technique [0002] Apple snails (Pomacea), also known as big bottle snails and apple snails, are international vicious pests. The snail is native to the Amazon River Basin in South America and was introduced to Asia in the early 1980s. It was abandoned due to poor management and poor taste; serious hazard. Apple snails have extremely strong fecundity, spread and spread quickly, and have become serious agricultural pests in most provinces and regions south of the Yangtze River. Today, apple snails are widely distributed in most provinces and regions south of 30° north latitude in my country, including Zhejiang, Fujian, Guangdong, Hainan, Guangxi, Yunnan, Guizhou, Hunan, Jiangxi, Chongqing, Sichuan, Anhui, etc., with a huge population d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/10C07K14/435
CPCC07K14/43504Y02A90/40
Inventor 刘光富俞晓平张蓬军申屠旭萍许益鹏杨倩倩郝培应
Owner CHINA JILIANG UNIV
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