Pomacea canaliculata stress-tolerance-related gene trehalose-6-phosphate synthase (TPS), and coding protein and cloning method thereof

A technology of trehalose synthase and apple snail, applied in genetic engineering, glycosyltransferase, plant gene improvement, etc., can solve the problem of no relevant research reports on the function and effect of trehalose, achieving high positive rate and strong credibility , highly reproducible effect

Active Publication Date: 2016-05-18
CHINA JILIANG UNIV
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although there have been many studies on the trehalose synthase gene, and a large number of cDNA sequences encoding trehalose synthase enzymes have also been cloned, the apple snail is a worldwide invasive alien organism, and the function and role of trehalose in the apple snail are currently unknown. There is no relevant research report yet

Method used

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  • Pomacea canaliculata stress-tolerance-related gene trehalose-6-phosphate synthase (TPS), and coding protein and cloning method thereof
  • Pomacea canaliculata stress-tolerance-related gene trehalose-6-phosphate synthase (TPS), and coding protein and cloning method thereof
  • Pomacea canaliculata stress-tolerance-related gene trehalose-6-phosphate synthase (TPS), and coding protein and cloning method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0039] (1) Take 50mg-100mg of fresh apple snail muscle tissue, wash it with DEPC water for 3-5 times, and quickly store it in liquid nitrogen;

[0040] (2) Grinding the muscle tissue of the apple snail into a fine powder under liquid nitrogen conditions, and then using Trizol reagent to extract the total RNA of the muscle tissue of the apple snail;

[0041] (3) The cDNA of the apple snail muscle tissue was synthesized using Sangon's M-MuLV first-strand cDNA synthesis kit. Stored at -20°C for later use.

Embodiment 2

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

[0043] Search the amino acid sequence of TPS gene in NCBI database, select the amino acid sequence of TPS of 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) is 60°C; Primers with low degeneracy, suitable Tm value and target fragment length will be sent to the company for synthesis. The designed primer sequences are as follows:

[0045] Upstream primer UP1: 5'-TCCACgaytaycayyt-3'

[0046] Downstream primer UP2: 5'-CCTTGGCC...

Embodiment 3

[0053] Example 3. Isolation of apple snail TPS gene

[0054] UP1 and UP2 were amplified by PCR with designed amalgamative primers to obtain a 738bp TPS intermediate fragment ( figure 1 ). The accession number of the intermediate fragment in GeneBanK is KR821168.1.

[0055] The PCR reaction system is 25 μL: 10xrTaqbuffer 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: pre-denaturation at 94°C for 3min; then 30 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 1min; extension at 72°C for 10°C.

[0056] With TPS5F, TPS5R primer pair, amplify the 5' end fragment of 2048bp ( figure 2 ).

[0057] With TPS3F, TPS3R primer pair, amplify the 3' end fragment of obtaining 420bp ( image 3 ).

[0058] The above amplified fragments were spliced ​​using DNAman software to obtain the full-length sequence of the apple snail TPS gene with a full length of 2152 bp. SEQ ID NO: 1 an...

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Abstract

The invention discloses a Pomacea canaliculata stress-tolerance-related gene trehalose-6-phosphate synthase (TPS), and a coding protein and a cloning method thereof, belonging to the technical field of biology. The invention also discloses application of the TPS gene. The TPS gene can enhance the low-temperature tolerance of Pomacea canaliculata in the Pomacea canaliculata invasion process. The protein and coding gene have important theoretical and practical meanings for the research of the stress-tolerance mechanism of the external invasion organism Pomacea canaliculata.

Description

technical field [0001] The invention relates to the technical field of freshwater molluscs, in particular to an apple snail stress-resistance-related gene trehalose synthase gene, its encoded protein and its cloning method. 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 density. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/10
CPCC12N9/1051C12N15/1096C12Y204/01015
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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