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TPR1 gene related to low-temperature tolerance of ampullaria gigas, encoded protein and application of TPR1 gene

A low-temperature tolerance and protein-coding technology, applied in the field of coding protein, TPR1 gene, can solve the problems of unrelated research reports on functions and effects, and achieve the effects of prolonging the incubation period, improving control efficiency, and strong repeatability

Active Publication Date: 2022-05-06
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been many studies on the TPR1 gene, and a large number of TPR1 cDNA sequences have also been cloned, but the apple snail is a worldwide invasive alien organism, there is no relevant research report on the function and role of the apple snail TPR1 gene.

Method used

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  • TPR1 gene related to low-temperature tolerance of ampullaria gigas, encoded protein and application of TPR1 gene
  • TPR1 gene related to low-temperature tolerance of ampullaria gigas, encoded protein and application of TPR1 gene
  • TPR1 gene related to low-temperature tolerance of ampullaria gigas, encoded protein and application of TPR1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1 Materials and methods

[0022] 1.1 Apple snails tested

[0023] The selected apple snails are young snails with a height of 5.0-10.5mm. Before the experiment, all individual apple snails were raised in tanks (40cm×25cm×28) in a constant temperature laboratory. 8h, the feed is fresh cabbage leaves.

[0024] 1.2 Main reagents

[0025] akaraMiniBest Universal RNA Extraction Kit (TakaRa), TakaRaMiniBESTAgarose Gel DNA Extraction Kit, Taq enzyme, SYBR Premix Ex Taq, RACE Kit User Manual were purchased from Clontech Company in the United States, and sequencing and primer synthesis were completed by Shanghai Sunny Bio-Express Co., Ltd.

[0026] Cloning of TPR1 gene of apple snail

[0027] 1) Take 10mg-50mg of apple snail mantle tissue, rinse with DEPC water 3-5 times, and store in liquid nitrogen;

[0028] 2) Extraction of total RNA: Grind the mantle tissue of the apple snail into powder under liquid nitrogen conditions, and then use TakaraMiniBest Universal RNA Extracti...

Embodiment 2

[0045] Example 2. Analysis of TPR1 expression in different tissues under different temperature stresses

[0046] Total RNA was extracted from different tissues, and the tissue expression of TPR1 gene under different temperature stress was detected by qPCR. The reaction volume is 25 μL. The reaction program was: pre-denaturation at 95°C for 1min; then 35 cycles at 95°C for 30s, 58°C for 45s, and 72°C for 1min; all data were statistically analyzed using Excel 2010. According to the method of Ct (2 -ΔΔCt method) to analyze the relative expression level of TPR1 gene. All data are marked in the form of mean ± SE. Results see figure 1 .

[0047] From figure 1 It can be seen that under heat shock conditions (36°C), TPR1 does not change much in various tissues compared with the control (26°C). Under cold shock conditions (6°C), compared with the control, the expression of TPR1 in all the tested tissues was significantly increased (P<0.05), and the expression in the mantle tissu...

Embodiment 3

[0048] Example 3: Effect of apple snail TPR1-dsRNA on the expression of target gene mRNA

[0049] (1) The apple snail TPR1-dsRNA obtained in the present invention was transfected into the apple snail by injection, and fed continuously for 5 days at 0°C.

[0050] (2) The mantle tissues of apple snails were collected at 0, 6, 12, 24, 72, and 120 hours after transfection, total RNA was extracted, and the mRNA expression level of apple snail TPR1 gene was detected by Real-time quantitative PCR.

[0051] The result is as figure 2 As shown, the expression level of TPR1 in the experimental group decreased significantly after 6 hours of transfection, and reached the lowest point after 24 hours of transfection. With the increase of transfection time, the interference effect decreased, and the expression level of TPR1 in the experimental group began to increase. But the expression of TPR1 in the experimental group was still significantly lower than that in the control group (P image 3...

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Abstract

The invention discloses a TPR1 gene related to low-temperature tolerance of pomacea canaliculata, an encoded protein and application of the TPR1 gene, and belongs to the technical field of biology. The method is rapid, effective and high in repeatability, and is an important supplement to the TPR1 gene family. The pomacea canaliculata is an important invasive organism, development of novel biopesticides is facilitated, and the low-temperature tolerance of the pomacea canaliculata is reduced by blocking expression of the TPR1 gene in the pomacea canaliculata, so that the purpose of controlling further north invasion of the pomacea canaliculata is achieved. Meanwhile, by interfering the expression of the TPR1 gene, the hatching rate of the eggs of the ampullaria gigas can be remarkably reduced, the hatching duration of the eggs can be prolonged, and therefore the harm of invasion of the ampullaria gigas is reduced. The method is of great significance to research and determination of biology, ecology and novel insecticides of the ampullaria gigas and research on a resistance mechanism of the ampullaria gigas to low-temperature tolerance.

Description

technical field [0001] The invention relates to the technical field of freshwater molluscs, in particular to a TPR1 gene related to low temperature tolerance of apple snails, encoded protein and application 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 density. seriousl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435A01N57/16A01P9/00
CPCC07K14/43504A01N57/16A01K2227/70A01K2207/05A01N25/00Y02A40/81C12N15/8273
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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