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Application of target gene in shellfish larva metamorphosis development process researched by RNA interference technology

A technology of RNA interference and technical research, which is applied to the application field of genes in the metamorphic development of shellfish larvae, can solve the problems of small individual shellfish larvae and cannot use injection methods, etc. Eliminate the effect of genetic interference

Active Publication Date: 2020-08-07
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RNAi is a new technology to study gene function. However, shellfish larvae are very small, about 80-210 μm, and injection methods cannot be used. Therefore, it is very important to explore the method of applying RNAi to shellfish larvae

Method used

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  • Application of target gene in shellfish larva metamorphosis development process researched by RNA interference technology
  • Application of target gene in shellfish larva metamorphosis development process researched by RNA interference technology
  • Application of target gene in shellfish larva metamorphosis development process researched by RNA interference technology

Examples

Experimental program
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Effect test

Embodiment 1

[0061] The phenotype observation experiment used a six-hole plate, with 100 razor clam larvae in each well, and 10ml of seawater (containing 2.5×10 5 of golden algae and 2.5 x 10 5 Chaetoceros), different concentrations of siRNA or DEPC reagent. The metamorphosis rate was observed every 4 hours. When the seawater group was completely metamorphosed, the metamorphosis rate and survival rate of each group were calculated. Finally, from the phenotype data, compared with the blank group and DEPC group, the metamorphosis rate of larvae in all siRNA groups decreased with the increase of siRNA concentration. It can be seen from the phenotype that during the development period of razor clam larvae, siRNA made the metamorphosis rate of razor clam ( figure 1 ) and survival rate ( figure 2 ) was significantly suppressed. It can be inferred that in the developmental stage of razor clam larvae, when siRNA silences the target gene-dopamine β-hydroxylase, its activity is inhibited, the n...

Embodiment 2

[0063] In this example, razor clam larvae from the shell top stage to the creeping stage were selected to collect samples at different time points after the disturbance. A total of 5 time points were designed, namely: 0h, 4h, 8h, 12h and 24h, and the corresponding DEPC groups were designed, and each group had three repetitions. 500ml volume beakers were used in the experiment, each containing 200ml seawater and razor clam larvae. Collect razor clam larvae at each time point, collect razor clam larvae with an enrichment net, pipette the larvae into a 1.5ml centrifuge tube, centrifuge and remove excess seawater, add RNA store and store in a -20°C refrigerator for later use Detection of gene expression.

[0064] Finally, from the results of gene expression: during the development of razor clam larvae from the top of the shell to the creeping stage, the inhibitory effect of siRNA on the target gene was detected at different soaking times ( image 3 ), thereby indicating the sile...

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Abstract

The invention provides an application of an RNA interference technology in researching a target gene in a shellfish larva metamorphosis development process. Specifically, sinonovacula constricta is selected as a research object; dopamine beta-hydroxylase is used as a target gene; siRNA is designed, a method for soaking larvae in vitro by adopting siRNA is employed. The metamorphosis rate, the survival rate and the target gene expression quantity change of larvae are detected; namely, in the sinonovacula constricta larva development period, siRNA effectively reduces the expression level of target genes, the metamorphosis rate and the survival rate of the larvae are remarkably reduced, so that the feasibility of the in-vitro siRNA soaking method is indicated; the in-vitro soaking method adopted by the invention breaks through the limitation of injecting siRNA in vivo, and realizes the application of an RNA interference technology in shellfish larva research; the experimental operation issimple and easy to implement, is not limited by experimental conditions, and can eliminate the interference of other genes, thereby more accurately implementing the research on the single target gene.

Description

technical field [0001] The invention belongs to the technical field of RNA interference, and in particular relates to the application of RNA interference technology to study target genes in the metamorphic development process of shellfish larvae. Background technique [0002] At present, due to the limitation of mollusc molluscs' individuality and feeding habits, the traditional method of introducing interfering strand RNA cannot meet the requirements of experiments, so there is no report on the research of RNA interference (RNA interference, RNAi) in the development of seawater mollusk larvae. In the existing RNAi research, the method of injection and feeding is mostly adopted. In aquatic animals, by injecting siRNA to interfere with grass carp hemorrhagic disease virus, to explore the means of treating the virus; injecting dsRNA to silence the target gene, and studying the effect of the gene on the tissue development of the species. Shellfish larvae have a process of meta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/6888
CPCC12N15/1137C12Q1/6888C12Q2600/124C12Q2600/158Y02A40/81
Inventor 牛东红李智李家乐李蓓蓓彭茂潇熊娅吴应晗李浩宇丁红兵
Owner SHANGHAI OCEAN UNIV
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