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RNAi used for regulating and controlling growth of silkworm pupae and application

A pupal stage, positive technology, applied in the fields of biotechnology and agricultural applications, can solve problems such as affecting the quality of silk and consuming large amounts of energy.

Inactive Publication Date: 2013-07-24
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this process not only consumes a lot of energy, but also seriously affects the quality of silk

Method used

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  • RNAi used for regulating and controlling growth of silkworm pupae and application
  • RNAi used for regulating and controlling growth of silkworm pupae and application
  • RNAi used for regulating and controlling growth of silkworm pupae and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0160] screening target genes

[0161] 1. Extraction of total RNA from silkworm

[0162] Sample: Bombyx mori Dazao strain (P50).

[0163] Methods: The total RNA of silkworm was extracted by conventional Trizol method, and purified by conventional methods. Use DNase to treat the obtained RNA to obtain a concentration ≥ 300 ng / μl, a total amount ≥ 6 μg, and an OD 260 / 280 1.8-2.2 for total RNA samples.

[0164] 2. Isolation of mRNA and synthesis of cDNA

[0165] The mRNA with polyA was isolated with magnetic beads with oligo-dT, and then the first strand of cDNA was synthesized with a random 6-mer and a Superscript II reverse transcriptase kit purchased from Invitrogen.

[0166] 3. Gene amplification and sequencing

[0167] Amplify with primers specific to the target gene, purify the obtained gene fragment, connect it to the PMD-18 vector (manufactured by Takara), transform into a commercially available host cell (Top10 strain), and use the blue-white screening method to ...

Embodiment 2

[0172] dsRNA synthesis

[0173] In this example, the inventors used MAGE kit to synthesize dsRNA that can be used in biological assays.

[0174] use T7Kit (purchased from AMBION, product number AM1334) was used to synthesize target gene dsRNA to obtain dsRNA that can be used for biological assays. Its brief principle is: at first use the total RNA of silkworm larvae tissue as template, obtain cDNA by reverse transcription, then amplify the structural domains of two genes with primers (see Table 1), purify its products, and use the purified products as Template, using the kit for in vitro transcription, after obtaining dsRNA, removing the DNA in the product, and performing purification to obtain the desired dsRNA.

[0175] The specific operation steps are as follows: use the DNA sequences of the two gene domains obtained above as templates, and operate according to the instructions in AM1334Kit:

[0176] 1) In vitro transcription to obtain dsRNA

[0177]

[0178] Add RN...

Embodiment 3

[0184] Applications of dsRNA

[0185] In this example, the inventors prepared the two kinds of dsRNA obtained in Example 2 into aqueous dsRNA solutions with a concentration of 200 ng / μl. 300 μl of each dsRNA aqueous solution was sprayed on mulberry leaves and fed to silkworms once a day for 5 consecutive days. Three groups of repeated experiments were set up, each group used 30 silkworms, and the dsRNA effect was identified every day.

[0186] The results showed that compared with the EYFP gene

[0187] (1) The dsRNA targeting two target genes (PTTH and Torso genes) has a lethal effect on silkworms in the pupal stage. Among them, in the dsRNA PTTH group, there were 20, 22 and 23 dead silkworms in the pupal stage, respectively, with an average of 21.7 , the mortality rate reached 72.3%. In the dsRNA Torso group, there were 19, 21 and 23 dead pupal silkworms in the three groups, with an average of 21, and the mortality rate reached 70%.

[0188] (2) dsRNA targeting two target ...

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Abstract

The invention relates to RNAi used for regulating and controlling growth of silkworm pupae and an application. Concretely, the invention discloses two gene segments based on the death of silkworm pupae due to a RNA interference technology, a composition containing dsRNA is sprayed on folium mori for feeding silkworm, so that the silkworm can be grown up at larval period and died at pupal period. The method is convenient, fast and accurate, can solve the problems of energy consumption and decreased silk quality due to cocoon drying treatment in sericulture industry, and provides references for preventing and controlling lepidoptera pests.

Description

technical field [0001] The invention belongs to the field of biotechnology and agricultural application, in particular, the invention relates to RNAi for regulating silkworm pupae development and its application. Background technique [0002] The silkworm (Bombyx mori) has been raised artificially in my country for nearly 5,000 years. It is an important economic insect, and the silk produced by the silkworm is a valuable textile raw material. The silkworm mainly produces silk in the pupal stage. The development cycle of the silkworm pupa stage is very short, and it will break out of the cocoon 9-14 days after silk spinning and cocooning. Once the silkworm cocoon is bitten, it will lose its economic value. A large amount of manpower and material resources are used to prolong the pupal period and increase the output of silk. [0003] In order to solve this problem, baking treatment is generally adopted in silk production at present. However, this process not only consumes a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N5/10C12N15/10A01K67/04
Inventor 苗雪霞李海超张浩王玉冰
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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