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Method for screening bombyx mori nuclear polyhedrosis virus disease resistance with BmIML-2 gene

A technology of nuclear polyhedron and viral disease, which is applied in the direction of biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of silkworm mutual infection and easy pollution of silkworm breeding environment, so as to reduce the probability of mutual cross-infection, The effect of shortening the test period and improving the resistance

Pending Publication Date: 2020-04-14
JIANGSU UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional screening method is to add virus to the silkworm in the 2nd or 3rd instar larval stage. This method is easy to pollute the silkworm breeding environment during the silkworm stage, resulting in mutual infection between silkworms.

Method used

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  • Method for screening bombyx mori nuclear polyhedrosis virus disease resistance with BmIML-2 gene
  • Method for screening bombyx mori nuclear polyhedrosis virus disease resistance with BmIML-2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Detection of the difference in transcription level of BmIML-2 gene in various tissues of larvae by RT-PCR

[0027] RNA was extracted from the head, midgut, fat body, silk gland, martensian tube, epidermis and trachea of ​​the 5th instar larvae on the 3rd day of the silkworm, and reverse transcribed to obtain cDNA. Using the cDNA obtained by reverse transcription as a template, specific primers were designed, wherein the forward primer (SEQ ID NO. 1): 5'-AGAGTTCGTGTGGCATCTA-3', and the reverse primer (SEQ ID NO. 2): 5'-ACCATATGAGAGGCAGGGTT- 3'. The silkworm BmGAPDH gene is used as an internal reference gene, and its upstream and downstream primer sequences are F (SEQ ID NO. 3): 5'-TTCATGCCACAACTGCTACA-3', R (SEQ ID NO. 4): 5'-AGTCAGCTTGCCATTAAGAG-3', and BmIML-2 is detected Differences in the transcription level of genes in different tissues of silkworm larvae (as attached figure 1 Shown).

Embodiment 2

[0028] Example 2 Treating several silkworm species with feeding nuclear polyhedrosis virus and detecting changes in BmIML-2 transcription level

[0029] When the silkworm larvae were first fed to mulberries, several silkworm varieties were used as experimental materials, and each variety was divided into two groups for treatment. The treatment group was fed with nuclear polyhedrosis virus (concentration 1×10 7 Pieces / mL) fresh mulberry leaves after soaking, and the control group was fed fresh mulberry leaves without virus soaking. After adding the poison, weed out the dead silkworms in time, until the cocoons are formed on the upper swarms, and weed out the dead pupae. Take each group of silkworm moths after seed production, extract fat body RNA and reverse transcription to obtain cDNA. Design specific primers (same as Example 1), and use the cDNA obtained by reverse transcription as a template to detect the difference in the transcription level of the silkworm BmIML-2 gene befo...

Embodiment 3

[0030] Example 3 Continued screening of silkworm varieties with significantly up-regulated BmIML-2 transcription level after feeding virus

[0031] According to the test results in Example 2, the silkworm eggs laid by the individual silkworm species whose BmIML-2 transcription level was significantly up-regulated after the addition of nuclear polyhedrosis virus were selected for seeding and subculture, and the subcultured silkworm species were repeatedly screened . That is, when the silkworm larvae were fed to the mulberry last time, repeat the virus feeding treatment in the previous step, use the RT-PCR method to detect the difference in the transcription level of the BmIML-2 gene before and after feeding the virus, and screen the excellent male and female species Subgeneration. After screening by the above method, the resistance of Baiyu CB variety to nuclear polyhedrosis virus is increased by 10-100 times. When the second instar silkworm is raised, Baiyu CB and Jingsong are ...

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Abstract

The invention discloses a method for screening bombyx mori nuclear polyhedrosis virus disease resistance with a BmIML-2 gene. According to the method, nuclear polyhedrosis virus is added during the last mulberry leaf feeding before mounting, and the transcription level of the BmIML-2 gene is adopted as the detection index to judge the resistance strength of bombyx mori to the virus. Compared withthe prior art, the method disclosed by the invention has the advantages that: (1) the virus is added before 5-instar mounting, so that the test period can be shortened, the workload is small, and theprobability of cross infection among bombyx mori is reduced; (2) the resistance of the bombyx mori variety screened by the method to the nuclear polyhedrosis virus is improved by 10-100 times; and (3)the bombyx mori variety screened by the method is stable in resistance and vigorous in physique after multi-generation feeding.

Description

Technical field [0001] The invention belongs to the technical field of silkworm breeding, and relates to a technology for breeding silkworm virus-resistant varieties by genetic engineering methods, specifically a method for screening silkworm nuclear polyhedrosis virus disease resistance by using BmIML-2 gene. Background technique [0002] As an important economic insect and model insect, the silkworm is of great significance in scientific research, silk production and the promotion of farmers’ income. Bombyx mori nucleopolyhedrosis virus, caused by the silkworm nuclear polyhedrosis virus, is the most harmful silkworm disease in sericulture production. The disease is extremely infectious and difficult to prevent and control. The economic loss caused by the silkworm disease accounts for 70% of the total loss of silkworm disease. ~80%. The breeding of silkworm nuclear polyhedrosis virus resistant varieties has always been an important research direction in the industry. The screen...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686
CPCC12Q1/6888C12Q1/686C12Q2600/124C12Q2600/158C12Q2600/166C12Q2521/107C12Q2545/101
Inventor 赵巧玲沈东旭郭际云佟美金夏定国裘智勇沈兴家
Owner JIANGSU UNIV OF SCI & TECH
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