Application of migratory locust metallothionein I-type gene LmMt I to grasshopper control

A metallothionein and migratory locust technology, which is applied in the biological field, can solve the problems of pesticide residues in agricultural products, harm of non-target organisms and natural enemy insects, and threats to insects, and achieves good specificity and high efficiency.

Inactive Publication Date: 2019-03-08
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, we mainly rely on chemical drugs to control migratory locusts. Long-term use of chemical insecticides leads to environmental pollution,

Method used

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  • Application of migratory locust metallothionein I-type gene LmMt I to grasshopper control
  • Application of migratory locust metallothionein I-type gene LmMt I to grasshopper control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] The acquisition and application of the metallothionein type I gene (LmMt I) sequence of migratory locust;

[0018] After transcriptome sequencing and using bioinformatics methods to search for the metallothionein type I gene of migratory locust, the gene was cloned and verified by sequencing using the migratory locust DNA as a template, and the sequence SEQ ID NO of the gene (LmMt I) was confirmed: 1;

[0019] Select the gene fragment of the open reading frame SEQ ID NO: 2 in the sequence of SEQ ID NO: 1;

[0020] According to the sequence characteristics of SEQ ID NO: 2, the primer premier5.0 software was used to design the upper and lower primers SEQ ID NO: 3 and SEQ ID NO: 4 containing the T7 promoter, and then according to T7 RiboMAX TM Express RNAi System Kit Instructions In vitro transcription and synthesis of dsRNA;

[0021] Use 1% agarose gel electrophoresis to detect the singleness of dsRNA, and then quantify it as the final concentration: 1.5 μg / μl;

[002...

Embodiment 2

[0026] The application of the sequence of metallothionein type I gene (LmMt I) of migratory locust;

[0027] The method for synthesizing dsRNA is as described in Example 1;

[0028] After detecting and confirming the singleness of dsRNA, quantify it to a final concentration of 1.5 μg / μl;

[0029] Select 100 healthy 5th-instar nymphs of migratory locusts, and divide them into control group and experimental group equally. Inject 5 μl of specific dsRNA intraperitoneally into the experimental group insects with a microsyringe, and inject the same amount of dsGFP into the control group locusts. Finally, the insects were placed in a 27-degree climate box, fed and observed with fresh rice leaves;

[0030] 24 and 48 hours after dsRNA injection, 20 worms were taken to detect the silencing efficiency of dsRNA on LmMt I. The results showed that the silencing efficiency of LmMt I gene reached 74.7% at 48 hours after dsRNA injection;

[0031] After 36 hours of dsRNA injection by the abov...

Embodiment 3

[0033] The application of the sequence of metallothionein type I gene (LmMt I) of migratory locust;

[0034] The method for synthesizing dsRNA is as described in Example 1;

[0035] Quantify the dsRNA to a final concentration of 2.0 μg / μl;

[0036] Select 90 healthy adult migratory locusts and divide them into control group and experimental group. Inject 4 μl of dsRNA into the insects of the experimental group with a micro-injector, and inject the same amount of dsGFP into the insects of the control group, and then place them in a 29-degree climate box. rice leaf feeding;

[0037] After 24 and 48 hours, 20 worms were taken immediately to detect the silencing efficiency of LmMt I was 77.3%;

[0038] A series of abnormal physiological behaviors were found in migratory locusts 36-48 hours after dsRNA injection. After 12 hours of injection of low-concentration copper solution (0.3 μg / μl) into the abdomen, the mortality rate of migratory locusts increased significantly compared w...

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Abstract

The invention discloses an application of a migratory locust metallothionein I-type gene LmMt I to grasshopper control. The high-expression metallothionein I-type gene is searched by a transcriptome sequencing and information biology method, the sequence SEQ ID NO: 1 of the gene is confirmed after clone sequencing verification, a gene segment (open reading frame) with the sequence of SEQ ID NO: 2is selected and used for synthesizing corresponding double stranded RNA (dsRNA), the double stranded RNA is injected into an abdominal cavity of a migratory locust by a micro-injector, and then a target gene LmMt I can be specifically silenced, so that the migratory locust suffers from in-vivo metal metabolic disturbance, food refusal, developmental retardation, digestive abnormity and body emaciation until death. The metal tolerance of the migratory locust injected with the double stranded RNA is greatly reduced, death rate is increased by 1.8-2.3 times after medicines of the same dose act onthe migratory locust, and the death rate of an experimental group is increased by 79.6% as compared with a control group after treatment with metal with the same concentration.

Description

technical field [0001] The invention relates to the application of a migratory locust metallothionein type I gene LmMt I in locust control and belongs to the field of biotechnology. Background technique [0002] Migratory locust is an important agricultural pest widely distributed all over the world. Due to its characteristics of migratory, gregarious, and omnivorous, once it swarms, it eats a huge amount and can fly long distances, which may cause serious damage to crops. At present, we mainly rely on chemical drugs to control migratory locusts. Long-term use of chemical insecticides leads to environmental pollution, pesticide residues in agricultural products, stresses pests to develop resistance, and causes harm to non-target organisms and natural enemy insects. Therefore, there is an urgent need to develop new, pollution-free, and specific control technologies to replace chemical control. [0003] Metal ions play an extremely important physiological role in organisms, s...

Claims

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Application Information

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IPC IPC(8): C12N15/89A01K67/033
CPCA01K67/0339A01K2217/05A01K2227/706C12N15/89
Inventor 刘耀明
Owner SHANXI UNIV
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