Preparation method and nucleic acid construct of male sterile lepidoptera insects

A technology of lepidopteran insects and nucleic acid constructs, applied in the field of a method of lepidopteran insects and its nucleic acid constructs, can solve problems such as not being male, and achieve the effect of promoting development and realizing controllability

Active Publication Date: 2019-08-13
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although both radiation and tetracycline can induce sterile insects, the target sterile insects for both are females, not males

Method used

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  • Preparation method and nucleic acid construct of male sterile lepidoptera insects
  • Preparation method and nucleic acid construct of male sterile lepidoptera insects
  • Preparation method and nucleic acid construct of male sterile lepidoptera insects

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The construction of embodiment 1 carrier

[0047] 1. The BmSer2 knockout plasmid PXL-BacII-IE1-DsRed2-U6-BmSer2sgRNA1-U6-BmSer2sgRNA2 was cloned by PCR method. Specific steps:

[0048]I. sgRNA-KnpI-F and sgRNA-R1, sgRNA-F1 and sgRNA-Overlap-R two pairs of primers respectively use the PXL-BacII-IE1-DsRed2-U6-U6 plasmid as a template to obtain the product by PCR, and then the product is 2 The volume ratio of :1 was used as a template, and sgRNA-KnpI-F and sgRNA-Overlap-R were used as primers to obtain the BmSer2sgRNA-1 fragment with restriction endonuclease KnpI homology arms by PCR. Similarly, use sgRNA-Overlap-F and sgRNA-R2, sgRNA-F2 and sgRNA-HindIII-R as the first round of PCR primers, and after the product is obtained, use sgRNA-Overlap-F and sgRNA-HindIII-R as the second round Primers, BmSer2sgRNA-2 fragments with restriction endonuclease HindIII homology arms were obtained by PCR.

[0049] Finally, the pXL-BacII-IE1-DsRed2-U6-U6 plasmid was digested with restri...

Embodiment 2

[0065] The acquisition of embodiment 2 transgenic silkworm

[0066] The Ser2 knockout plasmid PXL-BacII-IE1-DsRed2-U6-BmSer2sgRNA1-U6-BmSer2sgRNA2 prepared in Example 1 and the plasmid PHA3PIG capable of expressing Piggybac transposase were mixed in equal amounts and injected into silkworm primiparous eggs. The injection method was microinjection according to the method described by Kanda﹠Tamura (1991). After injection, it was sealed with non-toxic glue to prevent contamination, and then hatched in a sterile environment at 25°C, and the ant silkworm was reared. After mothization, the contemporary (G0 generation) silkworm moth was selfed, and the G1 generation ant silkworm obtained in the fluorescent The individual transgenic silkworms with red fluorescence were screened under a microscope (ie, the BmSer2 mutant intermediate strain).

[0067] Subsequently, the screened red fluorescent transgenic silkworm and green fluorescent transgenic silkworm Nos-Cas9 were reared and mated,...

Embodiment 3

[0068] The detection of embodiment 3 transgenic silkworms

[0069] Identification of gene mutations: 10 double fluorescent silkworms were selected during the ant silkworm stage to extract their genomes, using primers F1 and R1, F2 and R2, the target fragments were cloned by PCR and sequenced. Sequencing results showed that both "target 1" and "target 2" sequences were mutated in the BmSer2 gene in the double fluorescent silkworm.

[0070] Situation of eggs laid by double-fluorescent silkworms: 30 single-sex double-fluorescent silkworms were mated with wild-type silkworms at an ambient temperature of 25°C for 5 hours, then separated for 5 hours, and then laid eggs in the same environment for 36 hours, and the number of eggs laid and hatched were counted rate, see Figure 2. It can be seen that the hatching rate of eggs laid by BmSer2 mutation (i.e. double fluorescence) female silkworm and normal male silkworm is normal; the hatchability of eggs laid by BmSer2 mutation (i.e. dou...

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Abstract

The invention discloses a preparation method and nucleic acid construct of male sterile lepidoptera insects. The method comprises the steps that 1, the Ser2 gene knockout nucleic acid construct is built, wherein the nucleic acid construct comprises the following operable connection elements from the 5' terminal to the 3'terminal, and the operable connection elements include one U6 promoter, a first Ser2 genetic target, polyA, another U6 promoter, a second Ser2 genetic target and polyA; 2, the construct and a PHA3PIG plasmid capable of expressing Piggybac transposase are used for co-transformation of fresh lepidoptera insect eggs, a generation G0 is obtained and subjected to selfing, and a generation G1 is obtained; 3, the generation G1 and transgenic lepidoptera insects for expressing Cas9protein are mated to obtain a generation G2. According to the method, a CRISPR / Cas9 technology based on a PiggyBac transposon is used for successfully establishing the male sterile lepidoptera insects on the premise that normal mating behaviors are not affected, and the method has important value in prevention and treatment of lepidoptera pests through the male sterility technology.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for preparing male sterile Lepidoptera insects and a nucleic acid construct thereof. Background technique [0002] The silkworm is a typical model economic insect, one of the economic insects that feed on mulberry leaves and spin cocoons. The use of silkworm as the research object of insect pest control, especially the genetic control of Lepidoptera pests, will have a significant impact on the genetic control of agricultural and forestry pests, and will also be popularized and applied to other Lepidoptera insects. At the present stage, most of the sterile techniques for biological control of pests use radiation and tetracycline induction, but these two sterile techniques are time-consuming and labor-intensive and cannot produce stable genetic effect strains, and most of them release sterile males for pest control. However, these sterile males are less competitive than wild ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90A01K67/033
CPCC12N15/8509C12N15/902C12N9/22C12N9/6408A01K67/0339C12N2800/105C12N2810/10A01K2217/075A01K2227/706
Inventor 黄勇平徐霞何琳李恺张忠杰陈旭
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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