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DNA (deoxyribonucleic acid) molecule for expressing hairpin type RNA (ribonucleic acid) for suppressing sitobion avenae carboxylesterase and application of DNA molecule

A technology of Aphid spp reduced effect

Inactive Publication Date: 2014-02-19
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aphids harm wheat by sucking wheat juice as adults and nymphs, and the honeydew discharged on the leaves reduces photosynthesis and spreads a variety of viral diseases, including barley yellow dwarf virus, resulting in reduced yield and quality of wheat
[0003] At present, the control of wheat aphids or other pests mainly relies on chemical insecticides, although to a certain extent it can reduce the yield drop caused by insect pests, but the long-term use of chemical insecticides is not only expensive but also easy to cause corresponding damage to aphids. resistance, and at the same time lead to serious damage to aphid natural enemies and aggravate environmental pollution

Method used

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  • DNA (deoxyribonucleic acid) molecule for expressing hairpin type RNA (ribonucleic acid) for suppressing sitobion avenae carboxylesterase and application of DNA molecule
  • DNA (deoxyribonucleic acid) molecule for expressing hairpin type RNA (ribonucleic acid) for suppressing sitobion avenae carboxylesterase and application of DNA molecule
  • DNA (deoxyribonucleic acid) molecule for expressing hairpin type RNA (ribonucleic acid) for suppressing sitobion avenae carboxylesterase and application of DNA molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Obtaining of DNA Molecules Expressing Hairpin RNA that Inhibits Aphidida carboxylesterase

[0058] 1. Acquisition of the target fragment of SaCbE E4 of the aphids carboxylesterase gene

[0059] Design the following 2 pairs of primers P1-sF / P1-sR and P1-asF / P1-asR:

[0060] P1-sF: 5'- GGATCC AAGTATTGCGCAAGATGAGGGTC-3' (the underline is the Bam H I restriction site, and the following sequence is the 1-23rd position of sequence 1);

[0061] P1-sR: 5'- AAGCTT GCTACGTAAGAATAATTACCAC-3' (the underline is the Hind III restriction site, and the following sequence is the reverse complementary sequence of the 324-345th position of sequence 1).

[0062] P1-asF: 5'- GGTACC AAGTATTGCGCAAGATGAGGGTC-3' (the underline is the KpnI restriction site, and the following sequence is the 1-23rd position of sequence 1);

[0063] P1-asR: 5'- GAGCTC GCTACGTAAGAATAATTACCAC-3' (the underline is the Sac I restriction site, and the following sequence is the reverse complement...

Embodiment 2

[0086] Example 2, the acquisition and identification of RNAi transgenic wheat of SaCbE E4

[0087] 1. Acquisition of SaCbE E4 RNAi transgenic wheat

[0088] Select the young ears of the wheat variety Liaochun No. 10 12-15 days after flowering, sterilize the seeds, remove the immature embryos, place the scutellum up on the immature embryo inoculation medium (formula: MS basic medium + 2,4- D2mg / L + sucrose 30g / L + agar powder 7g / L, pH 5.8. Each concentration is the final concentration of the corresponding components in the medium), cultured in the dark at 25±1°C for 3-5 days to induce wheat callus. Select callus pieces with good growth status for gene gun co-transformation (mix pBAC-rbcs-CbE E4 and pBAC35SIH3 at a molar ratio of 3:1, wherein the pBAC35SIH3 plasmid carries the resistance gene bar of the herbicide Basta), and the transformed After the callus was cultured in the dark at 25±1°C for 2-3 days, it was transferred to the selection medium (recipe: immature embryo inocu...

Embodiment 3

[0099] Example 3, Study on the effect of RNAi transgenic wheat of SaCbE E4 on the control of the aphid

[0100] Aphids aphids population, three generations of aphids were raised indoors, during which no insecticides were ever applied. The indoor breeding temperature is 21±1°C, the relative humidity is 50-60%, and the photoperiod is L:D=16h:8h. Aphids were raised on aphid-sensitive wheat variety Jing 411, and fresh seedlings were replaced every two weeks for aphids.

[0101] 1. Analysis of the interference effect of SaCbE E4 RNAi transgenic wheat on the expression level of the carboxylesterase gene of A.

[0102] The Aphid aphid in the first instar nymph stage was used as the experimental material, and they were fed on T 3 Generation of SaCbE E4 RNAi transgenic lines SadsCbE1-5 and SadsCbE2-2 at the two-leaf one-heart seedling stage on 1 day, 3 days, 5 days (single plant isolation with gauze). Using qRT-PCR to analyze the expression level of carboxylesterase gene in the tube...

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Abstract

The invention discloses a DNA (deoxyribonucleic acid) molecule for expressing a hairpin type RNA (ribonucleic acid) for suppressing sitobion avenae carboxylesterase and application of the DNA molecule. The structure of the DNA molecule disclosed by the invention is SEQ (forward)-X-SEQ (reverse); the sequence of the SEQ (forward) is from a locus 1 to a locus 345 of a sequence 1; the sequence of the SEQ (reverse) is reversely complementary to that of the SEQ (forward); the X is an interval sequence between the SEQ (forward) and the SEQ (reverse); according to the sequence, the X is not complementary to the SEQ (forward) and the SEQ (reverse). An experiment shows that the DNA molecule is converted into an immature embryo callus of wheat to obtain a transgenic line; the transgenic line is subjected to an aphid feeding test; after sitobion avenae is fed on transgenic leaves, the carboxylesterase gene expression quantity and the enzymatic activity are obviously alleviated, and the rate of reproduction is reduced; furthermore, the tolerance of the sitobion avenae to a phoxim solvent is reduced by 20-30 percent. Therefore, the DNA molecule has a great significance for prevention and treatment of insects of agricultural production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a DNA molecule expressing a hairpin RNA that inhibits the aphidine aphidium carboxylesterase and an application thereof. Background technique [0002] Aphids are the main pests in wheat production, mainly including Metopolophium dirhodum, Schizaphis graminum, Rhopalosiphum padi and Sitobion avenae, The distribution is very wide, almost all over the world's wheat-producing countries. Aphids suck wheat sap as adults and nymphs to harm wheat, and the honeydew discharged on the leaves reduces photosynthesis and spreads a variety of viral diseases, including barley yellow dwarf virus, resulting in reduced yield and quality of wheat. [0003] At present, the control of wheat aphids or other pests mainly relies on chemical insecticides, although to a certain extent it can reduce the yield drop caused by insect pests, but the long-term use of chemical insecticides is not only expensive but al...

Claims

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Application Information

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IPC IPC(8): C12N15/113A01N57/16A01P7/04C12N15/82A01H5/00
Inventor 梁荣奇倪中福许兰杰段晓亮吕延华尤明山解超杰李保云彭惠茹姚颖垠杜金昆刘志勇胡兆荣孙其信
Owner CHINA AGRI UNIV
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