DNA molecule for expressing hairpin RNA for inhibiting macrosiphum avenae lipoprotein lipase and applications thereof

A wheat long tube aphid and expression cassette technology, applied in the field of DNA molecules, can solve the problems of high cost aphids, damage to wheat, and honeydew to reduce photosynthesis and the like

Inactive Publication Date: 2015-05-13
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 molecule for expressing hairpin RNA for inhibiting macrosiphum avenae lipoprotein lipase and applications thereof
  • DNA molecule for expressing hairpin RNA for inhibiting macrosiphum avenae lipoprotein lipase and applications thereof
  • DNA molecule for expressing hairpin RNA for inhibiting macrosiphum avenae lipoprotein lipase and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Obtaining of DNA Molecules Expressing Hairpin RNA that Inhibits Aphidhilipin Esterase

[0052] 1. Acquisition of the target fragment of the aphid lipoprotein esterase gene SaLPL

[0053] Design the following primers:

[0054] SaLPL-s1: 5'-CCTGTTCCTGAGAGGCGTCT-3' (positions 21-40 of Sequence 1);

[0055] SaLPL-a1: 5'-GCAACACCAGCTGAAAACGCTACTC-3' (reverse complement of positions 539-563 of Sequence 1).

[0056] The length of the amplified fragment was 543bp.

[0057] The 5th-instar Aphid aphid adults were taken, and the total RNA was extracted according to the Melab RNA extraction kit ( figure 1 ). In the figure, 28S and 18S are very clear, indicating that the extracted total RNA can be used for RT-PCR. The total RNA, primers P1-R and P1-F were used for RT-PCR, and the reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C for 40 s, 55°C for 40 s, 72°C for 90 s, 33 cycles; and finally 72°C for 10 min.

[0058] The results showed that the targ...

Embodiment 2

[0081] Example 2, the acquisition and identification of RNAi transgenic wheat of SaLPL

[0082] 1. Obtaining RNAi transgenic wheat of SaLPL

[0083] Select the young ears of the wheat variety Jinghua No. 1 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 blocks with good growth status for gene gun co-transformation (mix pBAC-rbcs-LPL and pBAC35SIH3 at a molar ratio of 3:1, wherein the pBAC35SIH3 plasmid carries the Basta resistance gene bar), and the transformed calli are 25± After 2-3 days of dark culture at 1°C, transfer to the screening medium (recipe: immature embryo inoculation medium + Basta 2-25mg / L. Each concentration i...

Embodiment 3

[0091] Example 3, Study on the control effect of SaLPL's RNAi transgenic wheat on the aphid

[0092] 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 30-40%, 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

[0093] 1. Analysis of the interference effect of SaLPL RNAi transgenic wheat on the expression of Aphidida lipin esterase gene

[0094] The first-year-old tube aphid was used as the experimental material, and it was fed in T 3 Generation of SaLPL on the RNAi transgenic line dsLPL-1 for 5 days. Using qRT-PCR to analyze the expression level of lipoprotein esterase gene in Aphid aphid. The specific operation is as follows:

[0095] Design the amplification primer pair SaLPL-s2 / SaLPL-a2 (amplified fragment size is 249bp) of ...

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Abstract

The invention discloses a DNA molecule for expressing hairpin RNA for inhibiting a macrosiphum avenae lipoprotein lipase and applications thereof. The structure of the DNA molecule provided by the invention is SEQ positive direction and -X-SEQ reverse direction, and the sequence of the SEQ positive direction is 21st-563rd position of a sequence 1 in a sequence table; the sequence if the SEQ reverse direction is reversely complemented to the sequence of the SEQ positive direction; and X is an interval sequence between the SEQ positive direction and the SEQ reverse direction, and in the sequence, X does not complement to the SEQ positive direction and the SEQ reverse direction. Proved by an experiment, the DNA molecule is converted to be wheat, to obtain transgene lines for carrying out a aphid feeding test, and after a macrosiphum avenae eats transgene leaves, a lipoprotein lipase gene expression level and reproductive rate decrease. Thus, the DNA molecule provided by the invention has an important meaning for controlling insects in agricultural production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a DNA molecule expressing a hairpin RNA inhibiting aphidia lipoprotein esterase and 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 also easy to cause...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A01N57/16A01P7/04C12N15/82A01H5/00
Inventor 梁荣奇倪中福许兰杰张小华侯起岭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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