DNA molecule for expressive inhibition of hairpin RNA of Sitobion avenae chitin synthetase 1 gene and application thereof

A technology for chitin synthase and Aphid vulgaris, which is applied in recombinant DNA technology, DNA/RNA fragments, applications, etc., can solve the problems of aggravated environmental pollution, killing of natural enemies of aphids, and high cost of aphid resistance.

Inactive Publication Date: 2016-10-12
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the control of wheat aphids or other pests mainly relies on chemical insecticides. Although it can reduce the yield drop caused by insect pests to a certain extent, long-term use of chemical insecticides is not only expensive but also easy to cause corresponding resistance to aphids. At the same time, it causes serious damage to natural enemies of aphids and aggravates environmental pollution

Method used

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  • DNA molecule for expressive inhibition of hairpin RNA of Sitobion avenae chitin synthetase 1 gene and application thereof
  • DNA molecule for expressive inhibition of hairpin RNA of Sitobion avenae chitin synthetase 1 gene and application thereof
  • DNA molecule for expressive inhibition of hairpin RNA of Sitobion avenae chitin synthetase 1 gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Example 1, Obtaining the target fragment expressing and inhibiting the chitin synthase 1 (CHS1) gene of the aphid

[0069] 1. Obtaining the target fragment

[0070] Design the following primers:

[0071] CHS1-F 5'-AACAGTACCAGTCACCATGTC-3'

[0072] CHS1-R 5'-CGACGAGCGCACTGATCTTC-3'

[0073] The length of the amplified fragment is 607bp.

[0074] Adults of Aphid agriculi were taken, and the total RNA of A. figure 1 Middle A), in the figure, 28S and 18S are very clear, indicating that the extracted total RNA can be used for RT-PCR. Utilize this total RNA, primer (CHS1-F and CHS1-R) to carry out RT-PCR, obtain about 607bp the target fragment ( figure 1 Middle B). Amplification program: initial denaturation at 95°C for 5 minutes before amplification, 35 cycles of PCR amplification, cycle program: denaturation at 95°C for 45 s, annealing at 56°C for 45 s, extension at 72°C for 50 s, after the last cycle, at 72°C Extend for 10 min and store at 4°C.

[0075] Recover the...

Embodiment 2

[0084] Embodiment 2, the acquisition of transgenic wheat

[0085] Explants: callus induced by wheat immature embryos;

[0086] Strain: Escherichia coli strain DH5α;

[0087] Vector: pBAC-rbcs-CHS1IR obtained from Example 1, pBAC35SIH3

[0088] Medium:

[0089] (a) Young embryo callus induction medium, the immature embryo inoculation medium is a solid medium obtained by adding 2,4-D to the MS medium, and the concentration of 2,4-D in the immature embryo inoculation medium is 2g / L, pH 5.8;

[0090] (b) screening medium, screening medium is the solid medium that the Basta concentration that adds Basta to obtain in MS medium is 0.2mg / L solid medium, and pH is 5.8;

[0091] (c) differentiation medium, the concentration of IAA, transzeatin and Basta obtained by adding IAA, transzeatin and Basta to MS medium is respectively 1mg / L, 10mg / L and 0.05mg / L Solid medium, pH 5.8;

[0092] (d) rooting medium for strong seedlings, the rooting medium for strong seedlings is a solid culture...

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Abstract

The invention discloses DNA molecule for expressive inhibition of hairpin RNA of Sitobion avenae chitin synthetase 1 gene and an application thereof. A DNA molecular structure for expressive inhibition of hairpin RNA of Sitobion avenae chitin synthetase 1 gene is shown as a formula (I) in the specification: SEQ forward sequence-X-SEQ reverse sequence (I); the SEQ forward sequence is 31 th-637th bit in the sequence 1; and the SEQ reverse sequence is reversely complemented with the SEQ forward sequence; X is a spacer sequence between the SEQ forward sequence and the SEQ reverse sequence, and X is neither complemented with the SEQ forward sequence nor complemented with the SEQ reverse sequence; a X sequence is the sequence containing FAD2 Intron1, and the FAD2 Intron1 sequence is the 664th-1793th bit of the sequence 1. The experiment proves that the transgenic lines through conversion of the DNA molecule to wheat can inhibiting breeding and ecdysis of Sitobion avenae, and the prevention and treatment on insects from agriculture production can be achieved.

Description

technical field [0001] The invention relates to a DNA molecule expressing a hairpin RNA inhibiting the chitin synthase 1 gene of the aphid aphid and its application in the field of biotechnology. 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. The damage of wheat aphids to wheat is almost throughout the whole growth period of wheat. Aphids suck wheat sap as adults and nymphs to harm wheat. The honeydew discharged on the leaves reduces photosynthesis and spreads a variety of viral diseases, including barley yellow dwarf virus, These will not only lead to a serious reduction in wheat yield, but also have a serious impact on the nutritional quality and processing quality of flour. The wheat aphid is the dominant species of wheat aphid, which seriously affects the...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/113A01H5/00
CPCC12N15/1137C12N15/8286C12N2310/14C12N2310/531
Inventor 梁荣奇赵艳杰卢立华王雪平倪中福尤明山解超杰李保云彭惠茹姚颖垠杜金昆辛明明胡兆荣孙其信
Owner CHINA AGRI UNIV
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