Cotton bollworm transferrin gene HaTrf and application thereof

A technology of transferrin and cotton bollworm, applied in the field of genetic engineering, can solve problems such as harmful genetic modification and achieve the effect of improving sensitivity

Active Publication Date: 2014-05-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The widespread use of chemical pesticides is very useful for the control of harmful organisms, but the long-term and large-scale use of chemical pesticides can cause harm to the environment and higher carnivores. The accumulation of chemical pesticides has caused damage to these The development of resistance to substances and, possibly in species higher on the evolutionary ladder, becoming mutagens and / or carcinogens, leading to irreversible and deleterious genetic modifications

Method used

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  • Cotton bollworm transferrin gene HaTrf and application thereof
  • Cotton bollworm transferrin gene HaTrf and application thereof
  • Cotton bollworm transferrin gene HaTrf and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Cloning of the full-length sequence of transferrin gene HaTrf of cotton bollworm

[0027] The full-length coding sequence of transferrin gene HaTrf in cotton bollworm has a total of 2052bp encoding a protein with a size of about 76KDa, and the first 20 amino acids of the sequence are found to be the signal peptide sequence (MVSKIKYIYLLVLACVCVQA) through the signal peptide online prediction software. The two topological loops at the N-terminal and C-terminal of transferrin in cotton bollworm form two domains with high homology, and each domain contains ferric ion binding sites, which can reversibly bind and release iron ions.

[0028] Extract the total RNA of cotton bollworm, reverse transcribe cDNA, use cDNA as a template, design degenerate primers according to the transferrin gene sequence of silkworm and Spodoptera litura, amplify the partial sequence of transferrin gene of cotton bollworm, and use the cDNA end to quickly clone Technology (RACE) was spliced ...

Embodiment 2

[0031] Example 2 Synthesis of HaTrf Gene dsRNA of Cotton Bollworm and Interference to HaTrf

[0032] According to the full length of the HaTrf gene of the cotton bollworm cloned in Example 1, primers F1 and R1 were designed to amplify a sequence of 667bp in length, and the dsRNA of the HaTrf gene was synthesized by an in vitro transcription kit, and the dsRNA was mixed with the cotton bollworm artificial diet Mix feeding 2nd instar cotton bollworm larvae.

[0033] More specifically, the dsRNA (15 μg / g or 35 μg / g) of the HaTrf gene transcribed in vitro by the MEGAscript T7 transcription kit was fed to the 2nd instar larvae after being mixed with the artificial diet of the cotton bollworm. Hours, 24 hours, 36h and 48h, the expression of HaTrf gene was found to be able to efficiently silence the expression of transferrin gene in cotton bollworm, which has a good target gene silencing effect ( image 3 ).

Embodiment 3

[0034] Example 3 The dsRNA of HaTrf gene can inhibit the growth and development of cotton bollworm

[0035] The dsRNA synthesized in vitro in Example 2 (35 μg / g double distilled water) was fed to the 2nd instar larvae of cotton bollworm by mixing with artificial feed, and its growth inhibition on cotton bollworm was recorded at the same time. HaTrf dsRNA compared with After 5 days of control treatment, the phenomenon of significantly inhibiting the birth of cotton bollworm ( Figure 4 ).

[0036] More specifically, the 2nd instar larvae of the cotton bollworm were treated with HaTrf dsRNA at a concentration of 35 μg / g, and the body weight changes of the cotton bollworm were recorded on day 0, day 1, day 3 and day 5.

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Abstract

The invention relates to the field of genetic engineering and particularly provides a cotton bollworm transferrin gene HaTrf. The amino acid sequence coded by the gene is shown in SEQ ID No.3, and the total-length coding sequence is shown in SEQ ID No.1. The dsRNA designed based on the gene sequence can remarkably inhibit the expression of the cotton bollworm transferrin gene and shows influence on the growth and development of cotton bollworm on the aspects of developmental retardation and increase of sensitivity to the plant secondary substance 2-tridecanone.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the transferrin gene HaTrf of cotton bollworm and its application. Background technique [0002] The widespread use of chemical pesticides is very useful for the control of harmful organisms, but the long-term and large-scale use of chemical pesticides can cause harm to the environment and higher carnivores. The accumulation of chemical pesticides has caused damage to these The development of resistance to substances, and possibly, in species higher on the evolutionary ladder, becoming mutagens and / or carcinogens, leading to irreversible and deleterious genetic modifications. Plant secondary substances have the advantages of good control effect on diseases, insect pests and weeds, and are safe for humans and animals, do not pollute the environment, have no residues, have strong specificity for diseases, insect pests and weeds, are easily degraded from nature, and are difficult ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/11C07K14/79A01N57/16A01P7/04
Inventor 高希武张雷尚庆利刘晓岚路瑶
Owner CHINA AGRI UNIV
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