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Helicoverpa armigera juvenile hormone binding protein (Ha-JHBP) and encoding gene and application thereof

Cotton bollworm, coding technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of slow development of transgenic plant resistance, etc., and achieve the effect of improving insect resistance

Active Publication Date: 2012-05-23
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Insect resistance to GM plants, although slow to develop, is a problem that cannot be ignored

Method used

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  • Helicoverpa armigera juvenile hormone binding protein (Ha-JHBP) and encoding gene and application thereof
  • Helicoverpa armigera juvenile hormone binding protein (Ha-JHBP) and encoding gene and application thereof
  • Helicoverpa armigera juvenile hormone binding protein (Ha-JHBP) and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 4

[0053] The sterile tobacco variety used in Example 4 is Nicotiana tabacum cv.Xanthi, and the public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences; References: A.NATO, S.BAZETOUX, Y.MATHIEUPhotosynthetic Capacities and Growth Characteristics of Nicotiana tabacum (cv. Xanthi) Cell Suspension Cultures Physiologia Plantarum Volume 41, Issue 2, pages 116-123, October 1977.

[0054] Cotton bollworm (Helicoverpa armigera): publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences; references: Wu KM, Lu YH, Feng HQ, Jiang YY, Zhao JZ. Suppression of cotton bollworm in multiple crops in China in areas with Bt toxin-containing cotton. Science. 2008 Sep 19;321(5896):1676-8.

[0055] LB medium: the solvent is water, containing yeast extract 5g / L, peptone 10g / L, NaCl 10g / L, and the pH is adjusted to 7.0 with 10M NaOH aqueous solution.

[0056] YEB medium: the solvent is water, containing b...

Embodiment 1

[0061] Embodiment 1, discovery of juvenile hormone-binding protein and its coding gene of cotton bollworm

[0062] 1. According to the JHBP gene sequence of known insects, the degenerate primers (P1 / P2) are designed as follows:

[0063] P1: 5'-AGTGA(C / T)ATA(A / G)AATGC(T / A)T(G / A)AGCAA-3';

[0064] P2: 5'-GG(C / T)TC(T / A)CCAA(T / A)GATTTC(A / G)CAAG-3'.

[0065] 2. Extract the total RNA of the 3rd instar cotton bollworm larvae, and obtain cDNA by RT-PCR in vitro reverse transcription.

[0066] 3. Using the cDNA in step 2 as a template, perform PCR amplification with the primers designed in step 1 to obtain a PCR amplification product.

[0067] 4. Cloning the PCR amplified product in step 3 into the pEASY-Blunt sequencing vector for sequencing. The sequencing results showed that the nucleotide sequence of the PCR amplified product was shown in sequence 5 in the sequence table.

[0068] 5. Extract the total RNA of the 3rd instar cotton bollworm for the primer extension experiment, add...

Embodiment 2

[0083] Embodiment 2, cotton bollworm larvae feeding test

[0084] 1. Construction of recombinant plasmid pLJP

[0085] 1. Using the recombinant plasmid pJHBP as a template, carry out PCR amplification with primers P7-BglII / P8-XhoI, and recover the PCR amplification product (the conserved segment of Ha-JHBP gene JHBP1-BX).

[0086] P7-BglII: 5'-GAAGATCTGATGTTGTGTATGA-3';

[0087] P8-XhoI: 5'-CCGCTCGAGTTAAAAGTCGTGGT-3'.

[0088] 2. The PCR amplification product of step 3 was double-digested with restriction enzymes BglII and XhoI, and the digested product was recovered.

[0089] 3. Digest the bacterial expression vector L4440 with restriction endonucleases BglII and XhoI, and recover the vector skeleton.

[0090] 4. Ligate the digested product of step 2 with the vector backbone of step 3 to obtain the recombinant plasmid pLJP. The starting vector of the recombinant plasmid pLJP is L4440, and the DNA shown in the 204-691 nucleotides of the sequence 2 of the sequence listing f...

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Abstract

The invention discloses a helicoverpa armigera juvenile hormone binding protein (Ha-JHBP) and an encoding gene and application thereof. The helicoverpa armigera juvenile hormone binding protein provided by the invention is a protein shown as (a) or (b), wherein (a) is constituted by an amino acid sequence shown as a sequence 1 in a sequence table, and (b) is obtained by performing substitution and / or deletion and / or addition of one or more amino acid residues on an amino acid residue sequence shown as a sequence 1, is relevant to helicoverpa armigera growth and is derived the sequence 1. The invention further provides an encoding gene of the protein and a functional fragment of the encoding gene. By establishing a dsRNA (double-stranded Ribonucleic Acid) plant expression vector in a way of taking a Ha-JHBP gene as a target gene and expressing a large quantity of dsRNAs of the Ha-JHBP in plants, the insect resistance of transgenic plants is enhanced. The Ha-JHBP has important application values in the fields of biological prevention and control of phytophagous insects, plant insect resistance gene engineering and molecular biology.

Description

technical field [0001] The invention relates to a cotton bollworm juvenile hormone-binding protein, its coding gene and application. Background technique [0002] Pests are an important factor restricting the high yield of crops. According to statistics, the average economic loss of various crops due to insect pests reaches 20% to 30%, and the annual loss is about hundreds of billions of dollars. Spraying chemical pesticides and biological insecticides is the main control method at present. Due to factors such as human beings' awareness of environmental protection and their own health, pest resistance, and pesticide residues, a large number of traditional pesticides are no longer suitable for the needs of modern agricultural development. [0003] The use of genetic engineering to breed insect-resistant new varieties has the advantages of continuous protection, abundant resources, specific toxicity, less environmental pollution, short cycle, low cost, and strong purpose. T...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/63C12N15/82C12N5/10C12N1/15C12N1/19C12N1/21C12N15/11A01N61/00A01P7/04
Inventor 朱祯王晓芳
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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