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A kind of gene recombination Corynesium fumigatus if01 GM and its application

A Corynebacterium and gene recombination technology, applied in the application, recombinant DNA technology, fungi and other directions, can solve the problems of genetically improved insect-producing fungal strains and other problems, and achieve good popularization and application value, fast insecticidal effect, and good The effect of continuous development promotion

Active Publication Date: 2016-09-28
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the use of RNAi technology to regulate pests is mainly focused on transgenic plants and dsRNA preparation technology.

Method used

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  • A kind of gene recombination Corynesium fumigatus if01 GM and its application
  • A kind of gene recombination Corynesium fumigatus if01 GM and its application
  • A kind of gene recombination Corynesium fumigatus if01 GM and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of recombinant plasmids

[0048] The target gene used in the present invention, the TLR7 gene of Bemisia tabaci, was obtained from the sequence of the transcriptome of Bemisia tabaci (Unigene database: Unigene20701_B4592195), and its nucleotide sequence is shown in SEQ ID NO:10.

[0049] The present invention transforms a partial fragment of the TLR7 gene of Bemisia tabaci (its nucleotide sequence is shown in SEQ ID NO.1) into a strain of Corynesporium fumigatus to obtain a genetically recombined strain of Corynesporium fumigatus. The specific operation steps are as follows:

[0050] 1. Obtain the target fragment

[0051] (1) The present invention designs the primers TLR7F and TLR7R of the target gene, that is, the TLR7 gene of Bemisia tabaci.

[0052] The nucleotide sequence of the primer TLR7F is shown in SEQ ID NO.2, and the nucleotide sequence of TLR7R is shown in SEQ ID NO.3.

[0053] (2) The total RNA of B. tabaci was extracted by the Tri...

Embodiment 2

[0065] Example 2 Construction and Screening of Recombinant Strains

[0066] 1. Obtain the protoplasts of Corynespora fumigatus IfB01 strain

[0067] The strain of Corynespora fumigatus IfB01 used has been disclosed in the patent application number 201210449949.0.

[0068] The steps to obtain protoplasts are as follows:

[0069] (1) The conidia of Corynespora fumigatus IfB01 strain were prepared with 0.05% (v / v) Tween-80 solution to a concentration of 10 8 per mL of spore suspension.

[0070] (2) Take 1 mL of the suspension and inoculate it in 100 mL of potato dextrose liquid medium (PD), and incubate at 25 °C with shaking at 150 rpm for 30 h.

[0071] (3) Take the bacterial solution in a centrifuge tube, centrifuge at 5,000 rpm for 10 min, discard the supernatant, wash twice with 0.7 mol / L NaCl solution, and then centrifuge to obtain mycelium.

[0072] (4) Add a mixed enzymolysis solution of 1% (w / v) helicase and 2% (w / v) cellulase, enzymolyze the mycelia, and filter the e...

Embodiment 3

[0091] Example 3 Toxicity Test of Recombinant Corynespora fumigatus If01 GM

[0092] Under indoor conditions, isolated hibiscus leaves with 2nd instar B. tabaci nymphs (not less than 30 nymphs per leaf) were separated at 2.0×10 by dipping method. 7 conidia / mL, 1.0×10 7 conidia / mL, 5.0×10 6 conidia / mL, 2.5×10 6 conidia / mL, 1.25×10 6 conidia / mL conidia suspension of recombinant Corynesporium fumigatus If01 GM conidia suspension was soaked for 10 s, dried and placed in a Petri dish (wet filter paper at the bottom, wet cotton balls wrapped around the petiole base) , and then put the petri dish into light at 14:10 (L:D) and culture at a temperature of 25±1°C, and the experiment was repeated 3 times.

[0093] Observe and record the death of nymphs every 2 days, and calculate the mortality and half-lethal concentration (LC) 50 ) and half-lethal time (LT 50 ).

[0094] The results are shown in Table 1, Table 2 and Table 3. The results showed that the spore concentration w...

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Abstract

The invention discloses a genetically recombined Corynespora fumigatus If01 GM ( Isariafumosorosea If01 GM). The strain uses the B-type whitefly TOLL-like receptor gene 7 (TLR7) as the target gene, uses the pSilent‑1 plasmid to construct a forward and reverse vector containing the TLR7 gene fragment, and transforms it through polyethylene glycol (PEG4000)-mediated transformation. The protoplasts of the wild Corynespora fumigatus strain were finally obtained through positive screening to obtain the recombinant strain. The gene recombinant strain can silence the transcription of the TLR7 gene of type B whitefly, the pathogenicity to the 2nd instar whitefly nymph is increased by about 50% compared with the original strain, and the insecticidal speed is 1-2 days faster. The recombinant strain has been preserved in the China Center for Type Culture Collection with the preservation date of May 13, 2014, and the preservation number CCTCC NO: M 2014199.

Description

technical field [0001] The invention belongs to the technical field of biological pesticides. More specifically, it relates to a gene recombinant Corynespora fumigatus If01 GM and its application. Background technique [0002] Bemisia tabaci ( Bemisia tabaci Gennadius) is a worldwide important agricultural and forestry pest with a wide range of hosts. It is estimated that there are more than 200 species of host plants, such as Cucurbitaceae, Solanaceae, Cruciferous vegetables, cotton, tobacco and other important economic crops. The main target of lice. In addition, whitefly can transmit more than 100 kinds of viruses, such as tomato yellow leaf curl virus and other viruses of Geminiviridae, which have a devastating impact on crop production. At present, Bemisia tabaci has developed a high level of resistance to most of the commonly used pesticides, and the serious problem of pesticide pollution has been widely criticized. Biological control has become an important means...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80A01N63/04A01P7/04C12R1/645
Inventor 胡琼波李霖金丰良翁群芳
Owner SOUTH CHINA AGRI UNIV
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