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Transgenic biocontrol fungus for interfering expression of diaphorina citri kuwayama vitellogenin gene as well as preparation method and application of transgenic biocontrol fungus

A vitellogenin and citrus psyllid technology, which is applied in the field of genetic engineering and pest control, can solve the problems of low Intron matching degree, not many, and poor dsRNA release effect, and achieve the effect of improving virulence and shortening the soaking time

Active Publication Date: 2020-12-08
SOUTHWEST UNIVERSITY
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Problems solved by technology

However, there are also transferred genes that do not perform the RNAi function. Zhang Fengzhen transferred the brown planthopper gene into rice, but did not find any RNAi effect on brown planthopper (Zhang Fengzhen, 2013)
In the experiment, there are many reasons why RANi doe

Method used

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  • Transgenic biocontrol fungus for interfering expression of diaphorina citri kuwayama vitellogenin gene as well as preparation method and application of transgenic biocontrol fungus
  • Transgenic biocontrol fungus for interfering expression of diaphorina citri kuwayama vitellogenin gene as well as preparation method and application of transgenic biocontrol fungus
  • Transgenic biocontrol fungus for interfering expression of diaphorina citri kuwayama vitellogenin gene as well as preparation method and application of transgenic biocontrol fungus

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0042] Example 1 Construction and transformation of filamentous fungal expression vector

[0043] In order to more intuitively observe the expression of the vector in fungi in the evaluation of transgenic strains, the one-step cloning method was selected according to the principle of homologous recombination to connect the PtrpC promoter + EGFP gene fragment to the original filamentous fungal expression vector pRCy1 in the laboratory , a filamentous fungal expression vector with the reporter gene EGFP (enhanced green fluorescent protein) was obtained and named pRCy3. The citrus psyllid transcriptome database was screened, and the conserved sequences DcVg 1 and DcVg 2 of the citrus psyllid vitellogenin gene were found. The primers were designed according to the principle of homologous recombination, and the forward fragment of the target gene was transferred into the pRCy3 vector by one-step cloning method. Between the promoter and the intron, the forward detection primers were...

Example Embodiment

[0083] Example 2 Agrobacterium-mediated transformation of the ihpRNA expression cassette into Lecanococcus attenuata

[0084] The successfully constructed ihpRNA expression cassette was transformed by the freeze-thaw method, and the positive clone of the Agrobacterium strain AGL1 was verified by medium screening, primer amplification and enzyme digestion, and the Agrobacterium strain containing the ihpRNA expression cassette was obtained. Subsequently, further using the acetosyringone-induced culture method, the Agrobacterium was induced for 6 hours and then co-cultured with the spore suspension of Lecanococcus attenuata for about 60 hours, then transferred to the hygromycin selective medium for continued cultivation, and separated and purified , PCR detection, it was found that the ihpRNA expression cassette had been successfully integrated into the genome of Lecanococcus attenuata. The sequencing results showed that the strain of Lecanococcus attenuata contained the ihpRNA e...

Example Embodiment

[0112] The actual effect of embodiment 3 transgenic strains

[0113]On the basis of the completion of the genetic modification of Leicoccus attenuosa, its biological characteristics and modification effects were evaluated. The results showed that the growth rate and unit sporulation of the transgenic strains La::DcVg 1a and La::DcVg 1b were not significantly different from those of the wild-type strain, indicating that the transfer of the citrus psyllid vitellogenin gene had no significant effect on the attenuated wax. There was no significant effect on the biological characteristics of the scale bacteria. After continuous culture on PDA medium without antibiotics for 10 generations, hygromycin detection primers were used for amplification, and positive bands were amplified at 1030bp, indicating that the Agrobacterium-mediated transformation method has good stability.

[0114] The citrus psyllid female adults were immersed for 15s with the spore suspension of transgenic leech...

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Abstract

The invention discloses a diaphorina citri kuwayama vitellogenin gene RNAi vector as well as a preparation method and application thereof. The RNAi vector comprises a skeleton vector and a hairpin structure, wherein the hairpin structure is an ihpRNA expression cassette, the structure of the ihpRNA expression cassette is a PtrpC promoter + a forward target gene segment + intron + a reverse targetgene segment + a TtrpC terminator structure, and the target gene segment is a diaphorina citri kuwayama vitellogenin gene conserved sequence DcVg1a or DcVg1b; and the skeleton vector is a filamentousfungus expression vector. The invention also discloses a transgenic biocontrol fungus as well as a preparation method and application thereof. The transgenic biocontrol fungus is obtained by converting the biocontrol fungus through the RNAi vector. Experiments prove that the obtained transgenic lecanicillium attenuatum can effectively improve the toxicity of the transgenic lecanicillium attenuatumto diaphorina citri kuwayama and shorten the dip-dyeing time of the diaphorina citri kuwayama.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and pest control, in particular to a transgenic biocontrol fungus that interferes with the expression of the citrus psyllid vitellogenin gene, and a preparation method and application thereof. Background technique [0002] Citrus Huanglongbing is a devastating citrus disease, and Diaphorina citri Kuwayama is the most important transmission vector of citrus Huanglongbing. At present, chemical control is still the main method in the prevention and control of citrus psyllids, but chemical control can easily cause problems such as pesticide residues, environmental pollution, and pest resistance. Biological control is an important supplementary way to overcome the shortcomings of chemical control. Microbial (filamentous fungi) control is an important way of biological control. It mainly uses entomopathogenic fungi to infect pests, penetrate their body walls and germinate in their bodies. A...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/12C12N15/113C12N1/15A01N63/30A01P7/04C12R1/645
CPCC07K14/43563C12N15/80C12N15/113A01N63/30C12N2310/14
Inventor 冉春杨娟生于士将丛林刘浩强李鸿筠
Owner SOUTHWEST UNIVERSITY
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