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Transgenic biocontrol fungus interfering with citrus psyllid vitellogenin gene expression and its preparation method and application

A technology of vitellogenin and biocontrol fungi, which is applied in the fields of genetic engineering and pest control, can solve the problems of low matching degree of Intron, not many, and the transferred gene does not perform RNAi function, etc., so as to improve the virulence and shorten the infection time. Effect

Active Publication Date: 2022-05-06
SOUTHWEST UNIV
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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 does not perform its function, and there are many uncertain factors. Some researchers believe that it may be caused by poor dsRNA release effect or low matching degree of Intron in RNAi vector. There are not many reports, and the reasons need more scientific research to find out

Method used

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  • Transgenic biocontrol fungus interfering with citrus psyllid vitellogenin gene expression and its preparation method and application
  • Transgenic biocontrol fungus interfering with citrus psyllid vitellogenin gene expression and its preparation method and application
  • Transgenic biocontrol fungus interfering with citrus psyllid vitellogenin gene expression and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0042] Construction and transformation of embodiment 1 filamentous fungus expression vector

[0043] In order to observe the expression of the vector in fungi more intuitively in the evaluation of transgenic strains, a 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 In , a filamentous fungal expression vector with reporter gene EGFP (Enhanced Green Fluorescent Protein) was obtained, 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, and 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 a one-step cloning method Between the promoter and the intron, use forward detection primers for ba...

Embodiment 2

[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...

Embodiment 3

[0112] The actual effect of embodiment 3 transgenic strains

[0113]On the basis of completing the genetic transformation of Lecanococcus attenuata, its biological characteristics and transformation effects were evaluated. The results found that the growth rate and unit sporulation of transgenic strains La::DcVg 1a and La::DcVg 1b were not significantly different from those of wild-type strains, which indicated that the transfer of the citrus psyllid vitellogenin gene had no significant effect on the attenuate wax There was no significant effect on the biological characteristics of scale fungus. After 10 generations of continuous culture on PDA medium without antibiotics, hygromycin detection primers were used to amplify, and positive bands could be amplified at 1030 bp, indicating that the Agrobacterium-mediated transformation method has good stability.

[0114] The citrus psyllid female adults were submerged in the spore suspension of the transgenic Lecanococcus attenuata o...

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Abstract

The invention discloses a citrus psyllid vitellogenin gene RNAi carrier and its preparation method and application. The RNAi carrier includes a backbone carrier and a hairpin structure, and the hairpin structure is an ihpRNA expression cassette. The structure is PtrpC promoter+forward target gene segment+intron+reverse target gene segment+TtrpC terminator structure, the target gene segment is the conserved sequence DcVg1a or DcVg1b of the citrus psyllid vitellogenin gene; the backbone vector is filamentous Fungal expression vector. Also disclosed is a transgenic biocontrol fungus and its preparation method and application. The transgenic biocontrol fungus is obtained by using the RNAi vector to transform the biocontrol fungus. Experiments have confirmed that the obtained transgenic Lecanococcus attenuata can effectively improve its virulence to citrus psyllids and shorten the time of infecting psyllids.

Description

technical field [0001] The invention relates to the technical fields 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 its preparation method and application. Background technique [0002] Citrus huanglongbing is a devastating disease of citrus. The citrus psyllid (Diaphorina citri Kuwayama) is the most important vector of citrus huanglongbing. Controlling citrus psyllid is a key measure to control citrus huanglongbing. At present, chemical control is still the main method in the control of citrus psyllids, but chemical control can easily cause problems such as pesticide residues, environmental pollution, and insecticide resistance. Biological control is an important supplementary way to overcome the shortcomings of chemical control. Microbial (filamentous fungus) control is an important way of biological control. It mainly uses entomopathogenic fungi to inf...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 UNIV
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