阅读说明：本技术 一种新型控蚜剂、RNAi与球孢白僵菌联合应用控蚜的方法 (Novel aphid control agent, RNAi (ribonucleic acid interference) and beauveria bassiana combined application aphid control method ) 是由 王进军 牛金志 张君 尚峰 叶超 张永军 于 2020-05-28 设计创作，主要内容包括：本发明涉及害虫防治和基因工程技术领域,公开了一种新型控蚜剂：包含蚜虫的dsRNA与球孢白僵菌孢子；还公开了一种RNAi与球孢白僵菌联合应用控蚜的方法,是采用所述的新型控蚜剂处理植株防治蚜虫。本发明将生防真菌和RNAi技术联合应用开发出一种新型的控蚜剂,该控蚜剂有显著的增效机制,能克服单独使用真菌或RNAi技术控蚜的缺陷,控蚜能力能达到80％。使用该新型控蚜剂直接在田间对农作物进行喷雾处理即可有效防治蚜虫。本发明适用范围广,只需要合成蚜虫的不同目标基因的dsRNA,就可以将相应的dsRNA与球孢白僵菌的孢子悬浮液混合一起进行喷雾处理,该方法可广泛应用于豌豆蚜、桃蚜以及褐色桔蚜等蚜虫。(The invention relates to the technical field of pest control and genetic engineering, and discloses a novel aphid control agent: dsRNA containing aphid and beauveria bassiana spore; also discloses a method for controlling aphids by combining RNAi and beauveria bassiana, which is to treat plants by adopting the novel aphid control agent to control aphids. The invention develops a novel aphid controlling agent by combining the biocontrol fungus and the RNAi technology, the aphid controlling agent has an obvious synergistic mechanism, can overcome the defect of controlling aphids by singly using the fungus or the RNAi technology, and the aphid controlling capacity can reach 80 percent. The novel aphid control agent can be directly used for spraying crops in the field to effectively control aphids. The method has wide application range, can mix the corresponding dsRNA with the spore suspension of the beauveria bassiana for spray treatment only by synthesizing the dsRNA of different target genes of the aphid, and can be widely applied to aphids such as pea aphid, peach aphid, brown orange aphid and the like.)

1. A novel aphid control agent is characterized in that: dsRNA containing aphid and beauveria bassiana spore.

2. The novel aphid control agent according to claim 1, characterized in that: the aphid control agent is a mixed solution of dsRNA solution of aphids and beauveria bassiana spore suspension.

3. The novel aphid control agent according to claim 1, characterized in that: the dsRNA of the aphid refers to dsRNA or a plurality of dsRNA combinations corresponding to one or more genes of gene4714, gene15782 and gene20844 of the aphid.

4. A novel aphid control agent according to claim 3, characterized in that: the aphids refer to pea aphids or green peach aphids or brown orange aphids.

5. The novel aphid control agent according to claim 4, characterized in that: the dsRNA is Apds15782 or Apds20844 or Apds4714+ Apds15782 or Apds15782+ Apds20844 or Apds4714+ Apds20844 or Mpds4714+ Mpds15782 or Acds4714+ Acds 15782.

6. A method for controlling aphids by combining RNAi and beauveria bassiana is characterized in that: the novel aphid control agent of any one of claims 1 to 5 is used for treating plants to control aphids.

7. The method of claim 6, wherein: the novel aphid control agent of any one of claims 1 to 5 is used for carrying out spray treatment on plants to control aphids.

Technical Field

The invention relates to the technical field of pest control and genetic engineering, in particular to a novel aphid control agent, an RNAi (ribonucleic acid interference) and beauveria bassiana combined application aphid control method.

Background

The damage of aphids to plants includes both direct feeding and virus transmission. At present, the harm and drug resistance of aphids are increasingly serious in production, and the development of novel aphid control strategies and technologies is urgently needed. Beauveria bassiana Bassiaana is a biological control microorganism with wide application, has the characteristics of wide host range, strong control toxicity and the like, and has great application potential in pest control. Among them, Beauveria bassiana is most widely used in corn borers and pine caterpillars.

RNA interference (RNAi), an important gene silencing means widely used in recent years, refers to a highly conserved phenomenon during evolution, induced by double-stranded RNA (dsRNA), that specifically degrades homologous RNA with high efficiency, and specifically degrades mRNA of a corresponding sequence through the mediation of dsRNA. Since the expression of a specific gene can be specifically inhibited using the RNAi technology, the technology has been widely used in the fields of exploring gene functions and gene therapy. The RNAi technology has the potential of being developed into a new generation of pesticide due to the advantages of strong specificity, no pollution, multiple gene targets and the like.

At present, beauveria bassiana or RNAi technology is generally adopted to control pests independently, and the problem of slow effect of fungus exists when beauveria bassiana is used independently. The effect of one control method used alone is poor, and no report of combining the two methods for controlling aphids is found.

Disclosure of Invention

The invention aims to solve the problems and provides a novel aphid control agent, a method for controlling aphids by combining RNAi and beauveria bassiana.

In order to achieve the purpose, the invention adopts the technical scheme that:

a novel aphid control agent comprises dsRNA of aphid and beauveria bassiana spore.

The aphid control agent is a mixed solution of dsRNA solution of aphids and beauveria bassiana spore suspension.

Preferably, the dsRNA of the aphid refers to dsRNA or a plurality of dsRNA combinations corresponding to one or more genes of gene4714, gene15782 and gene20844 of the aphid.

Preferably, the aphid is a pea aphid or a green peach aphid or a brown orange aphid.

Further preferably, the dsRNA is Apds15782 or Apds20844 or Apds4714+ Apds15782 or Apds15782+ Apds20844 or Apds4714+ Apds20844 or Mpds4714+ Mpds15782 or Acds4714+ Acds 15782.

A method for controlling aphids by combining RNAi and beauveria bassiana is to treat plants to control aphids by adopting any one of the novel aphid control agents.

Preferably, the novel aphid control agent is used for carrying out spray treatment on plants to control aphids.

The invention has the beneficial effects that: the invention develops a novel aphid controlling agent by combining the biocontrol fungus and the RNAi technology, the aphid controlling agent has an obvious synergistic mechanism, can overcome the defect of controlling aphids by singly using the fungus or the RNAi technology, and the aphid controlling capacity can reach 80 percent. The novel aphid control agent can be directly used for spraying crops in the field to effectively control aphids. The method has wide application range, can mix the corresponding dsRNA with the spore suspension of the beauveria bassiana for spray treatment only by synthesizing the dsRNA of different target genes of the aphid, and can be widely applied to aphids such as pea aphid, peach aphid, brown orange aphid and the like.

Drawings

FIG. 1 is a survival rate curve of the pea aphids in the combined aphid control experiment of the pea aphids by single gene interference of Apgene4714, Apgene20844 and Apgene15782 and Beauveria bassiana.

FIG. 2 is a pea aphid survival rate curve of a pea aphid multi-gene interference and beauveria bassiana combined aphid control experiment.

FIG. 3 shows the result of experiments for verifying beauveria bassiana infection by preserving moisture and culturing of a dead body of a pea aphid.

FIG. 4 is a survival rate curve of the pea aphids in the experiment of combining single gene interference of the pea aphids Apgene14740, Apgene14741, Apgene16968 and Apgene15105 with beauveria bassiana.

FIG. 5 is a survival rate curve of myzus persicae in a combined aphid control experiment of myzus persicae double-gene interference and beauveria bassiana.

FIG. 6 is a survival rate curve of brown orange aphid in the experiment of combining interference of brown orange aphid double genes and beauveria bassiana.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting.

The experimental procedures in the following examples are conventional unless otherwise specified.

The main reagent sources are as follows:

2 XTaq Master Mix (near shore protein science and technology limited, China)

reagent (Invitrogen corporation, USA), gel recovery and purification kit (Takara corporation, Japan)

TranscriptAId T7 High Yield transformation Kit (Thermo fisher scientific, USA), comprising:

5×TranscrptAid Reaction Bμffer、TranscriptAid EnzymeMix。

PrimeScript^TMRT Reagent Kit (Takara, Japan)