CN107858405B

CN107858405B - A method for determining the effect of exogenous dsRNA on lady beetle toxicity

Info

Publication number: CN107858405B
Application number: CN201710949193.9A
Authority: CN
Inventors: 潘慧鹏; 杨春晓; 邱宝利; 吴建辉; 桑文; 王兴民; 金丰良
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2017-10-12
Filing date: 2017-10-12
Publication date: 2021-09-24
Anticipated expiration: 2037-10-12
Also published as: CN107858405A

Abstract

The invention discloses a method for determining the toxic influence of exogenous dsRNA on ladybug. Specifically, exogenous dsRNA is uniformly mixed into a sucrose solution and then directly fed to ladybug for 2-3 days, and then the ladybug is fed by pea aphids; then detecting and analyzing the expression quantity change of the target gene in the ladybug, and observing the biological change of the ladybug to evaluate the toxicity of the exogenous dsRNA to the ladybug; the ladybug comprises harmonia axyridis, coccinella septempunctata or coccinella dodecasaceus. The method can effectively determine the direct toxic influence of the exogenous dsRNA on the harmonia axyridis, the coccinella septempunctata or the coccinella dodecasaceus and the like, is simple and feasible, has good effectiveness and sensitivity, and has important significance and application prospect on the function research of related genes and the environmental risk evaluation of related RNAi transgenic crops.

Description

Method for determining toxic influence of exogenous dsRNA on ladybug

Technical Field

The invention belongs to the technical field of insect molecular biology research. More particularly, the invention relates to a method for determining the toxicity influence of exogenous double-stranded RNA (dsRNA) on the ladybug (harmonia axyridis, seven-star ladybug or twelve-star ladybug), which comprises a method for determining the toxicity influence of the dsRNA expressed by exogenously synthesized dsRNA or RNAi transgenic crops on the ladybug (harmonia axyridis, seven-star ladybug or twelve-star ladybug).

Background

The agricultural transgenic technology is taken as a core technology of the second green revolution, and the pattern of modern agriculture is rapidly remodeled. In 1996, insect-resistant crops transformed with Bacillus thuringiensis (Bt) insecticidal protein gene began to be commercially planted, and up to 28 countries with Bt plants planted in 2015, the annual planting area was nearly 27 million acres (http:// www.isaaa.org /). The countries where the transgenic crops were grown were ranked by the area of the transgenic crop grown, and china was listed after usa, brazil, argentina, india and canada, ranking 6 th. In 2016, the central document No. 1 emphasizes that 'the development and supervision of agricultural transgenic technology are enhanced and the agricultural transgenic technology is carefully popularized on the basis of ensuring safety'. The 'thirteen five' national science and technology innovation plan published in 2016, 8 months and 8 days is clear, a series of national major science and technology specialities including transgenes can be implemented quickly, the core key technology needs to be continuously overcome in the 'thirteen five' period, and the industrialization process of novel transgenic insect-resistant cotton, insect-resistant corn and other major products is promoted. Therefore, the planting area of the transgenic crops in China can be further increased in the future.

RNA interference (RNAi) refers to the phenomenon of highly conserved, double-stranded RNA (dsRNA) induced highly specific degradation of homologous mRNA by species during evolution (Fire et al 1998). In the last 20 years, the technology and products of RNAi have rapidly developed and received unprecedented attention in the scientific community, the industrial community, and the public community. In agriculture, RNAi is considered as a new method of pest control with potential applications (Gordon and Waterhouse, 2007; Huvenne and Smighe, 2010). Insect-resistant transgenic crops based on RNAi technology have been demonstrated to be effective methods for controlling lepidopteran, coleopteran, and hemipteran pests, such as corn rootworm Diabrotica virgifera virgifera (Baum et al 2007); potato beetle Leptinotarsadecemlineata (Zhang et al.2015); helicoverpa armigera (Mao et al 2007); asiatic corn borer Ostrinia furnacalis (Wu et al.2016); bemisia tabaci (Thakur et al 2014; Shukla et al 2016); myzuspicate (Guo et al.2014); schizaphis graminum (Zhang et al 2015); sitobion avenae (Xu et al 2014); nilaparvata lugens (Zha et al.2011) and the like. RNAi transgenic crops with good insect resistance characters have been developed successfully, indicating that the commercial application of the RNAi transgenic crops becomes possible. Although Bt crops are still effective at controlling lepidopteran and coleopteran pests (Wu et al.2008; Tabashnik et al.2008), Bt crops are not effective against piercing-sucking mouthparts pests such as aphids, leafhoppers, plant hoppers and whiteflies, which have been continuously outbreak to be harmful in recent years. Therefore, the development of RNAi transgenic crops will provide a new approach to control including such pests (Price and gateway, 2008), and also provide an important rotation for Bt crops controlling lepidopteran and coleopteran pests.

Ecological risks associated with RNAi transgenic crops are a concern prior to commercial application. Among them, similar to the content of risk assessment of Bt crops (Duan et al 2002), the potential influence of RNAi insect-resistant crops on the natural enemy insects of the ladybug also becomes an important content of environmental safety assessment (Lundgren and Duan, 2013; Roberts et al 2015). Harmonia axyridis, Coccinella septempunctata and Coccinella dodecaptera are predatory natural enemy insects quite common to various agricultural ecosystems, and adults and larvae of the insects can prey on eggs, small larvae, phyllocladium larvae and the like of various aphids, scale insects, psyllids and moths. It also has the habit of feeding pollen during the flowering period of the crop. Thus, in the crop E.ladybug may be indirectly exposed to RNAi transgenic crop expressed dsRNA through prey; direct exposure to crop-expressed dsRNA by feeding pollen. Therefore, the natural enemy insects as indicator organisms are widely used for the environmental safety research of transgenic insect-resistant plants. However, there is currently no technical system available for assessing the direct toxic effect of dsRNA on ladybug.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects of the traditional evaluation technology and system for the toxicity of exogenous dsRNA to the ladybirds such as harmonia axyridis, seven-star ladybug or twelve-star ladybug, and provides a method for simply and sensitively detecting the direct toxicity influence of the exogenous dsRNA to the ladybug.

The invention aims to provide a method for determining the toxic effect of exogenous dsRNA on ladybug.

Another object of the invention is to provide application of the method in evaluating the toxic effect of exogenous dsRNA on ladybug.

The above purpose of the invention is realized by the following technical scheme:

a method for determining the influence of exogenous dsRNA on the toxicity of ladybug comprises the steps of uniformly mixing the exogenous dsRNA into a sucrose solution, directly feeding the ladybug, detecting and analyzing the expression change of a target gene after the ladybug eats, and observing the biological change of the ladybug to evaluate the toxicity of the exogenous dsRNA on the ladybug.

Preferably, the method for determining the influence of exogenous dsRNA on the toxicity of the ladybug comprises the steps of uniformly mixing the exogenous synthetic dsRNA into a sucrose solution, feeding the ladybug larvae which are just hatched for 1 day for 2-3 days (preferably 2 days), feeding the ladybug larvae with the pea aphids, detecting and analyzing the expression quantity change of target genes at different time after the ladybug is eaten, and observing the biological change of the ladybug to evaluate the toxicity of the exogenous dsRNA on the ladybug.

More preferably, the method for determining the toxic effect of the exogenous dsRNA on the ladybug is to feed the ladybug larvae which are just hatched for 1 day for 2 days after the exogenous dsRNA is uniformly mixed into a sucrose solution, and then feed the ladybug larvae with the pea aphids, wherein the treatment group is formed; meanwhile, a sucrose solution mixed with potassium arsenate is used as a positive control, and a sucrose solution mixed with beta-glucuronidase gene dsRNA derived from plants is used as a negative control; the potential toxicity of dsRNA can be evaluated by comparing the change of the expression quantity of the target gene in the ladybug of the treatment group and the control group and observing and counting the biological change of the ladybug.

Preferably, the ladybug is harmonia axyridis, coccinella septempunctata and/or coccinella dodecasaceus.

In addition, the final concentration of the sucrose solution is preferably 10 to 15%.

More preferably, the final concentration of the sucrose solution is 10%.

As mentioned above, the feeding system for feeding the ladybug (harmonia axyridis, seven-star ladybug or twelve-star ladybug) is as follows: the ladybug larvae which had just hatched for 1 day were provided with a 10% sucrose solution mixed with dsRNA for 2 days, after which the ladybug was fed with the pea aphids. The specific feeding method comprises the following steps: placing a sucrose solution mixed with dsRNA in a container, providing the solution for 2 days; and then shearing the broad bean seedlings covered with the pea aphids into sections, and putting the sections into a culture dish for sufficient supply.

As a specific embodiment, the specific feeding method comprises the following steps: mu.l of the sucrose solution mixed with dsRNA was placed in a 5cm diameter and 1.3cm high petri dish with a pipette gun to provide 4. mu.l of sucrose solution for 2 days; and then shearing the broad bean seedlings covered with the pea aphids into sections, and putting the sections into a culture dish for sufficient supply.

As a reference case, the sequences of dsHA, dsCS or dsCM, respectively, were not synthesized in vitro with the exogenous dsRNA as shown in Table 1.

In addition, the concentration of the potassium arsenate in the positive control (the sucrose solution mixed with the potassium arsenate) is preferably 50-100 ng/mu l.

More preferably, the concentration of potassium arsenate in the positive control (sucrose solution mixed with potassium arsenate) is 100 ng/. mu.l.

Preferably, the above measurements are performed at 23 ℃ in an incubator with 50% RH and 14L:10D light. 30 1 st larvae per treatment test were used for gene expression level testing, and the test was repeated 3 times.

Preferably, the method for measuring the change in the expression level of the target gene comprises: collecting ladybug (harmonia axyridis, seven-star ladybug or twelve-star ladybug)

samples

3, 5, 7 and 9 days after feeding, respectively, collecting 3 biological repeated samples at each time point, and analyzing target gene expression amount change by using RT-qPCR. Wherein, RT-qPCR primers of the target gene and the reference gene are shown in Table 2.

Preferably, the biological parameters are survival and developmental duration. Specifically, each observation was performed 1 time at 9 am and 9 pm every day. The tests were carried out at 23 ℃ in an incubator with 50% RH, light 14L: 10D.

In addition, the application of the method in the aspect of evaluating the toxicity influence of the exogenous dsRNA on the ladybug (harmonia axyridis, seven-star ladybug or twelve-star ladybug) is also within the protection scope of the invention.

The invention has the following beneficial effects:

the invention provides a method for determining the toxic influence of exogenous dsRNA on harmonia axyridis, seven-star ladybug or twelve-star ladybug, and the like, wherein the exogenous dsRNA is uniformly mixed into a sucrose solution and then directly fed to ladybug larvae for 2 days, and then the exogenous dsRNA is fed by pea aphids; and then detecting and analyzing the expression quantity change of the target gene after the ladybug takes the feed, and observing the biological change of the ladybug to evaluate the toxicity of the exogenous dsRNA to the ladybug. The method can effectively determine the direct toxic influence of the exogenous dsRNA on the ladybug, is simple and feasible, has good effectiveness and sensitivity, and has important significance and application prospect in the research of related gene functions and the environmental risk evaluation of related RNAi transgenic crops.

Drawings

FIG. 1 is the expression profile of the v-ATPaseA gene in harmonia axyridis at 3, 5, 7, 9 days after dsRNA feeding of example 1; RP49 and GAPDH were used as reference genes (Table 1), and the expression level of v-ATPaseA gene in untreated (day 1 incubation) harmonia axyridis was set to 1; the values in the graph are mean + standard error, and different letters indicate that there is a significant difference in expression level between the treated and control groups (P < 0.05).

FIG. 2 is the survival rate of harmonia axyridis in the treatment group and the control group after dsRNA feeding of example 1; the values in the graph are mean + standard error, and different letters indicate that there is a significant difference in expression level between the treated and control groups (P < 0.05).

FIG. 3 is the effect of dsRNA feeding of example 1 on developmental duration of harmonia axyridis; different letters indicate that there was a significant difference in expression level between the treated and control groups (P < 0.05).

FIG. 4 is the expression profile of the v-ATPaseA gene in E.septempunctata at 3, 5, 7, 9 days after dsRNA feeding of example 2; EF1A and Actin are used as internal reference genes; the expression level of the v-ATPaseA gene in untreated (day 1 incubation) coccinella septempunctata was set to 1; the values in the graph are mean + standard error; different letters indicate that there was a significant difference in expression level between the treated and control groups (P < 0.05).

FIG. 5 is the survival rate of E.septemfasciatus in the treatment group and the control group after dsRNA feeding of example 2; the values in the graph are mean + standard error, and different letters indicate that there is a significant difference in expression level between the treated and control groups (P < 0.05).

FIG. 6 is the effect of dsRNA feeding of example 2 on development epochs of 1 st and 2 nd instar ladybug; different letters indicate that there was a significant difference in expression level between the treated and control groups (P < 0.05).

FIG. 7 is the expression profile of the v-ATPase A gene in E.dodecascens at 3, 5, 7, 9 days after dsRNA feeding in example 3; EF1A and Actin are used as internal reference genes; the expression level of the v-ATPaseA gene in untreated (day 1 incubation) E.dodecascens was set to 1; the values in the graph are mean + standard error; different letters indicate that there was a significant difference in expression level between the treated and control groups (P < 0.05).

FIG. 8 is the survival rate of E.dodecarinus in the treatment group and the control group after dsRNA feeding of example 3; the values in the graph are mean + standard error, and different letters indicate that there is a significant difference in expression level between the treated and control groups (P < 0.05).

FIG. 9 is the effect of dsRNA feeding of example 3 on developmental history of 1 and 2 instar ladybug; different letters indicate that there was a significant difference in expression level between the treated and control groups (P < 0.05).

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1

In the embodiment, the harmonia axyridis is taken as a research object, and the v-ATPaseA gene is taken as an example, so that the method disclosed by the invention is used for determining the direct toxic effect of exogenous dsRNA on harmonia axyridis.

A simple and sensitive determination method for detecting direct toxicity influence of exogenous dsRNA on ladybug is characterized in that dsHA or dsCS or dsCM synthesized in vitro is uniformly mixed into a sucrose solution to feed the ladybug for 2 days, then sufficient pea aphids are provided, the sucrose solution mixed with potassium arsenate is used as a positive control, the sucrose solution mixed with dsGUS is used as a negative control, and potential toxicity of dsRNA is evaluated by comparing changes of v-ATPaseA gene expression quantity in ladybug of a treatment group and a control group and changes of important biological parameters such as ladybug survival rate, development period and the like.

Example 2

In this example, coccinella septempunctata was used as a subject of study, and the study was conducted in the same manner as in example 1.

Example 3

In this example, ladybug twelve pointed as a subject, and the rest of the study was conducted in the same manner as in example 1.

Specifically, the specific examination and measurement methods of examples 1 to 3 are as follows:

1. design of exogenous dsRNA (sequences as shown in table 1):

(1) dsrnas (dsHA, dsCS, dsCM) consisting of the nucleotides shown in table 1 and the nucleotides shown in the reverse complement thereof;

(2) dsrna consisting of the nucleotides shown in table 1 and the nucleotides shown in the reverse complement thereof (dsgus);

TABLE 1dsRNA sequences

Primer name	Sequence 5 '-3' (wherein T7 ═ TAATACGACTCACTATAGG)	Serial number
			dsHA-F	TAATACGACTCACTATAGGGAGATCTCTTTTCCCATGTGTCCA	SEQIDNO.1
dsHA-R	TAATACGACTCACTATAGGGAGAGCATCTCGGCCAGAC	SEQIDNO.2
			dsCS-F	TAATACGACTCACTATAGGGAGATCCCTTTTCCCATGTGT	SEQIDNO.3
dsCS-R	TAATACGACTCACTATAGGGAGAGCATCTCGGCCAGAC	SEQIDNO.4
			dsCM-F	TAATACGACTCACTATAGGGAGATCTCTTTTCCCATGT	SEQIDNO.5
dsCM-R	TAATACGACTCACTATAGGGAGAGCATCTCGGCCAGAC	SEQIDNO.6
			dsGUS-F	TAATACGACTCACTATAGGGAGAGGGCGAACAGTTCCTGATTA	SEQIDNO.7
dsGUS-R	TAATACGACTCACTATAGGGAGAGGCACAGCACATCAAAGAGA	SEQIDNO.8

2. Construction of toxicity assay System

Establishing and measuring the direct toxicity of the exogenous dsRNA to the tested insects, firstly, a proper method needs to be found for feeding the dsRNA to the tested insects. Based on the habit of ladybug sucking sucrose solution, we developed a ladybug feed based on sucrose solution. In the feeding system, 2-day sucrose solution mixed with dsRNA is provided for ladybug which is just hatched for 1 day, and then the ladybug is fed by the pea aphid. The pea aphid providing method is used for providing sufficient supply for cutting broad bean seedlings covered with pea aphids into sections.

Based on the established ladybug feeding system, a determination system of the influence of the exogenous dsRNA on the ladybug is established, and the determination system comprises the following steps: 1) negative control treatment: sucrose solution mixed with dsGUS was used as a negative control, and the total amount of dsRNA was 16. mu.g; 2) mixing with sucrose solution of dsHA or dsCS or dsCM, wherein the total amount of dsRNA is 16 μ g; 3) a sucrose solution mixed with potassium arsenate was used as a positive control, and the concentration of potassium arsenate was 100 ng/. mu.l. Through the established determination system, potassium arsenate which is known to be toxic to the ladybug is uniformly mixed into the sucrose solution, and the ladybug is fed according to the method, so that the influence of the potassium arsenate on the biology of the ladybug is researched; the method is used for verifying the effectiveness and the sensitivity of the established ladybug toxicity determination method, namely whether a test system can show the toxicity of a tested compound.

For each ladybug, approximately 30 1 st larvae were tested for gene expression level testing for each treatment, and the test was repeated 3 times. 3, 5, 7 and 9 days after feeding dsRNA, respectively taking insects, taking 5 as a sample, collecting 3 biological repeated samples at each time point, and using RT-qPCR to research the change of the v-ATPaseA gene expression level (the RT-qPCR primers of the v-ATPaseA gene and the internal reference gene are shown in Table 2). Test at 23 ℃, 50% RH, illumination 14L:10D in an incubator. After obtaining the data, analyzing and comparing the difference of the expression quantity of the different v-ATPaseA genes of the ladybug of the treatment group and the negative control group by adopting a proper biometric method.

TABLE 2RT-qPCR primers

For each ladybug, approximately 30 1 st larvae were tested per treatment for observation of the biological property change test, which was repeated 3 times. Biological parameter indicators include survival and developmental history. Each observation was 1 time at 9 am and 9 pm daily. The assay was carried out at 23 ℃, 50% RH, 14L light: 10D in an incubator. After the data are obtained, the difference of different life parameters of the ladybug of the comparative treatment group and the negative control group is analyzed by adopting a proper biometric method.

3. As a result, all of the harmonia axyridis individuals died within 2 days of taking the positive control containing the potassium arsenate sucrose solution. The detection system for detecting the toxicity influence of the ladybug has high sensitivity and can be effectively used for detecting the potential negative influence of the exogenous dsRNA on the ladybug.

Meanwhile, the results of the treatment group and the negative control (dsGUS) which take the sucrose solution containing the exogenous dsRNA (dsHA or dsCS or dsCM) are respectively shown in figures 1-9, and the results show that: the method of feeding dsRNA in vitro can inhibit the expression of v-ATPaseA gene in the ladybug, so that the ladybug generates a lethal effect. The effectiveness and sensitivity of the method of the invention is also demonstrated.

4. The following conclusions can be drawn from the experimental results:

(1) in the feeding system established by the invention, the sucrose solution can be used as a carrier to transfer exogenous dsRNA to ladybug (harmonia axyridis, seven-star ladybug or twelve-star ladybug);

(2) the effectiveness and the sensitivity of the established method for determining the toxicity of the harmonia axyridis (harmonia axyridis, seven-star or twelve-star) are verified by using potassium arsenate as a positive control;

(3) the invention provides a simple and feasible test method capable of effectively determining the toxic influence of exogenous dsRNA on ladybug (harmonia axyridis, seven-star ladybug or twelve-star ladybug).

Sequence listing

<110> southern China university of agriculture

<120> method for determining toxicity influence of exogenous dsRNA on ladybug

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 43

<212> DNA

<213> dsHA-F(dsHA-F)

<400> 1

taatacgact cactataggg agatctcttt tcccatgtgt cca 43

<210> 2

<211> 38

<212> DNA

<213> dsHA-R(dsHA-R)

<400> 2

taatacgact cactataggg agagcatctc ggccagac 38

<210> 3

<211> 40

<212> DNA

<213> dsCS-F(dsCS-F)

<400> 3

taatacgact cactataggg agatcccttt tcccatgtgt 40

<210> 4

<211> 38

<212> DNA

<213> dsCS-R(dsCS-R)

<400> 4

taatacgact cactataggg agagcatctc ggccagac 38

<210> 5

<211> 38

<212> DNA

<213> dsCM-F(dsCM-F)

<400> 5

taatacgact cactataggg agatctcttt tcccatgt 38

<210> 6

<211> 38

<212> DNA

<213> dsCM-R(dsCM-R)

<400> 6

taatacgact cactataggg agagcatctc ggccagac 38

<210> 7

<211> 43

<212> DNA

<213> dsGUS-F(dsGUS-F)

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taatacgact cactataggg agagggcgaa cagttcctga tta 43

<210> 8

<211> 43

<212> DNA

<213> dsGUS-R(dsGUS-R)

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taatacgact cactataggg agaggcacag cacatcaaag aga 43

<210> 9

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<212> DNA

<213> HA v-ATPase A RT-qPCR F(HA v-ATPase A RT-qPCR F)

<400> 9

gagttgggtc ctggtattat gg 22

<210> 10

<211> 22

<212> DNA

<213> HA v-ATPase A RT-qPCR R(HA v-ATPase A RT-qPCR R)

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agttctggac aaacaaggta ca 22

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<211> 20

<212> DNA

<213> HA RP49 RT-qPCR F(HA RP49 RT-qPCR F)

<400> 11

gccgtttcaa gggacagtat 20

<210> 12

<211> 22

<212> DNA

<213> HA RP49 RT-qPCR R(HA RP49 RT-qPCR R)

<400> 12

tgaatccagt aggaagcatg tg 22

<210> 13

<211> 20

<212> DNA

<213> HA GAPDH RT-qPCR F(HA GAPDH RT-qPCR F)

<400> 13

tgactacagt tcacgcaacc 20

<210> 14

<211> 23

<212> DNA

<213> HA GAPDH RT-qPCR R(HA GAPDH RT-qPCR R)

<400> 14

gatgactttg gttacagcct ttg 23

<210> 15

<211> 22

<212> DNA

<213> CS v-ATPase A RT-qPCR F(CS v-ATPase A RT-qPCR F)

<400> 15

ccgtcatctc ccaatctctt tc 22

<210> 16

<211> 22

<212> DNA

<213> CS v-ATPase A RT-qPCR R(CS v-ATPase A RT-qPCR R)

<400> 16

cggtttgacc ttcgatctct ac 22

<210> 17

<211> 21

<212> DNA

<213> CS EF1A RT-qPCR F(CS EF1A RT-qPCR F)

<400> 17

cctgaagtgg aagacgaaga g 21

<210> 18

<211> 22

<212> DNA

<213> CS EF1ART-qPCR R(CS EF1ART-qPCR R)

<400> 18

agaaggaaag gctgatggta aa 22

<210> 19

<211> 22

<212> DNA

<213> CS Actin RT-qPCR F(CS Actin RT-qPCR F)

<400> 19

gcgtaacctt cgtagattgg ta 22

<210> 20

<211> 22

<212> DNA

<213> CS Actin RT-qPCR R(CS Actin RT-qPCR R)

<400> 20

caagctgtac tctccctgta tg 22

<210> 21

<211> 21

<212> DNA

<213> CM v-ATPase A RT-qPCR F(CM v-ATPase A RT-qPCR F)

<400> 21

ttgactggag gcgacattta c 21

<210> 22

<211> 22

<212> DNA

<213> CM v-ATPase A RT-qPCR R(CM v-ATPase A RT-qPCR R)

<400> 22

cttccaggtt cggctatgta tg 22

<210> 23

<211> 21

<212> DNA

<213> CM EF1A RT-qPCR F(CM EF1A RT-qPCR F)

<400> 23

tgaattcgaa gccggtatct c 21

<210> 24

<211> 20

<212> DNA

<213> CM EF1ART-qPCR R(CM EF1ART-qPCR R)

<400> 24

cgccgacaat gagttgtttc 20

<210> 25

<211> 21

<212> DNA

<213> CM Actin RT-qPCR F(CM Actin RT-qPCR F)

<400> 25

cttcccgacg gtcaagttat c 21

<210> 26

<211> 21

<212> DNA

<213> CM Actin RT-qPCR R(CM Actin RT-qPCR R)

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gcaggattcc atacccaaga a 21

Claims

1. A method for determining the toxic influence of exogenous dsRNA on ladybug is characterized in that the ladybug is harmonia axyridis, seven-star ladybug and/or twelve-star ladybug; uniformly mixing exogenous dsRNA into a sucrose solution, feeding ladybug larvae which are just hatched for 1 day for 2 days, and feeding the ladybug larvae with pea aphids, wherein the treatment group is formed; meanwhile, a sucrose solution mixed with potassium arsenate is used as a positive control, and a sucrose solution mixed with beta-glucuronidase gene dsRNA derived from plants is used as a negative control; respectively taking ladybug samples 3, 5, 7 and 9 days after dsRNA feeding is started, and analyzing the change of the expression quantity of the target gene v-ATPase A by using RT-qPCR; the potential toxicity of dsRNA can be evaluated by comparing the change of the expression level of the target gene in the ladybug of the treatment group and the control group and observing and counting the survival rate and the development period of the ladybug.

2. The method according to claim 1, wherein the final concentration of the sucrose solution is 10-15%.

3. The method of claim 1, wherein the concentration of potassium arsenate in the positive control is 50-100 ng/μ l.

4. The method according to claim 1, wherein the RH content at 23 ℃ and 50% RH, light level 14L:10D in an incubator.

5. Use of the method of any one of claims 1 to 4 for evaluating the toxic effect of exogenous dsRNA on ladybug.