CN110935036A - Method for evaluating influence of RNAi transgenic product on Italian bees - Google Patents

Abstract

The invention discloses a method for evaluating the influence of RNAi transgenic products on Italian bees, which comprises the following steps of firstly synthesizing dsRNA: step 1: designing a primer: using SMV-P3-RNAi transgenic disease-resistant soybean B5B9013 as a test material, designing a primer according to a forward interference fragment sequence of an SMV-P3 gene in the B5B9013 soybean and a GFP gene sequence, and amplifying to obtain a DNA template for synthesizing dsRNA; step 2: reverse transcription of dsRNA: and synthesizing dsRNA by adopting a MEGAscript transcription kit. According to the invention, SMV-P3-RNAi gene resistance soybean B5B9013 is used as a test material, a primer is designed according to a forward interference fragment sequence and a GFP gene sequence of an SMV-P3 gene in the B5B9013 soybean, a DNA template for synthesizing dsRNA is obtained through amplification, a MEGAscript transcription kit is used for synthesizing dsRNA, and an influence test of the dsRNA on bees is carried out.

Description

Method for evaluating influence of RNAi transgenic product on Italian bees

Technical Field

The invention relates to the technical field of transgenic plants, in particular to a method for evaluating influence of RNAi transgenic products on Italian bees.

Background

RNAi interference (RNAi) is a eukaryotic regulatory system triggered by double-stranded RNA (dsRNA), specifically inhibiting the expression of homologous genes by using small-molecule RNA as a medium through a series of endogenous intrinsic gene expression regulation mechanisms (Hommon, 2002). RNAi interference is ubiquitous in animals, plants, fungi and insects, has the characteristics of high efficiency, specificity, systematicness, heritability and the like, and is widely applied to the research fields of gene function analysis, crop transgenic technology, molecular treatment of diseases and tumors and the like. In the field of crop transgenic research, researchers knock out or close a designated gene by utilizing high specificity interfered by RNAi, precisely change certain characters of a transformant, and can achieve the improvement purposes of improving yield, enhancing resistance or increasing nutritional value and the like.

Since the discovery of the 90 s in the 20 th century, RNAi technology has been widely applied to the field of crop biotechnology breeding as a highly efficient gene suppression (geneknockdown) technology, and has made remarkable progress in breeding for disease and insect resistance, abiotic stress resistance, quality improvement and the like, and RNAi interference with products such as corn and potato has entered the stage of commercial application (strong wood, 2018). Baum and the like design target dsRNA according to a V-ATPase A gene of American Western Corn Rootworm (WCR), and the target dsRNA can kill the WCR (Baum, 2007) through an RNA interference approach by a plurality of modes of directly feeding the WCR by dsRNA, feeding the WCR by transgenic corn field indirect insects, feeding the WCR by transgenic corn root systems and the like. The RNAi fragment of the P3 gene of the soybean mosaic virus SC-3 strain is introduced into a cultivated soybean variety by eastern Yang and the like, and the transgenic soybean plant is found to have good resistance to a plurality of soybean mosaic virus strains such as SC-3, SC7, SC15, SC18, SMV-R and the like and watermelon mosaic virus under field conditions, and the resistance character can be stably inherited (Yang, 2018).

Although RNAi technology began to be discovered and applied as early as 90 s in the past century, the scientific community has been a major debate about the safety of RNAi in transgenic crops due to the lack of thorough understanding of the mechanism of action of RNAi in interfering transgenic crops, the metabolism of exogenous RNA in mammals and the role of interfering. Such as gene level transfer, effects on non-target organisms and biodiversity, etc.

RNAi crops present a potential risk to non-target organisms that are directly or indirectly exposed to dsRNA or siRNA due to off-target binding. The substantial impact of RNAi crops on non-target organisms generally requires that the following conditions be met: expressing the dsRNA by the transgenic crops; the non-target organism directly or indirectly takes dsRNA for feeding; dsRNA is not degraded in non-target organisms; the exposure is sufficient to trigger endogenous RNAi machinery; resulting in degradation or inhibition of expression of a particular mRNA in a non-target organism; this mRNA affects the survival of non-target organisms, leading to significant changes in population numbers (Roberts, 2015). It follows that off-target binding does not necessarily significantly reduce non-target gene expression, nor does inhibition of gene expression necessarily result in significant negative effects.

Silkworm (Bombyx mori) is an important economic insect in China, silk industry has very important economic and social status in China, three areas of southwest, northeast and southwest become main production areas (Lijiaqin and the like, 2018) of silkworm in China, and due to the wide planting of crops such as corn, soybean and the like, the phenomenon of silkworm breeding and crop production synchronization exists in many places. Therefore, in the process of silkworm breeding, the silkworm may be directly contacted with the crop pollen with high expression quantity of exogenous dsRNA, and when the safety evaluation of RNAi transgenic crops is carried out, the influence of the expressed dsRNA on the economic insect silkworm needs to be evaluated.

Disclosure of Invention

The present invention aims to provide a method for evaluating the influence of RNAi transgenic products on Italian bees, so as to solve the problems in the background art.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for evaluating the effect of RNAi transgenic products on Italian bees, comprising the following steps:

step 1: designing a primer: using SMV-P3-RNAi transgenic disease-resistant soybean B5B9013 as a test material, designing a primer according to a forward interference fragment sequence of an SMV-P3 gene in the B5B9013 soybean and a GFP gene sequence, and amplifying to obtain a DNA template for synthesizing dsRNA;

step 2: reverse transcription of dsRNA: synthesis of dsRNA was performed using MEGAscript transcription kit (Applied Biosystems Inc., Foster City, Calif.).

Further, the reaction system of step 2 is as follows: 10 × Reaction Buffer 2 μ L, Enzyme Mix 2 μ L, template DNA (i.e., PCR product) 0.2 μ g, ATP solution, CTP solution, GTP solution, UTP solution each 2 μ L, and Nuclean-free water to 20 μ L.

Further, after mixing well, reacting at 37 ℃ for 16 hours, measuring the concentration of the synthesized dsRNA, and freezing and storing at-80 ℃ for later use.

Further, the test method for the influence of the dsRNA on the bees comprises the following steps:

and (3) experimental design: dissolving 500g of sucrose in a proper amount of distilled water, then dissolving to 1L for standby, and setting three treatments;

the test steps are as follows: during the test, randomly grabbing newly-taken Italian bees into bee cages, placing 10 adult bees in each cage, repeating each treatment for 4 times, starving for 2 hours before the test starts, checking the death number of the Italian bees every day, cleaning the dead Italian bees out of the bee cages, changing the feed and weighing every day, finishing the test after feeding for 14 days, keeping the whole test at 25 +/-2 ℃, and controlling the humidity to be between 50% and 80% under the illumination conditions of 0h L:24h D (except observation and recording);

survey recording: during the test period, the death number of Italian bees (the death is judged if no response is caused by touching the insect body with a writing brush) is recorded every day, the food intake of each cage of bees is recorded when the feed is replaced, the death rate or the survival rate (%) of each Italian bee adult is calculated and analyzed by adopting SPSS Statics 20 software according to the recorded data, and the survival rate curve of the Italian bees is compared and analyzed by adopting a log-rank detection method.

Further, the three processes include:

negative control group: uniformly mixing GFP gene dsRNA with a concentration of 25 mug/mL in 500g/L sucrose solution;

test dsRNA treatment groups: uniformly mixing synthetic SMV-P3 gene dsRNA with the concentration of 25 mu g/mL into 500g/L of sucrose;

positive control group: 10mg/mL of SBTI (soybean trypsin inhibitor) is uniformly mixed in 500g/L of sucrose solution.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, SMV-P3-RNAi gene resistance soybean B5B9013 is used as a test material, a primer is designed according to a forward interference fragment sequence and a GFP gene sequence of an SMV-P3 gene in the B5B9013 soybean, a DNA template for synthesizing dsRNA is obtained through amplification, a MEGAscript transcription kit is used for synthesizing dsRNA, and an influence test of the dsRNA on bees is carried out.

Drawings

FIG. 1 is a graph showing the natural survival curves of Italian bee 14 in the test of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to FIG. 1, a method for evaluating the effect of RNAi transgenic products on Italian bees first performs dsRNA synthesis comprising the steps of:

step 1: synthesis of dsRNA

Designing a primer: a DNA template for synthesizing dsRNA is obtained by using SMV-P3-RNAi transgenic disease-resistant soybean B5B9013 as a test material and amplifying according to a forward interference fragment sequence of an SMV-P3 gene in the B5B9013 soybean and a GFP gene sequence, wherein the primer sequence is shown in Table 1, and the underlined sequence is a T7 polymerase promoter binding sequence.

TABLE 1 primer sequences for dsRNA synthesis

Step 2: reverse transcription of dsRNA: synthesis of dsRNA was performed using MEGAscript transcription kit (Applied Biosystems Inc., Foster City, Calif.). The reaction system is as follows: 10 × Reaction Buffer 2 μ L, enzyme mix 2 μ L, template DNA (i.e., PCR product) 0.2 μ g, ATP solution, CTP solution, GTP solution, and UTP solution 2 μ L each, and Nuclean-free water was added to 20 μ L.

After mixing well, reacting for 16h at 37 ℃, measuring the concentration of the synthesized dsRNA, and freezing and storing at-80 ℃ for later use.

The test method for the influence of dsRNA on bees comprises the following steps:

and (3) experimental design: dissolving 500g of sucrose in a proper amount of distilled water, then dissolving to 1L for standby, and setting three treatments:

(1) negative control group: uniformly mixing GFP gene dsRNA with a concentration of 25 mug/mL in 500g/L sucrose solution;

(2) test dsRNA treatment groups: uniformly mixing synthetic SMV-P3 gene dsRNA with the concentration of 25 mu g/mL into 500g/L of sucrose;

(3) positive control group: 10mg/mL of SBTI (soybean trypsin inhibitor) is uniformly mixed in 500g/L of sucrose solution.

The test steps are as follows: during the test, randomly grabbing newly-taken Italian bees into bee cages, placing 10 adult bees in each cage, repeating each treatment for 4 times, starving for 2 hours before the test starts, checking the death number of the Italian bees every day, cleaning the dead Italian bees out of the bee cages, changing the feed and weighing every day, finishing the test after feeding for 14 days, keeping the whole test at 25 +/-2 ℃, and controlling the humidity to be between 50% and 80% under the illumination conditions of 0h L:24h D (except observation and recording);

survey recording: during the test period, the death number of Italian bees (the death is judged if no response is caused by touching the insect body with a writing brush) is recorded every day, the food intake of each cage of bees is recorded when the feed is replaced, the death rate or the survival rate (%) of each Italian bee adult is calculated and analyzed by adopting SPSS Statics 20 software according to the recorded data, and the survival rate curve of the Italian bees is compared and analyzed by adopting a log-rank detection method.

The results and analysis of this test are as follows:

the survival rates of Italian bees treated during the test are shown in Table 1, and the survival curves are shown in FIG. 1(χ)²30.643, df 2, P0.647). The results showed that feeding sucrose water containing 25. mu.g/mL of dsRNA of soybean SMV-P3 gene did not significantly affect the survival rate of bees compared to feeding dsRNA containing 25. mu.g/mL of GFP gene (see Table 2).

Table 2 italian bee survival (%)

As can be seen from Table 2 above in conjunction with FIG. 1, the survival rates of Italian bees in the RNAi groups were higher than those in the other two groups from day 1 to day 11, and the survival rates of Italian bees in the control groups were higher than those in the other two groups from day 12 to day 14.

In conclusion, the invention takes SMV-P3-RNAi gene disease-resistant soybean B5B9013 as a test material, designs a primer according to a forward interference fragment sequence of an SMV-P3 gene in the B5B9013 soybean and a GFP gene sequence, amplifies to obtain a DNA template for synthesizing dsRNA, synthesizes dsRNA by adopting a MEGAscript transcription kit (Applied Biosystems Inc., Foster City, CA), and tests the influence of the dsRNA on bees.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims (5)

1. A method for evaluating the influence of RNAi transgenic products on Italian bees, firstly synthesizing dsRNA, and is characterized by comprising the following steps:

step 1: designing a primer: using SMV-P3-RNAi transgenic disease-resistant soybean B5B9013 as a test material, designing a primer according to a forward interference fragment sequence of an SMV-P3 gene in the B5B9013 soybean and a GFP gene sequence, and amplifying to obtain a DNA template for synthesizing dsRNA;

step 2: reverse transcription of dsRNA: synthesis of dsRNA was performed using MEGAscript transcription kit (Applied Biosystems Inc., Foster City, Calif.).

2. The method of claim 1, wherein the response of step 2 is as follows: 10 × ReactionBuffer 2 μ L, Enzyme Mix 2 μ L, template DNA (i.e., PCR product) 0.2 μ g, ATP solution, CTP solution, GTP solution, UTP solution each 2 μ L, and Nuclean-free water to 20 μ L.

3. The method of claim 2, wherein the response is carried out at 37 ℃ for 16 hours after mixing, the concentration of the synthesized dsRNA is measured, and the product is frozen at-80 ℃ for storage.

4. The method of claim 1, wherein the test for the effect of dsRNA on bees comprises:

and (3) experimental design: dissolving 500g of sucrose in a proper amount of distilled water, then dissolving to 1L for standby, and setting three treatments;

the test steps are as follows: during the test, randomly grabbing newly-taken Italian bees into bee cages, placing 10 adult bees in each cage, repeating each treatment for 4 times, starving for 2 hours before the test starts, checking the death number of the Italian bees every day, cleaning the dead Italian bees out of the bee cages, replacing feeds and weighing every day, finishing the test after feeding for 14 days, keeping the whole test at 25 +/-2 ℃, and controlling the humidity to be between 50% and 80% under the illumination condition of 0hL:24h D (except observation and recording);

survey recording: during the test period, the death number of Italian bees (the death is judged if no response is caused by touching the insect body with a writing brush) is recorded every day, the food intake of each cage of bees is recorded when the feed is replaced, the death rate or the survival rate (%) of each Italian bee adult is calculated and analyzed by adopting SPSS Statics 20 software according to the recorded data, and the survival rate curve of the Italian bees is compared and analyzed by adopting a log-rank detection method.

5. The method of claim 4, wherein the three treatments comprise:

negative control group: uniformly mixing GFP gene dsRNA with a concentration of 25 mug/mL in 500g/L sucrose solution;

test dsRNA treatment groups: uniformly mixing synthetic SMV-P3 gene dsRNA with the concentration of 25 mu g/mL into 500g/L of sucrose;

positive control group: 10mg/mL of SBTI (soybean trypsin inhibitor) is uniformly mixed in 500g/L of sucrose solution.

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