CN117778376A - Parasitic wasp antenna separation method for extracting RNA - Google Patents

Abstract

The invention provides a parasitic wasp antenna separation method for extracting RNA, which comprises the following steps: picking parasitic wasps, putting the parasitic wasps into ice cubes frozen at the temperature of-80 to-70 ℃, performing freezing treatment for 70-100 seconds, and vibrating for 7-13 seconds to obtain pretreated parasitic wasps; placing the pretreated parasitic wasp on ice cubes, performing microscopic examination, separating and screening antennae, and adding antennae into Trizol solution to obtain target antennae. The invention provides a method for freezing parasitic wasps by ice cubes frozen in a refrigerator at the temperature of minus 80 ℃ and then separating antennae, which can realize high falling rate of antennae on the premise of ensuring the integrity of other body parts of the parasitic wasps, and provides a method for carrying out contact angle microscopic examination and screening on ice, and storing by using Trizol solution antennae in the collecting process, thereby reducing the degradation speed of the RNA of the antennae of the parasitic wasps in the collecting, storing and transporting processes, obviously improving the purity of the RNA of the parasitic wasps, and displaying good other quality indexes.

Description

Parasitic wasp antenna separation method for extracting RNA

Technical Field

The invention relates to the technical field of separation and extraction, in particular to a parasitic wasp antenna separation method for extracting RNA.

Background

Parasitic wasps are an important natural enemy insect of various pests of hymenoptera, lepidoptera, coleoptera and diptera, and play an important role in green control of crop pests. When parasitic wasps are utilized to biologically control pests, 5 links such as host habitat selection, host positioning, host acceptance, host adaptation and host adjustment are necessary stages for the parasitic wasps to successfully parasitize, and the control effect of the parasitic wasps depends on the ability of the parasitic wasps to find and position the hosts to a great extent. At present, semiochemicals are generally considered to be important cues for parasitic bees to find hosts, these chemical signals usually coming from the host itself, the host habitat, the host manure, the host return, the airway volatiles, etc., and complexes between several of these.

The smell sense is one of the main means for the insects to sense the external environment, and plays an important role in host positioning, information communication and the like. The sensors on the antennae, feet, ovipositors and other organs of insects and related proteins form an extremely sensitive and highly specific olfactory sensing system. Since the antenna is the most organ of distribution sensor, so at present research on the olfaction positioning of parasitic bees is concentrated on the aspect of antenna, and therefore, the efficient and rapid separation of the parasitic bees antenna is concerned about the smooth progress of related research work.

Usually, the separation and extraction processes of the antennae of the tiny insects can be completed under a microscope. Because the antenna of the parasitic wasp is very fine, the amount of the antenna needed when extracting the antenna RNA substance of the parasitic wasp is large, the parasitic wasp is cut by a surgical knife one by one under a microscope according to a common method, which is time-consuming and labor-consuming and has low efficiency, so the cutting work of the antenna cannot be completed at all according to the common method. The patent' quick separation method of the antenna of the tiny coleopteran insect (ZL 201510426675.7) is simplified in description of the antenna separation method of the tiny coleopteran insect. However, when the antenna of the parasitic wasps (especially the Aphis japonica Coccophagus japonicus Compere) is separated by the method for RNA extraction, the content of the extracted RNA is low, and sometimes the extracted RNA cannot be extracted, so that the requirement of the test cannot be met.

Disclosure of Invention

In view of the above, the present invention aims to provide a rapid separation method for parasitic wasp antennas, which is low in cost, time-saving, labor-saving, simple and easy to implement, and capable of improving the RNA extraction rate, so as to provide effective help for experiments involving a large amount of parasitic wasp antennas in operation.

The technical scheme of the invention is realized as follows:

a parasitic wasp antenna separation method for extracting RNA, comprising the steps of:

s1: picking parasitic wasps, putting the parasitic wasps into ice cubes frozen at the temperature of-80 to-70 ℃, performing freezing treatment for 70-100 seconds, and vibrating for 7-13 seconds to obtain pretreated parasitic wasps; namely, picking 40-50 parasitic bees with strong vitality into a 50mL transparent centrifuge tube by using a writing brush, putting ice cubes into an ultralow temperature refrigerator at-80 ℃ in advance for refrigerating for 12 hours, immediately immersing the centrifuge tube into the ice cubes frozen from the refrigerator at-80 ℃ for freezing, rapidly putting the centrifuge tube on a vortex oscillator for vibrating for 10-13 seconds, taking out the centrifuge tube at any time, observing under a stereoscopic microscope, enabling more than 90% of insect antennae of the centrifuge tube to separate without other tissues falling off, pouring the bodies of the small bees with all the falling antennae into a sterile culture dish from the centrifuge tube, observing that the separated insect antennae are adhered to the inner wall of the centrifuge tube, and sweeping the centrifuge tube into a new sterile culture dish (ice cubes) containing 3-4 drops of PBS by using a sterile brush;

s2: placing the pretreated parasitic wasp on ice cubes, performing microscopic examination, separating and screening antennae, and adding antennae into Trizol solution to obtain target antennae.

Placing the culture dish on ice cubes, performing microscopic examination under a stereoscopic vision, separating and screening the hornets washed by the PBS solution, pure insect tentacles were collected in a degranolase centrifuge tube containing Trizol reagent added in advance and used for RNA extraction and the like.

Further, the time of the freezing treatment is 70 to 80 seconds.

Further, the frequency of the oscillation is 3000-4000 r/min.

Further, the time of the oscillation is 10-13 s.

Further, the number of times of repeating the operation step S1 is 1 to 2.

Further, in step S1, before the freezing treatment, adding a trehalose solution until the parasitic wasps are immersed; the trehalose has stable property, is not easy to be destroyed in a low-temperature environment, has nonspecific protection effect on parasitic wasps in the freezing process, and is beneficial to reducing the damage degree of other body parts.

Further, the mass concentration of the trehalose solution is 10-20 g/L.

Further, in step S2, PBS buffer is added to the pretreated parasitic wasps.

Further, the parasitic wasp is of the genus Aphis.

Further, the aphidius is a japanese aphidius gifuensis Coccophagus japonicus Compere.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention provides a method for freezing parasitic wasps by using ice cubes frozen in a refrigerator at the temperature of minus 80 ℃ and then separating antennae, which can realize high falling rate of antennae on the premise of ensuring the integrity of other body parts of the parasitic wasps, and has the effect obviously superior to the treatment of freezing by using liquid nitrogen.

(2) According to the invention, the contact angle microscopic examination and screening are carried out on the ice cream, and the contact angle of the Trizol solution is used for preservation in the collecting process, so that the degradation speed of the parasitic wasp contact angle RNA in the collecting, storing and transporting processes is reduced, the purity of the parasitic wasp contact angle RNA is obviously improved, and other quality indexes are also well shown.

Drawings

FIG. 1 is a graph showing the effect of freezing on the RNA integrity of the horn of Aphis japonica;

fig. 2 is a graph showing the effect of the preservation mode during the antenna collection on the integrity of the antenna RNA of the japanese aphidius gifuensis.

Detailed Description

In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.

The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.

Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.

Example 1

A parasitic wasp antenna rapid separation method for RNA extraction comprises the following steps:

1. picking 40-50 parasitic wasps with strong vitality into a 50mL transparent centrifuge tube by using a writing brush;

2. immersing the centrifuge tube into ice cubes frozen by a refrigerator at the temperature of-80 to-70 ℃ immediately for freezing for 70-100 seconds;

3. then rapidly placing the centrifugal tube on a vortex oscillator to oscillate for 10-13 s, wherein the oscillation frequency is 3000-4000 r/min;

4. taking out the centrifuge tube, placing the centrifuge tube on an ice table in a culture dish, then placing the centrifuge tube under a stereoscopic microscope for observation, and repeating the procedures of the step 2 and the step 3 for 1 time if necessary, so that more than 90% of the feeler of insects is separated and other tissues are not separated;

5. the whole fallen bee bodies of the insect antennae are poured into a sterile culture dish from a centrifugal tube, the separated insect antennae are adhered to the inner wall of the centrifugal tube, and the separated insect antennae are stuck to the wall of the centrifugal tube and swept into a new sterile culture dish (ice cubes) containing 3-4 drops of PBS (ice cubes) by using a sterile brush.

6. Placing the culture dish on ice cubes, performing microscopic examination under a stereoscopic vision mirror, and separating and screening the hornets of the hornets;

7. pure insect tentacles were collected in a degranolase centrifuge tube with 100. Mu.L of Trizol added in advance for RNA extraction.

Test example 1

Test setup: collecting 50 head of Aphis japonica with strong vitality in a centrifuge tube, immediately immersing the centrifuge tube in ice cubes frozen by a refrigerator at-80 ℃ for freezing, setting the freezing time gradient to 60s, 80s and 100s, then vortex-shaking for 13s (the shaking frequency is 4000 r/min), taking out the centrifuge tube, placing the centrifuge tube on an ice table in a culture dish, observing under a stereoscopic microscope, taking all fallen Aphis japonica bodies out of the centrifuge tube into the culture dish, finding antennae adhered to the inner wall of the centrifuge tube, and sweeping the antennae of the Aphis japonica into a new sterile culture dish (ice cubes) containing 3-4 drops of PBS by using a sterile brush. Recording the average shedding amount of the parasitic wasps antenna in different freezing time and the number of broken insects in other body parts, and calculating the average antenna shedding rate and the breakage rate of other body parts. The test was repeated 3 times.

TABLE 1 influence of the freezing time on the antenna shedding rate and other body part breakage rate of Aphis japonica

The result shows that after the Japanese aphidius gifuensis is frozen for 60-100 s and then vortex oscillated for 13s, the antennal falling rate is gradually increased from 11.2% to 100% along with the extension of the freezing time; when frozen for 60-80 seconds, other body parts of the Japanese aphidius gifuensis are not broken, but when frozen for 100 seconds, 8.0% of the bodies of the Aphis gifuensis are broken, which is not beneficial to subsequent experiments. The freezing time of 80s is favorable for the shedding of the antenna of the Japanese aphidius gifuensis and the subsequent collection work.

Test example 2

Test setup: collecting 50 heads of Aphis japonica with strong vitality, putting the Aphis japonica into a centrifuge tube, immediately immersing the centrifuge tube into ice cubes frozen by a refrigerator at the temperature of-80 ℃ for freezing for 80 seconds, then vortex-vibrating, wherein the vibration frequency is 4000r/min, the vibration time gradient is 7s, 10s, 13s and 16s, taking out the centrifuge tube, putting the centrifuge tube into a culture dish on an ice table, then observing and taking out the centrifuge tube under a stereo microscope, pouring the bodies of the Aphis japonica with all the tentacles falling out into the culture dish from the centrifuge tube, and observing that the separated insect tentacles are adhered to the inner wall of the centrifuge tube, and sweeping the centrifuge tube onto a new sterile culture dish (ice cubes) containing 3-4 drops of PBS by using a sterile brush. The experiment is repeated for 3 times, the average shedding amount of the parasitic wasp antennae treated in different concussion times and the number of broken insects of other body parts are recorded, and the average antenna shedding rate and the breakage rate of other body parts are calculated.

TABLE 2 influence of concussion time on the antenna shedding rate and other body part breakage rate of Aphis japonica

The result shows that the shedding rate of the antenna of the Aphis japonica is increased along with the increase of the oscillation time within the oscillation time range of 7-16 s until 100%, but the breakage rate of other parts of the body of the Aphis japonica reaches 60.0% when the oscillation time reaches 16s, which is not beneficial to the subsequent test. The oscillation time of 13s is favorable for the shedding of the bee antennae and the subsequent collection.

Test example 3

Test setup: the test sets 3 treatments:

treatment 1: the centrifuge tube in which 50 head of the high-activity Japanese aphidius gifuensis are collected is put into, namely, all the centrifuge tube is immersed in ice cubes which are just knocked out, and the ice cubes are frozen for 80 seconds.

And 2, immediately immersing all centrifuge tubes in which 50 head of powerful Japanese aphidius gifuensis are collected into ice cubes frozen by a refrigerator at the temperature of minus 80 ℃ for 80 seconds.

Treatment 3: the centrifuge tube, in which 50 head of the vigorous Aphis japonica are collected, is placed in liquid nitrogen and cooled for 80s.

The method comprises the steps of treating a bee sample in the above 3 modes, performing vortex vibration for 13s according to the vibration frequency of 4000r/min, taking out a centrifuge tube, placing the centrifuge tube in a culture dish on an ice table, observing and taking out the centrifuge tube under a stereoscopic microscope, pouring the bodies of the bees with all the antenna falling off into the culture dish from the centrifuge tube, observing that the separated insect antenna is adhered to the inner wall of the centrifuge tube, and sweeping the separated insect antenna into a new sterile culture dish (ice cubes) containing 3-4 drops of PBS by using a sterile brush, wherein the new sterile culture dish is adhered to the tube wall. The experiment is repeated for 3 times, the average shedding amount of the parasitic wasp antennae processed by different freezing modes and the number of insects broken by other body parts are recorded, and the average antenna shedding rate and the breakage rate of other body parts are calculated.

TABLE 3 influence of different freezing modes on the antenna shedding rate and other body part breakage rates of Aphis japonica

The results show that when the Aphis japonica is frozen by using an ice freezing mode and ice freezing by using an ice freezing refrigerator at the temperature of minus 80 ℃, the contact falling rate of the Aphis japonica is respectively 10 percent and 98 percent, and the breakage rate of other body parts is 0; the shedding rate of the feeler of the Japanese aphidius gifuensis after the liquid nitrogen freezing treatment reaches 100%, but the breakage rate of other parts of the body of the bee reaches 80.0%, so that the collection of the feeler is seriously influenced, the sampling time is prolonged, and the subsequent test is not facilitated, and the RNA quality is influenced. The method for freezing the bee antennae in ice cubes frozen by a refrigerator at the temperature of-80 ℃ is more beneficial to falling off of the bee antennae and subsequent collection.

Test example 4

Test setup: collecting 50 heads of Aphis japonica with strong vitality in 2 centrifuge tubes respectively, immediately immersing 1 centrifuge tube in ice cubes frozen in a refrigerator at-80 ℃ for 80 seconds, freezing the other 1 centrifuge tube in liquid nitrogen for 80 seconds, then vortex vibrating for 13 seconds, taking out the centrifuge tube, placing the centrifuge tube on an ice table in a culture dish, pouring bodies of the Aphis japonica with all falling off insect antennae out of the centrifuge tube into the culture dish, sweeping the antennae adhered to the tube wall into a new sterile culture dish containing 3-4 drops of PBS by using a sterile brush, then placing the culture dish ice table under a stereoscopic microscope for separation and screening, recording the number of the bees and the falling off number of the antennae, and calculating the falling off rate of the antennae of the bees. 1000 heads of Aphis japonica are collected for each treatment, the antennae are soaked in a degranulation tube containing 100 mu L of Trizol solution, total RNA is extracted by the same Trizol method, the influence of two freezing methods on RNA quality is compared, the test is repeated 3 times, and an average RQN value is recorded.

The results of FIG. 1 show that the quality of RNA extracted from the tentacles obtained by separating the frozen ice cubes from the refrigerator at-80 ℃ is better than the quality of RNA extracted from the tentacles obtained by separating the frozen ice cubes from the Aphis japonica (RQN value 8.2), and the quality of RNA extracted from the tentacles obtained by separating the frozen ice cubes from the Aphis japonica with liquid nitrogen (RQN value 5.3) shows that the shedding of the tentacles of the ice cubes and the reduction of the degradation rate of RNA are more favorable after the ice cubes are frozen and treated on the ice cubes.

Test example 5

Test setup: collecting 50 heads of high-activity Aphis japonica, putting the centrifugal tube into a centrifugal tube, immediately immersing the centrifugal tube into ice cubes frozen from a refrigerator at the temperature of minus 80 ℃ for freezing for 80 seconds, then vortex oscillating for 13 seconds at the oscillating frequency of 4000r/min, taking out the centrifugal tube, placing the centrifugal tube on an ice table in a culture dish, pouring the bodies of the Aphis japonica, which are all shed off by the feeler, into the culture dish from the centrifugal tube, sweeping the feeler adhered to the tube wall into a new sterile culture dish containing 3-4 drops of PBS (phosphate buffer solution), dividing the two groups into two groups, placing one group on ice for microscopic examination, and placing the other group at room temperature for microscopic examination to obtain washed feeler feelers, collecting 1000 heads of Aphis japonica in each treatment, immersing the feeler into the RNase containing 100 mu L of Trizol solution, extracting the total RNA amount by using the same Trizol method, comparing the influence of the two microscopic examination methods on the quality of RNA, repeating the experiment three times, and finally recording whether the average RQN value has change.

TABLE 4 RQN values of RNA obtained from ice cubes and microscopic antenna at room temperature

The result shows that a group of RQN values (8.2) of the microscopic examination at room temperature are slightly lower than RQN values (7.7) of the microscopic examination on ice cubes through two microscopic examination treatment modes, so that the microscopic examination on ice cubes can effectively prevent RNA degradation, ensure the integrity of RNA, and facilitate subsequent experiments.

Test example 6

In order to clearly determine the influence of Trizol on the RNA integrity of the Aphis japonica in the process of antenna collection and preservation, 50 bees with strong vitality are collected and put into a centrifuge tube, the centrifuge tube is immediately and completely immersed into ice cubes frozen from a refrigerator at-80 ℃ for 80 seconds, then vortex vibration is carried out for 13 seconds, the vibration frequency is 4000r/min, the centrifuge tube is taken out and placed on an ice table in a culture dish, the bodies of the bees with all insect antennae falling off are poured into the culture dish from the centrifuge tube, the antennae adhered to the tube wall are swept into a new sterile culture dish containing 3-4 drops of PBS by using a sterile brush, the fresh sterile culture dish is subjected to microscopic examination under a stereoscopic microscope, the antennae are screened, then the antennae are touched into 2 groups, 1000 Aphis japonica is collected in total treatment, one group of antennae are collected into a norrnase which is added into 100 mu L of Trizol preservation solution in advance, one group of the system is directly put into the norrnase, the centrifuge tube is used for extracting total RNA, then the RNA quality is detected by using the Trizol method, and the average centrifuge tube is compared for 3 times, and the average RQN changes are recorded.

As shown in the results of FIG. 2, the RQN value (8.2) obtained by extracting the antenna with Trizol solution during the collection and preservation process is slightly higher than that obtained by not using Trizol solution (7.9) during the process, which shows that the antenna is directly collected in the RNase centrifuge tube containing Trizol to effectively prevent RNA degradation, ensure the integrity of RNA and facilitate the subsequent experiment.

Example 2

A parasitic wasp antenna rapid separation method for RNA extraction comprises the following steps:

1. picking 50 heads of high-activity aphidius gifuensis, immersing the aphidius gifuensis in a trehalose solution with the mass concentration of 10g/L for 2min, and taking out the aphidius gifuensis to a 50mL transparent centrifuge tube;

2. immersing the centrifuge tube into ice cubes frozen by a refrigerator at the temperature of minus 80 ℃ for freezing for 80 seconds;

3. then rapidly placing the centrifugal tube on a vortex oscillator to oscillate for 13 seconds, wherein the oscillation frequency is 3000-4000 r/min;

4. taking out the centrifuge tube, placing the centrifuge tube on an ice table in a culture dish, and then placing the centrifuge tube under a stereoscopic microscope for observation;

5. the whole fallen bee bodies of the insect antennae are poured into a sterile culture dish from a centrifugal tube, the separated insect antennae are adhered to the inner wall of the centrifugal tube, and the separated insect antennae are stuck to the wall of the centrifugal tube and swept into a new sterile culture dish (ice cubes) containing 3-4 drops of PBS (ice cubes) by using a sterile brush.

6. Placing the culture dish on ice cubes, performing microscopic examination under a stereoscopic vision mirror, and separating and screening the hornets of the hornets;

7. pure insect tentacles were collected in a degranolase centrifuge tube with 100. Mu.L of Trizol added in advance for RNA extraction.

Through detection, the shedding rate of the antenna of the Aphis japonica is 98.7%, and the breakage rate of other body parts is 0%.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (10)

1. A parasitic wasp antenna separation method for extracting RNA, comprising the steps of:

s1: picking parasitic wasps, putting the parasitic wasps into ice cubes frozen at the temperature of-80 to-70 ℃, performing freezing treatment for 70-100 seconds, and vibrating for 7-13 seconds to obtain pretreated parasitic wasps;

s2: placing the pretreated parasitic wasp on ice cubes, performing microscopic examination, separating and screening antennae, and adding antennae into Trizol solution to obtain target antennae.

2. The method for isolating parasitic wasp's antenna for extracting RNA as claimed in claim 1, wherein the time of the freezing treatment is 70-80 s.

3. The method for separating parasitic wasp's antenna for extracting RNA according to claim 1, wherein the frequency of said oscillation is 3000-4000 r/min.

4. The method for isolating parasitic wasp's antenna for extracting RNA as claimed in claim 1, wherein the time of said shaking is 10-13 s.

5. The method for separating parasitic wasp' S antenna for extracting RNA according to claim 1, wherein the number of times of repeating the operation step S1 is 1 to 2.

6. The method according to claim 1, wherein the step S1 further comprises adding a trehalose solution to the immersed parasitic bees before the freezing treatment.

7. The method for separating parasitic wasp's antenna for extracting RNA according to claim 6, wherein the mass concentration of the trehalose solution is 10-20 g/L.

8. The method according to claim 1, wherein in step S2, PBS buffer is added to the pretreated parasitic wasps.

9. The method according to any one of claims 1 to 8, wherein the parasitic wasp is of the genus cerclage.

10. The method of claim 9, wherein the aphidius genus is japanese aphidius gifuensis Coccophagus japonicus Compere.