CN111264475A - Microinjection method for small insect adults of whiteflies - Google Patents
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- 241000238631 Hexapoda Species 0.000 title claims abstract description 54
- 238000000520 microinjection Methods 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 38
- 241000258937 Hemiptera Species 0.000 title claims abstract description 20
- 238000002347 injection Methods 0.000 claims abstract description 66
- 239000007924 injection Substances 0.000 claims abstract description 66
- 239000001963 growth medium Substances 0.000 claims abstract description 36
- 239000011521 glass Substances 0.000 claims abstract description 11
- 241000254127 Bemisia tabaci Species 0.000 claims description 13
- 206010002091 Anaesthesia Diseases 0.000 claims description 10
- 230000037005 anaesthesia Effects 0.000 claims description 10
- 150000001875 compounds Chemical class 0.000 claims description 8
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- 239000003086 colorant Substances 0.000 claims description 7
- 238000007789 sealing Methods 0.000 claims description 6
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- 241000018137 Trialeurodes vaporariorum Species 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
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- 241001470017 Laodelphax striatella Species 0.000 claims description 3
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- A—HUMAN NECESSITIES
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
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Abstract
The invention relates to a microinjection method for small whitefly insect adults, which adopts a glass microinjection needle to carry out injection on a microscopic platform and comprises the following steps: preparing a glass micro-injection needle; the insect body is anesthetized before injection; placing the anesthetized insect body in a V-shaped groove of a special insect fixed culture medium flat plate for injection, and performing microinjection; placing the injected insect body on a feeding culture medium in a leaf feeding culture dish, placing the insect body in a light culture box for feeding for 10-14 hours, inverting the leaf feeding culture dish to enable the front side of the leaf to face upwards, and continuing feeding; the method has scientific and reasonable operation links for microinjecting the whitefly insects, obviously improves the injection efficiency, reduces the test error, can inject 80 whitefly adults and 40 planthopper adults per hour, and improves the injection speed by about 3-4 times compared with the conventional injection method; greatly reduces the death rate of the insects after injection and reduces the test error.
Description
Technical Field
The invention relates to the field of entomology, in particular to a microinjection method for whitefly small insect adults.
Background
Microinjection technology is a practical technology widely applied in the field of life science at present, and is mainly applied in the research fields of microbiology, medicine, entomology and the like. The microinjection technique is an operation technique in which a solution is injected into animals, plants, and microorganisms by microscopic observation using an injection needle. Microinjection techniques are widely used for the injection of adults or nymphs of small insects, as well as many eggs. Microinjection techniques are complex to operate and, especially for small insects, require high operating requirements. At present, microinjection technologies disclosed by various research institutions are mainly used for cells or large and medium animals and plants, and are not specially used for adult whitefly insects or other small-sized insect adults.
When small insects such as whitefly, plant hopper, parasitic wasp, partial egg or nymph and the like are researched for the functions of insect genes and insect symbiotic bacteria, double-stranded RNA, small interfering RNA, antibiotics, small guide RNA, protein, compounds and the like need to be injected for living body research, and strict requirements are provided for the number and survival rate of injected insects in the research process. For example, gene interference test research, particularly, injecting different treated worms in the same time period so as to reduce errors, and the worms are required to survive for a longer time after injection, so that the current injection method and the injected worm feeding method cannot meet the requirements.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention aims to provide the microinjection method for the small-sized whitefly insects, the operational link of microinjection of the whitefly insects is scientific and reasonable, the injection efficiency is obviously improved, the death rate of the injected insects is greatly reduced, the test error is reduced, and an effective technical method is provided for the research field of the small-sized insects.
The invention aims to realize the microinjection method for the small-sized whitefly insects by adopting the following technical scheme, the microinjection method for the small-sized whitefly insects adopts a glass microinjection needle to inject on a microscopic platform, the microscopic platform comprises a stereoscopic microscope, an air pump injection system, a microscopic operating frame, a needle pulling instrument, a needle grinding instrument and a shockproof platform, and the technical key points are as follows: the microinjection of the whitefly insect adult comprises the following steps:
(1) preparing a glass micro-injection needle: the inner diameter of the glass micro-injection needle is 4.5-5.5 mu m, the length of the needle point of the glass micro-injection needle is 5.5-6.5mm, and the length of the injection needle is 4-4.5 cm;
(2) and (3) insect anesthesia before injection: anaesthetizing the insect body to be injected on ice, wherein the anaesthesia time of bemisia tabaci is 20-40 minutes, the anaesthesia time of greenhouse trialeurodes vaporariorum is 50-70 minutes, the anaesthesia time of brown planthopper is 4-6 minutes, and the anaesthesia time of laodelphax striatellus is 10-15 minutes;
(3) microinjection: fixing an ice box on a microscope base moving platform, placing a fixed culture medium on the ice box, placing the anesthetized insect body in a V-shaped groove of a special insect fixed culture medium injection flat plate by using a small brush, and performing microinjection;
the formulation of the insect fixed culture medium special for injection is 4 percent of agar gel and 0.04 percent of compound colorant; the compound colorant is sky blue or fruit green; one to three V-shaped grooves are arranged on the special insect fixed culture medium flat plate for injection, the width of each V-shaped groove is 0.5-1mm, and the depth of each groove is 0.5-1 mm;
(4) feeding after injection: picking out the injected insect body by using a small brush, putting the insect body on a feeding culture medium in a leaf feeding culture dish, sealing the insect body by using a sealing film, putting the insect body in a light culture box for feeding for 10 to 14 hours, inverting the leaf feeding culture dish to enable the front side of the leaf to face upwards, and continuing feeding;
the culture dish cover of the leaf feeding culture dish is provided with a hole with the diameter of 2-4cm, and a 100-mesh gauze is covered outside the hole; the formula of the feeding culture medium is 1.5% of agar culture medium and 1% of PBS solution, young leaves of tomatoes, cottons or tobaccos are paved on the feeding culture medium, and the leaves are paved in a culture dish with the back faces upward and compacted; placing the culture dish with the prepared leaves in a 27 ℃ illumination incubator, and inverting for 12 h;
further, the parameters of the needle drawing instrument are HEAT: 265, FIL: 3, VEL: 30, DEL: 225, PUL: 150.
further, the needle grinding angle of the needle grinding instrument is 45 degrees.
The invention has the beneficial effects that:
(1) the method for microinjecting the whitefly insects has scientific and reasonable operation links, obviously improves the injection efficiency, reduces the test error, can inject 80 whitefly adults and 40 planthopper adults per hour, and improves the injection speed by about 3-4 times compared with the conventional injection method.
(2) The method greatly reduces the death rate of the injected insects, has simple operation, can be applied to various biological species, and provides an effective technical method for the research field of molecular biology of small insects.
Drawings
FIG. 1 is a diagram showing the arrangement of V-shaped grooves in a culture dish;
FIG. 2 is a sectional view of a V-shaped groove in a medium;
in fig. 1-2, the respective structure names are: a culture dish 1, a culture medium 2 and a V-shaped groove 3.
Detailed Description
Example 1 microinjection method of whitefly small insect adults
The injection subjects were: bemisia tabaci, trialeurodes vaporariorum, nilaparvata lugens, laodelphax striatellus.
The injection method comprises the following steps:
(1) adjusting a microscopic platform: uncovering the dustproof covers of the microscope and the needle grinding instrument, and taking off the lens cover; turning on a light source; opening an air pump switch to confirm that the balance pressure (pc) is adjusted to 0;
the microinjection platform comprises: an Olympus stereomicroscope, an eppendorf air pump injection system, a Japanese luxuriant microscopic control frame, a Sutter needle drawing instrument, a needle grinding instrument and a win-win brand shockproof platform;
(2) fixing the ice box on a microscope base moving platform, putting the ice box with the thickness of 14.5 multiplied by 10 multiplied by 2cm, and putting a fixed culture medium on the ice box; adjusting the micromanipulator bracket to form an included angle of 45 degrees with the horizontal plane;
(3) the injection needle manufacturing method comprises the following steps: the injection needle is made of glass (or quartz) capillary and is drawn by a needle drawing instrument; the tip of the injection needle is required to be sharp enough, not easy to bend and have enough rigidity in principle; when a SUTTER P-2000 needle drawing instrument is used for drawing a WPI standard borosilicate glass capillary, the drawn needle point length is about 0.5 cm; and (3) modulation of parameters of the needle drawing instrument: HEAT: 265, FIL: 3, VEL: 30, DEL: 225, PUL: 150; after the injection needles are pulled, the injection needles are densely arranged on a culture dish stuck with a double-sided adhesive tape, and the damage of the injection needles caused by touching the needle points is avoided;
(4) grinding the opening of the injection needle, namely grinding the needle opening by using a Sutter BV-10 needle grinding instrument at a needle grinding angle of 45 degrees, uniformly dripping distilled water on the edge of a grinding disc, putting the needle point in the distilled water on the edge of the grinding disc, adjusting a fine adjustment knob to grind the needle, wherein when the distilled water is sucked into the injection needle, the needle is opened, and the grinding disc needs to be cleaned before and after the needle is ground;
(5) an Eppendorf microapplicator sample injection needle is used for absorbing liquid to be injected, the liquid is added into the injection needle, and the injection needle is inserted into the head of the injection rod and screwed tightly; the tail part of the air duct of the injection rod is clamped on the air pump connector in a buckling manner;
(6) adjusting air pump parameters, a position of a micro-control rod, a position and an angle of an injection needle; pressing the pressure plate, or observing whether the needle is opened through an "inject" button on the air pump;
(7) the insect to be injected is anesthetized on ice, the anesthesia time of bemisia tabaci is 30 minutes, the anesthesia time of greenhouse trialeurodes vaporariorum is 1 hour, the anesthesia time of brown planthopper is 5 minutes, and the anesthesia time of gray planthopper is 10-15 minutes. Gently placing the small hairbrush in a V-shaped groove of a special insect fixed culture medium flat plate for injection, and injecting;
referring to fig. 1-2, the formulation of the insect fixed culture medium special for injection is 4% of agar gel and 0.04% of compound colorant (Americor sky blue edible compound colorant); 1 to 3V-shaped grooves are arranged in the flat plate of the insect fixed culture medium special for injection, the width of each V-shaped groove is 0.5 to 1mm, the depth of each groove is 0.5 to 1mm, long plastic strips with V-shaped sections float on the unset culture medium, and after the culture medium is solidified, the plastic strips are taken out to obtain the insect fixed culture medium;
(8) picking out the injected insect body by using a small brush, putting the insect body on a feeding culture medium in a leaf feeding culture dish, sealing the insect body by using a sealing film, putting the insect body in a light culture box for feeding for 10 to 14 hours, inverting the leaf feeding culture dish to enable the front side of the leaf to face upwards, and continuing feeding;
the blade feeding culture dish is a plastic culture dish, the specification is 3.5cm in diameter, 1 hole with the diameter of 2cm is formed in the culture dish cover of the blade feeding culture dish, and a 100-mesh gauze is covered outside the hole; the formula of the feeding culture medium is 1.5% of agar culture medium and 1% of PBS solution, the height of the feeding culture medium is 2-7mm, namely the using amount of the culture medium is determined according to the using days of a culture dish; young leaves of tomato cotton or tobacco are arranged on the breeding culture medium, and the leaves are fully paved in a culture dish and compacted according to the upward direction of the back of the leaves; the culture dish with the prepared leaf blade is placed in a light incubator at 27 ℃ and inverted for 12 hours.
By adopting the injection method, 40-80 adult whitefly insects can be injected in each hour, and the injection speed is improved by about 3-4 times compared with the conventional injection method.
Example 2
Influence of different microinjection methods on injection efficiency and survival rate in Bemisia tabaci PGRP gene silencing test
Treatment 1: microinjection method of example 1 was used
And (3) treatment 2: injecting by a conventional technical method, wherein the conventional injection method refers to Tissue-specific genetic engineering by RNA interference in the whiteflyBemisia tabaci(Gennadius).Ghanim et al. BMC Genomics 2013, 14:160
Results of injection efficiency investigations:
the number of injected polypide in 1 hour of treatment is 80;
the number of worms injected per hour for treatment 2 was 20.
And survival rate survey results:
at 24 hours after injection, the survival rate of the bemisia tabaci treated 1 is about 80-100%, and the survival rate of the bemisia tabaci treated 2 is about 50-60%; the survival rate of the treatment 1 is improved by 30-40% compared with that of the treatment 2;
at 48 hours after injection, the survival rate of the bemisia tabaci is about 65-75%, and the survival rate of the bemisia tabaci treated 2 is about 35%; the survival rate of the treatment 1 is improved by 30-40% compared with that of the treatment 2;
the survival rate of the bemisia tabaci is about 50% -65% 72 hours after injection, and the survival rate of the bemisia tabaci treated 2 is about 20% -30%; the survival rate of the treatment 1 is improved by 30-35% compared with that of the treatment 2;
at 144 hours after injection, the survival rate is about 30 percent, and the survival rate of the bemisia tabaci of the treatment 2 is 0; the survival rate of the treatment 1 is improved by 30 percent compared with that of the treatment 2;
by adopting the injection method of the treatment 1, the test result of the PGRP gene of the bemisia tabaci 144 hours after injection is obtained, the data is complete, and meanwhile, the test error is greatly reduced.
The principle of the invention is as follows:
(1) as whitefly insects are small in size and mostly off-white or brown in color, identification errors are easily caused under the microinjection visual field, and the injection efficiency is reduced. The method adopts a special insect fixed culture medium for injection, and color difference can be artificially set by adding the compound colorant in the culture medium, so that the color contrast is enhanced, and the injection efficiency is improved.
(2) A strip-shaped V-shaped groove is formed in the special insect fixing culture medium for injection, an insect body is clamped into the V-shaped groove, and the V-shaped groove formed by the hardness of 4% of agar gel in the ice box is harmless to the insect body and can play a role in fixing.
(3) The leaf feeding culture medium is adopted to feed the injected polypide, so that the optimal feeding condition is provided for the polypide with a wound after injection, the polypide survival rate is improved, and the experimental error is reduced.
(4) The prepared leaf feeding culture dish is placed in a 27 ℃ illumination culture box and is inverted for 12 hours, and then is used for feeding the injected insects, so that the purpose that the insect survival is prevented from being influenced by toxins generated by leaf wounds is achieved.
Claims (4)
1. A microinjection method for adult small insects such as whiteflies adopts a glass microinjection needle to inject on a microscope platform, the microscope platform comprises a stereomicroscope, an air pump injection system, a micromanipulation control frame, a needle pulling instrument, a needle grinding instrument and a shockproof platform, and is characterized in that: the microinjection of the whitefly insect adult comprises the following steps:
(1) preparing a glass micro-injection needle: the inner diameter of the glass micro-injection needle is 4.5-5.5 mu m, and the length of the needle point of the glass micro-injection needle is 5.5-6.5 mm;
(2) and (3) insect anesthesia before injection: anaesthetizing the insect body to be injected on ice;
(3) microinjection: fixing an ice box on a microscope base moving platform, placing a fixed culture medium on the ice box, placing the anesthetized insect body in a V-shaped groove of a special insect fixed culture medium injection flat plate, and performing microinjection;
the formulation of the insect fixed culture medium special for injection is 4 percent of agar gel and 0.04 percent of compound colorant; the compound colorant is sky blue or fruit green; one to three V-shaped grooves are arranged on the special insect fixed culture medium flat plate for injection, the width of each V-shaped groove is 0.5-1mm, and the depth of each groove is 0.5-1 mm;
(4) feeding after injection: placing the injected insect body on a feeding culture medium in a leaf feeding culture dish, sealing with a sealing film, feeding in a light incubator for 10-14 hours, inverting the leaf feeding culture dish to enable the front side of the leaf to face upwards, and continuing feeding;
the culture dish cover of the leaf feeding culture dish is provided with a hole with the diameter of 2-4cm, and a 100-mesh gauze is covered outside the hole; the formula of the breeding culture medium is 1.5% of agar culture medium and 1% of PBS solution, young leaves of any one of tomatoes, cottons or tobaccos are paved on the breeding culture medium, and the leaves are paved in a culture dish and compacted with the back faces of the leaves facing upwards; the prepared leaf feeding culture dish is placed in a light incubator at 27 ℃ and is inverted for 12 hours for use.
2. The microinjection method of claim 1, wherein the microinjection method comprises the following steps: the time for anaesthetizing the insect before injection comprises 20-40 minutes for anaesthetizing Bemisia tabaci, 50-70 minutes for anaesthetizing greenhouse whitefly, 4-6 minutes for anaesthetizing Nilaparvata lugens and 10-15 minutes for anaesthetizing Laodelphax striatellus.
3. The microinjection method of claim 1, wherein the microinjection method comprises the following steps: the parameters of the needle drawing instrument are HEAT: 265, FIL: 3, VEL: 30, DEL: 225, PUL: 150.
4. the microinjection method of claim 1, wherein the microinjection method comprises the following steps: the needle grinding angle of the needle grinding instrument is 45 degrees.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113693030A (en) * | 2021-08-26 | 2021-11-26 | 华南农业大学 | Method for artificially transfecting exogenous insect symbiotic bacteria Wolbachia into diaphorina citri |
| CN117721157A (en) * | 2024-02-08 | 2024-03-19 | 北京市农林科学院 | Single-needle double microinjection method for ladybug embryo and microinjection quartz microneedle used by same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117721157A (en) * | 2024-02-08 | 2024-03-19 | 北京市农林科学院 | Single-needle double microinjection method for ladybug embryo and microinjection quartz microneedle used by same |
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