CN101220377A - Method for horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius - Google Patents
Method for horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius Download PDFInfo
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- CN101220377A CN101220377A CNA2008100140406A CN200810014040A CN101220377A CN 101220377 A CN101220377 A CN 101220377A CN A2008100140406 A CNA2008100140406 A CN A2008100140406A CN 200810014040 A CN200810014040 A CN 200810014040A CN 101220377 A CN101220377 A CN 101220377A
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Abstract
The invention relates to a method for the horizontal transfection of bemisia tabaci of the exogenous endosymbiotic bacteria, which pertains to the field of agricultural biotechnology. The preparation method includes: the purification of the exogenous endosymbiotic bacteria Wolbachia, the collection and fixation of a bemisia tabaci receptor, the horizontal transfection of the exogenous endosymbiotic bacteria Wolbachia to the bemisia tabaci receptor, the collection and fixation of an injection individual and the detection of the endosymbiotic bacteria. The invention is simple and quick, which can successfully transfect the exogenous endosymbiotic bacteria to a bemisia tabaci individual, and a bemisia tabaci offspring has higher infection rate.
Description
(1) technical field
The present invention relates to the method for horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius, belong to agricultural biological technical field.
(2) background technology
Biotic intrusion tends to the economic security of a country, ecological safety, social safety are constituted a threat to.The direct economic loss that China causes because of biotic intrusion every year reaches hundreds billion of units, 13 kinds of alien species such as Hemp Eupatorium, pine wood nematode wherein, and the annual financial loss that production causes to agriculture, forestry, animal husbandry, fisheries just reaches 57,400,000,000 yuan.Current, the Bemisia tabaci of exotic invasive China (Type B) is broken out at wide geographic area and is caused disaster, it is the unique since the dawn of human civilization worldwide invasion insect titled with " super insect " appellation of International Technology circle, it not only directly draws water, the secretion honeydew influences photosynthesis of plant, and can propagate more than 110 kind of virus disease, cause enormous economic loss for every year invaded geographic agriculture production; In addition, it can also be competed and replace indigenous Bemisia tabaci or other indigenous insects, influences the ecosystem.The catastrophe mechanism of further investigation exotic invasive species and anti-control techniques thereof are the bases and key of this global problem of science reply biotic intrusion.At present, the international conference of holding at a kind of Agricultural pests is also few, and international Bemisia tabaci conference has been held 4 times up to now.In recent years, endosymbiosis bacterium becomes one of hot issue of domestic and international Bemisia tabaci catastrophe Mechanism Study to the influence and the mechanism thereof of Type B Bemisia tabaci, and this is for utilizing endosymbiosis bacterium control Bemisia tabaci (Type B) population to have important potential value.
Because endosymbiosis bacterium can not external a large amount of cultivations, simultaneously Bemisia tabaci adults and pseudo-pupa individuality less (＜1mm), this becomes the bottleneck that technology was studied and utilized to endosymbiosis bacterium and Bemisia tabaci mutually.Just because of the existence of above-mentioned technical bottleneck, for a long time, all adopt the method for antibiotic treatment to confirm the fitness influence of endosymbiosis bacterium to the research of Bemisia tabaci endosymbiosis bacterium both at home and abroad to host's Bemisia tabaci.There is multiple shortcoming in this method, and for example: 1) microbiotic suppresses and can not eradicate the purpose endosymbiosis bacterium, and is the endosymbiosis bacterium that suppresses all, thereby can not determine the biological function of concrete certain endosymbiosis bacterium; 2) the purpose endosymbiosis bacterium can not be changed over to the Bemisia tabaci population, thereby hinder research the potential utility value of endosymbiosis bacterium.In the experimentation, the method that the use hold-down bars is fixed other insects is improper for miniature insect, slides easily because miniature insect is individual; The method that double faced adhesive tape is fixed other insects is improper for miniature insect, causes its death because double faced adhesive tape clings these insects easily.Therefore, how exogenesis endosymbiosis bacterium transfection timely and effectively being arrived in the Bemisia tabaci body, is that the problem that presses for solution in the research is made and utilized to current endosymbiosis bacterium and Bemisia tabaci mutually.In addition, when carrying out other tests of Bemisia tabaci at present both at home and abroad, adopt postparalytic Bemisia tabaci adults as the RNAi test injection, and Bemisia tabaci adults is revived soon, bad fixing, and the Bemisia tabaci adults after fixing is dead easily, also to be influence to Bemisia tabaci experimentize needs the problem that solves in the process for this.
(3) summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of method with horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius is provided.
The technical system of horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius is with behind the stripped purifying of rice moth (Corcyra cephalonica) endosymbiosis bacterium Wal Pasteur's body (Wolbachia) among the present invention, utilize the microinjection technique transfection in the Bemisia tabaci body, biological study shows that this endosymbiosis bacterium can vertical transmission.
The noun note:
SPG damping fluid: be 218mM sucrose, 3.8mM KH
2PO
4, 7.2mM K
2HPO
4, 4.9mM L-paddy amino acid, the damping fluid of mixed configuration, pH=7.2.
A kind of method with horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius, comprise the cultivation of the pseudo-pupa acceptor of Bemisia tabaci after the preparation, injection, injection of the pseudo-pupa acceptor of preparation, Bemisia tabaci of exogenesis endosymbiosis bacterium Wal Pasteur's body (Wolbachia), it is characterized in that the preparation concrete steps of the pseudo-pupa acceptor of Bemisia tabaci are as follows:
Double faced adhesive tape simultaneously is attached on the clean slide glass,, exposes the glue face of 1mm at the gummed paper of the about 1mm of the edge of double faced adhesive tape excision; Gather pseudo-pupa of Bemisia tabaci and back and upwards be fixed on this glue face, promptly get the pseudo-pupa acceptor of Bemisia tabaci.
Preferably, the preparation concrete steps of described exogenesis endosymbiosis bacterium Wal Pasteur's body (Wolbachia) are as follows:
The ovum of rice moth is utilized SPG damping fluid rinsing 4-8 time; In sterile tube, use the SPG damping fluid that the ovum of rice moth is carried out homogenate, the centrifugal 5-10min of 300g, supernatant liquor is transferred in the clean 0.2mL sterile tube, and the centrifugal 10-20min of 12000g removes supernatant liquor, to precipitate with the SPG damping fluid and to suspend and at the centrifugal 5min of 300g, get supernatant, be the endosymbiosis bacterium of purifying, at room temperature preserve the endosymbiosis bacterium of purifying standby.
Preferably, described injecting step is specific as follows:
The cut-off footpath is less than the syringe needle of 20 μ m; On IM 300 injection instrument, the exogenesis endosymbiosis bacterium Wal Pasteur's body that makes is injected into respectively in the pseudo-pupa acceptor of the Bemisia tabaci that makes, the pseudo-pupa of Bemisia tabaci behind the horizontal transfection.
Preferably, the concrete steps of the cultivation of the pseudo-pupa acceptor of described injection back Bemisia tabaci are as follows:
The pseudo-pupa of Bemisia tabaci behind the horizontal transfection is put in the weather incubator of 27 ℃ of constant temperature, humidity (RH) 60%-80%; Cultivated 24 hours, and gathered the emergence adult then;
Preferred, in the preparation process of exogenesis endosymbiosis bacterium Wal Pasteur's body (Wolbachia), the described room temperature preservation time is 0-5 hour.
The application of a kind of endosymbiosis bacterium Wal Pasteur's body (Wolbachia) in preventing and treating Bemisia tabaci.
In the above-mentioned steps, except that specified otherwise, all adopt this area routine techniques.
Can obtain containing the Bemisia tabaci of exogenesis endosymbiosis bacterium Wal Pasteur's body (Wolbachia) by the inventive method, and can set up the offspring population, this population can vertical transmission.
The present invention selects the acceptor of the pseudo-pupa of Bemisia tabaci as injection first, has solved the shortcoming of using adult to be difficult for injection as injection worm attitude for a long time; Founded the pseudo-pupa fixing means of miniature insect first, overcome the shortcoming of using easy problem of sliding of hold-down bars polypide and double faced adhesive tape to cling adult easily, made that polypide surviving rate and the exogenesis endosymbiosis bacterium infection rate after the injection improves greatly.Present method is simple, quick, the system that makes up can satisfy the demand that Wal Pasteur's body (Wolbachia) and Bemisia tabaci are made Mechanism Study mutually, for utilizing Wal Pasteur's body (Wolbachia) and other endosymbiosis bacteriums control Bemisia tabaci population or utilizing RNAi technical study Bemisia tabaci population that the good technical platform is provided.
(4) description of drawings
Fig. 1 has the slide glass that double faced adhesive tape does not stick the pseudo-pupa of Bemisia tabaci;
Fig. 2 sticks the slide glass side-view that the pseudo-pupa of Bemisia tabaci is arranged;
Wherein: 1, slide glass, 2, double faced adhesive tape, 3, the pseudo-pupa of Bemisia tabaci, 4, the double faced adhesive tape paper, 5, the double faced adhesive tape adhesive tape.
(5) embodiment
Below in conjunction with embodiment the present invention is further elaborated, but content that the present invention protects is not limited only to this.
Embodiment 1:
A kind of method with horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius comprises the cultivation of the pseudo-pupa acceptor of Bemisia tabaci after the preparation, injection, injection of the pseudo-pupa acceptor of preparation, Bemisia tabaci of exogenesis endosymbiosis bacterium Wal Pasteur's body (Wolbachia), and concrete steps are as follows:
Ovum with rice moth is the donor of fungal component Wal Pasteur's body (Wolbachia), the ovum of 30 μ L rice moths is placed in the 200uL sterile tube, with SPG damping fluid (218mM sucrose, the 3.8mM KH of 100uL
2PO
4, 7.2mM K
2HPO
4, 4.9mM L-paddy amino acid, pH7.2) rinsing is 4 times; In the sterile tube of 0.2mL, use the SPG damping fluid of 100uL that ovum is carried out homogenate, the centrifugal 5min of 300g transfers to the centrifugal 10min of 12000g in the new pipe (0.2mL) with supernatant liquor, and centrifugal back supernatant liquor is removed, and will precipitate and use the SPG damping fluid to suspend again;
With the centrifugal 5min of suspension 300g, get supernatant liquor, room temperature preservation is used for injection and uses; Injection in the 5h; The preparation of the pseudo-pupa acceptor of Bemisia tabaci, concrete steps are as follows:
Double faced adhesive tape simultaneously is attached on the clean slide glass,, exposes the glue face of 1mm at the gummed paper of the about 1mm of the edge of double faced adhesive tape excision; Gather pseudo-pupa of Bemisia tabaci and back and upwards be fixed on this glue face, promptly get the pseudo-pupa acceptor of Bemisia tabaci.As shown in Figure 2;
Pin device (Narishige PN-30, Narishige Scientific Instrument Lab., Tokyo are drawn in use, Japan) will draw pin (TW100F-4, World Precision Instruments, Inc., Sarasota FL) pull out syringe needle, card grinding makes syringe needle less than 20 μ m; (Narishige Scientific Instrument Lab., Tokyo inject on Japan) at IM 300 injection instrument;
Injection back individuality is put in the culture dish of preserving moisture, and is placed in the weather incubator of 27 ℃ of constant temperature, humidity (RH) 60%-80%; Cultivated 24 hours, and gathered the emergence adult then; Make the eclosion rate of the pupa that obtains in this way improve 55.5% (table 1) than method before improving.
The eclosion rate of the pupa that table 1 different fixing method obtains relatively
| Method | The injection number | The emergence number | Eclosion rate |
| Contrast method is improved one's methods | 504 2141 | 40 297 | 0.093986 0.141062 |
The detection of endosymbiosis bacterium, concrete steps are as follows:
27 ℃ of constant temperature are in the weather incubator of humidity (RH) 60%-80%, in the little worm cage of imago breeding on host plant of sprouting wings; Utilize the technical system of optimizing (table 2) to carry out biological study, and its offspring's (table 4) of adult (table 3) is carried out the detection of endosymbiosis bacterium.
Table 2 is through the survival rate after the different treatment processs
| Treatment process | Injection pupa number | Emergence number (%) | |
| 1 2 3 4 5 6 7 8 | Syringe needle diameter (external diameter)＞20 μ m (pseudo-pupa) syringe needle diameter (external diameter)≤20 μ m (pseudo-pupa) injection pupa be non-pseudo-pupa (≤20 μ m) injection pupa be pseudo-pupa (≤20 μ m) injection back use paraffin oil to seal to be placed on after the wound injection cultivate in the biochemical incubator injection back culture temperature after 16-20 ℃ of injection culture temperature at 20-25 ℃ | 69 75 179 85 320 219 433 449 | 0(0) 10(13.3％) 0(0) 19(22.4％) 0(0) 0(0) 14(3.2％) 74(16.6％) |
By a large amount of tests, we find that the height estimation of eclosion rate is relevant with the syringe needle diameter and the culture temperature of the size in the length of time of injecting pupa, entry needle, and the length of time of pupa is big more, and the eclosion rate after the injection is high more; The diameter of syringe needle is more little, and the eclosion rate after the injection is high more; Optimal temperature can provide eclosion rate.Therefore, the basic optimum condition of the microinjection of Bemisia tabaci pupa is syringe needle as far as possible little (≤20 μ m); The injection pupa is preferably selected pseudo-pupa (be characterized as blood-shot eye illness occurs, individuality is bigger); Culture temperature is preferably constant in 20-25 ℃, changes smaller.
Table 3 injection result statistics
| Inject time | Injection pupa number | Eclosion rate | The raising rate | Wal Pasteur's body (Wolbachia) verification and measurement ratio |
| 2007.4.9 2007.4.10 2007.4.17 2007.4.18 2007.4.19 2007.4.20 2007.4.30 2007.5.3 2007.5.9 2007.5.14 | 34 40 103 46 98 30 38 33 22 60 | 32.4％ (11/34) 15％(6/40) 2.9％ (3/103) 21.7％ (10/46) 14.3％ (14/98) 3.3％ (1/30) 23.7％ (9/38) 3.0％ (1/33) 22.7％ (5/22) 3.3％ (2/60) | 81.8％(9/11) 66.7％(4/6) 66.7％(2/3) 70％(7/10) 57.1％(8/14) 100％(1/1) 44.4％(4/9) 100％(1/1) 60％(3/5) 50％(1/2) | 27.2％(3/11) 0(0/6) 100％(1/1) 30％(3/10) 14.3％(2/14) 0(0/1) 75％(3/4) 100％(1/1) 40％(2/5) 50％(1/2) |
By above table, eclosion rate is between 2.9%-32.4% as can be seen, and most of about 20%, average eclosion rate is 14.3%.Wal Pasteur's body (Wolbachia) average detected rate is 43.7%.By this test as can be seen, the verification and measurement ratio of the eclosion rate of the pupa of process injection and Wal Pasteur's body (Wolbachia) has certain regularity.
Table 4F
0Produce quantity and adult Wal Pasteur's body (Wolbachia) infection rate of adult
| Raise the date | Life-span (my god) | Biotype | Wol has or not | Offspring's pupa number | Emergence number (rate) | Wol has or not | Detect number (infection rate) |
| 10.10 10.10 10.10 | 2 15 6 | B B B | + + + | 57 102 15 | 50(87.71％) 96(94.12％) 11(73.33％) | + - + | 10/10(100％) 9/10(90％) 8/10(80％) |
By above table, the offspring has higher infection rate 80%-100% as can be seen, and this illustrates that this technology can satisfy the research of endosymbiosis bacterium to bemisia tabaci gennadius horizontal transfection.
Claims (5)
1. the method for a horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius, comprise the cultivation of the pseudo-pupa acceptor of Bemisia tabaci after the preparation, injection, injection of the pseudo-pupa acceptor of preparation, Bemisia tabaci of exogenesis endosymbiosis bacterium Wal Pasteur's body, it is characterized in that the preparation concrete steps of the pseudo-pupa acceptor of Bemisia tabaci are as follows:
Double faced adhesive tape simultaneously is attached on the clean slide glass,, exposes the glue face of 1mm at the gummed paper of the edge of double faced adhesive tape excision 1mm; Gather pseudo-pupa of Bemisia tabaci and back and upwards be fixed on this glue face, promptly get the pseudo-pupa acceptor of Bemisia tabaci.
2. the method for horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius as claimed in claim 1 is characterized in that, the preparation concrete steps of described exogenesis endosymbiosis bacterium Wal Pasteur's body are as follows:
The ovum of rice moth is utilized SPG damping fluid rinsing 4-8 time; In sterile tube, use the SPG damping fluid that the ovum of rice moth is carried out homogenate, the centrifugal 5-10min of 300g, supernatant liquor is transferred in the clean 0.2mL sterile tube, and the centrifugal 10-20min of 12000g removes supernatant liquor, to precipitate with the SPG damping fluid and to suspend and at the centrifugal 5min of 300g, get supernatant, be the endosymbiosis bacterium of purifying, at room temperature preserve the endosymbiosis bacterium of purifying standby.
3. the method for horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius as claimed in claim 1 is characterized in that, described injecting step is specific as follows:
The cut-off footpath is less than the syringe needle of 20 μ m; On IM 300 injection instrument, the exogenesis endosymbiosis bacterium Wal Pasteur's body that makes is injected into respectively in the pseudo-pupa acceptor of the Bemisia tabaci that makes, the pseudo-pupa of Bemisia tabaci behind the horizontal transfection.
4. the method for horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius as claimed in claim 1 is characterized in that, the concrete steps of the cultivation of the pseudo-pupa acceptor of described injection back Bemisia tabaci are as follows:
The pseudo-pupa of Bemisia tabaci behind the horizontal transfection is put in the weather incubator of 27 ℃ of constant temperature, humidity (RH) 60%-80%; Cultivated 24 hours, and gathered the emergence adult then.
5. the method for horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius as claimed in claim 2 is characterized in that, in the preparation process of exogenesis endosymbiosis bacterium Wal Pasteur's body, the described room temperature preservation time is 0-5 hour.
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| CN102212522A (en) * | 2011-04-29 | 2011-10-12 | 山东省农业科学院高新技术研究中心 | Method for preventing and treating bemisa babaci by utilizing RNA (Ribose Nucleic Acid) interference technology |
| CN102349473A (en) * | 2011-08-11 | 2012-02-15 | 广州沃巴克生物科技有限公司 | Method for quickly and effectively transfecting mosquito by using wolbachia |
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| CN102212522B (en) * | 2011-04-29 | 2012-08-22 | 山东省农业科学院高新技术研究中心 | Method for preventing and treating bemisa babaci by utilizing RNA (Ribose Nucleic Acid) interference technology |
| CN102349473B (en) * | 2011-08-11 | 2013-06-19 | 郑小英 | Method for quickly and effectively transfecting mosquito by using Wolbachia |
| CN102349473A (en) * | 2011-08-11 | 2012-02-15 | 广州沃巴克生物科技有限公司 | Method for quickly and effectively transfecting mosquito by using wolbachia |
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| CN103387945B (en) * | 2012-05-09 | 2015-01-07 | 中国农业科学院植物保护研究所 | Extraction method of Tobacco whitefly endosymbiont mycetocytes |
| CN105532581A (en) * | 2015-12-16 | 2016-05-04 | 中国计量学院 | In-vitro study method of insect symbiotic bacterium transovarial vertical transmission |
| CN105532581B (en) * | 2015-12-16 | 2019-04-05 | 中国计量大学 | A method of insect fungal component is studied in vitro through ovum vertical transmission |
| WO2018213970A1 (en) * | 2017-05-22 | 2018-11-29 | 广州威佰昆生物科技有限公司 | Brown planthopper and production method therefor |
| CN111264475A (en) * | 2020-03-09 | 2020-06-12 | 沈阳农业大学 | Microinjection method for small insect adults of whiteflies |
| 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 |
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