CN110241179A - The method that the distribution of fungal component Cardinium in planthopper different tissues is positioned - Google Patents
The method that the distribution of fungal component Cardinium in planthopper different tissues is positioned Download PDFInfo
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Abstract
The invention discloses the methods that the distribution to fungal component Cardinium in planthopper different tissues is positioned, and belong to technical field of agro-ecology.The present invention dissects first and isolates the different tissues of planthopper；Then go out the oligonucleotide probe of three sections of specificity with fluorescent marker according to the 16S rRNA sequence design of Cardinium；Finally Cardinium present in planthopper is hybridized using specific probe, quickly the Cardinium in planthopper tissue accurately can be positioned by the scanning of laser confocal microscope.Localization method of the present invention has specificity, sensibility, feasibility and the validity of height, while cost-saved.The present invention is conducive to preferably study the interaction relationship between Cardinium and host, it helps the strategy of research compacting pest population and the mechanism for resisting viral transmission.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to divide fungal component Cardinium in planthopper different tissues
The method that cloth is positioned.
Background technique
Cardinium is a kind of positioned at the intracellular Gram-negative Endosymbiotic bacterium that can carry out maternal propagation.It
Be widely present in many arthropods, in some important agricultural pests such as planthopper, tetranychid, Bemisia tabaci and parasitic wasp
It was found that the presence of Cardinium.They can reproduction to host and development regulate and control, including cytoplasm incompatibility
(Cytoplasmic incompatibility, CI), single-female generation (parthenogenesis inducing), feminize
(Feminization) and gametocide (Male-killing).Wherein, it is by the most important reproductive phenotypes that Cardinium is induced
CI.The generation of CI is related with infecting for fungal component, and when the female adult being uninfected by mates with the male worm of infection, generated offspring is not
It being capable of normal incubation.It is widely distributed in the various tissues of insect that the generation of these phenotypes mainly has benefited from Cardinium.
Wolbachia is another endosymbiosis bacterium, is also widely present in arthropod, and Wolbachia plants some
System has been transfected in mosquito and drosophila.People have understood them in place by positioning to the Wolbachia in host
Distribution in main tissue, it is determined that the 100% vertical transmission rate that they have, to be conducive to preferably research compacting
The strategy of pest population and the mechanism for resisting viral transmission.However, distribution of the Cardinium in some important pests tissues
But it is not described well.
White backed planthopper Sogatella furcifera (Horv á th) and brown paddy plant hopper Nilaparvata lugensIt is
Two kinds can migrating property over long distances pest, the rice of Asia cultivation region is caused to seriously endanger.White backed planthopper can be with natural sense
Dye Cardinium and the CI for inducing host strong；Although brown paddy plant hopper does not infect Cardinium under natural conditions, by manually turning
The method of dye can also stablize infection Cardinium.
However, the distribution of Cardinium in the tissue is accurately positioned not yet in two kinds of planthoppers.Therefore, we
It is badly in need of a kind of method to position the Cardinium being distributed in planthopper, to be conducive to preferably study fungal component and elder brother
Interaction relationship between worm.
Summary of the invention
The present invention provides the method positioned to the distribution of fungal component Cardinium in planthopper different tissues, utilizes
This method can intuitively understand the dependent interaction relationship of Cardinium and planthopper.
To the method that the distribution of fungal component Cardinium in planthopper different tissues is positioned, following step is specifically included
It is rapid:
(1) dissection and separation of planthopper tissue: planthopper adult is collected, carefully solution cuts plant hopper group under the microscope
It knits, they is separated in the centrifuge tube equipped with 1 × PBS；
(2) design of oligonucleotide probe: going out three sections according to the 16S rRNA sequence design of Cardinium has fluorescence mark
The oligonucleotide probe of the specificity of note；
(3) Cardinium hybridizes in oligonucleotide probe and planthopper tissue: by the tissue of step (1) dissection with 4%
Paraformaldehyde fix, cleaning is placed in hybridization solution overnight, after cleaning respectively with phalloidine and DAPI to cytoskeleton and
Nuclear targeting, with anti-fluorescence quencher mounting after cleaning again；
(4) observation under laser confocal microscope: adjusting the excitation wavelength and launch wavelength of laser confocal microscope,
It, can be to fungal component Cardinium in planthopper different tissues respectively to Cardinium, cytoskeleton and cell Nuclear receptor co repressor
Distribution is accurately positioned.
Further, planthopper described in (1) is white backed planthopper or brown paddy plant hopper.
Further, tissue described in (1) is one of salivary gland, ovary, spermary, enteron aisle or a variety of.
Further, in described (2), the oligonucleotide probe with specificity of three sections of fluorescent markers are as follows:
C162:5'-ATC TTT CCA GCA TGC GCT-3'(SEQ ID NO.1)；
C587:5'-CAA TCG CAG TTC TAG CGT TA-3'(SEQ ID NO.2)；
C997:5'-GCA CCT TGT ATT CCG TCC-3'(SEQ ID NO.3)；
SEQ ID NO.1~3 is held in sequence oligonucleotide probe 5 ' with Cy5 and is marked.
Further, in described (3), oligonucleotide probe and planthopper organize in Cardinium hybridize, specifically include
Following steps:
1. 4% paraformaldehyde is fixed: with 1 × PBS by tissue wash 2 times of dissection, removing residual liquid, about 1ml is added
The fixed 30min of 4% paraformaldehyde tissue, remove paraformaldehyde, then cleaned 2-3 times with 1 × PBS；
2. dark in nucleic acid hybridization solution stay overnight: three sections of specific oligonucleotides are added to the nucleic acid hybridization solution prepared and visit
Needle is added it to and is filled in organized centrifuge tube, and hybridization solution did not had tissue, this operating process need to be protected from light, in 46 DEG C of water
It is dark in bath to stay overnight；
3. cleaning hybridization solution: washing hybridization solution, take 2 × SSC (0.015% (w/v) DTT), 1 × SSC respectively
(0.015% (w/v) DTT), 0.5 × SSC (0.015% (w/v) DTT) respectively cleaning 1 time, then washed 1 time with PBS, it is placed in tissue
It is centrifuged bottom of the tube；
4. contaminating cytoskeleton: by phalloidine with 1 × PBS dilute 200 times, into centrifuge tube be added 700ml dilution into
Row dyeing, when dyeing, is 1 hour a length of, is then cleaned 3 times with 1 × PBS, is operated again in the case where being protected from light；
5. contaminating nucleus: DAPI is added into centrifuge tube, liquid did not had tissue, it is placed in dark 1 hour, with 1 ×
PBS is cleaned 3 times, is operated in the case where being protected from light；
6. anti-fluorescence quencher mounting: tissue being placed on glass slide, good position is adjusted, anti-fluorescence quencher envelope is added
Piece.
Further, it is described 2. in, the method for preparing hybrid liquid, using as following formula:
Further, it is described 2. in, the dilution ratio of probe and hybridization solution is 1:50~200, it is preferred that probe with hybridize
The dilution ratio of liquid is 1:100.
Further, in described (4), used excitation wavelength and launch wavelength are as follows:
Cadinium:Cy5,645~670nm；
Nucleus: DAPI, 359~461nm；
Cytoskeleton: FITC, 494~518nm.
The beneficial effects of the invention are that:
The present invention is led to by the different tissues of dissection planthopper using three sections of Cardinium specific oligonucleotide probes
The observation for crossing laser co-focusing positions Cardinium present in different tissues in planthopper.
The present invention by the detection method of Cardinium in three kinds of planthoppers (Cardinium in PCR qualitative detection planthopper,
Cardinium and Electronic Speculum observe Cardinium present in planthopper in qPCR quantitative detection planthopper) it combines,
Can with fast qualitative, it is quantitative, intuitively Cardinium present in the various tissues of planthopper is detected and is positioned.
Simultaneously the invention avoids the method dyed by immunohistochemistry to Cardinium, can save a large amount of
Expense.
Method of the present invention to Cardinium positioning has the specificity of height, sensibility, feasibility and effectively
Property, i.e., it is cost-saved, and intuitively the distribution of Cardinium can positioned within a short period of time.
The present invention also helps the relationship for intuitively observing Cardinium and host cell, this is conducive to preferably study
Interaction relationship between Cardinium and host, it helps preferably research is with suppressing pest population tactful and supports
The mechanism of antiviral propagation.
Detailed description of the invention
Fig. 1 a~Fig. 1 d is respectively distribution shape of the Cardinium in white backed planthopper ovarian duct, spermary, salivary gland and enteron aisle
Condition.
Fig. 2 a~Fig. 2 d is respectively distribution shape of the Cardinium in brown paddy plant hopper ovarian duct, spermary, salivary gland and enteron aisle
Condition.
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1: the distribution of fungal component Cardinium is positioned in Whitebacked Planthopper different tissues.
(1) dissection and separation of white backed planthopper tissue
It collects 20 mature white backed planthopper female adults and male worm, the plant hopper that will be dissected is placed in clean culture dish,
With dissecting forceps, carefully solution cuts the tissue such as salivary gland, ovary, spermary, enteron aisle under microscope, be transferred to containing 1 ×
In the test tube of PBS.
(2) design of oligonucleotide probe
It is visited according to the oligonucleotides that the 16S rRNA sequence design of Cardinium goes out three sections of specificity with fluorescent marker
Needle is marked at 5 ' ends of probe sequence with Cy5, is SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively.
(3) oligonucleotide probe hybridizes with Cardinium in white backed planthopper
Oligonucleotide probe hybridizes with Cardinium in white backed planthopper, specifically includes the following steps:
(1) 4% paraformaldehyde is fixed
With 1 × PBS by tissue wash 2 times of dissection, residual liquid is removed, the 4% paraformaldehyde tissue that about 1ml is added is solid
Determine 30min, removes paraformaldehyde, then cleaned 2-3 times with 1 × PBS；
(2) dark in nucleic acid hybridization solution to stay overnight
Nucleic acid hybridization solution is prepared first, and hybridization solution is specifically formulated referring to following table.
Into prepared nucleic acid hybridization solution be added three sections of designed specific oligonucleotide probes, middle probe with it is miscellaneous
The dilution ratio for handing over liquid is 1:100.Carefully mixing liquid, which is added to, deposits in organized centrifuge tube, and hybridization solution did not had white backward flight
Lice tissue.This operating process is protected from light, and centrifuge tube is placed in 46 DEG C of water-baths dark stay overnight；
(3) hybridization solution is cleaned
Hybridization solution is washed, takes 2 × SSC (0.015% (w/v) DTT), 1 × SSC (0.015% (w/ respectively with liquid-transfering gun
V) DTT), the tissue wash of 0.5 × SSC (0.015% (w/v) DTT) Whitebacked Planthopper, each cleaning 1 time, then number is washed with 1 × PBS
Time, until all remaining hybridization solutions are removed, clean white backed planthopper tissue is deposited in test tube bottom.Whole process is being protected from light lower progress
Operation.
(4) cytoskeleton is contaminated
1 × PBS of phalloidine is diluted 200 times, 700ml dilution is added into centrifuge tube and is dyed.It will centrifugation
Pipe, which is put into, to be wanted slowly to shake on bed, and the time is 1 hour.Remove residual liquid, is cleaned 3 times with 1 × PBS.Whole process in the case where being protected from light into
Row operation.
(5) dye dye nucleus
DAPI is added into centrifuge tube, liquid did not had tissue.Centrifuge tube is placed in dark 1 hour at room temperature, then
It is cleaned 3 times with PBS.Whole process is operated in the case where being protected from light.
(6) anti-fluorescence quencher mounting
White backed planthopper tissue is placed on glass slide, good position is adjusted, anti-fluorescence quencher is added, covered is used in combination
Nail sheet for oil seal.
(4) observation under laser confocal microscope
The excitation wavelength for adjusting laser co-focusing, is respectively scanned Cardinium, cytoskeleton and nucleus,
It can be accurately positioned, as shown in Figure 1, wherein Fig. 1 a~Fig. 1 d is respectively Cardinium in white backed planthopper ovarian duct, essence
Distribution situation in nest, salivary gland and enteron aisle is Cardinium at arrow.
Used excitation wavelength and launch wavelength:
Cadinium:Cy5,645~670nm；
Nucleus: DAPI, 359~461nm；
Cytoskeleton: FITC, 494~518nm.
Embodiment 2: the distribution of fungal component Cardinium in brown paddy plant hopper different tissues is positioned.
(1) dissection and separation of brown paddy plant hopper tissue
It collects 20 mature brown paddy plant hopper female adults and male worm, the plant hopper that will be dissected is placed in clean culture dish, aobvious
With dissecting forceps, carefully solution cuts the tissue such as salivary gland, ovary, spermary, enteron aisle under micro mirror, is transferred to the examination containing PBS
Guan Zhong.
(2) design of oligonucleotide probe
It is visited according to the oligonucleotides with specificity that the 16S rRNA sequence design of Cardinium goes out three sections of fluorescent markers
Needle is marked at 5 ' ends of probe sequence using Cy5, is SEQ ID NO.1, SEQ ID NO.2 and SEQ ID respectively
NO.3。
(3) oligonucleotide probe hybridizes with Cardinium in brown paddy plant hopper
Oligonucleotide probe hybridizes with Cardinium in brown paddy plant hopper, specifically includes the following steps:
(1) 4% paraformaldehyde is fixed
With 1 × PBS by tissue wash 2 times of dissection, residual liquid is removed, the 4% paraformaldehyde tissue that about 1ml is added is solid
Determine 30min, then remove paraformaldehyde, is cleaned 2-3 times with 1 × PBS；
(2) dark in nucleic acid hybridization solution to stay overnight
Nucleic acid hybridization solution is prepared first, and hybridization solution is specifically formulated referring to following table.
Into prepared nucleic acid hybridization solution be added three sections of designed specific oligonucleotide probes, middle probe with it is miscellaneous
The dilution ratio for handing over liquid is 1:100.Carefully mixing liquid is added in the centrifuge tube for having brown paddy plant hopper tissue, and hybridization solution did not had
Tissue.This operating process is protected from light, and centrifuge tube is placed in 46 DEG C of water-baths dark stay overnight；
(3) hybridization solution is cleaned
Hybridization solution is washed, takes 2 × SSC (0.015% (w/v) DTT), 1 × SSC (0.015% (w/ respectively with liquid-transfering gun
V) DTT), 0.5 × SSC (0.015% (w/v) DTT) is to the tissue wash of brown paddy plant hopper, each cleaning 1 time, then washes number with 1 × PBS
Time, until all hybridization solutions are removed, clean brown paddy plant hopper tissue is deposited in test tube bottom.Whole process is operated in the case where being protected from light.
(4) cytoskeleton is contaminated
1 × PBS of phalloidine is diluted 200 times, 700ml dilution is added into centrifuge tube and is dyed.It will centrifugation
Pipe, which is put into, to be wanted slowly to shake on bed, and the time is 1 hour.Remove residual liquid, is cleaned 3 times with 1 × PBS.Whole process in the case where being protected from light into
Row operation.
(5) dye dye nucleus
DAPI is added into centrifuge tube, liquid did not had tissue.Centrifuge tube is placed in dark 1 hour at room temperature, then
It is cleaned 3 times with 1 × PBS.Whole process is operated in the case where being protected from light.
(6) anti-fluorescence quencher mounting
Brown paddy plant hopper tissue is placed on glass slide, good position is adjusted, is added anti-fluorescence quencher, covered and with referring to
Nail polish mounting.
(4) observation under laser confocal microscope
The excitation wavelength for adjusting laser co-focusing, is respectively scanned Cardinium, cytoskeleton and nucleus,
It can be accurately positioned, as shown in Fig. 2, Fig. 2 a~Fig. 2 d is respectively Cardinium in brown paddy plant hopper ovarian duct, spermary, saliva
Distribution situation in gland and enteron aisle is Cardinium at arrow.
Used excitation wavelength and launch wavelength:
Cadinium:Cy5,645~670nm；
Nucleus: DAPI, 359~461nm；
Cytoskeleton: FITC, 494~518nm.
Sequence table
<110>Agricultural University Of Nanjing
<120>method that the distribution of fungal component Cardinium in planthopper different tissues is positioned
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atctttccag catgcgct 18
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
caatcgcagt tctagcgtta 20
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gcaccttgta ttccgtcc 18
Claims (8)
1. the method that the distribution of fungal component Cardinium is positioned in pair planthopper different tissues, which is characterized in that including with
Lower step:
(1) dissection and separation of planthopper tissue: collecting planthopper adult, and carefully solution cuts plant hopper tissue under the microscope,
They are separated in the centrifuge tube equipped with 1 × PBS；
(2) design of oligonucleotide probe: going out three sections according to the 16S rRNA sequence design of Cardinium has fluorescent marker
The oligonucleotide probe of specificity；
(3) Cardinium hybridizes in oligonucleotide probe and planthopper tissue: by the tissue of step (1) dissection with 4% it is more
Polyformaldehyde is fixed, and cleaning is placed in hybridization solution overnight, respectively with phalloidine and DAPI to cytoskeleton and cell after cleaning
Nuclear staining, with anti-fluorescence quencher mounting after cleaning again；
(4) observation under laser confocal microscope: adjusting the excitation wavelength and launch wavelength of laser confocal microscope, respectively
It, can be to the distribution of fungal component Cardinium in planthopper different tissues to Cardinium, cytoskeleton and cell Nuclear receptor co repressor
It is accurately positioned.
What 2. the distribution according to claim 1 to fungal component Cardinium in planthopper different tissues was positioned
Method, which is characterized in that (1) planthopper described in is white backed planthopper or brown paddy plant hopper.
What 3. the distribution according to claim 1 to fungal component Cardinium in planthopper different tissues was positioned
Method, which is characterized in that (1) tissue described in is one of salivary gland, ovary, spermary, enteron aisle or a variety of.
What 4. the distribution according to claim 1 to fungal component Cardinium in planthopper different tissues was positioned
Method, which is characterized in that in (2), the oligonucleotide probe with specificity of three sections of fluorescent markers are as follows:
C162:5’-ATC TTT CCA GCA TGC GCT-3’(SEQ ID NO.1),
C587:5’-CAA TCG CAG TTC TAG CGT TA-3’(SEQ ID NO.2),
C997:5'-GCA CCT TGT ATT CCG TCC-3'(SEQ ID NO.3)；
SEQ ID NO.1~3 is held in sequence oligonucleotide probe 5 ' with Cy5 and is marked.
What 5. the distribution according to claim 1 to fungal component Cardinium in planthopper different tissues was positioned
Method, which is characterized in that in (3), oligonucleotide probe and planthopper organize in Cardinium hybridize, specifically include
Following steps:
1. 4% paraformaldehyde is fixed: with 1 × PBS by tissue wash 2 times of dissection, residual liquid is removed, more than 4% that 1ml is added
Polyformaldehyde tissue fixes 30min, removes paraformaldehyde, then cleaned 2-3 times with 1 × PBS；
2. dark in nucleic acid hybridization solution stay overnight: three sections of specific oligonucleotide probes are added to the nucleic acid hybridization solution prepared,
It adds it to and fills in organized centrifuge tube, hybridization solution did not had tissue, this operating process need to be protected from light, in 46 DEG C of water-baths
Middle dark is overnight；
3. cleaning hybridization solution: washing hybridization solution, take 2 × SSC (0.015% (w/v) DTT), 1 × SSC (0.015% (w/ respectively
V) DTT), 0.5 × SSC (0.015% (w/v) DTT) respectively cleaning 1 time, then washed 1 time with PBS, so that tissue is placed in centrifugation bottom of the tube；
4. contaminating cytoskeleton: 1 × PBS of phalloidine being diluted 200 times, 700ml dilution is added into centrifuge tube and is contaminated
Color, when dyeing, are 1 hour a length of, are then cleaned 3 times with 1 × PBS, are operated again in the case where being protected from light；
5. contaminating nucleus: DAPI being added into centrifuge tube, liquid did not had tissue, was placed in dark 1 hour, clear with 1 × PBS
It washes 3 times, is operated in the case where being protected from light；
6. anti-fluorescence quencher mounting: tissue being placed on glass slide, good position is adjusted, anti-fluorescence quencher mounting is added.
What 6. the distribution according to claim 5 to fungal component Cardinium in planthopper different tissues was positioned
Method, which is characterized in that it is described 2. in, the method for preparing hybrid liquid, using as following formula:
。
What 7. the distribution according to claim 5 to fungal component Cardinium in planthopper different tissues was positioned
Method, which is characterized in that it is described 2. in, the dilution ratio of probe and hybridization solution is 1:50~200, it is preferred that probe with hybridize
The dilution ratio of liquid is 1:100.
What 8. the distribution according to claim 1 to fungal component Cardinium in planthopper different tissues was positioned
Method, which is characterized in that in (4), used excitation wavelength and launch wavelength are as follows:
Cadinium:Cy5,645~670nm；
Nucleus: DAPI, 359~461nm；
Cytoskeleton: FITC, 494~518nm.
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| ZHEN ZENG 等: "Bacterial endosymbiont Cardinium cSfur genome sequence provides insights for understanding the symbiotic relationship in Sogatella furcifera host", 《BMC GENOMICS》 * |
| 周晓飞: "烟粉虱内共生菌Wolbachia和Cardinium与宿主的互作及其对宿主的生物学效应", 《中国博士学位论文全文数据库 (农业科技辑)》 * |
| 张开军 等: "白背飞虱中的 Wolbachia 和 Cardinium 双重感染特性", 《昆虫学报》 * |
| 朱路雨等: "Wolbachia和Cardinium对皮氏叶螨生殖的影响及在寄主体内的定位", 《昆虫学报》 * |
| 武建国 等: "《医学实验诊断学进展 卷2 临床基因诊断》", 31 May 2002, 东南大学出版社 * |
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