CN103088142B - Method for detecting egg laying amount of insects by heat shock protein 90 gene - Google Patents
Method for detecting egg laying amount of insects by heat shock protein 90 gene Download PDFInfo
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- CN103088142B CN103088142B CN201310031965.2A CN201310031965A CN103088142B CN 103088142 B CN103088142 B CN 103088142B CN 201310031965 A CN201310031965 A CN 201310031965A CN 103088142 B CN103088142 B CN 103088142B
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Abstract
The invention discloses a method for detecting the egg laying amount of insects by a heat shock protein 90 gene, which provides application of a substance which is used for detecting the expression quantity of heat shock protein 90 encoding genes of insect populations eating different substances to detection of the egg laying amount of insect populations eating different substances, wherein the amino acid sequence of heat shock protein 90 is a sequence 2 in the sequence table. Experiments prove that the detection method provided by the invention can be used for detecting the egg laying amount of the insects and particularly the egg laying amount of arma chinensis with different nutrition sources, and the egg laying amount of the insects can be detected within only 2 to 3 days by the detection method while the detection requires 30 to 60 days by a traditional biological method. The detection method provided by the invention has the advantages that materials have wide sources and are easily available, and the operation is simple as well as time-saving and labor-saving, thus being suitable for popularization and application in insect commodity inspection and detection.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method that detects insect spawning amount with heat shock protein pane gene.
Background technology
Biological control of insect pests is being brought into play irreplaceable vital role in agroforestry Sustainable development.A large amount of productions of natural enemy insect are the most basic in biological control is especially also most important link.Predatory natural enemy insect Candle-sticks stinkbug (Armachinensis) can be controlled from multiple agriculture and forestry injurious insects such as lepidopteran, Coleoptera, Hemiptera, Homoptera, Hymenopteras, especially it can also control alien insect pest colorado potato bug and fall webworms, so Candle-sticks stinkbug is the good natural enemy insect commodity of a kind of application prospect.
As commodity, reducing in a large number production cost is the maximized approach of people's profit-push.In natural enemy insect Candle-sticks stinkbug produces, just can apply different food and produce Candle-sticks stinkbug, for example, different insect prey, containing the artificial diet of insect composition with not containing the artificial diet of insect composition, and then minimizing production cost.But the biological characteristics of applying different foodstuffs production Candle-sticks stinkbug is out uneven, some can reach the effect of good Control pests after discharging, have after release effect not remarkable.The Candle-sticks stinkbug of feeding due to different food is difficult to distinguish in phenotype, so these natural enemy insects are to be difficult to their biological characteristics of assessment before release.
The egg laying amount of Candle-sticks stinkbug adult is a very important index evaluating natural enemy insect biological characteristics.Have the Candle-sticks stinkbug population breeding of higher egg laying amount fast, the doubling time is short, can control rapidly in a short time insect.This can greatly reduce the cost of pest control, increases rate of profit.On the contrary, the Candle-sticks stinkbug that egg laying amount is lower is slow owing to breeding, and the doubling time extends, and the time of therefore controlling insect can relatively lag behind, and this has just increased cost accounting greatly, has reduced profit.At present, for the detection method of the commodity Candle-sticks stinkbug egg laying amount of different food sources or different businessmans be still whole adult stage or artificial its egg laying amount of specific time build-in test number, this method wastes time and energy expensive.Therefore need a kind of method of rapid detection natural enemy insect commodity biological characteristics-egg laying amount.
In insect body, the maturation of the ovocyte that heat shock protein 90 (heatshockprotein90) participation is mediated by progesterone.
Summary of the invention
An object of the present invention is to provide the purposes for detection of the material of heat shock protein 90 encoding gene expression amounts.
The invention provides the application in detecting insect spawning amount for detection of the material of heat shock protein 90 encoding gene expression amounts;
The aminoacid sequence of described heat shock protein 90 is the sequence 2 in sequence table.
In above-mentioned application, described insect is insect individuality or insect population;
Described insect population is specially and takes food different substances insect population; The described different substances insect population that takes food is further specially the insect population that takes food artificial diet or the insect population that takes food prey;
The nucleotides sequence of described heat shock protein 90 encoding genes is classified sequence 1 in sequence table or the sequence 1 in sequence table as from 5 ' end 2-331 position Nucleotide.
Described insect is specially Candle-sticks stinkbug.
Above-mentioned artificial diet are prepared as follows: live pig liver 60g, soyflour 10g, water 100ml, egg 20ml, casein food grade 2g, sucrose 8g, VC0.2g, choline chloride 60 0.4g, calcium pantothenate 8mg, folic acid 0.1mg, niacinamide 0.2mg, gentamicin 3.9mg; Concrete feeding method is shown in embodiment 2;
Above-mentioned prey is tussah (Antheraea pernyi) pupa; Concrete feeding method is shown in embodiment 2.
In above-mentioned application, the described material for detection of heat shock protein 90 encoding gene expression amounts is following 1)-3) in any one:
1) primer pair A: described primer pair is comprised of primer 1 and primer 2; The nucleotides sequence of described primer 1 is classified sequence 3 in sequence table as; The nucleotides sequence of described primer 2 is classified sequence 4 in sequence table as;
2) the RT-PCR reagent that contains described primer pair A;
3) test kit that contains described primer pair A or described RT-PCR reagent.
Another object of the present invention is to provide a kind of primer pair A.
Primer pair A provided by the invention, is comprised of primer 1 and primer 2;
The nucleotides sequence of described primer 1 is classified sequence 3 in sequence table as; The nucleotides sequence of described primer 2 is classified sequence 4 in sequence table as.
The RT-PCR reagent that contains above-mentioned primer pair A is also the scope of protection of the invention; Above-mentioned RT-PCR reagent is specifically comprised of water, RT-PCR amplification buffer, magnesium ion, dNTPs, above-mentioned primer pair A, fluorescence dye (SYBR GreenI) and Taq enzyme;
The final concentration of each primer in described primer pair A in described RT-PCR reagent is specially 0.2-1 μ M, and the final concentration of each primer in described primer pair A in described RT-PCR reagent is further specially 0.25 μ M.
The test kit that contains above-mentioned primer pair A or above-mentioned RT-PCR reagent is also the scope of protection of the invention.
Mentioned reagent box also comprises internal reference primer pair B, and described internal reference primer pair B is comprised of primer 3 and primer 4; The nucleotides sequence of described primer 3 is classified the sequence 6 in sequence table as; The nucleotides sequence of described primer 4 is classified the sequence 7 in sequence table as.
The 3rd object of the present invention is to provide a kind of method that detects insect spawning amount.
Method provided by the invention, comprises the steps:, with above-mentioned primer pair A or above-mentioned RT-PCR reagent or above-mentioned test kit, insect A to be measured and B are carried out to RT-PCR amplification;
If the expression amount of the heat shock protein pane gene of described insect A is higher than described insect B, described insect A egg laying amount is higher than described insect B.
In aforesaid method, described insect is insect individuality or insect population;
The cDNA that the template of described RT-PCR amplification is insect;
Described insect is specially Candle-sticks stinkbug.
In an embodiment of the present invention, described insect A is the insect population that takes food prey; Described insect B is the insect population that takes food artificial diet.
The 4th object of the present invention is to provide a kind of albumen or its encoding gene.
The aminoacid sequence of albumen provided by the invention is the sequence 2 in sequence table;
The nucleotides sequence of the encoding gene of albumen provided by the invention is classified sequence 1 in sequence table or the sequence 1 in sequence table as from 5 ' end 2-331 position Nucleotide.
Of the present inventionly experiment showed, that detection method provided by the present invention can detect by detecting heat shock protein pane gene expression amount the egg laying amount of Candle-sticks stinkbug of the insect spawning amount, particularly Different Nutrition source of different population.Experiment showed, with the egg laying amount of detection method detection insect of the present invention and only need 2-3 days.And need about 30-60 days with traditional biological method.The material source that detection method of the present invention relates to is wide, easily buys, simple to operate, saves time, laborsaving, is suitable for applying in insect commodity inspection detects.
Accompanying drawing explanation
Fig. 1 is Candle-sticks stinkbug beta-actin internal reference amplification curve
Fig. 2 is Candle-sticks stinkbug beta-actin internal reference melt curve analysis
Fig. 3 is Candle-sticks stinkbug heat shock protein pane gene amplification curve
Fig. 4 is Candle-sticks stinkbug heat shock protein pane gene melt curve analysis
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Heat shock protein 90 and the discovery of encoding gene and the design of primer special of embodiment 1, detection insect spawning amount
Research finds, heat shock protein pane gene is a kind of good molecule marker that detects insect spawning amount, so heat shock protein 90 (the heat shock protein90) expression of encoding gene of the present invention by Candle-sticks stinkbug is used for detecting the egg laying amount of Candle-sticks stinkbug.
The nucleotides sequence of heat shock protein 90 encoding genes of Candle-sticks stinkbug is classified sequence 1 in sequence table or the sequence 1 in sequence table as from 5 ' end 2-331 position Nucleotide, and the aminoacid sequence of this albumen is the sequence 2 in sequence table.
The primer that is designed for this encoding gene of amplification according to heat shock protein 90 encoding genes of Candle-sticks stinkbug is as follows:
Upstream primer: GAAGGTGAGGATGATAAGCCCAA(sequence 3);
Downstream primer: GTCCAGATCGGCTTTGTCTTG(sequence 4).
The application in detecting Candle-sticks stinkbug egg laying amount of embodiment 2, heat shock protein 90 or encoding gene or primer special
Candle-sticks stinkbug (the Arma chinensis adopting in following experiment; be documented in Taxonomic and bionomic notes onArma chinensis (Fallou) (Hemiptera:Pentatomidae:Asopinae; Zootaxa; 3382:41-52; 2012; public Ke Cong Plant Protection institute, Chinese Academy of Agricultral Sciences obtains) according to the difference that takes food material, be divided into two groups, 50 every group:
Take food artificial diet group Candle-sticks stinkbug: the Candle-sticks stinkbug of the artificial diet of feeding every adult (27 ± 1 ° of C, 16:8 (L:D), 75 ± 5%RH) every day 160 μ l artificial diet of feeding, until the natural death of Candle-sticks stinkbug;
Take food prey group Candle-sticks stinkbug: every pair of adult of the Candle-sticks stinkbug of the prey of feeding (27 ± 1 ° of C, 16:8 (L:D), 75 ± 5%RH) prey of feeding, was taken food situation every 7-15 days according to prey and change a prey, until the natural death of Candle-sticks stinkbug;
Artificial diet make a living pork liver 60g, soyflour 10g, water 100ml, egg 20ml, casein food grade 2g, sucrose 8g, VC0.2g, choline chloride 60 0.4g, calcium pantothenate 8mg, folic acid 0.1mg, niacinamide 0.2mg, gentamicin 3.9mg;
Prey is tussah (Antheraea pernyi) pupa (commercially available).
One, the acquisition of Candle-sticks stinkbug cDNA
1, Candle-sticks stinkbug RNA extracting
Step is as follows:
Each group Candle-sticks stinkbug sample is moved into and added in the mortar of appropriate liquid nitrogen, fast, firmly grind to form homogenate, move in 1.5ml centrifuge tube.
Add 1000 μ lTrizol in 1.5ml centrifuge tube, standing 5 minutes.
Add 200 μ l chloroforms, vortex vibration 10 seconds, standing 5 minutes, put into whizzer, 12,000g4 ℃ is centrifugal 15 minutes.
Supernatant liquor is transferred in new 1.5ml centrifuge tube, adds isopyknic Virahol, vibration mixes, and places 1 hour standing 10 minutes of room temperature for-20 ℃.
Put into whizzer, 12,000g, 4 ℃ are centrifugal 10 minutes.
Abandon supernatant liquor, Virahol is eliminated, add the dehydrated alcohol of 1ml75%, 12,000g, 4 ℃ are centrifugal 5 minutes.
Abandon supernatant liquor, add DEPC to process water after precipitation is dry ,-80 ℃ of preservations, obtain RNA.
2, reverse transcription
Adopt Superscript Ш Reverse Transcriptasekit test kit (Invitrogen18080044) to carry out reverse transcription:
A) get the pipe of the eppendorf without RNA enzyme of a sterilizing, each sample adds component as shown in table 1 below to obtain mix1;
Table 1 adds component 1 for reverse transcription
| Component | Volume | Source |
| Upto5μgtotalRNA | 5μl | ? |
| Primer(50μMoligio(dt) | 0.5μl | Invitrogen |
| Randomprimer | 0.5μl | Invitrogen |
| 10mMdNTPMix | 1μl | Invitrogen |
| DEPC-treatedwater | 5μl | ? |
| Total | 12μl | ? |
B) mix165 ℃ of temperature bathed 5 minutes, then put immediately 1 minute on ice;
C) in mix1, add composition as shown in table 2 below, obtain mix2 totally 20 μ l systems;
Table 2 adds component 2 for reverse transcription
D) process 5 minutes for 25 ℃;
E) process 60 minutes for 50 ℃;
F) process 15 minutes for 70 ℃, be placed into immediately on ice;
G) obtain cDNA, and this cDNA can preserve half a year at-20 ℃.
Two, RT-PCR amplification
In RT-PCR amplification system, the volume of each component is as shown in table 3 below, adopts SYBR (R) Green I NucleicAkit (InvitrogenS7567):
Table 3 is the volume of each component in pcr amplification system
PCR reaction conditions is as shown in table 4:
Table 4 is PCR reaction conditions
| Circulation | Loop cycle |
| 95 ℃, 2 minutes | ? |
| 40 circulations | The first step sex change: 95 ℃, 10 seconds |
| ? | Second step annealing: 60 ℃, 30 seconds |
| Solubility curve | 60 ℃-95 ℃, within every 5 seconds, increase by 0.5 ℃ |
Using Candle-sticks stinkbug beta-actin (ACTB) as reference gene (sequence 5), and the primer of this reference gene that increases is as follows:
Internal reference upstream primer: TGTCCAAGCAGGAGTACGAC(sequence 6);
Internal reference downstream primer: GGTTCCTCTTTGGAGTCCGATT(sequence 7).
If take food heat shock protein pane gene average expression amount in the Candle-sticks stinkbug group of prey higher than the Candle-sticks stinkbug group that takes food artificial diet, take food the Candle-sticks stinkbug group egg laying amount of prey higher than the Candle-sticks stinkbug group that takes food artificial diet.
As Figure 1-4, Fig. 1 is two groups of Candle-sticks stinkbug beta-actin internal reference amplification curves to result; Fig. 2 is two groups of Candle-sticks stinkbug beta-actin internal reference melt curve analysis; Fig. 3 is two groups of Candle-sticks stinkbug heat shock protein pane gene amplification curves; Fig. 4 is Candle-sticks stinkbug heat shock protein pane gene melt curve analysis; Can find out, amplification curve is level and smooth, reproducible; Solubility curve is simple spike, illustrates and there is no non-specific amplification, and fluorescent quantitation result is accurate.
Candle-sticks stinkbug beta-actin (ACTB) reference gene ct value is as shown in table 5:
Table 5 is each group Candle-sticks stinkbug beta-actin (ACTB) reference gene ct value
Candle-sticks stinkbug heat shock protein pane gene quantitative result is as shown in table 6:
Table 6 is each group Candle-sticks stinkbug heat shock protein pane gene quantitative result
From the above results, find, compare with the Candle-sticks stinkbug that takes food insect prey, the expression amount of heat shock protein pane gene of Candle-sticks stinkbug that takes food artificial diet is extremely low, can't detect; And this heat shock protein pane gene can be detected in taking food the Candle-sticks stinkbug of insect prey, obviously raise; This proof, takes food the egg laying amount that the egg laying amount of the Candle-sticks stinkbug group of insect prey organizes than the Candle-sticks stinkbug that takes food artificial diet high.
By traditional biological method, (method according to above-mentioned two groups is fed, and collect every day and respectively organize the pieces of an egg that Candle-sticks stinkbug produces, counting, until the natural death of Candle-sticks stinkbug) detect the egg laying amount of feed group Candle-sticks stinkbug and prey group Candle-sticks stinkbug, the Candle-sticks stinkbug that takes food the Candle-sticks stinkbug of insect prey and take food artificial diet in life average egg laying amount is respectively 458 and 213; Can find out, this detected result is consistent with the result detecting by traditional biological method, proves that method of the present invention is correct.
Result shows, the variation of the expression amount of the heat shock protein pane gene of Candle-sticks stinkbug that can rapid detection Different Nutrition source by detection method of the present invention, judges the difference of the egg laying amount of live body population with this.
The material that detection heat shock protein pane gene therefore of the present invention is expressed can be used as molecule marker for detection of the egg laying amount that takes food the Candle-sticks stinkbug population in Different Nutrition source; And for the primer of the heat shock protein pane gene that increases or RT-PCR reagent, can be used as the component of the egg laying amount test kit that detects the Candle-sticks stinkbug population that takes food Different Nutrition source;
Above-mentioned RT-PCR reagent is by ultrapure water, 10 * pcr amplification buffer, magnesium ion, dNTPs, upstream primer, SYBR(SYBR Green I fluorescence dye), downstream primer, Taq enzyme form; Wherein the final concentration of magnesium ion in RT-PCR reagent is 1.25mM; In dNTPs, each final concentration in RT-PCR reagent is 0.25mM; Upstream primer and downstream primer are 0.25 μ M at the final concentration in RT-PCR reagent; The final concentration of Taq enzyme in RT-PCR reagent is 0.05u/ μ l.
Claims (9)
1. the application in detecting insect spawning amount for detection of the material of heat shock protein 90 encoding gene expression amounts;
The aminoacid sequence of described heat shock protein 90 is the sequence 2 in sequence table;
Described insect is specially Candle-sticks stinkbug;
The described material for detection of heat shock protein 90 encoding gene expression amounts is following 1)-3) in any one:
1) primer pair A: described primer pair is comprised of primer 1 and primer 2; The nucleotides sequence of described primer 1 is classified sequence 3 in sequence table as; The nucleotides sequence of described primer 2 is classified sequence 4 in sequence table as;
2) the RT-PCR reagent that contains described primer pair A;
3) test kit that contains described primer pair A or described RT-PCR reagent.
2. application according to claim 1, is characterized in that: described insect is insect individuality or insect population; The nucleotides sequence of described heat shock protein 90 encoding genes is classified sequence 1 in sequence table or the sequence 1 in sequence table as from 5 ' end 2-331 position Nucleotide.
3. a primer pair A, is comprised of primer 1 and primer 2;
The nucleotides sequence of described primer 1 is classified sequence 3 in sequence table as; The nucleotides sequence of described primer 2 is classified sequence 4 in sequence table as.
4. the RT-PCR reagent that contains primer pair A claimed in claim 3; Described RT-PCR reagent is specifically comprised of water, RT-PCR amplification buffer, magnesium ion, dNTPs, primer pair A claimed in claim 3, fluorescence dye and Taq enzyme;
The final concentration of each primer in described primer pair A in described RT-PCR reagent is specially 0.2-1 μ M.
5. RT-PCR reagent according to claim 4, is characterized in that: the final concentration of each primer in described primer pair A in described RT-PCR reagent is 0.25 μ M.
6. the test kit that contains the RT-PCR reagent described in primer pair A claimed in claim 3 or claim 4 or 5.
7. test kit according to claim 6, is characterized in that: described test kit also comprises internal reference primer pair B, and described internal reference primer pair B is comprised of primer 3 and primer 4; The nucleotides sequence of described primer 3 is classified the sequence 6 in sequence table as; The nucleotides sequence of described primer 4 is classified the sequence 7 in sequence table as.
8. detect a method for insect spawning amount, comprise the steps:, with the RT-PCR reagent described in primer pair A claimed in claim 3 or claim 4 or 5 or the test kit described in claim 6 or 7, insect A to be measured and B are carried out to RT-PCR amplification;
If the expression amount of the heat shock protein pane gene of described insect A is higher than described insect B, the egg laying amount of described insect A is higher than described insect B;
Described insect is specially Candle-sticks stinkbug.
9. method according to claim 8, is characterized in that: described insect is insect individuality or insect population;
The cDNA that the template of described RT-PCR amplification is insect.
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| CN102771670A (en) * | 2012-08-03 | 2012-11-14 | 中国农业科学院植物保护研究所 | Artificial diet for predative natural enemy insect-true bugs |
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Non-Patent Citations (7)
| Title |
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| Accession No: GU235994, Antheraea pernyi heat shock protein 90 mRNA, complete cds;Cao,J. et al;《GeneBank database》;20100401;FEATURES和ORIGIN部分 * |
| Accession No:ADD91573, Antheraea pernyi heat shock protein 90 mRNA, complete cds;Cao,J. et al;《GeneBank database》;20100401;FEATURES和ORIGIN部分 * |
| Cao J. et al.Accession No: GU235994 |
| Cao J. et al.Accession No:ADD91573 |
| 捕食性天敌——蠋蝽;郑友贤等;《昆虫天敌》;19850228;第6卷(第2期);第87-90页 * |
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| 高卓等.蠋蝽(Ａｒｍａ ｃｈｉｎｅｎｓｉｓ)生物学特性研究.《黑龙江大学工程学报》.2011,第2卷(第4期),第72-77页. * |
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