CN103060466B - Method for detecting self-mutilation rate of insects by using phylaxin gene - Google Patents
Method for detecting self-mutilation rate of insects by using phylaxin gene Download PDFInfo
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Abstract
The invention discloses a method for detecting the self-mutilation rate of insects by using a phylaxin gene. The invention provides an application of a substance for detecting the expression level of a phylaxin-like protein-coding gene in insect populations fed with different substances in detection of the self-mutilation rate of the insect populations fed with different substances; and the amino acid sequence of a phylaxin-like protein is sequence 2 in a sequence table. According to the method disclosed by the invention, experiments prove that the detection method provided by the invention can be used for detecting the self-mutilation rate of the insects, in particular to the self-mutilation rate of arma chinensis with different nutrient sources; and the detection of the self-mutilation rate of the insects by the detection method disclosed by the invention only requires 2-3 days. However, the use of a traditional biological method requires about 30-60 days. The detection method disclosed by the invention has the advantages of wide range of involved material sources, easiness in purchase, simplicity in operation, and time-saving and labor-saving properties, and is suitable for popularization and application in commodity inspection and detection of the insects.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of method that phylaxin gene detects insect autotomy rate.Background technology
Biological control of insect pests plays irreplaceable vital role in agroforestry Sustainable development.A large amount of productions of natural enemy insect are the most basic in biological control especially is also most important link.Predatory natural enemy insect Candle-sticks stinkbug (Armachinensis) can control 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, and therefore Candle-sticks stinkbug is the good natural enemy insect commodity of a kind of application prospect.
As commodity, a large amount of minimizing production cost is the maximized approach of people's profit-push.Different food just can be applied to produce Candle-sticks stinkbug in natural enemy insect Candle-sticks stinkbug produces, such as, 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 applying different foodstuffs production Candle-sticks stinkbug is out uneven, after some release, the effect of good Control pests can be reached, have then effect is not remarkable upon discharge.Candle-sticks stinkbug due to different fed diets is difficult to distinguish in phenotype, and therefore these natural enemy insects are be difficult to their biological characteristics of assessment before releasing.
In kind, autotomy phenomenon is a problem very serious in predatory natural enemy insect a large amount of group breeding process.It is numerous that the insect population that autotomy rate is high is difficult to a large amount of expansion in a short time, and therefore the doubling time extends greatly, and then increases feeding cost.With in the Candle-sticks stinkbug population of artificial diet, autotomy rate is higher.But be still the number of testing its autotomy rate in feeding process for the detection method of the commodity Candle-sticks stinkbug autotomy rate of different food source or different businessman, this method wastes time and energy expensive.Do not carry out biological characteristis to detect fast commodity with regard to being difficult to.
Alexin is stinkbug class defence rival and a kind of important weapon hiding natural enemy; alexinic a large amount of release can greatly reduce the autotomy phenomenon of natural enemy insect in natural enemy feeding process; and when discharging natural enemy to field or protecting field pest control, the harm of the hostile Candle-sticks stinkbug in sky can be reduced.
Summary of the invention
An object of the present invention is to provide the purposes of the material for detection type alexin protein encoding gene expression amount.
The material that the invention provides for detection type alexin protein encoding gene expression amount is detecting the application in insect autotomy rate;
The aminoacid sequence of described class alexin protein (defensin-like protein) 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 specially the insect population taking food artificial diet or the insect population taking food prey further;
The nucleotides sequence of described class alexin protein encoding gene is classified as the sequence 1 in sequence table or the sequence 1 in sequence table from 5 ' end 81-272 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 type alexin protein encoding gene expression amount is following 1)-3) in any one:
1) primer pair A: described primer pair is made up of primer 1 and primer 2; The nucleotides sequence of described primer 1 is classified as sequence 3 in sequence table; The nucleotides sequence of described primer 2 is classified as sequence 4 in sequence table;
2) the RT-PCR reagent containing described primer pair A;
3) test kit containing 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 made up of primer 1 and primer 2;
The nucleotides sequence of described primer 1 is classified as sequence 3 in sequence table; The nucleotides sequence of described primer 2 is classified as sequence 4 in sequence table.
RT-PCR reagent containing above-mentioned primer pair A is also the scope of protection of the invention; Above-mentioned RT-PCR reagent is specifically made up 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 bar primer in described RT-PCR reagent in described primer pair A is specially 0.2-1 μM, and the final concentration of each bar primer in described RT-PCR reagent in described primer pair A is specially 0.25 μM further.
Test kit containing 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 made up of primer 3 and primer 4; The nucleotides sequence of described primer 3 is classified as the sequence 6 in sequence table; The nucleotides sequence of described primer 4 is classified as the sequence 7 in sequence table.
3rd object of the present invention is to provide a kind of method detecting insect autotomy rate.
Method provided by the invention, comprises the steps: to carry out RT-PCR amplification with above-mentioned primer pair A or above-mentioned RT-PCR reagent or above-mentioned test kit to insect A and B to be measured;
If the expression amount of the class alexin protein gene of described insect A is higher than described insect B, then the autotomy rate of described insect A is lower than described insect B.
In aforesaid method, described insect is insect individuality or insect population;
The template of described RT-PCR amplification is the cDNA of insect;
Described insect is specially Candle-sticks stinkbug.
In an embodiment of the present invention, described insect A is the insect population taking food prey; Described insect B is the insect population taking food artificial diet.
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 as the sequence 1 in sequence table or the sequence 1 in sequence table from 5 ' end 81-272 position Nucleotide.
Experiment of the present invention proves, detection method provided by the present invention can detect the insect autotomy rate of different population by detection type alexin protein gene expression amount, the particularly autotomy rate of the Candle-sticks stinkbug in Different Nutrition source, the autotomy rate detecting insect by detection method of the present invention only needs 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 class alexin protein gene amplification curve
Fig. 4 is Candle-sticks stinkbug class alexin protein gene melt curve analysis
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, the class alexin protein detecting insect autotomy rate and the discovery of encoding gene thereof and the design of primer special
Research finds, phylaxin gene is a kind of good molecule marker detecting insect autotomy rate, and therefore the present invention detects the autotomy rate of Candle-sticks stinkbug by the expression of class alexin protein (defensin-like protein) encoding gene detecting Candle-sticks stinkbug.
The nucleotides sequence of the class alexin protein encoding gene of Candle-sticks stinkbug is classified as the sequence 1 in sequence table or the sequence 1 in sequence table from 5 ' end 81-272 position Nucleotide, and the aminoacid sequence of this albumen is the sequence 2 in sequence table.
The primer being designed for this encoding gene of amplification according to the class alexin protein encoding gene of Candle-sticks stinkbug is as follows:
Upstream primer: TGCGCTAGTTCACGTGCCTT(sequence 3);
Downstream primer: AGCGGTACTGTCCCTAGGTAAT(sequence 4).
Embodiment 2, class alexin protein or its encoding gene or primer special are detecting the application in Candle-sticks stinkbug autotomy rate
Candle-sticks stinkbug (the Arma chinensis adopted in following experiment; be documented in Taxonomic and bionomic notes onArma chinensis (Fallou) (Hemiptera:Pentatomidae:Asopinae; Zootaxa; 3382:41-52; 2012; the public can obtain from Plant Protection institute, Chinese Academy of Agricultral Sciences) be divided into two groups according to the difference taking food material, often organize 50:
Take food artificial diet group Candle-sticks stinkbug: Candle-sticks stinkbug (27 ± 1 ° of C, 16:8 (L:D), 75 ± 5%RH) adult every day every of artificial diet of feeding is fed 160 microlitre artificial diet, until the natural death of Candle-sticks stinkbug;
Take food prey group Candle-sticks stinkbug: Candle-sticks stinkbug (27 ± 1 ° of C, 16:8 (L:D), 75 ± 5%RH) often pair of adult of prey of feeding is fed a prey, is changed a prey, until the natural death of Candle-sticks stinkbug every 7-15 days according to prey by the situation of taking food;
Artificial diet are made 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 of Candle-sticks stinkbug sample is moved into and adds in the mortar of appropriate liquid nitrogen, grind to form homogenate fast, firmly, move in 1.5ml centrifuge tube.
Add in 1000 μ lTrizol to 1.5ml centrifuge tubes, leave standstill 5 minutes.
Add 200 μ l chloroforms, vortex vibrates 10 seconds, and leave standstill 5 minutes, put into whizzer, 12,000g4 DEG C centrifugal 15 minutes.
Be transferred to by supernatant liquor in new 1.5ml centrifuge tube, add isopyknic Virahol, vibration mixing, place 1 hour for-20 DEG C, room temperature leaves standstill 10 minutes.
Put into whizzer, 12,000g, 4 DEG C centrifugal 10 minutes.
Abandon supernatant liquor, eliminated by Virahol, add the dehydrated alcohol of 1ml75%, 12,000g, 4 DEG C centrifugal 5 minutes.
Abandon supernatant liquor, add DEPC process water after drying to be precipitated ,-80 DEG C of preservations, obtain RNA.
2, reverse transcription
Superscript Ш Reverse Transcriptasekit test kit (Invitrogen18080044) is adopted to carry out reverse transcription:
A) eppendorf without RNA enzyme getting a sterilizing manages, and each sample adds component as shown in table 1 below and obtains mix1;
Table 1 adds component 1 for reverse transcription
| Component | Volume | Source |
| Up to5μg total RNA | 5μl |
| Primer(50μM oligio(dt) | 0.5μl | Invitrogen |
| Random primer | 0.5μl | Invitrogen |
| 10mM dNTP Mix | 1μl | Invitrogen |
| DEPC-treated water | 5μl | |
| Total | 12μl |
B) mix165 DEG C of temperature bath 5 minutes, 1 minute is then put immediately 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) 25 DEG C process 5 minutes;
E) 50 DEG C process 60 minutes;
F) 70 DEG C process 15 minutes, are placed into immediately on ice;
G) obtain cDNA, and this cDNA can preserve half a year at-20 DEG C.
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 NucleicA kit (Invitrogen S7567):
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 DEG C, 2 minutes | |
| 40 circulations | The first step sex change: 95 DEG C, 10 seconds |
| Second step is annealed: 60 DEG C, 30 seconds | |
| Solubility curve | 60 DEG C-95 DEG C, within every 5 seconds, increase by 0.5 DEG C |
Using Candle-sticks stinkbug beta-actin (ACTB) as reference gene (sequence 5), 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 the class alexin protein gene average expression amount taken food in the Candle-sticks stinkbug group of prey is higher than the Candle-sticks stinkbug group taking food artificial diet, then take food the Candle-sticks stinkbug group autotomy rate of prey lower than the Candle-sticks stinkbug group taking food artificial diet.
As Figure 1-4, Fig. 1 is Candle-sticks stinkbug beta-actin internal reference amplification curve to result; Fig. 2 is Candle-sticks stinkbug beta-actin internal reference melt curve analysis; Fig. 3 is Candle-sticks stinkbug class alexin protein gene amplification curve; Fig. 4 is Candle-sticks stinkbug class alexin protein gene melt curve analysis; Can find out, amplification curve is level and smooth, reproducible; Solubility curve is simple spike, illustrates do not have 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 of Candle-sticks stinkbug beta-actin (ACTB) reference gene ct value
Candle-sticks stinkbug class alexin protein gene quantification result is as shown in table 6:
Table 6 is each group of Candle-sticks stinkbug class alexin protein gene quantification result
Find from the above results, compared with the Candle-sticks stinkbug taking food insect prey, the expression amount taking food the class alexin protein gene of the Candle-sticks stinkbug of artificial diet is extremely low, can't detect; And such alexin protein gene obviously raises in the Candle-sticks stinkbug taking food insect prey; This proves, takes food the Candle-sticks stinkbug group autotomy rate of insect prey lower than the Candle-sticks stinkbug group taking food artificial diet.
(fed according to the method for above-mentioned two groups by traditional biological method, every day, record was by the adult quantity of companion's imago feeding, with by the ghost polypide that stays after taking food for card, counting, until the whole natural death of Candle-sticks stinkbug adult population, calculate autotomy rate) detect the autotomy rate of feed group Candle-sticks stinkbug and prey group Candle-sticks stinkbug, the Candle-sticks stinkbug group taking food insect prey and take food artificial diet Candle-sticks stinkbug group in life on average autotomy rate be respectively 7% and 17%; Can find out, this detected result is consistent with the result detected by traditional biological method, proves that method of the present invention is correct.
Result shows, can the change of expression amount of class alexin protein gene of Candle-sticks stinkbug in rapid detection Different Nutrition source by detection method of the present invention, judges the difference of the autotomy rate of live body population with this.
Therefore the material of detection type alexin protein of the present invention genetic expression can as molecule marker for detecting the autotomy rate of the Candle-sticks stinkbug population taking food Different Nutrition source; And can as detecting the component that take food the autotomy rate test kit of the Candle-sticks stinkbug population in Different Nutrition source for the primer of the class alexin protein gene that increases or RT-PCR reagent;
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 μM 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 material for detecting protein coding gene expression amount shown in the sequence 2 in sequence table is detecting the application in Candle-sticks stinkbug autotomy rate;
Shown in the described sequence 2 for detecting in sequence table, the material of protein coding gene expression amount is following 1)-3) in any one:
1) primer pair A: described primer pair is made up of primer 1 and primer 2; The nucleotides sequence of described primer 1 is classified as sequence 3 in sequence table; The nucleotides sequence of described primer 2 is classified as sequence 4 in sequence table;
2) the RT-PCR reagent containing described primer pair A;
3) test kit containing described primer pair A or described RT-PCR reagent.
2. application according to claim 1, is characterized in that: described Candle-sticks stinkbug is Candle-sticks stinkbug individuality or Candle-sticks stinkbug population; The nucleotides sequence of protein coding gene shown in sequence 2 in described sequence table is classified as the sequence 1 in sequence table or the sequence 1 in sequence table from 5 ' end 81-272 position Nucleotide.
3. an oligonucleotide sequence group, be primer pair A, described primer pair A is made up of primer 1 and primer 2;
The nucleotides sequence of described primer 1 is classified as sequence 3 in sequence table; The nucleotides sequence of described primer 2 is classified as sequence 4 in sequence table.
4. the RT-PCR reagent containing primer pair A according to claim 3; Described RT-PCR reagent is made up of water, RT-PCR amplification buffer, magnesium ion, dNTPs, primer pair A according to claim 3, fluorescence dye and Taq enzyme;
The final concentration of each bar primer in described RT-PCR reagent in described primer pair A is 0.2-1 μM.
5. RT-PCR reagent according to claim 4, is characterized in that: the final concentration of each bar primer in described RT-PCR reagent in described primer pair A is 0.25 μM.
6. contain the test kit of primer pair A according to claim 3 or the RT-PCR reagent described in 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 made up of primer 3 and primer 4; The nucleotides sequence of described primer 3 is classified as the sequence 6 in sequence table; The nucleotides sequence of described primer 4 is classified as the sequence 7 in sequence table.
8. detect a method for Candle-sticks stinkbug autotomy rate, comprise the steps: to carry out RT-PCR amplification with the RT-PCR reagent described in primer pair A according to claim 3 or claim 4 or 5 or the test kit described in claim 6 or 7 to Candle-sticks stinkbug A and B to be measured;
If the expression amount of protein coding gene shown in the sequence 2 in the sequence table of described Candle-sticks stinkbug A is higher than described Candle-sticks stinkbug B, then the autotomy rate of described Candle-sticks stinkbug A is lower than described Candle-sticks stinkbug B.
9. method according to claim 8, is characterized in that: described Candle-sticks stinkbug is Candle-sticks stinkbug individuality or Candle-sticks stinkbug population; The template of described RT-PCR amplification is Candle-sticks stinkbug cDNA.
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| CN101724703A (en) * | 2009-12-25 | 2010-06-09 | 山西大学 | Oxya specificity molecular marker DNA sequence and application thereof |
| CN101940270A (en) * | 2010-09-27 | 2011-01-12 | 中国农业科学院植物保护研究所 | Artificial feed for natural enemy insects |
| 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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| CN101724703A (en) * | 2009-12-25 | 2010-06-09 | 山西大学 | Oxya specificity molecular marker DNA sequence and application thereof |
| CN101940270A (en) * | 2010-09-27 | 2011-01-12 | 中国农业科学院植物保护研究所 | Artificial feed for natural enemy insects |
| 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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