CN103875608B - Transgenic pest-resistant rice is to predator Mirid, cyrtorhinus method for evaluating safety - Google Patents
Transgenic pest-resistant rice is to predator Mirid, cyrtorhinus method for evaluating safety Download PDFInfo
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Abstract
The present invention relates to a kind of transgenic pest-resistant rice to predator Mirid, cyrtorhinus method for evaluating safety, by Tier-1 toxicity assay, three grades of nutritional tests, albumen transfer law, predation functional responses and population density compare 5 parts and form with population dynamic.Each several part evaluates the Bt albumen of Transgenic Bt Rice expression respectively to the genotoxic potential of Mirid, cyrtorhinus; Evaluate Transgenic Bt Rice by the impact of prey mediation on Mirid, cyrtorhinus; Evaluate approach and degree that Mirid, cyrtorhinus is exposed to Bt albumen; Evaluate Transgenic Bt Rice to the impact of Mirid, cyrtorhinus population.Finally, the ecological consequence that the plantation evaluating described genetically modified crops according to the aggregation of data of 5 aspects produces this non-target organism.The present invention establishes the evaluation method of set of system, science, to improving genetically modified crops to the influence research of non-target organism and determine that the ecological security that genetically modified crops are planted is significant.
Description
Technical field
The invention belongs to genetically modified crops technical field of ecological risk assessment, be specifically related to a kind of transgenic pest-resistant rice to predator Mirid, cyrtorhinus method for evaluating safety.
Background technology
Paddy rice is one of most important cereal crops in the world, and the whole world has more than 30 hundred million populations to take rice as food.Simultaneously paddy rice is again one of maximum cereal crops of insect pest, every year due to rice leaf roller, bore causing harm of the lepidopterous insects such as moth property snout moth's larva, cause great production loss (Cheng, 1996; Nathan, 2006).In production reality, the control of lepidoptera pest depends on chemical pesticide for a long time, and the abuse of chemical pesticide not only adds the production cost of paddy rice and contaminated environment, also easily makes insect produce resistance, reduces the drug effect (Suetal, 2003) of agricultural chemicals.Therefore, the lepidopterous resistant variety of high resistance be a kind of economy, effectively, the means of prevention of environmental protection.But, in paddy rice and wild rice, be difficult to the anti-source finding high resistance lepidopterous insects.Cultivate the rice varieties of high resistance lepidoptera pest, need by means of Extrinsic Anti-insect Genes.Bacillusthuringiensis (Bt) insecticidal proteins is used as the history that biological source insecticide had five more than ten years, has stable control efficiency to lepidoptera pest.To be Transgenic Bt Rice be at present controls the main insect-proof rice of lepidoptera pest.
But, while the utilization of insect-resistant transgenic plants brings interests to the mankind, also there is necessarily potential ecological risk.The ecological security evaluation of transgenic anti-insect plants concentrates on: the resistance management on target pest, the impact on non-target organism and bio-diversity, genetic drift problem and the impact etc. on edaphon.The potential impact of transgenic anti-insect plants to non-target organism is the pith about genetically modified crops environmental risk assessment.At present, international risk assessment worker and genetically modified plants regulatory authorities extensively adopt " by different level appraisement system " to carry out genetically modified plants affect aspect research work (Romeisetal., 2008 on non-target organism; Wang is round, and 2011).This appraisement system is exactly select suitable biological subject, then carries out successively from laboratory test to half field trial, then to the system evaluation that field trial is carried out by different level stage by stage.
Brown planthopper Nilaparvatalugens
being one of topmost insect in temperate zone and Tropical Rice field, is non-target plant-feed insect important in Bt Transgenic Rice planting process.Mirid, cyrtorhinus is the important predator of rice fulgorid ovum phase and nymph, obviously can reduce population quantity (Chenetal., 1992 of rice fulgorid; Louetal., 2013; Sigsgaard, 2007), it can absorb Bt insecticidal proteins by predation process, has the possibility that there is ecological risk, so, sets up set of system evaluation Transgenic Bt Rice particularly important to the method whether Mirid, cyrtorhinus is safe.
Summary of the invention
The object of the invention is for above-mentioned present situation, aim to provide a kind of transgenic pest-resistant rice safe and effective, easy and simple to handle, with low cost to predator Mirid, cyrtorhinus method for evaluating safety.
The implementation of the object of the invention is, transgenic pest-resistant rice, to predator Mirid, cyrtorhinus method for evaluating safety, comprises following 5 parts:
(1) Tier-1 toxicity assay: evaluate the Bt albumen of Transgenic Bt Rice expression to the genotoxic potential of non-target organism Mirid, cyrtorhinus;
Mirid, cyrtorhinus is raised with artificial feeding device, access Mirid, cyrtorhinus examination worm, raise device black cloth during raising to cover, only expose the port having feed, and towards direction of illumination, keep relative moisture to reach more than 90%, then be placed in temperature 28 ± 1 DEG C, photoperiod L:D=14:10h, cultivate in the intelligent climatic cabinate of humidity 90 ± 5%;
With the Chemically defined diet of three kinds of forms as Mirid, cyrtorhinus feed namely: Chemically defined diet, the Bt protein concentration more than 10 times containing transgenic paddy rice leaf sheath tissue expression and the Chemically defined diet containing 40 μ g/ml potassium dihydrogen arsenates; With the rice seedling containing brown planthopper ovum by Mirid, cyrtorhinus raise to 2 age nymph, in single head access raising device, each process respectively connects Mirid, cyrtorhinus nymph 36, change feed every day and observed and recorded Mirid, cyrtorhinus is casted off a skin and survival condition, until sprout wings, new emerging adult is in 24 hours, and 2 ~ 3 identical sex adults merge rear ten thousand/scales/electronic balance weighing;
By the concentration of ELISA and sensitive insect raw survey technology monitored over time insecticidal proteins, stability and biologically active in test;
Select the nymphal development of Mirid, cyrtorhinus to go through phase, nymph survival rate, ratio of female and first emergence body weight as evaluating index, statistical analysis is carried out to it, to evaluate the Bt albumen of Transgenic Bt Rice expression to the toxic effect of Mirid, cyrtorhinus;
(2) three grades of nutritional tests: evaluate Transgenic Bt Rice by the safety effects of prey mediation to Mirid, cyrtorhinus by the food chain of paddy rice-brown planthopper-Mirid, cyrtorhinus;
1. using brown planthopper ovum as food
Brown planthopper female adult pest preoviposition period that access 2 being crossed in the glass tube that 6 ~ 7 15 age in days rice seedlings are housed, lays eggs 2 days, raises the nymph of Mirid, cyrtorhinus to adult respectively with different rice varieties, the seedling of laying eggs that is; In each glass tube within access hatching 24h, from the Mirid, cyrtorhinus nymph 1 of non-transgenic parent paddy rice, each kind, be respectively connect Mirid, cyrtorhinus nymph 32, Mirid, cyrtorhinus 1 ~ 3 age nymph, within every 2 days, change 1 seedling of laying eggs, change 1 seedling of laying eggs in every day in 4 ages to adult stage; Every morning observed and recorded Mirid, cyrtorhinus nymph survival and situation of casting off a skin, until nymph sprouts wings, in new emerging adult 24 hours, with ten thousand/scales/electronic balance weighing after 2 ~ 3 identical sex adults merge;
Select the nymphal development of Mirid, cyrtorhinus go through the phase, nymph survival rate, ratio of female and just emergence body weight as evaluating index, and carry out statistical analysis, evaluate the Transgenic Bt Rice that mediated by brown planthopper ovum to the safety effects of Mirid, cyrtorhinus;
2. using brown planthopper nymph as food
Be provided in different rice varieties every day, fasten hatch and the brown planthopper nymph taking food 24 ~ 48 hours as fed diets Mirid, cyrtorhinus, concrete operations are as follows: with containing brown planthopper ovum rice seedling by Mirid, cyrtorhinus raise to 2 age nymph, feed with different rice varieties again, the brown planthopper nymph of system, 2 ~ 3 Mirid, cyrtorhinus nymph every days every in age provided 10 brown planthopper nymphs, 4 age Mirid, cyrtorhinus provide 20 brown planthopper nymphs to every day every in adult stage, Mirid, cyrtorhinus nymph single head is raised and is covered with in the finger-type pipe of wet sponge moisturizing in bottom, each kind, system respectively connects Mirid, cyrtorhinus nymph 80, every morning observed and recorded Mirid, cyrtorhinus nymph survival and situation of casting off a skin, until nymph sprouts wings, in new emerging adult 24 hours, with ten thousand/scales/electronic balance weighing after 3 ~ 5 identical sex adults merge,
Select the nymphal development of Mirid, cyrtorhinus go through the phase, nymph survival rate, ratio of female and just emergence body weight as evaluating index, and carry out statistical analysis, evaluate the Transgenic Bt Rice that mediated by brown planthopper nymph to the safety effects of Mirid, cyrtorhinus;
(3) albumen transfer law: the approach and the degree that are exposed to Bt albumen by ELISA detection means qualification Mirid, cyrtorhinus;
The rice seedling put into the different rice varieties of 15 ages in days respectively in glass jar, being, access 20 bosom ovum brown planthopper female adult pest that corresponding rice material is raised, access after laying eggs 2 days 3 ~ 4 age Mirid, cyrtorhinus, after taking food 3 days, respectively to rice leaf sheath, brown planthopper ovum, Mirid, cyrtorhinus samples, each process repetition 4 ~ 5 times;
By different rice varieties, be raise hatching after 24 ~ 48 hours brown planthopper nymph with 3 ~ 4 age Mirid, cyrtorhinus access in the glass jar containing corresponding rice seedling simultaneously, every day changes brown planthopper, after taking food 3 days, respectively to rice leaf sheath, brown planthopper nymph and Mirid, cyrtorhinus sample, each process weight 4 ~ 5 times;
Adopt elisa technique, respectively the content of Bt albumen in above sample is detected, determine that the food chain Mirid, cyrtorhinus by different is exposed to the degree of Bt albumen;
(4) predation functional responses: by comparing the Mirid, cyrtorhinus predation parameter that Transgenic Bt Rice and non-transgenic parent control kind are raised, passes judgment on Transgenic Bt Rice to the impact of this non-target organism;
Mirid, cyrtorhinus raises a generation respectively at different rice material, sprout wings latter 2 days and the female worm of mating for test, Nature enemy 24 hours before test, puts into bottom and is covered with sponge and finger-type test tube containing rice seedling by Mirid, cyrtorhinus single head, 1 age brown planthopper nymph as prey; 5 BPH density process, are respectively 10,20,30,40,50, and each density process repeats 5 times, record the brown planthopper nymph number of prey after 24 hours;
Select the Zero-day Attack rate of Mirid, cyrtorhinus and process the prey time as evaluating index, and carrying out statistical analysis, evaluating Transgenic Bt Rice to the impact of Mirid, cyrtorhinus predation functional responses;
(5) population density compares with population dynamic: by comparing the occurrence dynamics of Transgenic Bt Rice and non-transgenic parent control rice field Mirid, cyrtorhinus, evaluates the plantation of Transgenic Bt Rice to the impact of this non-target organism population;
Adopt diagonal sampling method, every plot area is 150m
2, get 5 samples, every sample 0.25m at every turn
2, about 6 clumps of rice strains; Within after rice transplanting 30 days, start, with pest sucking device sampling, to paddy rice harvesting first 10 days, to sample 5 ~ 7 times altogether; Sampling is preserved with the alcohol of 75% immediately, carry out identifying and counting under stereoscope, to add up Mirid, cyrtorhinus population density and population dynamic, and statistical analysis is carried out to the otherness of parameter between different paddy rice field, evaluate Transgenic Bt Rice to the safety effects of Mirid, cyrtorhinus field population.
The present invention evaluates the Bt albumen of Transgenic Bt Rice expression to the genotoxic potential of non-target organism Mirid, cyrtorhinus by Tier-1 toxicity assay; Transgenic Bt Rice is evaluated by the impact of prey mediation on Mirid, cyrtorhinus by the food chain of paddy rice-brown planthopper-Mirid, cyrtorhinus; ELISA detection means qualification Mirid, cyrtorhinus is adopted to be exposed to approach and the degree of Bt albumen; By the comparison to Mirid, cyrtorhinus predation parameter, evaluate Transgenic Bt Rice and whether this non-target organism predation is had a negative impact; By comparing the occurrence dynamics of Transgenic Bt Rice and non-transgenic parent control rice field Mirid, cyrtorhinus, evaluate Transgenic Bt Rice to the impact of Mirid, cyrtorhinus population.Finally, the ecological consequence that the plantation comprehensively analyzing Transgenic Bt Rice according to the data of 5 aspects produces this non-target organism.
The present invention establishes the evaluation method of set of system, science, and to improving, the influence research of Transgenic Bt Rice to non-target organism is significant, to determining that the ecological security that Transgenic Bt Rice is planted is significant.
Accompanying drawing explanation
Fig. 1 is Mirid, cyrtorhinus artificial feeding device structural representation,
Fig. 2 takes food pure artificial feed and adds the Mirid, cyrtorhinus survivorship curve figure of Cry2Aa albumen and PA artificial feed,
Fig. 3,4 is T2A-1 rice leaf sheath tissue, brown planthopper ovum and take food brown planthopper ovum Mirid, cyrtorhinus body in Cry2Aa protein content figure,
Fig. 5,6 is T2A-1 rice leaf sheath tissue, brown planthopper nymph and take food brown planthopper nymph Mirid, cyrtorhinus body in Cry2Aa protein content figure,
The functional response figure of the Mirid, cyrtorhinus that Fig. 7,8 is T2A-1, bright extensive 63 rice materials are raised,
Fig. 9,10,11,12 be pest sucking device method Xiaogan 2011,2012,2013, the transgenic paddy rice that gathers of Suizhou 2011 with contrast paddy field Mirid, cyrtorhinus population dynamic figure.
Embodiment
Transgenic Bt Rice of the present invention is selected to turn cry2Aa trans-genetic hybrid rice T2A-1, it has very high resistance to lepidoptera pest, non-transgenic Juvenile stage is to extensive 63 paddy rice of illumination, and rice paddy seed provides by State Key Laboratory of Crop Genetic Improvent.Paddy rice is cultivated to about 15cm height for evaluation test with Yoshida nutrient solution in dish for cultivating (length × wide × height=25cm × 20cm × 3cm).Rice seedling breeding condition is temperature (26 ± 2 DEG C), relative moisture (80%).
Confession examination brown planthopper of the present invention picks up from rice field, Wuhan, Hubei, and sets up population in indoor respectively at T2A-1 and the bright rice seedling of extensive 63, raises 10 generations more than.Mirid, cyrtorhinus picks up from Xiaogan rice field, Hubei, raises 6 generations more than for test at indoor bright extensive 63 rice seedlings with brown planthopper ovum.Rearing conditions is temperature (28 ± 1 DEG C), relative moisture (70 ± 5%), Light To Dark Ratio (L14h:D10h).
Evaluation method of the present invention is by Tier-1 toxicity assay, and three grades of nutritional tests, albumen transfer law, predation functional responses and population density compare 5 key components and form with population dynamic.Be described in detail respectively below.
1, Tier-1 toxicity assay: evaluate the Bt albumen of Transgenic Bt Rice expression to the genotoxic potential of non-target organism Mirid, cyrtorhinus.
Mirid, cyrtorhinus is raised with artificial feeding device, Mirid, cyrtorhinus artificial feeding device is the bilateral glass tube 2 (see Fig. 1) of diameter × height=2cm × 12cm, its one end is feed chamber fitted, and feed chamber fitted is formed by parcel feed in the middle of double-deck Parafilm film (3).After access Mirid, cyrtorhinus examination worm, with rubber band, the nylon gauze 1 of quality exquisiteness is tied in the glass tube other end.Raise device black cloth during raising to cover, only expose the port having feed, and towards direction of illumination, keep relative moisture to reach more than 90%, then be placed in temperature 28 ± 1 DEG C, photoperiod L:D=14:10h, cultivate in the intelligent climatic cabinate of humidity 90 ± 5%.
With the Chemically defined diet of three kinds of forms as Mirid, cyrtorhinus feed namely: Chemically defined diet (negative control), containing 300 μ g/mlCry2Aa albumen artificial feed, containing 40 μ g/mlPA artificial feed (potassium dihydrogen arsenate, positive control); With the rice seedling containing brown planthopper ovum by Mirid, cyrtorhinus raise to 2 age nymph (because of from 1 age raising, lethality is very high), in single head access raising device, each process respectively connects Mirid, cyrtorhinus nymph 36, change feed every day and observed and recorded Mirid, cyrtorhinus is casted off a skin and survival condition, until sprout wings, new emerging adult is in 24 hours, and 2 ~ 3 identical sex adults merge rear ten thousand/scales/electronic balance weighing.
Described Chemically defined diet is the feed for the announcement of the patent No. 99108791.7 embodiment 2, and described amino acid contains following component: half deamination acid 80; Cystine 20; Asparagine 230; Glutamine 240; Arginine 175; Isoleucine 100; Glutamic acid 250; Serine 400; Histidine 100; Leucine 240; Lysine 240; Phenyl alanine 200; Threonine 130; Tyrosine 10; Valine 300; Methionine 110; γ-aminobutyric acid 10; Glycine 30; Alanine 130; Proline 100; Tryptophan 105; Aspartic acid 100.Described vitamin contains following component: vitamin h 0.025; Calcium pantothenate 5.0; Choline Chloride 50.0; Folic acid 1.0; Inositol 50; Nicotinic acid 15; Nicotinic acid serge trembles pungent 2.5; Vitamin b3 5.0; Thiamine hydrochloride 2.0; Vitamin C 100.Described mineral salt contain following component: CaCl
2﹒ 2H
2o3.115; CuCl
2﹒ 2H
2o0.268; FeCl
3﹒ 6H
2o2.228; MnCl
2﹒ 4H
2o0.793; ZnCl
20.396; MgCl
2﹒ 6H
2o200; KH
2pO
4500.Described sucrose concentration 9000.Above concentration unit is mg/100ml.
The Mirid, cyrtorhinus life table parameters taking food three kinds of foods is as shown in table 1,
Table 1
Note: in table, data are mean ± standard error, numeral observation sample number in bracket.
au checks;
bchi-square Test;
cstudent t checks.Numerical value heel
*represent significant difference (P<0.01) compared with the control.
As seen from Table 1, compared with control treatment, containing the Mirid, cyrtorhinus development duration in each length of time of 300 μ g/mlCry2Aa albumen food preparation, nymph survival rate, ratio of female and first emergence body weight there are no significant difference; And in positive control process, only have 2 age development duration there is no significant difference compared with the control, other development duration parameter equal significant prolongation compared with the control, and do not have Mirid, cyrtorhinus to be developed to adult.Kaplan-Meier survival analysis result shows: take food the survival rate there was no significant difference (P=0.640) between Mirid, cyrtorhinus containing 300 μ g/mlCry2Aa albumen artificial feed and control treatment; And take food containing 40 μ g/mlPA artificial feed Mirid, cyrtorhinus compared with the control, its survival rate significantly declines (P<0.001) (Fig. 2).In addition, utilize elisa technique to detect Mirid, cyrtorhinus and take food front and back, be respectively 98.39 ± 3.15 μ g/ml and 91.09 ± 3.71 μ g/ml containing Cry2Aa protein content in Cry2Aa albumen artificial feed, have dropped 7.42%, but its content be far above the expression of rice leaf sheath tissue.
The artificial feed of different disposal are suitably diluted, after adding TritonX-100 mixing in 0.1% ratio, is applied to maize leaf surface with writing brush, naturally after drying, each process connects rice leaf roller 2 instar larvae 15, repeats 4 times, records death condition after 48 hours.Result shows, before and after taking food, the lethality rate of artificial feed to rice leaf roller larvae containing Cry2Aa albumen is respectively 90% and 80%, and far above pure artificial feed 13.3%, wholely take food in process, Cry2Aa albumen all has higher insecticidal activity.
In sum, Mirid, cyrtorhinus is exposed to high dose and has the Cry2Aa albumen of insecticidal activity, shows that it is insensitive to Cry2Aa albumen.
2, three grades of nutritional tests: evaluate Transgenic Bt Rice by the safety effects of prey mediation to Mirid, cyrtorhinus by the food chain of paddy rice-brown planthopper-Mirid, cyrtorhinus.
1. using brown planthopper ovum as food
To 6 ~ 7 15 age in days rice seedlings are housed, access 2 brown planthopper female adult pest cross preoviposition period in the glass tube of diameter × height=3cm × 25cm, lay eggs 2 days, raise the nymph of Mirid, cyrtorhinus to adult with two rice varieties, the seedling of laying eggs that is respectively; In each glass tube within access hatching 24h, from the Mirid, cyrtorhinus nymph 1 of non-transgenic parent paddy rice, and seal with 100 order gauzes.Each kind, be respectively connect Mirid, cyrtorhinus nymph 32, Mirid, cyrtorhinus 1 ~ 3 age nymph, within every 2 days, change 1 seedling of laying eggs, change 1 seedling of laying eggs in every day in 4 ages to adult stage.Every morning observed and recorded Mirid, cyrtorhinus nymph survival and situation of casting off a skin, until nymph sprouts wings, in new emerging adult 24 hours, with ten thousand/scales/electronic balance weighing after 2 ~ 3 identical sex adults merge.
The Mirid, cyrtorhinus life table parameters that the brown planthopper ovum of raising with different rice material is food is as shown in table 2,
Table 2
Note: in table, data are mean ± standard error, numeral observation sample number in bracket.
With in a line, Transgenic Bt Rice T2A-1 with contrast paddy rice bright extensive 63 and carry out statistics and compare,
au checks;
bchi-square Test;
cstudent t checks.
From table 2, Transgenic Bt Rice T2A-1 compared with parent control, every life table parameters of Mirid, cyrtorhinus there are no significant difference.
2. using brown planthopper nymph as food
Be provided in two rice varieties every day, fasten hatch and the brown planthopper nymph taking food 24 ~ 48 hours as fed diets Mirid, cyrtorhinus, concrete operations are as follows: with the rice seedling containing brown planthopper ovum Mirid, cyrtorhinus raised to 2 age nymph (during with brown planthopper nymph for food, 1 age nymph have higher lethality, therefore selection 2 Mirid, cyrtorhinus in age is as initial subjects), feed with different rice varieties again, the brown planthopper nymph of system, 2 ~ 3 Mirid, cyrtorhinus nymph every days every in age provided 10 brown planthopper nymphs, 4 age Mirid, cyrtorhinus provide 20 brown planthopper nymphs to every day every in adult stage, Mirid, cyrtorhinus nymph single head is raised and is covered with wet sponge moisturizing in bottom, in 2cm × 12cm finger-type pipe, each kind, system respectively connects Mirid, cyrtorhinus nymph 80, every morning observed and recorded Mirid, cyrtorhinus nymph survival and situation of casting off a skin, until nymph sprouts wings, in new emerging adult 24 hours, with ten thousand/scales/electronic balance weighing after 3 ~ 5 identical sex adults merge.
The Mirid, cyrtorhinus life table parameters that the brown planthopper nymph raised with different rice material is food is as shown in table 3,
Table 3
Note: in table, data are mean ± standard error, numeral observation sample number in bracket.
With in a line, Transgenic Bt Rice T2A-1 with contrast paddy rice bright extensive 63 and carry out statistics and compare,
au checks;
bchi-square Test;
cstudent t checks.
From table 3, Transgenic Bt Rice T2A-1 compared with parent control, every life table parameters of Mirid, cyrtorhinus there are no significant difference.
3, Cry2Aa albumen is at the transfer law of paddy rice-brown planthopper-Mirid, cyrtorhinus food chain;
The rice seedling put into the different rice varieties of 15 ages in days respectively in 2L glass jar, being, access 20 bosom ovum brown planthopper female adult pest that corresponding rice material is raised, access after laying eggs 2 days 3 ~ 4 age Mirid, cyrtorhinus, after taking food 3 days, respectively to rice leaf sheath, brown planthopper ovum, Mirid, cyrtorhinus samples, leaf sheath about 30mg, about 200, brown planthopper ovum, Mirid, cyrtorhinus 20; Each process repetition 4 ~ 5 times.
By different rice varieties, be raise hatching after 24 ~ 48 hours brown planthopper nymph with 3 ~ 4 age Mirid, cyrtorhinus access in the 2L glass jar containing corresponding rice seedling simultaneously, every day changes brown planthopper, after taking food 3 days, respectively to rice leaf sheath, brown planthopper nymph and Mirid, cyrtorhinus sample, leaf sheath about 30mg, brown planthopper nymph about 30mg, Mirid, cyrtorhinus 20; Each process weight 4 ~ 5 times;
Respectively with two food chains that brown planthopper ovum and nymph are food, its Cry2Aa albumen transfer law is as shown in Fig. 3, Fig. 4, Fig. 5, Fig. 6, and in T2A-1 leaf sheath tissue, Cry2Aa protein content is far away higher than the content in brown planthopper nymph and Mirid, cyrtorhinus body; The Cry2Aa albumen of higher concentration is detected, far above brown planthopper nymph in the Mirid, cyrtorhinus body taking food brown planthopper ovum and nymph; The existence of Cry2Aa albumen is not detected in brown planthopper ovum.All process of check variety bright extensive 63 all do not detect Cry2Aa albumen.
Above result shows, Cry2Aa albumen is not successively decrease step by step in two food chain Different Nutrition levels, and Mirid, cyrtorhinus is exposed to the Cry2Aa albumen of higher concentration.
5, the impact of cry2Aa trans-genetic hybrid rice on Mirid, cyrtorhinus predation functional responses is turned.
Mirid, cyrtorhinus raises generation respectively at two kinds of rice material T2A-1 and bright extensive 63, sprout wings latter 2 days and the female worm of mating for test, Nature enemy 24 hours before test.Mirid, cyrtorhinus single head is put into bottom and is covered with sponge and containing the finger-type test tube of rice seedling, 1 age brown planthopper nymph as prey.5 BPH density process, are respectively 10,20,30,40,50, and each density process repeats 5 times, record the brown planthopper nymph number of prey after 24 hours.Result is as shown in Fig. 7, Fig. 8.
Mirid, cyrtorhinus from different rice material all meets Holling type Ⅱ reaction to the functional response of brown planthopper nymph.The statistical analysis of 2 parameters in Holling type Ⅱ reaction disc equation is shown, from Bt Transgenic Rice T2A-1 and bright extensive 63 Mirid, cyrtorhinus of raising of check variety paddy rice to the parameter of brown planthopper predation functional responses, Zero-day Attack rate and process prey time all there was no significant differences (table 4).
Table 4
Note: a: Zero-day Attack rate; Th: process prey time.Identical parameters compares employing student t inspection.
Above result shows: the Cry2Aa albumen mediated by brown planthopper affects without significance Mirid, cyrtorhinus predation functional responses.
6, the impact of cry2Aa trans-genetic hybrid rice on Mirid, cyrtorhinus field population is turned
Experimental scheme and field management
Test site selects rice workspace in Hubei Province to have two places representing meaning: Xiaogan and Suizhou, carries out field trial, adopt randomized complete-block design, every community 150m respectively at 2011 ~ 2013 years
2(10m × 15m), each rice material repeats 4 times.This rice transplanting single, 1 meter of wide isolation strip is left in minizone, and whole test region surrounding kind leaves protection row (0.5m).Rich water quality management operates routinely, but whole growth period duration of rice does not use any agricultural chemicals.
Sampling and species identification:
Pest sucking device: utilize China to contain Mount Taishan backpack ultra-low-volume sprayer, (1999) such as Liu Yufang are copied to convert, wherein additional structure leads cover by air inlet, collection worm net, two flexible pipes (Ф 60cm × 100cm), sample boxes (50 × 50 × 120cm) form, collection worm net is commercially available 100 order nylon gauzes, is placed in air outlet.
Investigation method: the pest sucking device of repacking is operated by two people, and a people bears pest sucking device, another people removes stage makeup and costume and collects worm net and settle sample boxes.Adopt diagonal sampling method, every plot area 150m
2, 5 samples are investigated in every community at every turn, every sample 0.25m
2, about 6 clumps of rice strains.Within about 30 days after rice transplanting, institute an inquiry, sampling in every about 10 days 1 time, to paddy rice harvesting first about 10 days, look-see 5 ~ 7 times.
Classification and qualification: sampling is preserved with the alcohol of 75% immediately, after bringing indoor back, removing foreign material, the arthropods chosen preserves with 80% alcohol again, then carries out identifying and counting under stereoscope.On this basis, statistics Mirid, cyrtorhinus population density and population dynamic, and statistical analysis is carried out to the otherness of parameter between two kinds of paddy rice fields.
Population density comparative result is as shown in table 5, and Rice District of Hubei Province rice fulgorid predator Mirid, cyrtorhinus generating capacity is also little, and on two kinds of paddy rice, population density difference is not all significantly (P>0.05).As can be seen from the population occurrence dynamics of difference sampling period also, black to educate green plant bug similar at the occurrence tendency of two kinds of paddy rice fields, and all sampling time point difference is all not remarkable; RepeatedmeasuresANOVA analysis result shows two kinds of paddy rice field Mirid, cyrtorhinus population dynamic there was no significant differences (Fig. 9, Xiaogan 2011 equally; Figure 10. Xiaogan 2012; Figure 11. Xiaogan 2013; 2011, Figure 12 Suizhou).
Visible, the plantation of Transgenic Bt Rice T2A-1 to field Mirid, cyrtorhinus population without negative effect.
Table 5
Note: in table, data are mean ± standard error.The same place identical time adopts student t inspection.
Population density and whole growth period duration of rice gather the mean of Mirid, cyrtorhinus quantity.
Comprehensive high dose Cry2Aa albumen feeding result of the test, three grades of nutritive life table parameters, albumen transfer law, predation functional responses and field investigation interpretation of result, demonstrate and turn the ecological security of cry2Aa trans-genetic hybrid rice to Mirid, cyrtorhinus.
Claims (3)
1. transgenic pest-resistant rice is to predator Mirid, cyrtorhinus method for evaluating safety, it is characterized in that comprising following 5 parts:
(1) Tier-1 toxicity assay: evaluate the Bt albumen of Transgenic Bt Rice expression to the genotoxic potential of non-target organism Mirid, cyrtorhinus;
Mirid, cyrtorhinus is raised with artificial feeding device, access Mirid, cyrtorhinus examination worm, raise device black cloth during raising to cover, only expose the port having feed, and towards direction of illumination, keep relative moisture to reach more than 90%, then be placed in temperature 28 ± 1 DEG C, photoperiod L:D=14:10h, cultivate in the intelligent climatic cabinate of humidity 90 ± 5%;
With the Chemically defined diet of three kinds of forms as Mirid, cyrtorhinus feed namely: Chemically defined diet, the Bt protein concentration more than 10 times containing transgenic paddy rice leaf sheath tissue expression and the Chemically defined diet containing 40 μ g/ml potassium dihydrogen arsenates; With the rice seedling containing brown planthopper ovum by Mirid, cyrtorhinus raise to 2 age nymph, in single head access raising device, each process respectively connects Mirid, cyrtorhinus nymph 36, change feed every day and observed and recorded Mirid, cyrtorhinus is casted off a skin and survival condition, until sprout wings, new emerging adult is in 24 hours, and 2 ~ 3 identical sex adults merge rear ten thousand/scales/electronic balance weighing;
By the concentration of ELISA and sensitive insect raw survey technology monitored over time insecticidal proteins, stability and biologically active in test;
Select the nymphal development of Mirid, cyrtorhinus to go through phase, nymph survival rate, ratio of female and first emergence body weight as evaluating index, statistical analysis is carried out to it, to evaluate the Bt albumen of Transgenic Bt Rice expression to the toxic effect of Mirid, cyrtorhinus;
(2) three grades of nutritional tests: evaluate Transgenic Bt Rice by the safety effects of prey mediation to Mirid, cyrtorhinus by the food chain of paddy rice-brown planthopper-Mirid, cyrtorhinus;
1. using brown planthopper ovum as food
Brown planthopper female adult pest preoviposition period that access 2 being crossed in the glass tube that 6 ~ 7 15 age in days rice seedlings are housed, lays eggs 2 days, raises the nymph of Mirid, cyrtorhinus to adult respectively with different rice varieties, the seedling of laying eggs that is; In each glass tube within access hatching 24h, from the Mirid, cyrtorhinus nymph 1 of non-transgenic parent paddy rice, each kind, be respectively connect Mirid, cyrtorhinus nymph 32, Mirid, cyrtorhinus 1 ~ 3 age nymph, within every 2 days, change 1 seedling of laying eggs, change 1 seedling of laying eggs in every day in 4 ages to adult stage; Every morning observed and recorded Mirid, cyrtorhinus nymph survival and situation of casting off a skin, until nymph sprouts wings, in new emerging adult 24 hours, with ten thousand/scales/electronic balance weighing after 2 ~ 3 identical sex adults merge;
Select the nymphal development of Mirid, cyrtorhinus go through the phase, nymph survival rate, ratio of female and just emergence body weight as evaluating index, and carry out statistical analysis, evaluate the Transgenic Bt Rice that mediated by brown planthopper ovum to the safety effects of Mirid, cyrtorhinus;
2. using brown planthopper nymph as food
Be provided in different rice varieties every day, fasten hatch and the brown planthopper nymph taking food 24 ~ 48 hours as fed diets Mirid, cyrtorhinus, concrete operations are as follows: with containing brown planthopper ovum rice seedling by Mirid, cyrtorhinus raise to 2 age nymph, feed with different rice varieties again, the brown planthopper nymph of system, 2 ~ 3 Mirid, cyrtorhinus nymph every days every in age provided 10 brown planthopper nymphs, 4 age Mirid, cyrtorhinus provide 20 brown planthopper nymphs to every day every in adult stage, Mirid, cyrtorhinus nymph single head is raised and is covered with in the finger-type test tube of wet sponge moisturizing in bottom, each kind, system respectively connects Mirid, cyrtorhinus nymph 80, every morning observed and recorded Mirid, cyrtorhinus nymph survival and situation of casting off a skin, until nymph sprouts wings, in new emerging adult 24 hours, with ten thousand/scales/electronic balance weighing after 3 ~ 5 identical sex adults merge,
Select the nymphal development of Mirid, cyrtorhinus go through the phase, nymph survival rate, ratio of female and just emergence body weight as evaluating index, and carry out statistical analysis, evaluate the Transgenic Bt Rice that mediated by brown planthopper nymph to the safety effects of Mirid, cyrtorhinus;
(3) albumen transfer law: the approach and the degree that are exposed to Bt albumen by ELISA detection means qualification Mirid, cyrtorhinus;
The rice seedling put into the different rice varieties of 15 ages in days respectively in glass jar, being, access 20 bosom ovum brown planthopper female adult pest that corresponding rice material is raised, access after laying eggs 2 days 3 ~ 4 age Mirid, cyrtorhinus, after taking food 3 days, respectively to rice leaf sheath, brown planthopper ovum, Mirid, cyrtorhinus samples, each process repetition 4 ~ 5 times;
By different rice varieties, be raise hatching after 24 ~ 48 hours brown planthopper nymph with 3 ~ 4 age Mirid, cyrtorhinus access in the glass jar containing corresponding rice seedling simultaneously, every day changes brown planthopper, after taking food 3 days, respectively to rice leaf sheath, brown planthopper nymph and Mirid, cyrtorhinus sample, each process repetition 4 ~ 5 times;
Adopt elisa technique, respectively the content of Bt albumen in above sample is detected, determine that the food chain Mirid, cyrtorhinus by different is exposed to the degree of Bt albumen;
(4) predation functional responses: by comparing the Mirid, cyrtorhinus predation parameter that Transgenic Bt Rice and non-transgenic parent control kind are raised, evaluates Transgenic Bt Rice to the impact of this non-target organism;
Mirid, cyrtorhinus raises a generation respectively at different rice material, sprout wings latter 2 days and the female worm of mating for test, Nature enemy 24 hours before test, puts into bottom and is covered with sponge and finger-type test tube containing rice seedling by Mirid, cyrtorhinus single head, 1 age brown planthopper nymph as prey; 5 BPH density process, are respectively 10,20,30,40,50, and each density process repeats 5 times, record the brown planthopper nymph number of prey after 24 hours;
Select the Zero-day Attack rate of Mirid, cyrtorhinus and process the prey time as evaluating index, and carrying out statistical analysis, evaluating Transgenic Bt Rice to the impact of Mirid, cyrtorhinus predation functional responses;
(5) population density compares with population dynamic: by comparing the occurrence dynamics of Transgenic Bt Rice and non-transgenic parent control rice field Mirid, cyrtorhinus, evaluates the plantation of Transgenic Bt Rice to the impact of this non-target organism population;
Adopt diagonal sampling method, every plot area is 150m
2, get 5 samples, every sample 0.25m at every turn
2, about 6 clumps of rice strains; Within after rice transplanting 30 days, start, with pest sucking device sampling, to paddy rice harvesting first 10 days, to sample 5 ~ 7 times altogether; Sampling is preserved with the alcohol of 75% immediately, carry out identifying and counting under stereoscope, to add up Mirid, cyrtorhinus population density and population dynamic, and statistical analysis is carried out to the otherness of parameter between different paddy rice field, evaluate Transgenic Bt Rice to the safety effects of Mirid, cyrtorhinus field population.
2. transgenic pest-resistant rice according to claim 1 is to predator Mirid, cyrtorhinus method for evaluating safety, it is characterized in that Mirid, cyrtorhinus artificial feeding device is the bilateral glass tube (2) of diameter × height=2cm × 12cm, its one end is feed chamber fitted, and feed chamber fitted is formed by parcel feed in the middle of double-deck Parafilm film (3).
3. transgenic pest-resistant rice according to claim 1 is to predator Mirid, cyrtorhinus method for evaluating safety, it is characterized in that in 2cm × 12cm finger-type test tube, each kind, is respectively connect Mirid, cyrtorhinus nymph 80.
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| CN104222024B (en) * | 2014-09-03 | 2016-06-08 | 华中农业大学 | The method of the blue or green hidden wing first of wing ant shape of artificial mass rearing |
| CN104798616B (en) * | 2015-04-29 | 2017-03-22 | 广西壮族自治区农业科学院水稻研究所 | Method for evaluating transgenic anti-herbicide rice safety based on brown planthopper honeydew |
| CN104813855A (en) * | 2015-04-29 | 2015-08-05 | 广西壮族自治区农业科学院水稻研究所 | Evaluation method of transgenic herbicide-resistant rice for environmental safety of cyrtohinus lividipennis |
| CN104823924B (en) * | 2015-05-08 | 2017-11-03 | 河南省农业科学院植物保护研究所 | Toxicity test method of the systemic insecticide to green plant bug |
| CN106093369B (en) * | 2016-06-29 | 2017-10-20 | 福建农林大学 | Method for evaluating safety of the Bt paddy rice to non-target insects brown paddy plant hopper |
| CN107828817B (en) * | 2017-09-29 | 2021-02-19 | 杭州瑞丰生物科技有限公司 | Method for preventing and treating hemiptera pests of crops by using Bt protein |
| CN108575912B (en) * | 2018-03-13 | 2020-11-13 | 中国农业科学院棉花研究所 | Method for evaluating influence of insecticidal protein on adult propylaea japonica and feeding device |
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