CN103931564B - Transgenic pest-resistant rice is to parasite Anagrus nilaparvatae method for evaluating safety - Google Patents
Transgenic pest-resistant rice is to parasite Anagrus nilaparvatae method for evaluating safety Download PDFInfo
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Abstract
The present invention relates to a kind of transgenic pest-resistant rice to parasite Anagrus nilaparvatae method for evaluating safety, there are 3 parts, Tier-1 toxicity assay: evaluate the Bt albumen of Transgenic Bt Rice expression to the genotoxic potential of non-target organism Anagrus nilaparvatae; Successive generation three grades of nutritional tests: evaluate Transgenic Bt Rice by the safety effects of host's mediation to Anagrus nilaparvatae by the food chain of paddy rice-brown planthopper-Anagrus nilaparvatae; Albumen transfer law: the approach and the degree that are exposed to the Bt albumen that Transgenic Bt Rice is expressed by ELISA detection means qualification Anagrus nilaparvatae; Finally, the ecological consequence that the plantation evaluating described genetically modified crops according to 3 aspect aggregation of data 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 parasite Anagrus nilaparvatae 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.Current Transgenic Bt Rice is the main insect-proof rice controlling 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.Anagrus nilaparvatae is the main ovum parasite of rice fulgorid, obviously can reduce the population quantity (Louetal. of rice fulgorid, 2013), in rice field, egg parasitoids is likely by the honeydew of hemipteran, pollen, nectar or other plant liquid are exposed to Bt albumen, there is the possibility that there is ecological risk, the impact of research transgenic paddy rice on Anagrus nilaparvatae is the pith that Study on Environmental Safety of Transgenic Crops is evaluated, so, set of system evaluation Transgenic Bt Rice is set up particularly important to the method whether Anagrus nilaparvatae 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 parasite Anagrus nilaparvatae method for evaluating safety.
The implementation of the object of the invention is, transgenic pest-resistant rice, to parasite Anagrus nilaparvatae method for evaluating safety, comprises following 3 parts:
(1) Tier-1 toxicity assay: evaluate the Bt albumen of Transgenic Bt Rice expression to the genotoxic potential of non-target organism Anagrus nilaparvatae;
1. biological activity determination is carried out to the Bt albumen that test batch Transgenic Bt Rice is expressed
Using Indian meal moth as test insect, the Bt albumen that Transgenic Bt Rice is expressed is added the feed preparing variable concentrations gradient in Indian meal moth artificial feed, after feed prepares, ELISA method detects the real content of this Bt albumen of variable concentrations gradient, and with pure feed in contrast, in every part of feed, access 40 Indian meal moth newly hatched larvaes, repeat 5 times, within 7 days, record larvae alive situation afterwards, calculate LC
50, the activity of the Bt albumen that Transgenic Bt Rice is expressed is judged with this;
2. the high dose Transgenic Bt Rice Bt albumen of expressing is on the impact of Anagrus nilaparvatae survival rate and fecundity, evaluates Anagrus nilaparvatae to the susceptibility of this Bt albumen;
With 10% hydromel as Anagrus nilaparvatae food, prepare three kinds of hydromels and be used for test, that is: pure 10% hydromel (w/v) as negative control, 10% hydromel containing the Bt albumen of more than 10 times of content expressing in Transgenic Bt Rice, 10% hydromel containing 10 μ g/mlPA as positive control;
Impact on Anagrus nilaparvatae survival rate: get in 4 hours and sprout wings and the Anagrus nilaparvatae queen bee 30 of mating, in every 10 accesses glass tube, seal with nylon wire, raise with above three kinds of hydromels respectively, sooner or later each replacing food, every day records its death condition respectively at early, middle and late, investigates the Bt albumen of high dose Transgenic Bt Rice expression to the impact of Anagrus nilaparvatae survival rate;
Impact on Anagrus nilaparvatae fecundity: raise respectively at the beginning of 20 with above three kinds of hydromels and sprout wings and the Anagrus nilaparvatae queen bee 24 hours of mating, then in single head access glass tube, to lay eggs 2 ~ 3 days, 15 age in days rice seedlings containing brown planthopper in glass tube; Seal with nylon wire after connecing worm, shift out parasitic wasp after 6 hours, record generation Anagrus nilaparvatae emergence quantity and sex every day, investigate the Bt albumen of high dose Transgenic Bt Rice expression to the impact of Anagrus nilaparvatae fecundity;
3. the Bt protein stability that the Transgenic Bt Rice in hydromel is expressed and biologically active;
The hydromel of the Bt albumen that front and back of feeding are expressed containing Transgenic Bt Rice is sampled respectively, repeat 3 times, ELISA detects this Bt protein content, meanwhile, the hydromel of the Bt albumen of being expressed containing Transgenic Bt Rice front and back of feeding is diluted to the concentration of 80 ± 10% lethality rates, is coated in Indian meal moth artificial feed surface, ventilating kitchen is after air-dry 2 hours, access 30 Indian meal moth newly hatched larvaes, repeat 3 times, after 7 days, record its death condition;
(2) successive generation three grades of nutritional tests: evaluate Transgenic Bt Rice by the safety effects of host's mediation to Anagrus nilaparvatae by the food chain of paddy rice-brown planthopper-Anagrus nilaparvatae;
1. Transgenic Bt Rice is on Anagrus nilaparvatae development duration and the impact in life-span
To newly in access glass tube, and seal with nylon wire by emergence Anagrus nilaparvatae list.The rice seedling of the different rice materials that 15 ages in days have been laid eggs 2 ~ 3 days by brown planthopper is housed in glass tube respectively.Until Anagrus nilaparvatae after parasitic 2 hours, shift out parasitic wasp, observe the emergence situation of Anagrus nilaparvatae of future generation, every 2 hour records once Anagrus nilaparvatae of future generation sprout wings time, quantity and sex, repeat above parasitic processes between corresponding rice material different generations.Each rice material establishes 40 repetitions;
Anagrus nilaparvatae adult point male and female for emergence raised in glass tube, often pipe 4, to feed 10% hydromel (w/v) with absorbent cotton, sooner or later respectively change a food, every day records its death condition respectively at early, middle and late;
2. Transgenic Bt Rice is on the impact of Anagrus nilaparvatae fecundity, ratio of female and survival rate
Will newly emergence Anagrus nilaparvatae list to be in access glass tube, with (2) 1. processing method, will add 10% hydromel (w/v) in absorbent cotton, and absorbent cotton is attached to Anagrus nilaparvatae that glass tube walls feeds, and sooner or later feeds once; Observe Anagrus nilaparvatae emergence quantity of future generation and sex every day, until sprout wings completely; After 7 days, dissect rice stem under stereoscope, the parasitic but Anagrus nilaparvatae ovum grain number of not sprouting wings of record, is become redness by the ovum of parasitism;
Select the development duration of Anagrus nilaparvatae continuous multi-generation, life-span, fecundity, ratio of female and survival rate as evaluating index, and carry out statistical analysis, evaluate the Transgenic Bt Rice that mediated by brown planthopper ovum to the safety effects of Anagrus nilaparvatae;
(3) albumen transfer law: the approach and the degree that are exposed to the Bt albumen that Transgenic Bt Rice is expressed by ELISA detection means qualification Anagrus nilaparvatae;
For different rice materials, respectively to 15 age in days rice leaf sheaths, originate in the brown planthopper ovum grain of rice leaf sheath and sample using brown planthopper ovum as the Anagrus nilaparvatae of host, leaf sheath 30 ± 1mg, the Anagrus nilaparvatae adult that 200, brown planthopper ovum and all generations collect, each process repeats 3 ~ 5 times;
Adopt elisa technique, respectively the content of the Bt albumen that Transgenic Bt Rice in above sample is expressed is detected, determine whether Anagrus nilaparvatae is exposed to the Bt albumen of Transgenic Bt Rice expression and the degree of its exposure by brown planthopper ovum approach.
The present invention evaluates the Bt albumen of Transgenic Bt Rice expression to the genotoxic potential of non-target organism Anagrus nilaparvatae by Tier-1 toxicity assay; Transgenic Bt Rice is evaluated by the impact of host's mediation on Anagrus nilaparvatae by the food chain of paddy rice-brown planthopper-Anagrus nilaparvatae; ELISA detection means qualification Anagrus nilaparvatae is adopted to be exposed to approach and the degree of the Bt albumen that Transgenic Bt Rice is expressed.Finally, the ecological consequence that the plantation comprehensively analyzing Transgenic Bt Rice according to the data of 3 aspects produces this non-target organism.
The present invention establishes the evaluation method of set of system, science in laboratory, 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 takes food pure hydromel and adds the Anagrus nilaparvatae survivorship curve figure of Cry2Aa albumen and PA hydromel.
Embodiment
Transgenic Bt Rice of the present invention is selected to turn cry2Aa trans-genetic hybrid rice T2A-1, express the one of Cry2Aa albumen system Bt albumen, it has very high resistance to lepidoptera pest, non-transgenic Juvenile stage is to extensive 63 paddy rice of illumination, and turning cry2Aa trans-genetic hybrid rice T2A-1 seed provides by State Key Laboratory of Crop Genetic Improvent.Bright extensive 63 paddy rice, turn cry2Aa trans-genetic hybrid rice T2A-1 and cultivate to after about 15cm height, for evaluation test with Yoshida nutrient solution in the dish for cultivating of length × wide × height=25cm × 20cm × 3cm.Rice seedling breeding condition is temperature (26 ± 2 DEG C), relative moisture (80%).
The Cry2Aa protein content of expressing due to Bt Transgenic Rice is limited, in view of the Cry2Aa albumen that Anagrus nilaparvatae will be made to be exposed to higher dosage, the applicant have purchased the Cry2Aa albumen that Beijing Le Shining Science and Technology Ltd. produces, the Cry2Aa albumen system same Bt albumen that the Cry2Aa albumen bought and Bt Transgenic Rice T2A-1 express, therefore the applicant is using this Cry2Aa albumen replacing Bt Transgenic Rice to express as trial target.
Of the present inventionly pick up from rice field, Wuhan, Hubei for examination brown planthopper, and in indoor respectively at turning cry2Aa trans-genetic hybrid rice T2A-1 and the bright rice seedling of extensive 63 sets up population, raise 10 generations more than.Anagrus nilaparvatae is provided by insect science research institute of Zhejiang University, raises 10 generations more than for test at the indoor TN1 rice seedling with brown planthopper ovum.Rearing conditions is temperature (28 ± 1 DEG C), relative moisture (70 ± 5%), Light To Dark Ratio (L14h:D10h).Indian meal moth sensitive strain is used for Cry2Aa protein active and measures.
Evaluation method of the present invention is by Tier-1 toxicity assay, and successive generation three grades of nutritional tests and albumen transfer law 3 key components form.Be described in detail respectively below.
(1) Tier-1 toxicity assay: evaluation turns the Cry2Aa albumen of cry2Aa gene pest-resistant paddy rice T2A-1 expression to the genotoxic potential of non-target organism Anagrus nilaparvatae.
1. biological activity determination is carried out to test batch Cry2Aa albumen
Using Indian meal moth as test insect, the Cry2Aa albumen of purchase is added in Indian meal moth artificial feed and prepare 5 concentration gradients, be respectively 2,10,20,30, the feed of 40 μ g/g, and with pure feed in contrast, after feed prepares, ELISA method detects the real content of variable concentrations gradient Cry2Aa albumen, 40 Indian meal moth newly hatched larvaes are accessed in every part of feed, repeat 5 times, within 7 days, record larvae alive situation afterwards, calculate LC
50, the activity of Cry2Aa albumen is judged with this.
Detect Cry2Aa protein content in Indian meal moth artificial feed by ELISA, the real content of 5 concentration gradients is respectively 0.29,8.96,14.29,24.74,32.10 μ g/g, and Cry2Aa albumen is to the LC of Indian meal moth newly hatched larvae
50be 14.89 μ g/g.Illustrate that the Cry2Aa albumen bought has good insecticidal activity.
2. high dose Cry2Aa albumen is on the impact of Anagrus nilaparvatae survival rate and fecundity, evaluates Anagrus nilaparvatae to the susceptibility of Cry2Aa albumen.
With 10% hydromel as Anagrus nilaparvatae food, prepare three kinds of hydromels and be used for test, that is: pure 10% hydromel (w/v) as negative control, 10% hydromel containing 300 μ g/mlCry2Aa, containing 10 μ g/mlPA(potassium dihydrogen arsenates) 10% hydromel as positive control.
Impact on Anagrus nilaparvatae survival rate: get in 4 hours and sprout wings and the Anagrus nilaparvatae queen bee 30 of mating, in every 10 accesses diameter × height=3cm × 25cm glass tube, with 100 order nylon wire sealings, raise with above three kinds of hydromels respectively, sooner or later each replacing food, every day is respectively at 8:00,14:00,20:00 records its death condition, investigates high dose Cry2Aa albumen to the impact of Anagrus nilaparvatae survival rate;
Kaplan-Meier survival analysis result shows: the survival rate between taking food Anagrus nilaparvatae containing 300 μ g/mlCry2Aa albumen hydromels and contrasting, there was no significant difference (P=0.287); And compared with the control, take food Anagrus nilaparvatae survival rate containing 10 μ g/mlPA hydromels and then significantly to decline (P<0.001) (see figure 1).
Impact on Anagrus nilaparvatae fecundity: raise at the beginning of 20 with above three kinds of hydromels and sprout wings and the Anagrus nilaparvatae queen bee 24 hours of mating, then, in single head access diameter × height=3cm × 25cm glass tube, to lay eggs 2 ~ 3 days, 15 age in days rice seedlings containing brown planthopper in glass tube; Seal with 100 order nylon wires after connecing worm, shift out parasitic wasp after 6 hours, record generation Anagrus nilaparvatae emergence quantity and sex every day, investigate high dose Cry2Aa albumen to the impact of Anagrus nilaparvatae fecundity; .
Observed result: the Anagrus nilaparvatae egg laying amount taking food pure 10% hydromel, the hydromel containing 300 μ g/mlCry2Aa albumen and the hydromel containing 10 μ g/mlPA is respectively 14.5 ± 1.84,16.1 ± 1.68,3.7 ± 0.63, Dunnetttest analysis result shows: compared with the control, take food the Anagrus nilaparvatae egg laying amount there was no significant difference (P=0.650) containing 300 μ g/mlCry2Aa albumen hydromels, the Anagrus nilaparvatae egg laying amount taken food containing 10 μ g/mlPA hydromels then significantly declines (P<0.001).
3. the Cry2Aa protein stability in hydromel and biologically active;
In order to ensure stability and the biologically active of Cry2Aa albumen in the hydromel that Anagrus nilaparvatae took food in 12 hours, to feeding, the hydromel of front and back containing Cry2Aa albumen samples respectively, repeat 3 times, ELISA detects its Cry2Aa protein content, simultaneously, by feeding, the hydromel of front and back containing Cry2Aa albumen dilutes 10 times, be coated in Indian meal moth artificial feed surface, ventilating kitchen, after air-dry 2 hours, accesses 30 Indian meal moth newly hatched larvaes, repeat 3 times, after 7 days, record its death condition.
In the hydromel do not taken food by Anagrus nilaparvatae, the extraction efficiency of Cry2Aa albumen is about 94.5%, and its content is 283.5 ± 7.4 μ g/ml; After tassel chalcid fly takes food 12 hours, in hydromel, Cry2Aa protein content have dropped 24.9%, is 212.9 ± 26.6 μ g/ml.Although before and after taking food, in hydromel, Cry2Aa protein content declines to some extent, its content far exceeds more than 10 times that turn the Cry2Aa protein content that cry2Aa trans-genetic hybrid rice T2A-1 expresses.
The raw result of surveying of Indian meal moth shows: the hydromel before Anagrus nilaparvatae takes food and containing Cry2Aa albumen after taking food, after being coated in Indian meal moth artificial feed surface, 87.8% and 86.7% are respectively to the lethality rate of Indian meal moth newly hatched larvae, far away higher than pure 10% hydromel (lethality rate 10.0%).
In sum, Anagrus nilaparvatae is exposed to high dose and has the Cry2Aa albumen of insecticidal activity, shows that it is insensitive to Cry2Aa albumen.
(2) successive generation three grades of nutritional tests: turn cry2Aa gene pest-resistant paddy rice T2A-1 by the safety effects of host's mediation to Anagrus nilaparvatae by the food chain evaluation of paddy rice-brown planthopper-Anagrus nilaparvatae.
1. cry2Aa gene pest-resistant paddy rice T2A-1 is turned on Anagrus nilaparvatae development duration and the impact in life-span
The new emergence Anagrus nilaparvatae list of being raised by TN1 paddy rice in access diameter × height=3cm × 25cm glass tube, and seals with 100 order nylon wires.T2A-1 or bright extensive 63 rice seedlings that 15 ages in days have been laid eggs 2 ~ 3 days by brown planthopper are housed in glass tube respectively.Until Anagrus nilaparvatae after parasitic 2 hours, shift out parasitic wasp, observe the emergence situation of Anagrus nilaparvatae of future generation, every 2 hour records once Anagrus nilaparvatae of future generation sprout wings time, quantity and sex, repeat above parasitic processes between corresponding rice material different generations.Each rice material establishes 40 repetitions;
Anagrus nilaparvatae adult point male and female for emergence raised in glass tube, often pipe 4, to feed 10% hydromel (w/v) with absorbent cotton, sooner or later respectively change a food, every day records its death condition respectively at 8:00,14:00,20:00.
The Anagrus nilaparvatae development duration that the brown planthopper ovum of raising with different rice material is host is as shown in table 1,
Table 1
Note: in table, data are mean ± standard error, numeral observation sample number in bracket.
With in a line, turn cry2Aa gene pest-resistant paddy rice T2A-1 and contrast paddy rice bright extensive 63 and adopt U to check to carry out statistics and compare.
From table 1, no matter male and female, each from generation to generation between turn cry2Aa gene pest-resistant paddy rice T2A-1 compared with parent control, the development duration of Anagrus nilaparvatae there are no significant difference.
The Anagrus nilaparvatae life-span that the brown planthopper ovum of raising with different rice material is host 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, turn cry2Aa gene pest-resistant paddy rice T2A-1 and contrast paddy rice bright extensive 63 and adopt U to check to carry out statistics and compare.
From table 2, no matter male and female, each from generation to generation between turn cry2Aa gene pest-resistant paddy rice T2A-1 compared with parent control, difference that life-span of Anagrus nilaparvatae, there are no significant.
2. cry2Aa gene pest-resistant paddy rice T2A-1 is turned on the impact of Anagrus nilaparvatae fecundity, ratio of female and survival rate
The new emergence Anagrus nilaparvatae list of being raised by TN1 paddy rice is in access glass tube (specification is the same), processing method with (2) 1., 10% hydromel (w/v) will be added in absorbent cotton, and absorbent cotton is attached to Anagrus nilaparvatae that glass tube walls feeds, sooner or later feed once; Observe Anagrus nilaparvatae emergence quantity of future generation and sex every day, until sprout wings completely; After 7 days, dissect rice stem under stereoscope, the parasitic but Anagrus nilaparvatae ovum grain number of not sprouting wings of record, is become redness by the ovum of parasitism.
The Anagrus nilaparvatae fecundity that the brown planthopper ovum of raising with different rice material is host 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, turn cry2Aa gene pest-resistant paddy rice T2A-1 and contrast paddy rice bright extensive 63 and adopt student t to check to carry out statistics and compare.
From table 3, each from generation to generation between turn cry2Aa gene pest-resistant paddy rice T2A-1 compared with parent control, the fecundity of Anagrus nilaparvatae there are no significant difference.
The Anagrus nilaparvatae ratio of female (%) that the brown planthopper ovum of raising with different rice material is host is as shown in table 4,
Table 4
Note: in table, data are mean ± standard error, numeral observation sample number in bracket.
With in a line, turn cry2Aa gene pest-resistant paddy rice T2A-1 and contrast paddy rice bright extensive 63 and adopt student t to check to carry out statistics and compare.
From table 4, each from generation to generation between turn cry2Aa gene pest-resistant paddy rice T2A-1 compared with parent control, the ratio of female of Anagrus nilaparvatae there are no significant difference.
The Anagrus nilaparvatae survival rate (%) that the brown planthopper ovum of raising with different rice material is host is as shown in table 5,
Table 5
Note: in table, data are mean ± standard error, numeral observation sample number in bracket.
With in a line, turn cry2Aa gene pest-resistant paddy rice T2A-1 and contrast paddy rice bright extensive 63 and adopt student t to check to carry out statistics and compare.
From table 5, each from generation to generation between turn cry2Aa gene pest-resistant paddy rice T2A-1 compared with parent control, the survival rate of Anagrus nilaparvatae there are no significant difference.
Two-way analysis of variance from generation to generation, kind and as shown in table 6 with the impact of interaction on Anagrus nilaparvatae fecundity, ratio of female and survival rate of kind from generation to generation,
Table 6
From table 6, kind, generation and kind × generation all do not make significant difference to Anagrus nilaparvatae fecundity and survival rate, and ratio of female only affects by generation significance.
Show through continuous multi-generation long-term observation result, the Cry2Aa albumen mediated by brown planthopper ovum affects without significance Anagrus nilaparvatae life table parameters.
3, Cry2Aa albumen is at the transfer law of paddy rice-brown planthopper-Anagrus nilaparvatae food chain
For two kinds of different rice materials, respectively to 15 age in days rice leaf sheaths, originate in the brown planthopper ovum grain of rice leaf sheath and sample using brown planthopper ovum as the Anagrus nilaparvatae of parasitism, leaf sheath 30 ± 1mg, the Anagrus nilaparvatae adult that 200, brown planthopper ovum and all generations collect, each process repeats 3 ~ 5 times.ELISA will clean polypide body surface PBST buffer solution before detecting, to remove the Cry2Aa albumen of body surface absorption.
In T2A-1 rice leaf sheath tissue, Cry2Aa protein content is 13.9 ± 1.2 μ g/g fresh weights, and lay eggs in grain with the brown planthopper that T2A-1 raises, the existence of Cry2Aa albumen do not detected, and Anagrus nilaparvatae becomes in polypide to detect 16.8 ± 2.1ng/gCry2Aa albumen.In this test system, in Anagrus nilaparvatae body, Cry2Aa albumen may derive from brown planthopper and take food the honeydew that T2A-1 paddy rice drains.
Comprehensive high dose Cry2Aa albumen feeding result of the test, continuous multi-generation three grades of nutritive life table parameters and albumen transfer law are comprehensively analyzed, and demonstrate and turn cry2Aa gene pest-resistant paddy rice T2A-1 to the ecological security of Anagrus nilaparvatae.
Claims (4)
1. transgenic pest-resistant rice is to parasite Anagrus nilaparvatae method for evaluating safety, it is characterized in that comprising following 3 parts:
(1) Tier-1 toxicity assay: evaluate the Bt albumen of Transgenic Bt Rice expression to the genotoxic potential of non-target organism Anagrus nilaparvatae;
1. biological activity determination is carried out to the Bt albumen that test batch Transgenic Bt Rice is expressed
Using Indian meal moth as test insect, the Bt albumen that Transgenic Bt Rice is expressed is added the feed preparing variable concentrations gradient in Indian meal moth artificial feed, after feed prepares, ELISA method detects the real content of this Bt albumen of variable concentrations gradient, and with pure feed in contrast, in every part of feed, access 40 Indian meal moth newly hatched larvaes, repeat 5 times, within 7 days, record larvae alive situation afterwards, calculate LC
50, the activity of the Bt albumen that Transgenic Bt Rice is expressed is judged with this;
2. the high dose Transgenic Bt Rice Bt albumen of expressing is on the impact of Anagrus nilaparvatae survival rate and fecundity, evaluates Anagrus nilaparvatae to the susceptibility of this Bt albumen;
With 10% hydromel as Anagrus nilaparvatae food, prepare three kinds of hydromels and be used for test, that is: the pure hydromel that w/v is 10% contains 10% hydromel of 10 μ g/ml potassium dihydrogen arsenates as positive control as in the Bt albumen containing Bt protein content more than 10 times expressed by Transgenic Bt Rice in negative control, 10% hydromel, 10% hydromel;
Impact on Anagrus nilaparvatae survival rate: get in 4 hours and sprout wings and the Anagrus nilaparvatae queen bee 30 of mating, in every 10 accesses glass tube, seal with nylon wire, raise with above three kinds of hydromels respectively, sooner or later each replacing food, every day records its death condition respectively at early, middle and late, investigates the Bt albumen of high dose Transgenic Bt Rice expression to the impact of Anagrus nilaparvatae survival rate;
Impact on Anagrus nilaparvatae fecundity: raise respectively at the beginning of 20 with above three kinds of hydromels and sprout wings and the Anagrus nilaparvatae queen bee 24 hours of mating, then in single head access glass tube, to lay eggs 2 ~ 3 days, 15 age in days rice seedlings containing brown planthopper in glass tube; Seal with nylon wire after connecing worm, shift out parasitic wasp after 6 hours, record generation Anagrus nilaparvatae emergence quantity and sex every day, investigate the Bt albumen of high dose Transgenic Bt Rice expression to the impact of Anagrus nilaparvatae fecundity;
3. the Bt protein stability that the Transgenic Bt Rice in hydromel is expressed and biologically active;
The hydromel of the Bt albumen that front and back of feeding are expressed containing Transgenic Bt Rice is sampled respectively, repeat 3 times, ELISA detects this Bt protein content, meanwhile, the hydromel of the Bt albumen of being expressed containing Transgenic Bt Rice front and back of feeding is diluted to the concentration of 80 ± 10% lethality rates, is coated in Indian meal moth artificial feed surface, ventilating kitchen is after air-dry 2 hours, access 30 Indian meal moth newly hatched larvaes, repeat 3 times, after 7 days, record its death condition;
(2) successive generation three grades of nutritional tests: evaluate Transgenic Bt Rice by the safety effects of host's mediation to Anagrus nilaparvatae by the food chain of paddy rice-brown planthopper-Anagrus nilaparvatae;
1. Transgenic Bt Rice is on Anagrus nilaparvatae development duration and the impact in life-span
To newly in access glass tube, and seal with nylon wire by emergence Anagrus nilaparvatae list; The rice seedling of the different rice materials that 15 ages in days have been laid eggs 2 ~ 3 days by brown planthopper is housed in glass tube respectively; Until Anagrus nilaparvatae after parasitic 2 hours, shift out parasitic wasp, observe the emergence situation of Anagrus nilaparvatae of future generation, every 2 hour records once Anagrus nilaparvatae of future generation sprout wings time, quantity and sex, repeat above parasitic processes between corresponding rice material different generations; Each rice material establishes 40 repetitions;
Anagrus nilaparvatae adult point male and female for emergence are raised in glass tube, often pipe 4, with absorbent cotton feed w/v be 10% hydromel, sooner or later respectively change a food, every day records its death condition respectively at early, middle and late;
2. Transgenic Bt Rice is on the impact of Anagrus nilaparvatae fecundity, ratio of female and survival rate
Will newly emergence Anagrus nilaparvatae list to be in access glass tube, with (2) 1. processing method, will add the hydromel that w/v is 10% in absorbent cotton, and absorbent cotton will be attached to Anagrus nilaparvatae that glass tube walls feeds, and sooner or later feed once; Observe Anagrus nilaparvatae emergence quantity of future generation and sex every day, until sprout wings completely; After 7 days, dissect rice stem under stereoscope, the parasitic but Anagrus nilaparvatae ovum grain number of not sprouting wings of record, is become redness by the ovum of parasitism;
Select the development duration of Anagrus nilaparvatae continuous multi-generation, life-span, fecundity, ratio of female and survival rate as evaluating index, and carry out statistical analysis, evaluate the Transgenic Bt Rice that mediated by brown planthopper ovum to the safety effects of Anagrus nilaparvatae;
(3) albumen transfer law: the approach and the degree that are exposed to the Bt albumen that Transgenic Bt Rice is expressed by ELISA detection means qualification Anagrus nilaparvatae;
For different rice materials, respectively to 15 age in days rice leaf sheaths, originate in the brown planthopper ovum grain of rice leaf sheath and sample using brown planthopper ovum as the Anagrus nilaparvatae of host, leaf sheath 30 ± 1mg, the Anagrus nilaparvatae adult that 200, brown planthopper ovum and all generations collect, each process repeats 3 ~ 5 times;
Adopt elisa technique, respectively the content of the Bt albumen that Transgenic Bt Rice in above sample is expressed is detected, determine whether Anagrus nilaparvatae is exposed to the Bt albumen of Transgenic Bt Rice expression and the degree of its exposure by brown planthopper ovum approach.
2. transgenic pest-resistant rice according to claim 1 is to parasite Anagrus nilaparvatae method for evaluating safety, it is characterized in that Transgenic Bt Rice is selected to turn cry2Aa trans-genetic hybrid rice T2A-1.
3. transgenic pest-resistant rice according to claim 2 is to parasite Anagrus nilaparvatae method for evaluating safety, it is characterized in that turn cry2Aa trans-genetic hybrid rice T2A-1 cultivates high to 15cm with Yoshida nutrient solution in the dish for cultivating of length × wide × height=25cm × 20cm × 3cm.
4. transgenic pest-resistant rice according to claim 1 is to parasite Anagrus nilaparvatae method for evaluating safety, it is characterized in that step (1) 2., (2) 1. in glass tube used be diameter × height=3cm × 25cm glass tube, nylon wire is 100 order nylon wires.
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