CN103931564A - Evaluation method of safety of genetically modified insect resistant rice relative to parasite anagrus nilaparvatae - Google Patents
Evaluation method of safety of genetically modified insect resistant rice relative to parasite anagrus nilaparvatae Download PDFInfo
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Abstract
The invention relates to an evaluation method of safety of genetically modified insect resistant rice relative to parasite anagrus nilaparvatae. The evaluation method includes three parts of Tier-1 toxicity determination test, wherein the potential toxicity of Bt proteins expressed by Bt-gene modified insect resistant rice on non-target organism anagrus nilaparvatae is evaluated; successive generation three-level nutrition test, wherein the influence of the Bt-gene modified insect resistant rice on the safety of the anagrus nilaparvatae through parasitic mediators is evaluated according to the food chain of rice-delphacidae-anagrus nilaparvatae; a protein transfer rule, wherein the way and degree that the anagrus nilaparvatae is exposed in the Bt proteins expressed by the Bt-gene modified insect resistant rice are identified through an ELISA detection means; finally, the ecological consequences generated by genetically modified crop cultivation on non-target organisms are evaluated synthetically according to the data of the three parts. The evaluation method is systematic and scientific, and has great significance for the research of the influence of genetically modified crops on the non-target organisms and ecological safety of genetically modified crop cultivation.
Description
Technical field
The invention belongs to genetically modified crops ecological risk assessment technical field, be specifically related to a kind of transgenic pest-resistant rice to parasite rice lice tassel chalcid fly method for evaluating safety.
Background technology
Paddy rice is one of most important cereal crops in the world, and there are more than 30 hundred million populations in the whole world taking rice as food.Paddy rice is again one of maximum cereal crops of insect pest simultaneously, eats into causing harm of the lepidopterous insects such as property snout moth's larva every year due to rice leaf roller, brill, causes 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 has not only increased the production cost of paddy rice, and contaminated environment, also easily makes insect produce resistance, reduces the drug effect (Su et al, 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 find the anti-source of high resistance lepidopterous insects.Cultivate the rice varieties of high resistance lepidoptera pest, need be by means of Extrinsic Anti-insect Genes.Bacillus thuringiensis (Bt) insecticidal proteins is used as biological source insecticide the history of five more than ten years, and lepidoptera pest is had to stable control efficiency.Transgenic Bt Rice is to control the main insect-proof rice of lepidoptera pest at present.
But, when the utilization of insect-resistant transgenic plants brings interests to the mankind, also there is certain 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 on edaphon etc.Transgenic anti-insect plants is the pith about genetically modified crops environmental risk assessment to the potential impact of non-target organism.At present, international risk assessment worker and genetically modified plants related management department extensively adopt " appraisement system by different level " to carry out genetically modified plants affects research work (Romeis et al., 2008 of aspect on non-target organism; Wang is round etc., and 2011).This appraisement system is exactly to select suitable biological subject, then carries out successively from laboratory test to half field trial, then the system evaluation of carrying out stage by stage by different level to field trial.
Brown planthopper Nilaparvata lugens
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.Rice lice tassel chalcid fly is the main ovum parasite of rice fulgorid, can obviously reduce population quantity (the Lou et al. 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 has ecological risk, research transgenic paddy rice is the pith that Study on Environmental Safety of Transgenic Crops is evaluated on the impact of rice lice tassel chalcid fly, so, the method for safety is particularly important to rice lice tassel chalcid fly to set up a set of system evaluation Transgenic Bt Rice.
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 rice lice tassel chalcid fly method for evaluating safety.
The implementation of the object of the invention is that transgenic pest-resistant rice, to parasite rice lice tassel chalcid fly method for evaluating safety, comprises following 3 parts:
(1) Tier-1 toxicity test test: the genotoxic potential of the Bt albumen that evaluation Transgenic Bt Rice is expressed to non-target organism rice lice tassel chalcid fly;
The Bt albumen of 1. test batch Transgenic Bt Rice being expressed carries out biological activity determination
Using Indian meal moth as test insect, the Bt albumen that Transgenic Bt Rice is expressed adds the feed of 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, after 7 days, record larvae alive situation, calculate LC
50, judge with this activity of Bt albumen that Transgenic Bt Rice is expressed;
2. the impact of the Bt albumen that high dose Transgenic Bt Rice is expressed on rice lice tassel chalcid fly survival rate and fecundity, evaluates the susceptibility of rice lice tassel chalcid fly to this Bt albumen;
With 10% hydromel as rice lice tassel chalcid fly food, prepare three kinds of hydromels for test, that is: pure 10% hydromel (w/v) as negative control, 10% hydromel that contains the above Bt albumen of expressing in Transgenic Bt Rice of 10 times of content, contain 10 μ g/ml PA 10% hydromel as positive control;
Impact on rice lice tassel chalcid fly survival rate: get in 4 hours and to sprout wings and 30 of the rice lice tassel chalcid fly queen bees of mating, in a glass tube of every 10 accesses, seal with nylon wire, raise with above three kinds of hydromels respectively, sooner or later a food of each replacing, every day is respectively at early, middle and late its death condition that records, and investigates the impact on rice lice tassel chalcid fly survival rate of Bt albumen that high dose Transgenic Bt Rice expresses;
Impact on rice lice tassel chalcid fly fecundity: raise respectively at the beginning of 20 and to sprout wings and the rice lice tassel chalcid fly queen bee of mating 24 hours with above three kinds of hydromels, then in single head access glass tube, contain brown planthopper lay eggs 2～3 days, 15 age in days rice seedlings in glass tube; Connect after worm with nylon wire sealing, after 6 hours, shift out parasitic wasp, record generation rice lice tassel chalcid fly emergence quantity and sex every day, investigate the impact on rice lice tassel chalcid fly fecundity of Bt albumen that high dose Transgenic Bt Rice expresses;
3. the Transgenic Bt Rice in hydromel is expressed Bt protein stability and biologically active;
Hydromel to the Bt albumen that contains Transgenic Bt Rice expression before and after feeding samples respectively, repeat 3 times, ELISA detects this Bt protein content, meanwhile, the hydromel of the Bt albumen that Transgenic Bt Rice expression is contained in the front and back of feeding is diluted to the concentration of 80 ± 10% lethality rates, is coated in Indian meal moth artificial feed surface, after air-dry 2 hours of ventilating kitchen, 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 food chain of paddy rice-brown planthopper-rice lice tassel chalcid fly and mediate the safety effects to rice lice tassel chalcid fly by host;
1. Transgenic Bt Rice is on rice lice tassel chalcid fly development duration and the impact in life-span
The rice lice tassel chalcid fly that will newly sprout wings is single in access glass tube, and seals with nylon wire.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 respectively in glass tube.Until rice lice tassel chalcid fly after parasitic 2 hours, shift out parasitic wasp, the emergence situation of observing rice lice tassel chalcid fly of future generation, every 2 hour records are time, quantity and the sex of the little bee eclosion of rice lice tassel of future generation once, between corresponding rice material different generations, repeats above parasitic processes.Each rice material is established 40 repetitions;
The little bee imago of rice lice tassel point male and female for emergence are raised in glass tube, and 4 of every pipes, with absorbent cotton 10% hydromel (w/v) of feeding, sooner or later respectively change a food, and every day is respectively at early, middle and late its death condition that records;
2. the impact of Transgenic Bt Rice on rice lice tassel chalcid fly fecundity, ratio of female and survival rate
The rice lice tassel chalcid fly that will newly sprout wings is single, and in access glass tube, with (2) 1. processing method, will add 10% hydromel (w/v) in absorbent cotton, and absorbent cotton is attached to the rice lice tassel chalcid fly of feeding in glass tube walls, sooner or later feeds once; Observe rice lice tassel chalcid fly emergence quantity of future generation and sex every day, until sprout wings completely; After 7 days, dissect rice stem under stereoscope, record rice lice tassel chalcid fly ovum grain number parasitic but that do not sprout wings, is become redness by parasitic ovum;
Select development duration, life-span, fecundity, ratio of female and the survival rate of rice lice tassel chalcid fly continuous multi-generation as evaluating index, and carry out statistical analysis, evaluate the safety effects of the Transgenic Bt Rice being mediated by brown planthopper ovum to rice lice tassel chalcid fly;
(3) albumen transfer law: the approach and the degree that are exposed to the Bt albumen of Transgenic Bt Rice expression by ELISA detection means qualification rice lice tassel chalcid fly;
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 the rice lice tassel chalcid fly using brown planthopper ovum as host samples, leaf sheath 30 ± 1mg, the little bee imago of rice lice tassel that 200, brown planthopper ovum and all generations collect, each processing repeats 3～5 times;
Adopt elisa technique, the content of Bt albumen of respectively Transgenic Bt Rice in above sample being expressed detects, determine rice lice tassel chalcid fly whether by brown planthopper ovum approach be exposed to Bt albumen that Transgenic Bt Rice expresses with and the degree that exposes.
The genotoxic potential of the Bt albumen that the present invention expresses by Tier-1 toxicity test test evaluation Transgenic Bt Rice to non-target organism rice lice tassel chalcid fly; Evaluate Transgenic Bt Rice by the food chain of paddy rice-brown planthopper-rice lice tassel chalcid fly and mediate the impact on rice lice tassel chalcid fly by host; Adopt ELISA detection means qualification rice lice tassel chalcid fly to be exposed to approach and the degree of the Bt albumen of Transgenic Bt Rice expression.Finally, the ecological consequence that the plantation of comprehensively analyzing Transgenic Bt Rice according to the data of 3 aspects produces this non-target organism.
The present invention has set up the evaluation method of a cover system, science in laboratory, significant on the impact research of non-target organism on improving Transgenic Bt Rice, significant to determining the ecological security of Transgenic Bt Rice plantation.
Brief description of the drawings
Fig. 1 takes food pure hydromel and adds Cry2Aa albumen and the rice lice tassel chalcid fly survivorship curve figure of PA hydromel.
Embodiment
Transgenic Bt Rice of the present invention selects to turn cry2Aa trans-genetic hybrid rice T2A-1, expressing Cry2Aa albumen is the one of Bt albumen, it has very high resistance to lepidoptera pest, non-transgenic parent selects extensive 63 paddy rice of throwing light on, 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 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%).
Because the Cry2Aa protein content that Bt Transgenic Rice is expressed is limited, in view of making rice lice tassel chalcid fly be exposed to the Cry2Aa albumen of higher dosage, the applicant has bought the Cry2Aa albumen that Beijing Le Shining Science and Technology Ltd. produces, the Cry2Aa albumen that the Cry2Aa albumen of buying and Bt Transgenic Rice T2A-1 express is same Bt albumen, therefore the applicant replaces the Cry2Aa albumen of Bt Transgenic Rice expression as trial target using this.
Of the present inventionly pick up from rice field, Wuhan, Hubei for examination brown planthopper, and indoor respectively at turning cry2Aa trans-genetic hybrid rice T2A-1 and bright extensive 63 rice seedling is set up population, more than 10 generations of raising.Rice lice tassel chalcid fly is provided by insect science research institute of Zhejiang University, raises more than 10 generations for test at the indoor TN1 rice seedling with brown planthopper ovum.Raising condition is temperature (28 ± 1 DEG C), relative moisture (70 ± 5%), Light To Dark Ratio (L14h:D10h).Indian meal moth sensitive strain is measured for Cry2Aa protein active.
Evaluation method of the present invention is tested by Tier-1 toxicity test, 3 key component compositions of successive generation three grades of nutritional tests and albumen transfer law.Be elaborated respectively below.
(1) Tier-1 toxicity test test: evaluate and turn Cry2Aa albumen that cry2Aa gene pest-resistant paddy rice T2A-1 the expresses genotoxic potential to non-target organism rice lice tassel chalcid fly.
1. test batch Cry2Aa albumen is carried out to biological activity determination
Using Indian meal moth as test insect, the Cry2Aa albumen of purchase is added in Indian meal moth artificial feed and prepares 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, in every part of feed, access 40 Indian meal moth newly hatched larvaes, repeat 5 times, after 7 days, record larvae alive situation, calculate LC
50, judge the activity of Cry2Aa albumen 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, the LC of Cry2Aa albumen to Indian meal moth newly hatched larvae
50be 14.89 μ g/g.Illustrate that the Cry2Aa albumen of buying has good insecticidal activity.
2. the impact of high dose Cry2Aa albumen on rice lice tassel chalcid fly survival rate and fecundity, evaluates the susceptibility of rice lice tassel chalcid fly to Cry2Aa albumen.
With 10% hydromel as rice lice tassel chalcid fly food, prepare three kinds of hydromels for test, that is: pure 10% hydromel (w/v) as negative control, 10% hydromel that contains 300 μ g/ml Cry2Aa, contain 10 μ g/mlPA(potassium dihydrogen arsenates) 10% hydromel as positive control.
Impact on rice lice tassel chalcid fly survival rate: get in 4 hours and to sprout wings and 30 of the rice lice tassel chalcid fly queen bees of mating, in diameter × height=3cm × 25cm glass tube of every 10 accesses, with 100 order nylon wire sealings, raise with above three kinds of hydromels respectively, sooner or later respectively change a food, every day is respectively at 8:00,14:00,20:00 records its death condition, investigates the impact of high dose Cry2Aa albumen on rice lice tassel chalcid fly survival rate;
Kaplan-Meier survival analysis result shows: the survival rate between taking food the rice lice tassel chalcid fly that contains 300 μ g/ml Cry2Aa albumen hydromels and contrasting, there was no significant difference (P=0.287); And compared with the control, take food contain 10 μ g/ml PA hydromels rice lice tassel chalcid fly survival rate significantly decline (P<0.001) (see figure 1).
Impact on rice lice tassel chalcid fly fecundity: the rice lice tassel chalcid fly queen bee of emergence and mating at the beginning of raising 20 with above three kinds of hydromels 24 hours, then in single head access diameter × height=3cm × 25cm glass tube, in glass tube, contain brown planthopper lay eggs 2～3 days, 15 age in days rice seedlings; Connect after worm with 100 order nylon wires sealings, after 6 hours, shift out parasitic wasp, record generation rice lice tassel chalcid fly emergence quantity and sex every day, investigate the impact of high dose Cry2Aa albumen on rice lice tassel chalcid fly fecundity; .
Observed result: take food pure 10% hydromel, be respectively 14.5 ± 1.84 containing the hydromel of 300 μ g/ml Cry2Aa albumen and containing the rice lice tassel chalcid fly egg laying amount of the hydromel of 10 μ g/ml PA, 16.1 ± 1.68,3.7 ± 0.63, Dunnett test analysis result shows: compared with the control, take food the rice lice tassel chalcid fly egg laying amount there was no significant difference (P=0.650) that contains 300 μ g/ml Cry2Aa albumen hydromels, take food the significantly decline (P<0.001) of rice lice tassel chalcid fly egg laying amount that contains 10 μ g/ml PA hydromels.
3. Cry2Aa protein stability and the biologically active in hydromel;
Stability and the biologically active of Cry2Aa albumen in the hydromel taking food in 12 hours in order to ensure rice lice tassel chalcid fly, the hydromel that contains Cry2Aa albumen before and after feeding is sampled respectively, repeat 3 times, ELISA detects its Cry2Aa protein content, simultaneously, by 10 times of the hydromel dilutions that contains Cry2Aa albumen before and after feeding, 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 not taken food by rice lice tassel chalcid fly, the extraction efficiency of Cry2Aa albumen is about 94.5%, and its content is 283.5 ± 7.4 μ g/ml; Tassel chalcid fly took food after 12 hours, and in hydromel, Cry2Aa protein content has declined 24.9%, was 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 the more than 10 times of Cry2Aa protein content that turn cry2Aa trans-genetic hybrid rice T2A-1 expression.
The raw result of surveying of Indian meal moth shows: the hydromel that contains Cry2Aa albumen before rice lice tassel chalcid fly takes food and after taking food, be coated in behind Indian meal moth artificial feed surface, the lethality rate of Indian meal moth newly hatched larvae is respectively to 87.8% and 86.7%, far away higher than pure 10% hydromel (lethality rate 10.0%).
In sum, rice lice tassel chalcid fly 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 food chain evaluation of paddy rice-brown planthopper-rice lice tassel chalcid fly and mediate the safety effects to rice lice tassel chalcid fly by host.
1. turn cry2Aa gene pest-resistant paddy rice T2A-1 to rice lice tassel chalcid fly development duration and the impact in life-span
The new emergence rice lice tassel chalcid fly that TN1 paddy rice is raised is single 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 respectively in glass tube.Until rice lice tassel chalcid fly after parasitic 2 hours, shift out parasitic wasp, the emergence situation of observing rice lice tassel chalcid fly of future generation, every 2 hour records are time, quantity and the sex of the little bee eclosion of rice lice tassel of future generation once, between corresponding rice material different generations, repeats above parasitic processes.Each rice material is established 40 repetitions;
The little bee imago of rice lice tassel point male and female for emergence are raised in glass tube, and 4 of every pipes, with absorbent cotton 10% hydromel (w/v) of feeding, sooner or later respectively change a food, and every day is respectively at 8:00,14:00, and 20:00 records its death condition.
The brown planthopper ovum of raising taking different rice materials as host's rice lice tassel chalcid fly development duration as shown in table 1,
Table 1
Note: in table, data are mean ± standard error, numeral observation sample number in bracket.
In a line, turn cry2Aa gene pest-resistant paddy rice T2A-1 and contrast paddy rice bright extensive 63 and adopt U inspection to carry out statistics comparison.
From table 1, no matter male and female, respectively turn cry2Aa gene pest-resistant paddy rice T2A-1 between from generation to generation and contrast and compare with parent, the development duration of rice lice tassel chalcid fly there are no significant difference.
The brown planthopper ovum of raising taking different rice materials as host's the rice lice tassel chalcid fly life-span as shown in table 2,
Table 2
Note: in table, data are mean ± standard error, numeral observation sample number in bracket.
In a line, turn cry2Aa gene pest-resistant paddy rice T2A-1 and contrast paddy rice bright extensive 63 and adopt U inspection to carry out statistics comparison.
From table 2, no matter male and female, respectively turn cry2Aa gene pest-resistant paddy rice T2A-1 between from generation to generation and contrast and compare with parent, difference that the life-span of rice lice tassel chalcid fly, there are no significant.
2. turn the impact of cry2Aa gene pest-resistant paddy rice T2A-1 on rice lice tassel chalcid fly fecundity, ratio of female and survival rate
The new emergence rice lice tassel chalcid fly that TN1 paddy rice is raised is single in access glass tube (specification is the same), processing method with (2) 1., to in absorbent cotton, add 10% hydromel (w/v), and absorbent cotton is attached to the rice lice tassel chalcid fly of feeding in glass tube walls, sooner or later feed once; Observe rice lice tassel chalcid fly emergence quantity of future generation and sex every day, until sprout wings completely; After 7 days, dissect rice stem under stereoscope, record rice lice tassel chalcid fly ovum grain number parasitic but that do not sprout wings, is become redness by parasitic ovum.
The brown planthopper ovum of raising taking different rice materials as host's rice lice tassel chalcid fly fecundity as shown in table 3,
Table 3
Note: in table, data are mean ± standard error, numeral observation sample number in bracket.
In a line, turn cry2Aa gene pest-resistant paddy rice T2A-1 and contrast paddy rice bright extensive 63 and adopt student t inspection to carry out statistics comparison.
From table 3, each turn cry2Aa gene pest-resistant paddy rice T2A-1 between from generation to generation and contrast and compare with parent, the fecundity of rice lice tassel chalcid fly there are no significant difference.
The brown planthopper ovum of raising taking different rice materials as host's rice lice tassel chalcid fly ratio of female (%) as shown in table 4,
Table 4
Note: in table, data are mean ± standard error, numeral observation sample number in bracket.
In a line, turn cry2Aa gene pest-resistant paddy rice T2A-1 and contrast paddy rice bright extensive 63 and adopt student t inspection to carry out statistics comparison.
From table 4, each turn cry2Aa gene pest-resistant paddy rice T2A-1 between from generation to generation and contrast and compare with parent, the ratio of female of rice lice tassel chalcid fly there are no significant difference.
The brown planthopper ovum of raising taking different rice materials as host's rice lice tassel chalcid fly survival rate (%) as shown in table 5,
Table 5
Note: in table, data are mean ± standard error, numeral observation sample number in bracket.
In a line, turn cry2Aa gene pest-resistant paddy rice T2A-1 and contrast paddy rice bright extensive 63 and adopt student t inspection to carry out statistics comparison.
From table 5, each turn cry2Aa gene pest-resistant paddy rice T2A-1 between from generation to generation and contrast and compare with parent, the survival rate of rice lice tassel chalcid fly there are no significant difference.
From generation to generation, kind and generation are as shown in table 6 on the impact of rice lice tassel chalcid fly fecundity, ratio of female and survival rate with the interaction of kind for two-way analysis of variance,
Table 6
From table 6, kind, generation and kind × generation all do not make significant difference to rice lice tassel chalcid fly fecundity and survival rate, and ratio of female is affected by significance from generation to generation only.
Show through continuous multi-generation long-term observation result, the Cry2Aa albumen being mediated by brown planthopper ovum affects without significance rice lice tassel chalcid fly life table parameters.
3, Cry2Aa albumen is at the transfer law of paddy rice-brown planthopper-rice lice tassel chalcid fly 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 as parasitic rice lice tassel chalcid fly using brown planthopper ovum, leaf sheath 30 ± 1mg, the little bee imago of rice lice tassel that 200, brown planthopper ovum and all generations collect, each processing repeats 3～5 times.ELISA will clean with PBST buffer solution polypide body surface 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 the brown planthopper of raising with T2A-1 is laid eggs in grain, the existence of Cry2Aa albumen do not detected, and rice lice tassel chalcid fly becomes 16.8 ± 2.1ng/g Cry2Aa albumen in polypide, to be detected.In this test system, in rice lice tassel chalcid fly 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, three grades of nutritive life table parameters of continuous multi-generation and albumen transfer law are comprehensively analyzed, and have proved to turn the ecological security of cry2Aa gene pest-resistant paddy rice T2A-1 to rice lice tassel chalcid fly.
Claims (4)
1. transgenic pest-resistant rice, to parasite rice lice tassel chalcid fly method for evaluating safety, is characterized in that comprising following 3 parts:
(1) Tier-1 toxicity test test: the genotoxic potential of the Bt albumen that evaluation Transgenic Bt Rice is expressed to non-target organism rice lice tassel chalcid fly;
The Bt albumen of 1. test batch Transgenic Bt Rice being expressed carries out biological activity determination
Using Indian meal moth as test insect, the Bt albumen that Transgenic Bt Rice is expressed adds the feed of 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, after 7 days, record larvae alive situation, calculate LC
50, judge with this activity of Bt albumen that Transgenic Bt Rice is expressed;
2. the impact of the Bt albumen that high dose Transgenic Bt Rice is expressed on rice lice tassel chalcid fly survival rate and fecundity, evaluates the susceptibility of rice lice tassel chalcid fly to this Bt albumen;
With 10% hydromel as rice lice tassel chalcid fly food, prepare three kinds of hydromels for test, that is: pure 10% hydromel (w/v) as negative control, 10% hydromel that contains the above Bt albumen of expressing in Transgenic Bt Rice of 10 times of content, contain 10 μ g/ml PA 10% hydromel as positive control;
Impact on rice lice tassel chalcid fly survival rate: get in 4 hours and to sprout wings and 30 of the rice lice tassel chalcid fly queen bees of mating, in a glass tube of every 10 accesses, seal with nylon wire, raise with above three kinds of hydromels respectively, sooner or later a food of each replacing, every day is respectively at early, middle and late its death condition that records, and investigates the impact on rice lice tassel chalcid fly survival rate of Bt albumen that high dose Transgenic Bt Rice expresses;
Impact on rice lice tassel chalcid fly fecundity: raise respectively at the beginning of 20 and to sprout wings and the rice lice tassel chalcid fly queen bee of mating 24 hours with above three kinds of hydromels, then in single head access glass tube, contain brown planthopper lay eggs 2～3 days, 15 age in days rice seedlings in glass tube; Connect after worm with nylon wire sealing, after 6 hours, shift out parasitic wasp, record generation rice lice tassel chalcid fly emergence quantity and sex every day, investigate the impact on rice lice tassel chalcid fly fecundity of Bt albumen that high dose Transgenic Bt Rice expresses;
3. the Transgenic Bt Rice in hydromel is expressed Bt protein stability and biologically active;
Hydromel to the Bt albumen that contains Transgenic Bt Rice expression before and after feeding samples respectively, repeat 3 times, ELISA detects this Bt protein content, meanwhile, the hydromel of the Bt albumen that Transgenic Bt Rice expression is contained in the front and back of feeding is diluted to the concentration of 80 ± 10% lethality rates, is coated in Indian meal moth artificial feed surface, after air-dry 2 hours of ventilating kitchen, 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 food chain of paddy rice-brown planthopper-rice lice tassel chalcid fly and mediate the safety effects to rice lice tassel chalcid fly by host;
1. Transgenic Bt Rice is on rice lice tassel chalcid fly development duration and the impact in life-span
The rice lice tassel chalcid fly that will newly sprout wings is single in access glass tube, and seals with nylon wire.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 respectively in glass tube.Until rice lice tassel chalcid fly after parasitic 2 hours, shift out parasitic wasp, the emergence situation of observing rice lice tassel chalcid fly of future generation, every 2 hour records are time, quantity and the sex of the little bee eclosion of rice lice tassel of future generation once, between corresponding rice material different generations, repeats above parasitic processes.Each rice material is established 40 repetitions;
The little bee imago of rice lice tassel point male and female for emergence are raised in glass tube, and 4 of every pipes, with absorbent cotton 10% hydromel (w/v) of feeding, sooner or later respectively change a food, and every day is respectively at early, middle and late its death condition that records;
2. the impact of Transgenic Bt Rice on rice lice tassel chalcid fly fecundity, ratio of female and survival rate
The rice lice tassel chalcid fly that will newly sprout wings is single, and in access glass tube, with (2) 1. processing method, will add 10% hydromel (w/v) in absorbent cotton, and absorbent cotton is attached to the rice lice tassel chalcid fly of feeding in glass tube walls, sooner or later feeds once; Observe rice lice tassel chalcid fly emergence quantity of future generation and sex every day, until sprout wings completely; After 7 days, dissect rice stem under stereoscope, record rice lice tassel chalcid fly ovum grain number parasitic but that do not sprout wings, is become redness by parasitic ovum;
Select development duration, life-span, fecundity, ratio of female and the survival rate of rice lice tassel chalcid fly continuous multi-generation as evaluating index, and carry out statistical analysis, evaluate the safety effects of the Transgenic Bt Rice being mediated by brown planthopper ovum to rice lice tassel chalcid fly;
(3) albumen transfer law: the approach and the degree that are exposed to the Bt albumen of Transgenic Bt Rice expression by ELISA detection means qualification rice lice tassel chalcid fly;
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 the rice lice tassel chalcid fly using brown planthopper ovum as host samples, leaf sheath 30 ± 1mg, the little bee imago of rice lice tassel that 200, brown planthopper ovum and all generations collect, each processing repeats 3～5 times;
Adopt elisa technique, the content of Bt albumen of respectively Transgenic Bt Rice in above sample being expressed detects, determine rice lice tassel chalcid fly whether by brown planthopper ovum approach be exposed to Bt albumen that Transgenic Bt Rice expresses with and the degree that exposes.
2. transgenic pest-resistant rice according to claim 1, to parasite rice lice tassel chalcid fly method for evaluating safety, is characterized in that Transgenic Bt Rice selection turns cry2Aa trans-genetic hybrid rice T2A-1.
3. transgenic pest-resistant rice according to claim 1 and 2, to parasite rice lice tassel chalcid fly method for evaluating safety, is characterized in that turning cry2Aa trans-genetic hybrid rice T2A-1 and cultivates to 15cm high 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 rice lice tassel chalcid fly 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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