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1.
转Cry1Ab基因水稻Bt01为一种新型的转基因水稻, 文章首先利用Southern blotting验证了外源基因Cry1Ab转入了Bt01中, 且为单拷贝, 再利用TAIL-PCR方法获得了其插入位点信息, 根据获得的Bt01的5′端插入位点序列, 设计了相应的定性与定量PCR检测体系的引物及探针, 实验结果显示, 定性PCR检测体系的最低检测极限(LOD)为10个拷贝, 定量PCR检测体系的LOD为5拷贝, 最低定量极限(LOQ)为10拷贝。同时为了验证建立的定量PCR体系的准确性, 利用该体系检测已知转基因水稻Bt01含量分别为3%和0.5%的样品, 定量结果分别为2.7%和0.47%。研究结果表明, 该转化体特异性定性与定量检测方法具有高度的特异性和良好的灵敏性, 为转基因水稻Bt01的身份识别和检测提供了有效的方法。 相似文献
2.
转Cry1Ab基因水稻Bt01为一种新型的转基因水稻,文章首先利用Southern blotting验证了外源基因Cry1Ab转入了Bt01中,且为单拷贝,再利用TAIL-PCR方法获得了其插入位点信息,根据获得的Bt01的5′端插入位点序列,设计了相应的定性与定量PCR检测体系的引物及探针,实验结果显示,定性PCR检测体系的最低检测极限(LOD)为10个拷贝,定量PCR检测体系的LOD为5拷贝,最低定量极限(LOQ)为10拷贝。同时为了验证建立的定量PCR体系的准确性,利用该体系检测已知转基因水稻Bt01含量分别为3%和0.5%的样品,定量结果分别为2.7%和0.47%。研究结果表明,该转化体特异性定性与定量检测方法具有高度的特异性和良好的灵敏性,为转基因水稻Bt01的身份识别和检测提供了有效的方法。 相似文献
3.
Effect of transgenic Bacillus thuringiensis rice lines on mortality and feeding behavior of rice stem borers (Lepidoptera: Crambidae) 总被引:1,自引:0,他引:1
Ten transgenic Bacillus thuringiensis Bt rice, Oryza sativa L., lines with different Bt genes (two Cry1Ac lines, three Cry2A lines, and five Cry9C lines) derived from the same variety Minghui 63 were evaluated in both the laboratory and the field. Bioassays were conducted by using the first instars of two main rice lepidopteran insect species: yellow stem borer, Scirpophaga incertulas (Walker) and Asiatic rice borer, Chilo suppressalis (Walker). All transgenic lines exhibited high toxicity to these two rice borers. Field evaluation results also showed that all transgenic lines were highly insect resistant with both natural infestation and manual infestation of the neonate larvae of S. incertulas compared with the nontransformed Minghui63. Bt protein concentrations in leaves of 10 transgenic rice lines were estimated by the sandwich enzyme-linked immunosorbent assay. The cry9C gene had the highest expression level, next was cry2A gene, and the cry1Ac gene expressed at the lowest level. The feeding behavior of 7-d-old Asiatic rice borer to three classes of Bt transgenic rice lines also was detected by using rice culm cuttings. The results showed that 7-d-old larvae of Asiatic rice borer have the capacity to distinguish Bt and non-Bt culm cuttings and preferentially fed on non-Bt cuttings. When only Bt culm cuttings with three classes of different Bt proteins (CrylAc, Cry2A, and Cry9C) were fed, significant distribution difference of 7-d-old Asiatic rice borer in culm cuttings of different Bt proteins also was found. In the current study, we evaluate different Bt genes in the same rice variety in both the laboratory and the field, and also tested feeding behavior of rice insect to these Bt rice. These data are valuable for the further development of two-toxin Bt rice and establishment of appropriate insect resistance management in the future. 相似文献
4.
Rice lines genetically modified with the crystal toxin genes from Bacillus thuringiensis (Bt) have experienced rapid development, with biosafety certificates for two Bt rice lines issued in 2009. There has still been no commercial release of these lines yet due to public concerns about human health and environmental risks. Some studies confirmed that Bt rice was as safe as conventional rice to non-target organisms when pesticides were not applied, however, pesticides are still required in Bt rice to control non-lepidopteran pests. In this study, we assessed the environmental effects of two Bt rice lines expressing either the cry1Ab/1Ac or cry2A genes, respectively, by using zooplanktons as indicator species under normal field management practices using pesticides when required. In the whole rice growing season, non-Bt rice was sprayed 5 times while Bt rice was sprayed 2 times, which ensured both rice achieved a normal yield. Field investigations showed that rice type (Bt and non-Bt) significantly influenced zooplankton abundance and diversity, which were up to 95% and 80% lower in non-Bt rice fields than Bt rice fields. Laboratory rearing showed that water from non-Bt rice fields was significantly less suitable for the survival and reproduction of Daphnia magna and Paramecium caudatum in comparison with water from Bt rice fields. Higher pesticide residues were detected in the water from non-Bt than Bt rice fields, accounting for the bad performance of zooplankton in non-Bt field water. Our results demonstrate that Bt rice is safer to aquatic ecosystems than non-Bt rice, and its commercialization will be beneficial for biodiversity restoration in rice-based ecosystems. 相似文献
5.
转基因抗虫玉米CM8101是经人工改造合成的转Bt抗虫基因Cry1Ab?Ma转基因玉米新品系,由我国自主研发,具有更优良的抗虫性,目前已进入生产性试验阶段,具有广阔的产业化应用前景。依据CM8101的5′端旁侧序列信息,设计并筛选出最佳引物探针组合,通过普通PCR和实时荧光PCR技术,建立了转基因抗虫玉米CM8101的两种特异性定性检测方法。结果显示,普通PCR检测方法检出限达0.1%,实时荧光PCR检测方法检出限达0.05%。这两种定性检测方法的建立为今后准确高效检测转基因抗虫玉米CM8101及其产品提供了新的参考方法。 相似文献
6.
Laboratory bioassays and field surveys were carried out to compare the resistance of three transgenic rice (Oryza sativa L.) lines including Bt-DL expressing a single gene cry1Ab, Bt-KF6 expressing stacked genes cry1Ac and CpTI genes and Bt-SY63 expressing a fusion gene cry1Ab/cry1Ac, respectively, to an important rice pest Chilo suppressalis (Walker). In addition, enzyme-linked immunosorbent assays (ELISA) were conducted to monitor the Bt protein expressions in rice leaves and stems at different rice growth stages. Results showed that all the transgenic rice lines exhibited significantly high resistance to the pest compared with their corresponding nontransformed isolines. Among the transgenic rice lines, Bt-SY63 and Bt-KF6 had higher resistance to C. suppressalis at early growth stage, but lower resistance at late stages, while the pest resistance of Bt-DL was relatively stable throughout the growing season. The results were consistent with ELISA results showing that Bt protein levels in Bt-SY63 or Bt-KF6 leaves decreased in late growth stages, but were relatively stable in Bt-DL at all growth stages. This demonstrates that the resistance to a pest by Bt plants is positively correlated with Cry protein expression levels in plant tissues. Compared with Bt-SY63 and Bt-KF6, the Bt protein expression levels were significantly lower in Bt-DL, while its resistance to C. suppressalis was the highest. This may suggest that C. suppressalis is more susceptible to Cry1Ab than to Cry1Ac. The data from the current study are valuable for decision-making for commercial use of Bt rice lines and development of appropriate pest control and resistance management strategies for the transgenic rice lines. 相似文献
7.
随着转基因技术的迅猛发展,转基因产品的安全性受到了广泛关注。转基因检测用有证标准物质在确保转基因产品定性、定量检测结果的可比性和可追溯性方面发挥着重要作用。但转基因蛋白质标准物质的开发相对缓慢,其中一个难点是制备高纯度的转基因蛋白质候选物。苏云金芽胞杆菌Bacillus thuringiensis cry1Ah1基因因其对亚洲玉米螟等鳞翅目害虫有很好的杀虫活性,已用于转基因抗虫作物的研制,并获得具有较好抗虫性状的转基因株系。为了研发Cry1Ah蛋白有证标准物质,亟需建立其制备及纯化体系。文中优化了利用Bt表达系统制备Cry1Ah蛋白的体系,利用离子交换色谱法和排阻色谱法逐级纯化的方法,获得了高纯度的Cry1Ah蛋白 (排阻色谱纯度:99.6%)。生物活性测定结果表明,纯化的Cry1Ah蛋白与原毒素对小菜蛾Plutella xylostella的杀虫活性没有显著差异。最后使用Edman降解法和质谱法确定了Cry1Ah蛋白活化后的氨基酸序列。综上所述,获得的Cry1Ah纯蛋白可用于蛋白质标准物质的研制。 相似文献
8.
随着转基因技术的迅猛发展,转基因产品的安全性受到了广泛关注。转基因检测用有证标准物质在确保转基因产品定性、定量检测结果的可比性和可追溯性方面发挥着重要作用。但转基因蛋白质标准物质的开发相对缓慢,其中一个难点是制备高纯度的转基因蛋白质候选物。苏云金芽胞杆菌Bacillus thuringiensis cry1Ah1基因因其对亚洲玉米螟等鳞翅目害虫有很好的杀虫活性,已用于转基因抗虫作物的研制,并获得具有较好抗虫性状的转基因株系。为了研发Cry1Ah蛋白有证标准物质,亟需建立其制备及纯化体系。文中优化了利用Bt表达系统制备Cry1Ah蛋白的体系,利用离子交换色谱法和排阻色谱法逐级纯化的方法,获得了高纯度的Cry1Ah蛋白(排阻色谱纯度:99.6%)。生物活性测定结果表明,纯化的Cry1Ah蛋白与原毒素对小菜蛾Plutella xylostella的杀虫活性没有显著差异。最后使用Edman降解法和质谱法确定了Cry1Ah蛋白活化后的氨基酸序列。综上所述,获得的Cry1Ah纯蛋白可用于蛋白质标准物质的研制。 相似文献
9.
利用蛋白质工程技术对Cry蛋白进行改造是创制新Bt蛋白的主要途径之一。Cry蛋白改造涉及结构域交换、定点突变、蛋白截断等多种方法。利用结构域交换、密码子优化方法对Bt基因进行合理化设计改造,获得新型Bt蛋白编码基因cryNAc,进一步利用农杆菌介导法转入吉林省水稻主栽品种吉粳88中,并开展了转基因后代的分子鉴定和抗虫性功能评价相关研究工作。分子检测结果表明cryNAc基因成功整合进入吉粳88基因组中,且稳定表达;CryNAc蛋白在各个发育时期根、茎、叶中的表达存在显著差异,灌浆期水稻叶片中蛋白表达量最高(2 959.73 ng/g),分蘖期茎中蛋白表达量最低(150.9 ng/g);田间接虫试验表明cryNAc转基因水稻抗二化螟的能力显著。上述结果表明cryNAc基因可作为新的cry基因用于作物遗传改良。 相似文献
10.
【背景】此研究为"十二五"转基因生物新品种培育国家项目中创建新的转基因棉花品种环境安全评价技术而设。【方法】以转双价双Bt抗虫基因(cry1Ac+cry2Ab)棉和转双价抗虫、抗除草剂基因(cry1Ac+EPSPS)棉为观察品种,非转基因棉赣棉11号为对照品种,在荒地用撒播和3cm深度播种2种方式,于2011年5月~2012年3月对棉花出苗率、株高、生育进程、棉吐絮瓣数、絮瓣脱落率、自生苗等生存竞争能力进行比较,检测、评价其杂草化的风险,并探讨、验证检测技术的可行性。【结果】在荒地条件下,以2种方式播种的转cry1Ac+cry2Ab基因棉和转cry1Ac+EPSPS基因棉与非转基因棉相比,上述各项指标的竞争能力总体上未表现显著优势。【结论与意义】转cry1Ac+cry2Ab基因棉和转cry1Ac+EPSPS基因棉在荒地条件下生长无杂草化风险。同时,研究证明,在荒地自然生态条件下,可以采用撒播和3cm深度播种方法检测新的转基因棉花品种在生存竞争能力上的杂草化风险,在测评上有互为参照效应,为定性评价新的转基因棉花品种的杂草化风险提供了保障。 相似文献
11.
Bt水稻杂交育种中转基因的遗传分析 总被引:12,自引:0,他引:12
利用PCR、GUS染色和Western印迹杂交技术检测了Bt水稻杂交后代群体,发现在394株GUS阳性株中,共有392株表达Bt蛋白,协同表达株率达99.49%。由此表明,在杂交后代中报告基因Gus和目的基因crylAb紧密连锁遗传与表达。本试验还发现,在BC1、BC1F2和粳粳交F2群体中转基因呈单基因显性遗传,而在籼粳交F2群体中偏离3:1分离。
Abstract:Improved histochemical staining for GUS activity,PCR and Western blotting were used to detect the population of Bt rice crossed to conventional rice varieties.A total of 392 plants expressing Bt toxin protein were found in 394 GUS positive plants.The result demonstrated that cry1Ab gene closely inherited and expressed with reporter gene gus.Therefore,it is possible to develop GUS-assisted-selection to preliminarily identify the Bt gene and study the inheritance of transgenes in (back)cross breeding.Mendelian segragation of reporter gene Gus was observed in F2,BC1 and BC1F2 progenies.Thus indicated that transgenes inherited as a single dominant gene in the progenies of Bt rice crossed to conventional rice varieties. 相似文献
12.
全球气候变化下转Bt水稻种植对土壤生态系统的影响仍是未知领域。利用野外开顶式气室(OTC)模拟气候变化,探讨了温度和CO2浓度([CO2])升高后种植转Bt(华恢1号)水稻对土壤活性碳氮和线虫群落的影响。结果表明:(1)温度和[CO2]升高条件下种植转Bt水稻显著影响土壤可溶性碳(DOC)、可溶性氮(DON)和硝态氮(NO-3-N)含量,同时DOC和DON受到升温和[CO2]升高与转Bt水稻的交互影响;在正常温度和[CO2]下,转Bt水稻显著降低了土壤微生物量碳氮含量(MBC、MBN),但在温度和[CO2]升高后转Bt水稻种植使土壤MBC和MBN含量显著高于亲本水稻。(2)在温度和[CO2]升高条件下,转Bt水稻土壤线虫总数呈高于亲本水稻的趋势,植食性线虫的比例随[CO2]和温度升高呈增大的趋势。此外,升温和转Bt水稻种植提高了土壤线虫群落的能流通道指数,而[CO2]升高和转Bt水稻种植则提高了土壤线虫群落的富集指数。总之,在模拟全球气候变化下,种植转Bt水稻在短期内对土壤活性碳氮和线虫群落均产生影响,但短期研究并未发现转Bt水稻种植对土壤生态系统的不利影响。 相似文献
13.
外源蛋白在环境中的残留与积累是转Bt基因作物环境安全评价的重要内容之一。我国已育成多个具有商业化前景的Bt稻品系,但目前多年种植Bt稻后Bt外源蛋白是否会在土壤中积累还不清楚。本研究在同一试验田连续9年种植了转cry1Ab/1Ac基因明恢63(华恢1号)和转cry2A基因明恢63水稻,采用酶联免疫吸附法(ELISA)跟踪监测了分蘖期和收获后60 d根际土中外源蛋白含量变化,试验第1年(2012年)和最后1年(2020年)还测定了苗期、开花期和成熟期根际土中外源蛋白含量。结果表明: 2012年,转cry1Ab/1Ac基因明恢63在苗期、分蘖期、开花期、成熟期和收获后60 d根际土中外源蛋白含量分别为1.25、1.77、1.97、1.71和0.30 ng·g-1,2020年分别为1.30、1.69、2.03、1.77和0.43 ng·g-1;2012年,转cry2A基因明恢63在苗期、分蘖期、开花期、成熟期和收获后60 d根际土中外源蛋白含量分别为0.91、1.52、1.53、1.37和0.12 ng·g-1,2020年分别为0.95、1.43、1.61、1.40和0.15 ng·g-1。多因素方差分析显示,时间效应对Bt外源蛋白积累不显著,而品种和生育期效应显著。Bt稻生长过程中根际土中可以检测出微量的Bt外源蛋白,但收获后60 d已经基本降解完毕,根际土中Bt外源蛋白含量不会随着种植时间的增加而累积。 相似文献
14.
以转Bt基因抗虫水稻T1C-19(含cry1C基因)和T2A-1(含cry2A基因)及其亲本水稻MH63为材料,用20和40 mg·L-1的三唑磷以及1、3和6 mg·L-1的溴氰菊酯喷雾分别处理稻株上的3龄褐飞虱若虫,研究了两种农药对转Bt基因抗虫水稻上褐飞虱再猖獗的影响.结果表明: 三唑磷处理对褐飞虱的若虫历期无显著性影响,溴氰菊酯则能显著降低若虫历期,但随着两种药剂处理浓度的升高,若虫的存活率降低、成虫的产卵量增加.在同一浓度农药药剂处理下,3个水稻品种上的褐飞虱若虫发育历期、若虫存活率、初羽雌成虫体质量、产卵量和卵孵化率等生态学参数均没有显著差异.表明褐飞虱在两种转Bt水稻上对三唑磷和溴氰菊酯诱导再猖獗的反应能力与其亲本水稻MH63没有差异. 相似文献
15.
In fields of genetically modified, insect-resistant rice expressing Bacillus thuringiensis (Bt) proteins, frogs are exposed to Bt Cry proteins by consuming both target and non-target insects, and through their highly permeable skin. In the present study, we assessed the potential risk posed by transgenic cry1Ca rice (T1C-19) on the development of a frog species by adding purified Cry1Ca protein or T1C-19 rice straw into the rearing water of Xenopus laevis tadpoles, and by feeding X. laevis froglets diets containing rice grains of T1C-19 or its non-transformed counterpart MH63. Our results showed that there were no significant differences among groups receiving 100 μg L–1 or 10 μg L–1 Cry1Ca and the blank control in terms of time to completed metamorphosis, survival rate, body weight, body length, organ weight and liver enzyme activity after being exposed to the Cry1Ca (P > 0.05). Although some detection indices in the rice straw groups were significantly different from those of the blank control group (P < 0.05), there was no significant difference between the T1C-19 and MH63 rice straw groups. Moreover, there were no significant differences in the mortality rate, body weight, daily weight gain, liver and fat body weight of the froglets between the T1C-19 and MH63 dietary groups after 90 days, and there were no abnormal pathological changes in the stomach, intestines, livers, spleens and gonads. Thus, we conclude that the planting of transgenic cry1Ca rice will not adversely affect frog development. 相似文献
16.
Bt水稻对田间非靶标害虫种群动态的影响 总被引:4,自引:0,他引:4
以转cry1Ab/cry1Ac基因水稻汕优63(以下简称汕优63/Bt)为材料,亲本汕优63(以下简称汕优63/CK)为对照,在田间自然感虫条件下研究汕优63/Bt对稻田几种非靶标害虫种群消长动态的影响。结果表明,汕优63/Bt上稻苞虫Pelopidas mathias(Fabricius)和稻眉眼蝶Mycalesis gotamaMoore幼虫数量都显著低于对照品种,但汕优63/Bt对稻苞虫的毒性强于稻眉眼蝶。汕优63/Bt上稻飞虱(白背飞虱Sogatellafurcifera(Horvath)和褐飞虱Nilaparvatalugens(Stal))混合种群数量通常与对照品种基本一致,在少数调查时间显著高于对照品种。相反,除少数调查时间与对照品种没有显著差异外,稻叶蝉(黑尾叶蝉Nephotettixcincticeps(Uhler)和二点黑尾叶蝉N.viriscens(Distant))混合种群数量通常显著高于对照品种。稻飞虱和叶蝉成为汕优63/Bt上的主要害虫。 相似文献
17.
为阐明转cry1Ab/cry1Ac基因水稻对大螟Sesamia inferens (Walker)作用的生理生化机制, 本研究用转cry1Ab/cry1Ac基因水稻茎秆饲喂大螟3龄和5龄幼虫, 采用酶活性测定方法研究了取食转Bt水稻对大螟幼虫体内3种保护酶SOD(superoxide dismutase)、 CAT(catalase)和POD(peroxidase)活性的影响。结果表明, 大螟3龄幼虫在取食转基因水稻24 h后SOD活性与对照相比提高了43.44%, 48 h后降至最低值; 在取食24 h后POD值达到最高值, 其酶活性比对照升高了29.22%, 最终在取食48 h后降至最低值, 并显著低于对照; 在取食转基因水稻4 h后, CAT活性升高了30.33%, 在取食48 h后, 与对照相比, CAT活性降低了27.01%; 5龄幼虫取食4 h后SOD活性显著高于对照水平, 36 h后降至最低值, 与对照相比, 活性下降了31.62%; 在取食8 h后POD活性达到最高值, 与对照相比, 升高了73.20%, 36 h后酶活性降至最低值; 在取食之初4 h CAT活性达到最高值, 与对照相比, 其值升高了75.73%, 在取食48 h后, 其活性与对照相比减少了7.55%。3龄幼虫与5龄幼虫相比, 对Bt的抗性水平较低, 自身防卫能力较差。结果说明, 在取食初期, 试虫体内保护酶活性升高, 以抵御Bt毒蛋白对虫体的伤害作用, 随着取食时间的延长, 保护酶活性迅速降低, 从而干扰虫体正常的代谢过程, 导致虫体出现中毒症状, 致使昆虫死亡。 相似文献
18.
Numerous genetically modified (GM) crops expressing proteins for insect resistance have been commercialized following extensive testing demonstrating that the foods obtained from them are as safe as that obtained from their corresponding non-GM varieties. In this paper, we report the outcome of safety studies conducted on a newly developed insect-resistant GM rice expressing the cry2A* gene by a subchronic oral toxicity study on rats. GM rice and non-GM rice were incorporated into the diet at levels of 30, 50, and 70 % (w/w), No treatment-related adverse or toxic effects were observed based on an examination of the daily clinical signs, body weight, food consumption, hematology, serum biochemistry, and organ weight or based on gross and histopathological examination. These results demonstrate that the GM rice with cry2A* gene is as safe for food as conventional non-GM rice. 相似文献
19.
Soo-Yun Park Si Myung Lee Jin-Hyoung Lee Hyeon-Seok Ko Soon Jong Kweon Seok-Cheol Suh Kong-Sik Shin Jae Kwang Kim 《Plant biotechnology reports》2012,6(1):29-37
Composition analysis of genetically modified crops is an important consideration in the assessment of food safety. Agb0101
(Oryza sativa L. cv. Nakdongbyeo) developed in Korea is a form of insect-resistant rice that contains a synthetic truncated cry1Ac gene isolated from Bacillus thuringiensis (Bt), and demonstrates high resistance to rice leaf folder under field conditions. Nutrients, anti-nutritive components,
and secondary metabolites of Agb0101 were analyzed and compared with those of its non-transgenic counterpart. The amounts
of proximates, amino acids, fatty acids, minerals, vitamins, trypsin inhibitors, phytic acid, and ferulic acid in brown rice
from Agb0101 were comparable to those of its non-transgenic counterpart. Statistical comparisons to test for equivalence showed
that all the analyzed components in the insect-resistant rice plants were substantially equivalent to those of its non-transgenic
counterpart. Furthermore, most of the measured values from Agb0101 were within the range of values reported for other commercial
rice varieties. 相似文献
20.
Bt水稻"克螟稻”花粉对家蚕生长发育的影响 总被引:13,自引:0,他引:13
本试验以家蚕为供试对象研究Bt水稻"克螟稻”花粉对家蚕生长发育的影响.结果发现,与正常无花粉处理相比,无论是非Bt水稻花粉处理,还是Bt水稻花粉处理,对初孵家蚕幼虫的致死率无多大影响,而对家蚕的体重有较大影响,其中三龄期家蚕体重存在极显著差异.还发现Bt水稻花粉处理组家蚕在3龄时期大小很不一致,最轻体重为18.1mg,而最重体重为183.8mg.这是由于采用人工抖粉桑叶上花粉浓度不均匀造成的.鉴于实际生态条件下桑叶上的花粉浓度可能远远低于试验条件,因此,在实际稻桑共作环境下,Bt水稻"克螟稻”花粉对家蚕生长发育可能不会造成太大的影响。
Abstract:The effect on the development of silkworm larvae of Bt transgenic rice pollen containing cry1 Ab gene from Bacillus thuringiensis were investigated.Compared with normal treatment,mortality of newly hatched silkworm lar vae in either Bt rice pollen or susceptible rice pollen treatment were not significantly different,while the variances of silkworm larvae weight at third instar were significant at 0.01level.In addition,the weight of each silkworm larva at third instar in Bt rice pollen treatment showed a big difference,the biggest and smallest silkworm larvae were 183.8rug and 18.lmg respectively,which was probably caused by the difference of Bt rice pollen concentration on the mulberry leaves.As pollen concentration on the mulberry leaves in actual field was lower than in lab,the influence on the development of newly hatched silkworm larvae of Bt rice pollen is not likely significant in actual ecological cnvi ronment. 相似文献