论文标题:Bt毒蛋白在“转Bt水稻—稻纵卷叶螟—纵卷叶螟绒茧蜂”食物链中的传递和作用机制
Biotransfer and Effect Mechanisms of Cry1Ab Insecticidal Protein in Rice Plant-Rice Leaf Folder-Apanteles Cypris Nixon Food Chain
论文作者
论文导师 陈学新；叶恭银；彭于发,论文学位 博士,论文专业 农业昆虫与害虫防治
论文单位 浙江大学,点击次数 96,论文页数 133页File Size10357K
2006-12-01论文网 http://www.lw23.com/lunwen_78424642/
transgenic Bt rice; rice leaf-folder; Apanteles cypris Nixon; biosafety assessment; non-target organism; natural enemy; feeding; midgut; haemolymph; proteinase; population dynamic; insecticidal protein; food chain; biotransfer; behavior response; arthropod; community; diversity index; species richness
水稻作为人类主要口粮之一，转基因水稻安全性倍受关注，有关转基因水稻生态安全性研究尤为必要。因此，在转基因水稻商业化生产之前，进行系统、科学的生态安全性评价刻不容缓。本论文以转crylAb基因粳稻纯合品系“克螟稻1号(KMD1)”和“克螟稻2号(KMD2)”为材料，通过室内外实验，评价了转基因抗虫水稻对靶标生物稻纵卷叶螟及其天敌纵卷叶螟绒茧蜂的生态安全性影响以及对稻田生境内节肢动物群落多样性的影响。结果如下： 1转crylAb基因抗虫粳稻对靶标害虫稻纵卷叶螟取食、生长及存活的影响 连续取食转crylAb基因的水稻后，不同龄期的稻纵卷叶螟幼虫食物的消耗量都显著的低于取食对照水稻“秀水11”的幼虫的消耗量。稻纵卷叶螟幼虫在对照水稻“秀水11”上连续取食时，食物的消耗量随着时间的延长而增加。当幼虫取食转Bt基因的水稻“KMD1”时，不同龄期的变化趋势有所不同，3龄幼虫取食水稻的食物消耗量随时间的变化也有所不同于4龄和5龄，3龄幼虫在取食后食物消耗量有先减再稍微增的趋势，而4龄和5龄老熟的幼虫则表现为先增加再略微减少的趋势。取食转crylAb基因的水稻后，稻纵卷叶螟3龄，4龄和5龄的幼虫体重都出现了负增长的趋势，取食对照水稻“秀水11”的幼虫则表现出体重的正增长。取食转Bt基因的水稻后，稻纵卷叶螟3龄，4龄和5龄的幼虫分别都表现出了较高的相对死亡率，其中3龄，4龄和5龄幼虫在取食转Bt基因水稻72小时后的相对死亡率分别为81％，77％和68％。表明低龄幼虫比高龄幼虫对转Bt基因水稻显得更加敏感。 2转crylAb基因抗虫粳稻对靶标害虫稻纵卷叶螟组织病理学效应 稻纵卷叶螟幼虫取食了转CrylAb基因的水稻后，中肠上皮细胞的线粒体先发生形态变化，随取食时间的延长，线粒体出现凝聚、内嵴破坏、空泡化等。内质网的变化也很明显，病变过程中伴随着粗糙内质网的肿胀、核糖体脱落等。此外，在病变过程中，细胞质内出现了粗糙内质网增多等现象。组织病变程度不一，有的细胞在病变早期就出现了空泡化。细胞核的变化较小，在处理后期出现细胞核拉长、核仁聚集等变化。杯状细胞的杯腔底部的细胞质突起变化很明显，表现为突起拉长，脱落成膜性结构，最后破碎，消失，线粒体则呈凝聚态随突起脱落或沿杯腔边沿单一排列。 3转crylAb基因抗虫粳稻对靶标害虫稻纵卷叶螟中肠血淋巴糖代谢和蛋白质浓度的影响 取食转crylAb基因水稻的稻纵卷叶螟幼虫，在取食12h，24h，36h后的5龄幼虫血淋巴的浓度显著低于对照，3龄幼虫和5龄幼虫其他取食时间的血淋巴的总蛋白浓度均低于取食对照水稻“秀水11”的幼虫血淋巴总蛋白浓度，但差异不显著。取食转Bt基因水稻的稻纵卷叶螟幼虫无论是3龄幼虫还是5龄幼虫血淋巴总糖含量均低于取食对照水稻秀水11，差异不显著。取食转Bt基因水稻的稻纵卷叶螟幼虫无论是3龄幼虫还是5龄幼虫血淋巴海藻糖的含量也均低于取食对照水稻“秀水11”的幼虫血淋巴中海藻糖含量，但差异不显著。 4转crylAb基因抗虫粳稻对靶标害虫稻纵卷叶螟中肠蛋白酶活性的影响 稻纵卷叶螟3龄幼虫在取食转crylAb基因水稻“KMD1”12h、24h、36h和48h后中肠总蛋白酶活性比对照有所上升，取食60小时后的总蛋白酶活性比对照低；在取食“KMD2”时，在12h和48h后比对照略升，而36h和60h后则比对照下降，取食24h后无明显变化。5龄幼虫在取食转Bt基因水稻“KMD1”12h、24h、48h和60h后中肠总蛋白酶活性比对照有所上升，取食36小时后的总蛋白酶活性和对照相近；在取食“KMD2”时，在12h、36h、48h和60h后比对照略升，而取食24h后比对照略为下降。 稻纵卷叶螟3龄幼虫在取食转crylAb基因水稻“KMD1”、“KMD2”分别在12h、24h、36h、48h和60h后中肠强碱性类胰蛋白酶活性比对照都有所下降，但无明显差异。5龄幼虫除了取食“KMD1”在36h后和取食“KMD2”60h后蛋白酶活性有上升外，其余时间均表现为下降。 弱碱性类胰蛋白酶活性以及类胰凝乳蛋白酶活性的变化与强碱性类胰蛋白酶活性变化相似，大部分时间段均呈现下降的趋势，除了弱碱性类胰蛋白酶活性在幼虫3龄取食“KMD1”12h和24h后，5龄取食“KMD2”48h后有所上升，以及类胰凝乳蛋白酶活性在幼虫3龄取食“KMD1”12h后，5龄取食“KMD2”24h，36h，60h后有所上升。 5纵卷叶螟绒茧蜂对转crylAb基因水稻及稻虫复合体挥发物的行为反应 在转crylAb水稻“KMD1”和对照水稻“秀水11”之间，纵卷叶螟绒茧蜂选择转基因水稻较多，但差异不显著，在另一转基因水稻“KMD2”上则表现明显的偏好。在转基因水稻或对照水稻和2龄稻纵卷叶螟幼虫的复合体中，纵卷叶螟绒茧蜂极显著选择对照和2龄稻纵卷叶螟幼虫的复合体。在转基因水稻或对照水稻和4龄稻纵卷叶螟幼虫的复合体中，纵卷叶螟绒茧蜂也是极显著选择对照和4龄稻纵卷叶螟幼虫的复合体。 6转crylAb基因抗虫粳稻对纵卷叶螟绒茧蜂生物学特性的影响 寄生在取食转基因水稻“KMD1”和“KMD2”的稻纵卷叶螟幼虫体内的纵卷叶螟绒茧蜂，其卵+幼虫历期与对照无明显差异，2龄寄主育出的雄蜂、雌蜂的寿命都明显短于对照，3龄寄主育出的只有雄峰的寿命明显短于对照。两个龄期寄主育出的绒茧蜂出茧率和羽化率都明显的低于对照；茧长与对照无明显差异；2龄寄主育出的蜂茧重与对照无明显差异，3龄寄主育出的蜂茧重明显低于对照；蜂重无论雌雄都显著低于对照：2龄寄主育出的雌雄蜂前翅长与对照无明显差异，而3龄寄主育出的雌雄蜂前翅长均显著低于对照。 7 Bt毒蛋白在“转Bt基因水稻—稻纵卷叶螟—纵卷叶螟绒茧蜂”食物链中的传递 ELISA结果表明，不同生育期“KMD1”和“KMD2”水稻叶鞘内均含有CrylAb杀虫蛋白，“KMD1”和“KMD2”在不同生育期间存在差异，其中苗期、分蘖期和孕穗期叶鞘内CrylAb杀虫蛋白的含量显著高于黄熟期。“KMD1”和“KMD2”上取食的稻纵卷叶螟幼虫整体中则可以检测到毒蛋白的存在。在取食“KMD1”的连续5天内，其体内的CrylAb杀虫蛋白含量表现出先下降再略上升的趋势。而在“KMD2”上，第3天的变化趋势和“KMD1”相反，其他基本一致，但均无显著差异。同时，在纵卷叶螟绒茧蜂幼蜂体内也检测到了CrylAb杀虫蛋白的存在。并且随着稻纵卷叶螟取食Bt水稻时间的延长，体内的毒蛋白有逐渐下降的趋势。单头纵卷叶螟绒茧蜂中毒蛋白的含量明显低于单头稻纵卷叶螟幼虫体内的含量。同时，我们也检测了解剖的稻纵卷叶螟幼虫的血淋巴、稻纵卷叶螟幼虫整体以及稻纵卷叶螟幼虫排泄物中杀虫蛋白的含量。随着取食时间的延长，稻纵卷叶螟幼虫的血淋巴、稻纵卷叶螟幼虫以及稻纵卷叶螟幼虫排泄物中杀虫蛋白的含量没有上升的趋势，反而有一定的下降。 8转Bt基因水稻对稻田生境内节肢动物群落多样性的影响 在3年3地点的调查中，Bt稻田和对照田间节肢动物物种总数没有明显差异，在杭州试验田中，Bt水稻(B系列)上节肢动物的物种总数略少于对照田块，但差异不显著；在建德试验田中，Bt水稻(TT系列)上节肢动物物种总数略高于对照田块，差异也不显著；富阳试验田块中Bt水稻(K系列)上节肢动物物种总数与对照田无明显差异。Bt水稻田和对照田在各参数上(丰富度，多样性指数，优势集中性指数和均匀度)均没有明显的差异。在功能团优势度上，除了03年杭州试验田外，其他试验田中群落中的植食性功能团占显著优势，寄生性功能团和腐食性功能团其次，在整个功能团组成上Bt稻田和对照田间没有明显差异。
Biosafety of transgenic crops has been becoming a focus in the world. Concerns on the biosafety of transgenic rice are necessary, because rice is one of staple foods of human. Biosafety assessments should be systematically and scientifically conducted before the commercialization of transgenic rice. So, a series of laboratory and field experiments were carried out in order to systematically evaluate the biosafety effects of the transgenic rice lines with cry1 Ab gene on both their target and non-target organisms. 1 Effects of transgenic Bt rice expressing a gene of cry1Ab on the food consumption, growth and survival of rice leaf-folder larvae The transgenic rice KMD1, expressing a synthetic Cry1Ab gene from Bacillus thuringiensis, showed effective resistance to the older (third- to fifth-instar) larvae of the rice leaf-folder (RLF), Cnaphalocrocis medinalis (Guenee) in laboratory bioassay. Significant declines were revealed in food consumption and growth of the older RLF nymphs fed on the cut-leaves of transgenic KMD1 plants. The increase rate of food consumption by larvae fed on KMD1 was drastically lower than those on Xiushui 11. Food consumption was varied with different instars when the larvae fed on the Bt rice. Those of four- and fifth-instar larvae were different compared to the third-instar, lower than those on the non-transgenic rice but still increased a litter when the feeding time prolonged. It is indicated that younger RLF larvae are more sensitive to Bt rice than older ones. Also, about 81%, 78% and 68% of the third-, fourth- and fifth-instar RLF larvae died within 72 hours bioassay period on KMD1 leaves, respectively. These results demonstrated that the transgenic Bt rice "KMD1" confers substantial protection against infestation with older RLF larvae. 2 Pathological effects of transgenic Bt rice expressing a gene of cry1Ab on the larval midgut of rice leaf-folders The pathological changes of the larval midgut of rice leaf-folders after feeding with Bt rice line KMD1 and KMD2 was studied using electron microscopy. Ultrastructural investigations of the anterior midgut showed cellular alternations in larvae, such as swelling of mitochondria, endoplasmic reticulum, and dilatation of intercellular spaces, fenestration or disorganization of the Golgi complex, and concentric arrangement of rough endoplasmic reticulum. The initial infected organelles were mitochondria. As the pathological process went on, the upper portions of the cells swelled, and cytoplasm in these regions decreased in electronic density and other organelles were damaged to a certain degree, such as wiredraw of nucleus, condensation of nuclear, and the increase of number of rough endoplasmic reticulum. As the infection proceed still further, pathological changes in the mitochondrial abundant areas of basal membrane enfoldings of columnar cells and cytoplasmic projections of goblet cells became more obvious. Finally the midgut epithelium cells disrupted and sloughed off from the basal membrane. 3 Sugar and protein content in the haemolymph of rice leaf-folder larvae after fed with the transgenic Bt rice Protein concentration in the haemolymph of rice leaf-folders (third- or fifth-instar) fed with KMD1 or KMD2 was lower than that of the control Xiushui 11, and the difference was not significant, while there was no significant difference among different times after treatment. Total sugar content in the haemolymph of rice leaf-folders fed with KMD1 or KMD2 was lower than that of the control, and the difference was not significant. Similarly, the trehalose content in the haemolymph of rice leaf-folders fed with KMD1 or KMD2 was lower than that of the control, and the difference was also not significant. 4 Influence of Bt rice on the digestible proteinase activity in the midgut of rice leaf-folders after fed with transgenic Bt rice The influence of Bt rice line KMD1 and KMD2 on the digestive enzymes of rice leaf-folders was tested. We measured the activities of some digestive enzymes in the larval midgut, including trypsin-like proteinase and chymotrypsin-like proteinase within different time after treatment. The activities of the total proteinase were higher than the controls, but not significantly. While the activities of the weak alkaline trypsin-like and the active alkaline trypsin-like enzymes generally trend to decline, the same as the chymotrypsin-like enzymes activies. 5 Effects of the health and damaged (by rice leaf-folders) transgenic Bt rice on the behavioral responses of Apanteles cypris Nixon The behavioral responses of Apanteles cypris Nixon to the transgenic Bt rice (KMD1 and KMD1) and the control rice Xiushui 11 were studied with a Y-tube olfactometer. The results indicated that health Bt rice and non-Bt rice all had attraction to Apanteles cypris Nixon compared with clean air; Apanteles cypris Nixon has preference to the KMD1 rice on health plants; among the damged Bt rice and non-Bt rice plants, Apanteles cypris Nixon preferred the volatiles of non Bt rice. 6 Impacts of transgenic rice lines on biological characteristics of Apanteles cypris Nixon The developments of Apanteles cypris Nixon were suppressed when A. cypris Nixon parasitized rice leaf-folder larvae fed with Bt rice. The results showed that the adult longevity of the male A. cypris Nixon, and female A. cypris Nixon parasitized in 2nd instar larvae of rice leaf-folder, cocooning rates and rates of eclosion, weights of adult wasp, weight of cocoon parasitized in third- instar larvae of rice leaf-folders, and the length of the front-wing were all lower than those parasitized in larvae fed with non-Bt rice Xiushui 11. No significant difference were observed on cocoon-larvae duration, the length of the cocoon, and the length of the front wings which parasitized in 2nd instar larvae RLF fed with Bt rice. 7 Biotransfer and bioaccumulation of Cry1Ab insecticidal protein in rice plant-rice leaf folder-Apanteles cypris Nixon food chain The concentration of Cry1Ab insecticidal protein expressed in the two homozygous transgenic Bacillus thuringiensis (Bt) rice lines, KMD1 and KMD2, were determined by enzyme-linked immunosorbent assay (ELISA) with the Pathoscreen kit for Bt cry1Ab/Ac protein (Agdia, USA). The results showed that the concentration of Cry1Ab expressed both in KMD1 and KMD2 at heading and maturing stage were significantly lower than that at seedling, tillering and booting stage. The quantitative cycle of Cry1Ab insecticidal protein in the food chain of transgenic rice plants and its target pest leaf folder, Cnaphalocrocis medinalis (Guenee) as well as the parasitoid wasp Apanteles cypris Nixon was analyzed using the kit. After being fed on KMD1 or KMD2 rice plants for 12 hours, Cry1Ab protein could be detected in the bodies of rice leaf folders. However, as the feeding time increased, the content of Cry1Ab remaining in RLF body did not show the tendency of increase. Also, Cry1Ab protein could be detected in the bodies of parasitoid wasp parasitizing in the RLF reared on KMD1 or KMD2 rice plants for increasing time, while their contents were less than those of RLF fed on KMD1 or KMD2 for the same time. It is clear that Cry1Ab protein could be transferred from transgenic rice to its target pest, RLF, and then to its parasitoid, Apanteles cypris Nixon in the food chain, but no bioaccumulation pattern of Cry1Ab protein was observed. Additionally, contents of Cry1Ab protein in the excretion of RLF were observed, and also did not show the tendency of increase with the feeding time increased. 8 Effects of the transgenic Bt rice on the arthropod abundance and diversity in the paddy fields In a 3-yr field experiment, possible effects of transgenic Bt rice on the arthropod communities under paddy-field condition were assessed in terms of arthropod guild dominance, family composition, dominance distribution of each guild, individuals of each guild and some common community indices (including species richness, Shannon-Weaver diversity index, evenness index and dominant concentration index). Our results overall suggested no significant differences between the Bt and control rice plots in such arthropod community specific parameters. The similarity of arthropod community between the Bt and control rice plots was apparently high. It is clear that Bt rice generally shows no marked negative effects on the arthropod community in paddy fields.
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