论文标题:转Bt基因水稻Cry1Ab杀虫蛋白的环境动态及其对非靶标昆虫的影响
Environmental Dynamics of Cry1Ab Expressed by Transgenic Bt Rice and Effects on Non-Target Insects in Rice Paddy Habitat
论文作者 白耀宇
论文导师 程家安,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 121,论文页数 152页File Size6484k
2004-05-01论文网 http://www.lw23.com/lunwen_756391212/ Bt水稻;克螟稻1号;克螟稻2号;华池B_6;Cry1Ab杀虫蛋白;生态风险;水稻土;降解;残留;土壤性质;温度;时空表达;水生昆虫;龟纹瓢虫;水稻花粉;适合度;褐飞虱;中华通草蛉;人工饲料;弹尾虫;灰橄榄长角跳虫;酯酶;同工酶;尖钩宽黾蝽;功能反应;种群密度;生长发育;繁殖;寿命
Bt rice,KMD1,KMD2,HuachiB_6,CrylAb insecticidal protein,ecological risk,paddy soil,fitness,degradation,physical and chemical propertity of soil,temperature,spatial-temporal dynamics,aquatic insect,Propylaea japonica,rice pollen,Nilaparvata lugens,Chrysoperla sinica,artifical diet,esterases,isoenzyme,Microvelia horvathi,functional responses,Entomobrya griseoolivata,population density,development,reproduction,longevity
自1986年世界上第一例转Bt基因植物问世以来，这些转入了外源Bt杀虫基因植物潜在的生态风险一直是人们关注的焦点。本文以转Bt cry1Ab基因水稻克螟稻1号(KMD1)、克螟稻2号(KMD2)和华池B_6为研究材料，通过室内和田间研究，对其所表达的杀虫蛋白在环境中的动态以及对一些非靶标昆虫的影响进行了研究，取得了如下结果： 1．Bt水稻Cry1Ab杀虫蛋白在稻田土壤中的降解动态及其影响因子 比较了KMD2叶片干粉中的Cry1Ab杀虫蛋白在三种水稻土即青紫泥田(Blue clayey paddy soil)、黄筋泥田(Pale paddy soil on quaternary red clay)和黄松田(Marine-fluvigenic yellow loamy paddy soil)中的降解动态，以及华池B_6叶片、KMD1稻茎和叶片中的Cry1Ab杀虫蛋白在这三种土壤淹水和非淹水条件下的降解动态，研究了土壤微生物、土壤含水量、土壤pH值和温度对KMD2粉碎叶片中Cry1Ab降解的影响。结果表明，各Bt水稻供试组织中Cry1Ab杀虫蛋白在水稻土中的降解均以前期较快，中后期降解速度缓慢，Cry1Ab残留量和时间之间关系符合一级化学降解动力学指数方程，半消减期为1.8～11.6d。Cry1Ab蛋白的降解动态与土壤类型密切相关，在青紫泥田中降解最快，其次为黄筋泥田，黄松田中降解最慢。淹水可显著加快水稻组织残体的腐解，从而促进Cry1Ab蛋白的降解。土壤微生物、土壤含水量、土壤pH和温度与Cry1Ab蛋白降解速率呈显著的相关性。发现土壤微生物是影响Bt水稻Cry1Ab蛋白在土壤中降解的主要因子之一。pH较低的酸性土壤不利于Cry1Ab蛋白的降解，Cry1Ab蛋白在中性和碱性土壤中降解较快。低温不利于Cry1Ab蛋白的降解，温度越高降解越快。 2．Bt水稻组织中Cry1Ab杀虫蛋白表达及其在田间残留的时空动态 用ELISA技术检测了KMD1和KMD2各生育期不同组织中cry1Ab基因的表达水平，测定了水稻收割后不同时间植株地表残体(茎和叶)、根系及土壤中Cry1Ab的含量。结果表明，KMD1和KMD2生长期间cry1Ab基因在茎、叶片、根系、花粉及种子中均有表达。分蘖初期至成熟期，KMD1茎、叶片和根系中Cry1Ab蛋白的表达量分别为3.74～7.50μg／g、3.78～9.13μg／g和0.51～0.56μg／g；KMD2分别为3.97～8.25μg／g、4.19～8.84μg／g和0.47～0.51μg／g；KMD1和KMD2花粉干粉中Cry1Ab蛋白平均含量分别为13.12和31.4μg／g；中文摘要KMDI根系分泌物中Cry1Ab蛋白的含量为1.18~21.85n岁株/d。在收割后160d，KMDI植株地表残体和根系中的Cry1Ab蛋白平均残留量分别为0.29林留g和0.07协留g，KMDZ分别为0.38林岁g和。.09林留g。在Bt水稻生长期间，或在收割后其植株残体(包括根系)腐解过程中，向土壤中释放的Cry1Ab蛋白量均很低，低于试剂盒的检测下限0.5n岁g(土壤鲜重)。3.Bt水稻Cry1Ab杀虫蛋白在水体中的残留及其对水生昆虫的影响 以KMDI和KMDZ成熟后期的茎叶为材料，测定了其经浸水处理后残体中和水体中Cry IAb杀虫蛋白的含量:调查了华池B6分孽初期、分桑盛期和分粟末期稻田主要几类(鞘翅目、摇蚊科和蟀蟒目)水生昆虫的数量。结果表明，KMDI和KMDZ茎叶经浸水lood后，水体中Cry1Ab杀虫蛋白平均含量分别为1.52ng/mL和1.osng/mL，160d后分别为1.22ng/mL和1.33ng/mL。除分桑末期华池B6稻田中的蟀蟒目昆虫数量显著高于对照田(嘉早935)外，其它各类昆虫在两类稻田间均无显著差异。这表明，Bt水稻植株组织残体释放的Cry1Ab杀虫蛋白可在水体中残留和积累，但含量低，不会对稻田主要水生昆虫种群数量产生显著的影响。4.Bt水稻对龟纹瓢虫取食适合度的影响 龟纹瓢虫尸八￥少laeaj即onica是稻田重要的捕食性天敌昆虫，具有取食稻花补充营养的习性。本研究通过龟纹瓢虫直接取食Bt水稻花粉以及通过捕食Bt水稻上饲养的稻褐飞虱NIIaPa洲ata lugens两种途径，评价了Bt水稻cry1Ab杀虫蛋白对该飘虫生命参数的影响。结果表明，龟纹瓢虫取食Bt水稻花粉其幼虫和蛹生长发育和成虫生殖参数与取食非转基因水稻花粉的相比无显著差异;捕食Bt水稻上饲养的褐飞虱(其成虫体内可检测到Cry1Ab杀虫蛋白)后，其幼虫和蛹生长发育和成虫生殖参数与捕食非转基因水稻上的稻褐飞虱处理相比无显著差异。这表明，龟纹瓢虫直接取食Bt水稻花粉，或通过褐飞虱这一营养联系，Bt水稻Cry1Ab杀虫蛋白均未能对其适合度产生显著影响。5.Bt水稻花粉对中华通草蛤产卵和成虫寿命的影响 中华通草岭Chrysoperla sinica是多种农林害虫的重要天敌，成虫嗜食植物花粉和花蜜。本研究分别用含5%的KMDI、KMDZ和对照XSll花粉的人工饲料饲喂中华通草岭初羽化成虫，发现两类Bt水稻花粉对该虫产卵前期、产卵量和成虫寿命等生命参数均无显著影响。人工饲料中水稻花粉的比例加大后(上升至20%和80%)，KMDI、KMDZ和XSn花粉处理中的草岭均出现产卵前期延长、产卵量减少和产卵天数缩短的趋势，且在同一花粉比例下这些参数在三个花粉处理间均无显著差异。表明Bt水稻花粉对中华中文摘要通草岭的产卵和成虫寿命无显著影响。6.Bt水稻对弹尾虫的影响 调查了Bt水稻生长季节和收割后稻田弹尾虫的发?
Since the emergence of the first transgenic Bt plant in 1986, an increasing attention has been paid on the potential ecological risk brought about by the release of the transgenic Bt plants. Laboratory and field studies using two transgenic Bt rice, KMD1 and KMD2, containing a synthetic crylAb from Bacillus thuringiensis under the control of maize ubiquitin promoter, were carried out to investigate the degradation dynamics of CrylAb from Bt rice in environment and the relevant ecological risks to some non-target insects. The results are as follows:1. Degradation dynamics of CrylAb insecticidal protein from transgenic Bt rice in paddy soils and the relevant environment factorsThree types of paddy soils, i.e., blue clayey paddy soil, pale paddy soil on quaternary red soil and marine-fluvigenic yellow loamy paddy soil, were used. The degradation dynamics of CrylAb from ground dry powders of KMD2 leaf-blades in each type of soil, and the degradation dynamics of CrylAb from Huachi B6 leaf-blades and CrylAb from KMD1 stems and leaf-blades in flooded and unflooded soil of each type, were determined. Moreover, the effects of soil microbe, water content, pH value of soil and temperature on the degradation were also studied. It was found that, in each type of soils, the degradation rate of CrylAb was high during early experimental stage, but it slowed down steadily during middle and later stages, which could be described by exponential equations, with the half-life period of degradation determined as 1.8-11.6 d. Degradation rate of CrylAb protein from Bt rice tissue was closely related to the soil types, with the highest rate in blue clayey paddy soil, and the lowest in marine-fluvigenic yellow loamy paddy soil. Watering accelerated the degradation of rice tissues significantly, and thus enhanced the degradation of CrylAb protein within them. The degradation of CrylAb was significantly affected by soil microbe, water contents, pH value and temperature of soils. The soil microbe was determined as a primary factor affecting CrylAb degradation in paddy soils. CrylAb degradation rates were getting higher as the increase of pH value and temperatures under the experimental conditions.2. CrylAb content in Bt rice plant at varous growing stages, and degradation of CrylAb in plant in paddy habitatsBy using standard ELISA (Enzyme-linked immunosorbent) test kits of US EPA, the seasonal dynamics of Bt insecticidal protein expression in different plant tissues of two transgenic rice lines, KMD1 and KMD2, crylAb and the temporal and spatial patterns of CrylAb degradation and residue in the tissue litter, were determined in the greenhouse and paddy. The results showed that CrylAb was detected in all tissues of KMD, but its concentrations varied with the plant part and growing stages. From early tillering stage to mature stage, the concentrations of CrylAb in the stem, leaf and root of KMD1 were 3.74~ 7.50g/g, 3.78~9.13g/g and 0.51~0.56g/g, respectively, and those of KMD2 were 3.97~8.25g/g, 4.19~8.84g/g and 0.47~0.51g/g, respectively. The mean CrylAb concentrations in dry pollens of KMD1 and KMD2 were 13.12g/g and 31.40g/g, respectively. CrylAb in the root exudation of KMD1 reached 1.18~21.85 ng/plant-d. At 160d after harvest, the mean concentrations of CrylAb in tissue litter and root of KMD1 were 0.29g/g and 0.07g/g, respectively, and those of KMD2 were 0.38g/g and 0.09g/g, respectively. The concentrations of CrylAb at soil surface and root rhizosphere released from decomposition of tissue litter and root exudation were less than the limit of detection with 0.5ng/g fresh soil.3. Concentrations of CrylAb insecticidal protein in water released from Bt rice tissue litter and its impacts on aquatic insectsThe stems and leaves of KMD1 and KMD2 at late mature stage were submerged in water, and the CrylAb concentrations in these plant tissues as well in the water were determined. The population densities of major aquatic insect groups were surveyed at early, mid- and late tillering stages of Huachi 6 and Jiazao 935.
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