【作者】 陈洋；

【导师】 郭予元； 叶恭银；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2010， 博士

【摘要】 水稻是世界上最为重要的粮食作物之一,随着科学技术的发展,已有抗虫、抗病、抗除草剂和改良品质等方面的基因应用于水稻中,转基因水稻的生态安全性研究成为人们关注的焦点。转基因植物的生态安全性评价主要包括了对非靶标生物的影响、对天敌的影响和对靶标害虫的抗性治理。本文主要以三类转基因水稻和水稻重要害虫褐飞虱为研究对象,对三类转基因水稻对其非靶标生物褐飞虱的生长发育和繁殖的继代效应影响进行了评价,并对其机理进行了初步分析,主要结果如下：1转cry1Ab/vip3H基因水稻抗螟虫性转cry1Ab/vip3H基因水稻G6H1、G6H2、G6H3、G6H4、G6H5.和G6H6对二化螟和大螟的室内生测结果表明,取食转cry1Ab/vip3H水稻的二化螟和大螟在48 h和96 h的死亡率要显著高于取食对照非转基因水稻Xiushui 110,48 h和96 h取食面积也显著低于对照,并且在’168 h时死亡率达到100%。2008年和2009年田间调查结果表明：G6H1、G6H2、G6H3、G6H4、G6H5和G6H6在田间都表现出很高的抗性,其中G6H4和G6H5被螟虫的为害率不到5%。Western blot结果表明Cry1Ab/Vip3H蛋白在水稻植株中是融合表达,通过对Cry1Ab蛋白的含量测定来衡量Cry1Ab/Vip3H蛋白在水稻不同时期的表达量,蛋白含量在各个时期在6个转基因水稻品种中不同时期的表达量相对稳定,无明显规律。TOPSIS分析表明：G6H1在田间和实验室内对螟虫的抗性最好。2转cry1Ab/vip3H基因水稻G6H1对褐飞虱生物学影响G6H1对褐飞虱生长发育与繁殖的继代效应评价结果表明,不论是在苗期和成株期,褐飞虱的若虫生长发育和产卵量都不受水稻品种的影响,即取食G6H1与取食其非转基因亲本Xiushui 110相比,褐飞虱的生长发育和繁殖没有受到影响。田间调查也表明,在G6H1田间和Xiushui 110田间的褐飞虱的若虫、成虫和成虫若虫总密度没有显著差异。3转cry1Ab基因水稻KMD2对褐飞虱生物学影响在苗期,取食转cry1Ab基因粳稻(KMD2)与其亲本非转基因水稻(Xiushui11)的褐飞虱相比,其若虫发育时间受到水稻类型和代别的显著影响;成虫寿命受到代别和水稻类型与代别互作的显著影响；产卵量受到水稻类型和代别的显著影响。在成株期,褐飞虱若虫发育时间受到水稻类型和水稻类型与代别互作的影响；产卵量受到水稻类型和代别的显著影响。t测验结果表明不论是苗期还是成株期,取食KMD2褐飞虱的若虫生长发育显著延迟,并且产卵量显著下降,这就表明KMD2抑制了褐飞虱的生长发育和繁殖。2008和2009年田间调查结果表明,KMD2田间褐飞虱的种群数量始终要低于Xiushui 11,并且若虫密度和成虫若虫总密度在2008年三个取样时间和2009年4个取样时间达到显著差异。KMD2田间植株在拔节期、抽穗期、孕穗期和成熟期的褐飞虱分蘖为害率显著低于Xiushui 11;卵块数在成熟期要显著的低；卵数在抽穗期、孕穗期和成熟期均显著低。褐飞虱在田间KMD2植株上的平均产卵位置大致上随着时间的推移而升高(除了分蘖期),而在Xiushui 11植株上的平均产卵位置也随着水稻的生长而升高,并且褐飞虱在KMD2和Xiushui 11上的平均产卵位置存在显著差异。在分蘖期、拔节期、抽穗期和灌浆期,在KMD2上褐飞虱卵的位置要比其在Xiushui 11上的高,其中在分蘖期抽穗期和灌浆期达到显著水平。4转纤维素酶基因水稻KMD2对褐飞虱生物学影响对转纤维素酶基因水稻Z8-1、Z8-4、Z9-1、Z9-2、CBD-1或CBD-3进行褐飞虱集团鉴定法进行抗鉴定发现,不论是苗期和成株期它们都对褐飞虱表现出易感性,且Z9-2最为明显一些。与敏感品系TN1相比,Z9-2其体内的纤维素酶活性要高一些,而其亲本非转基因水到Xiushui 11体内的酶活性要低一些。转纤维素酶基因水稻Z9-2与其亲本非转基因水稻Xiushui 11相比,在苗期,褐飞虱若虫发育没有受到水稻类型、代别和水稻类型与代别互作的显著影响,产卵量受到代别的显著影响；在成株期,除了褐飞虱雄若虫受到代别的显著影外,雌若虫和总若虫发育时间、成虫寿命都没有受到水稻类型、代别和稻类型与代别互作的影响；产卵量受到代别的显著影响。2008年和2009年对Z9-2和Xiushui 11田间褐飞虱种群密度进行了调查,结果表明,在2008年,褐飞虱的若虫数量和成虫若虫总数量受到水稻品种的显著影响, Z9-2田间的褐飞虱数量要高于Xiushui 11田间褐飞虱的数量,并且在其中3个取样时间达到显著差异；在2009年,褐飞虱的若虫数量和成虫若虫总数量受到取样时间的显著影响,Z9-2田间的褐飞虱数量要高于Xiushui 11田间褐飞虱的数量,并且在其中1个取样时间达到显著差异。5 KMD2与Xiushui 11及其褐飞虱诱导后基因表达差异利用基因芯片技术对转cry1Ab基因水稻(KMD2)与其亲本非转基因水稻(Xiushui 11)及其褐飞虱诱导后基因表达谱进行了初步的分析。KMD2与Xiushui 11相比,有324个基因探针的mRNA水平发生了变化,表达量上调的水稻基因有177个,下调的探针有147个。KEGG Pathway分析表明,这324个差异基因参与到18条途径中,并且Go Ontology分析表明,这324个差异基因最相关的5个生物过程：氨基酸及其衍生物的代谢、类脂化合物代谢、光合作用、次生代谢和内源性胁迫反应。褐飞虱诱导后的KMD2与褐飞虱诱导后的Xiushui11相比,有317个基因探针的mRNA水平发生了变化,表达量上调的水稻基因有124个,下调的探针有193个。KEGG Pathway分析表明,这317个差异基因参与到31条途径中,并且Go Ontology分析表明与317个差异基因最相关得7个生物过程是非生物胁迫反应、碳水化合物代谢、氨基酸及其衍生物的代谢、根瘤发生与形成和UTP(糖合成)、GTP(蛋白合成)和CTP(磷脂合成)。更多还原

【Abstract】 Rice is the one of the most important food resource in the world. With the rapid development of technology, we have obtained the transgenic rice with insect resistance, disease resistance, herbicides resistance and/or quality improved characteristic, and more and more people focus on the ecological safety assessment of transgenic rice. The ecological safety assessments of transgenic plant mainly include the effect on non-target pests, the effect on natural nememies and the resistance management. The effeects of three transgenic rices on the rice secondary pest the brown planthopper (BPH), Nilaparvata lugens stal, were evaluated, and the mechanism was primary analysis. The results are as follows:1 Resistance of transgenic crylAb/vip3H gene rice to stem borersBioassay result indicated that the transgenic crylAb/vip3H rice lines G6H1, G6H2, G6H3, G6H4, G6H5 and G6H6 showed high resistance to stem borers including striped stem borer (SSB), Chilo suppresalis (Walker) and purplish stem borer (PSB), Sesamia inferens (Walker). After 48 h and 96 h ingestion, the mortality of SSB and PSB that fed on six transgenic rice lines was significantly higher than that fed on its non-transgenic parental line Xiushui 110, and the cumulate feeding area was significantly lower. After 168 h ingestion, the mortality of SSB and PSB that fed on six transgenic rice lines arrived 100%. In 2008 and 2009, when the field test was conducted under natural infestation, the G6H1, G6H2, G6H3 and G6H6 were highly resistant to SSB and PSB, and were completely undamaged on all sample dates. For the transgenic lines G6H4 and G6H5, their plants were undamaged before the sample date of 8 September, while were encountered to a few damage with less than 4%of plants and 0.5% of tillers. The Western blot analysis confirmed the presence of the CrylAb and Vip3H fusion protein in the six transgenic rice lines. The CrylAb protein was assayed to estimate the concentration of the fusion protein of CrylAb/Vip3H expressed in transgenic rice plants. In the flag leaves and main stems for each transgenic line, the level of CrylAb protein significantly changed with rice developmental stages. It is estimated that G6H1 showed best resistance to stem borers according TOPSIS analysis.2 Effect of transgenic cry1Ab/vip3H rice G6H1 on N. lugensThe nymph developmental duration and reproduction was not affected by rice type for 1st,2nd and 4th generation neither in seedling or adult stage. Compared with fed on Xiushui 110, the nymph duration, adult longevity and egg production of BPH that fed on G6H1 showed no significant difference. And the field investigation showed that the average density of nymph, adult and total of nymph and adult were similar between the G6H1 and Xiushui 110.3 Effect of transgenic crylAb rice KMD2 on N. lugensAt seedling stage, the nymph duration of BPH feeding on transgenic cry1Ab rice KMD2 and its nontransgenic parental rice Xiushui 11 was significantly affected by rice type and generation; adult longevity was significantly affected by generation and rice type with generation interaction; egg production was affected by rice type and generation. At adult stage, the nymphe duration of BPH was affected by rice type and rice type with generation interaction; egg production was affected by rice type and generation. t-test result indicated that the nymph duration was significantly prolonged and the egg production was signifantly reduced when the BPH fed on the KMD2 at both seedling and adult stage. This result revealed that the KMD2 suppress the development and reproduction of the BPH. The field investigation indicated that the average density of BPH in the KMD2 paddy was lower than that in Xiushui 11 paddy at all the sample times in 2008 and 2009. The average density of nymph and total of nymph and adult showed significantly difference between KMD2 and Xiushui 11 paddy both in three sample times in 2008 and four sample times in 2009. By comparison with the non-Bt rice, both percentage of tillers with eggs and number of eggs per tiller in the Bt rice were significantly lower from jointing to mature stage for the former and from heading to mature stage for the later. The relative height of oviopsition on the Bt rice was significantly higher than that on the non-Bt rice at the tillering, heading and filling stage of rice, while was significantly lower than that on the non-Bt rice at the mature stage of rice.4 Effect of transgenic cellulase rice on N. lugensResistance of transgenic cellulase rice lines (Z8-1、Z8-4、Z9-1、Z9-2、CBD-1 and CBD-3) to BPH was evaluated using the Modified Seedbox Screening Technique (MSST). All of the transgenic lines showed sensitive to BPH both at seedling and adult stage, and the Z9-2 was the most sensitive variety among the six varieties. Compared with the accepted rice variety that sensitve to BPH, the activity of cellulase in Z9-2 was higher, and its non-transgenic rice line Xiushui 11 was lower. At seedling stage, the egg production of BPH feeding on transgenic rice Z9-2 and its non-transgenic parental rice Xiushui 11 was significantly affected by generation and nymph developmental duration and adult longevity was not affected by rice type, generation and rice type with generation interaction. At adult stage, the male nymphe duration and egg production of BPH were significantly affected by generation. The field investigation indicated that the average density of BPH in the Z9-2 paddy was higher than that in Xiushui 11 paddy at most sample times in 2008 and 2009. In 2008, the average density of nymph and total of nymph and adult was affected by rice type, and Z9-2 showd significantly higher population at three sample tines. In 2009, the average density of BPH was affected by the sample time. And the average density of nymph in Z9-2 paddy was higher than that in Xiushui 11 paddy at one sample time.4 The gene expression profiling of KMD2 and Xiushui 11, BPH induced KMD2 and BPH induced Xiushui 11The gene expression profiling of KMD2, Xiushui 11, BPH induced KMD2 and BPH induced Xiushui 11 were performed using gene chip method. Compared with its non-transgenic parental rice Xiushui 11, transcriptional levels of 324 genes in KMD2 were different, in which the numbers of upregulated and.downregulated genes were 177 and 147 respectively. KEGG Pathway analysis indicated that the 324 genes were involved in 18 pathways. And Go Ontology analysis showed that the most five correlative biological processes were amino acid and derivative metabolism, lipid metabolism, photosynthesis, secondary metabolism and response to endogenous stimulus. Compared with its BPH induced Xiushui 11, transcriptional levels of 317 genes in BPH induced KMD2 were different, in which in which the numbers of upregulated and downregulated genes were 124 and 193 respectively. KEGG Pathway analysis indicated that the 317 genes were involved in 31 pathways. And Go Ontology analysis showed that the most seven correlative biological processes were response to abiotic stimulus, carbohydrate metabolism, amino acid and derivative metabolism, nodulation, UTP biosythesis, CTP biosythesis and GTP biosythesis.更多还原