Impacts of Environmental Factors on Degradation of CrylAb Insecticidal Protein in Leaf-Blade Powders of Transgenic Bt Rice

The determination of the environmental fate of Bt insecticidal protein released by Bt rice plants in paddy soils is a key issue in its ecological risk assessment. In this study, the impacts of soil water content, pH, and temperature on the degradation of CrylAb protein expressed in the leaves of Bt rice KMD2 were studied in the laboratory. Three types of paddy soils were used, i.e., blue clayey paddy soil, pale paddy soil on quaternary red soil, and marine-fluvigenic yellow loamy paddy soil. Ground powders of KMD2 leaf blades were mixed with each type of soil, and degradation dynamics of Cry lAb were measured using enzyme-linked immunosorbent assay （ELISA）. The degradation rate of CrylAb was high at the early experimental stage, but slowed down steadily at middle and later stages, which could be described by exponential equations, with the half-life period of degradation determined as 1.8-4.0 d. The soil water content, pH, and temperature could affect the degradation of CrylAb, but the effects of soil pH and temperature were relatively greater. In general, CrylAb degradations were slower under lower soil pH and temperature conditions, especially for marine-fluvigenic yellow loamy paddy soil.

Impact Assessments of Transgenic cry1Ab Rice on the Population Dynamics of Five Non-Target Thrips Species and Their General Predatory Flower Bug in Bt and Non-Bt Rice Fields Using Color Sticky Card Traps

A 2-yr field study was conducted to assess the effects of transgenic japonica rice（KMD1 and KMD2） with a synthetic cry1 Ab gene from Bacillus thuingiensis Berliner on population dynamics and seasonal average densities of five thrips species including Stenchaetothrips biformis（Bagnall）,Frankliniella intonsa（Trybom）,F.tenuicornis（Uzel）,Haplothrips aculeatus（Fabricius）,Haplothrips tritici（Kurd） and their general predatory flower bug,Orius similis Zheng as compared to the parental control rice line using the white,blue and yellow sticky card traps.Population dynamics and seasonal average densities of these five thrips species and their general predatory flower bug were not significantly affected by rice type.Additionally,the white sticky card trap was suggested to be the most suitable for monitoring the population of these five thrips species and their general predator.These results show that our tested Bt rice lines do not interrupt the population of non-target thrips species and their general predatory flower bug in the field,and also cannot result in more occurrence of these thrips species in the rice ecosystem.

Development of Insect-Resistant Hybrid Rice by Introgressing the Bt Gene from Bt Rice Huahui 1 into Ⅱ-32A/B,a Widely Used Cytogenic Male Sterile System

Huahui 1 is an elite transgenic male sterile restorer line of wild rice abortive-type that expresses a Bacillus thuringiensis （Bt） δ-endotoxin and provides effective and economic control oflepidopteran insects. To exploit Huahui 1 to develop a new Bt rice, the insertion site of the Bt gene was determined by thermal asymmetric interlaced PCR （TAIL-PCR）. Bt was located in the promoter region ofLOC. Os10g10360, approximately 5.35 Mb from the telomere of the short arm of chromosome 10. For the first time, a Bt cytoplasmic male sterile （CMS） system was developed by introgressing Bt from Huahui 1. The recipient CMS system used consisted of Indonesia paddy rice-type II-32B （maintainer line） and II-32A （male sterile line）. Marker-assisted selection was used to increase selection efficiency in the backcrossing program. In BC5F1, the Bt plant 85015-8 was selected for further analyses, as it had the highest SSR marker homozygosity. In addition, the linkage drag of the foreign Bt gene in 85015-8 was minimized to 8.01-11.46 Mb. The foreign Bt gene was then delivered from 85015-8 into II-32A. The resultant Bt II-32A and Bt II-32B lines were both resistant to lepidopteran in field trials, and agronomic traits were not disturbed. The maintainability of II-32B, and the male sterility and general combining ability of II-32A, were not affected by the Bt introgression. This study demonstrates a simple and fast approach to develop Bt hybrid rice.

Risk Assessment and Biosafety Studies of Transgenic Bt Rice （Oryza sativa L.）

Transgenic crops having alien genes from different sources are getting popularity. A Rice （Oryza sativa L.） containing gene from Bacillus thuringiensis, a soil bacterium, was assessed to study the potential risks of transgenic plants on environment. The crop was found resistant to target insect pests. Rice variety Basmati-370 transformed with two insecticidal genes, crylAc and cry2A, was grown under field conditions for several years. Data were collected at different stages of the plant growth. Fate of Cry protein in soil, effect of Bt protein on non-target insects, risks of vertical and horizontal gene flow were evaluated. No potential hazard was found at all levels. Bt protein was unstable and degraded significantly in soil within 30 days after harvesting the crop. No harmful effects were found on non-target insects （insects other than order lepidoptera）. Maximum gene flow of 0.02% was observed at close spacing and no evidence of horizontal gene transfer to Rhizobium spp. was found. In conclusion, the transgenic rice plants transformed with Bt genes have no harmful effects on the environment.

Effect of vegetation of transgenic Bt rice lines and their straw amendment on soil enzymes, respiration, functional diversity and community structure of soil microorganisms under field conditions

With the development of transgenic crops, there is an increasing concern about the possible adverse effects of their vegetation and residues on soil environmental quality. This study was carried out to evaluate the possible effects of the vegetation of transgenic Bt rice lines Huachi B6 （HC） and TT51 （TT） followed by the return of their straw to the soil on soil enzymes （catalase, urease, neutral phosphatase and invertase）, anaerobic respiration activity, microbial utilization of carbon substrates and community structure, under field conditions. The results indicated that the vegetation of the two transgenic rice lines （HC and TT） and return of their straw had few adverse effects on soil enzymes and anaerobic respiration activity compared to their parent and distant parent, although some transient differences were observed. The vegetation and subsequent straw amendment of Bt rice HC and TT did not appear to have a harmful effect on the richness, evenness and community structure of soil microorganisms. No different pattern of impact due to plant species was found between HC and TT. It could be concluded that the vegetation of transgenic Bt rice lines and the return of their straw as organic fertilizer may not alter soil microbe-mediated functions.

常见水稻病虫害胁迫对转cry1C基因抗虫水稻Bt蛋白表达量的影响

【目的】明确转Bt基因抗虫水稻在病虫害胁迫下对Bt蛋白表达量的影响。【方法】以Bt水稻T1C-19(表达Cry1C蛋白)为研究对象,探究褐飞虱、白叶枯病等6种水稻常见病虫害胁迫下其体内Bt蛋白的表达量变化。【结果】褐飞虱取食引起T1C-19水稻叶片中Bt蛋白含量降低,而黑尾叶蝉取食显著降低了T1C-19水稻叶鞘中的Bt蛋白含量,白叶枯病侵染导致Bt水稻叶鞘中的Bt蛋白含量显著上升。二化螟、水稻普通矮缩病、稻瘟病的胁迫对转基因水稻T1C-19叶片和叶鞘中的Bt蛋白含量均无显著影响。【结论】病虫害胁迫因种类不同对Bt水稻中Bt蛋白表达量的影响有所不同。这将为Bt水稻的抗虫效果评价提供数据基础,同时为Bt水稻病虫害综合治理提供科学依据。

Modified accumulation of selected heavy metals in Bt transgenic rice

Safety assessment of genetically modified crops generally does not take into account the potential hazard of altered patterns of heavy metal accumulation in plants. A pot experiment was conducted under greenhouse conditions to evaluate the impact of heavy metal amendments on the accumulation of Cd, Cu, Pb and Zn in a Bt transgenic rice Ke-Ming-Dao （KMD） and its wild-type Xiushui 11 （Xs11）. In control soils, significant difference was only found in contents of Cu （p 〈 0.01） and Pb （p 〈 0.05） in straw between KMD and Xs11. At three levels of Cd amendments （5, 10, and 20 mg/kg）, the Cd contents in grain and straw of KMD were significantly higher than those of Xs11, and all grain Cd contents were significantly higher than the International Criteria （0.2-0.4 mg/kg） as specified by the Codex Alimentarius Commission （CAC）. These results implied that it may be unsafe for growing Bt transgenic rice in heavily Cd-polluted areas. No significant difference in Zn was found between the two varieties with the exception of roots at Zn amendment level of 600 mg/kg, while Pb contents in KMD were much higher in the straw at the lead amendment level of 1000 mg/kg and in the root at 250 mg Pb/kg. Data on the heavy metal accumulation patterns for the genetically modified rice may be used for the selection of growing areas as well as for plant residue management for Bt rice.

Breeding and Characterization of a New Rice Restorer Line Containing Bt Gene

Bt5198, a new rice restorer line containing Bt gene, was developed from the cross and backcross of the elite restorer line Chenghui 177 with Bt Minghui 63, a transgenic Bt restorer line. The inbred lines were evaluated using PCR amplification, test paper evaluation, insect resistance evaluation in both the laboratory and paddy fields, nursery evaluation of rice blast resistance and pedigree selection of agronomic traits. Larval mortalities on Bt5198 and Bt Minghui 63 were 100% when rice culms were inoculated with the eggs of the striped stem borer （SSB） in the laboratory. Bt5198 was highly resistant against SSB and the yellow stem borer （YSB） under field conditions. The F1 hybrids derived from Bt5198 and four cytoplasmic male sterile （CMS） lines were also highly resistant to SSB and YSB and had a significant heterosis. Two-year evaluation of rice blast resistance confirmed that the resistance levels of Bt5198 to leaf blast and neck blast were similar to those of Chenghui 177 and significantly better than those of Bt Minghui 63. Seed germination ability and pollen yield of Bt5198 were similar with Chenghui 177, suggesting that the introduction of the Bt gene into the new restorer line had no significant effects on seed vitality or the yield of seed production. To identify the presence of the Bt gene, it was effective to combine test paper examination with the evaluation of insect-resistance, both in the laboratory and under field conditions.

Preparative Purification and Bioassay of Bt Toxin from Cry1Ab Transgenic Rice

A method of extracting and purifying Cry1Ab protein(Bt toxin) from Cry1Ab transgenic rice was established. Most of the Bt toxin present in the tissue of Cry1Ab transgenic rice was extracted effectively with a solution of 50 mmol/LNa_2CO_3 and NaHCO_3. The crude protein containing Bt toxin was obtained after the pretreatment of Cry1Ab transgenic rice with ultra-filtration, ammonium sulfate precipitation and centrifugation. The dialysed crude protein was futher separated on DEAE Sephadex A-50 columns and Sephadex G-150 columns. The protein bound on DEAE Sephadex A-50 gel was eluted with buffer solution B(10 mmol/L tris-HCl buffer+1.0 mmol/L EDTA, pH=8.0) mixed with 0.1, 0.3, 0.5 and 0.8 mol/L NaCl in a discontinuous gradient elution mode. The peak of the Bt toxin eluted from the columns was identified by ELISA and bioassayed with larvae of tobacco hornworms and silkworms. The purity and the bioactivity of the Bt toxin were determined by means of SDS-PAGE and larvicidal assay, respectively. The purity of the Bt toxin obtained by this method is high, and its insecticidal activity is retained after the toxin is purified.

Studies on Rapid Quantitative Analysis of Bt Toxin by Using Envirologix Kits in Transgenic Rice

Investigations were done on the usefulness of Envirologix CrylAb/CrylAc Plate kits for quantitative analysis of Bt toxin content in transgenic rice grains. Two transgenic rice lines: Kemingdao 1 (KMD1) and Kemingdao 2 (KMD2), transformed with a crylAb gene, and their parental variety, cv. Xiushui 11, were used as positive and negative samples. Results showed that the correlation coefficients as high as 0. 985 - 0. 998, significant at probability level of 0. 05 or 0.01, were obtained for linear regression equations by using the appended calibrators of the kits. No significant differences were detected for values of same rice sample obtained from different trials or by using different lots of the product (kit). The detectable Bt toxin content by this method could be as low as 0.5 ng/g. The Envirologix Kit could be useful for rapid quantitative detection of Bt toxin in rice grains because of its preciseness, simplicity and time saving.

Expression Characteristics and Borer Resistance of Bt Gene of Transgenic cry1C~* Super Japonica Rice

[Objective] The paper was to study the expression characteristics and borer resistance of Bt gene of Songjing 9 （1C^＊）. [Method] With super japonica rice Songjing 9 as the receptor, transgenic crylC^＊ super japonica rice Songjing 9 （1C^＊） regulated by ubi promoter was created by agrobacterium-mediated genetic transformation method. Ten strains of transgenic super japonica rice formed from different transformation events were planted in the field; the Tt-mRNA of leaf, stem-sheath and young spikelet at booting stage were detected by real-time fluorescent quantitative PCR; the Bt protein content of leaf, stem-sheath and young spikelet and harvested brown rice were detected by enzyme-linked immunosorbent assay （ELISA）; the white head rate damaged by Chilo suppressalis was manually investigated. [Result] The Bt-mRNA and Bt protein content of various organs and Bt protein content of mature brown rice at booting stage of Songjing 9 （1C^＊） significantly differed among strains; there was no correlation relationship in Bt-mRNA and Bt protein content between leaf and stem-sheath, between leaf and young spikelet, and between stem-sheath and young spikelet; there was no relation between Bt-mRNA or Bt protein content of leaf, stem-sheath and young spikelet and Bt protein content of brown rice; there was positive correlation between Bt-mRNA and Bt protein content in the same organ such as leaf, stem-sheath and young spikelet at booting stage; such strains as Songjing 9 （1C^＊）, whether the Bt gene expression was high or low, showed good resistance against C.suppressalis. This indicated that there were differences in Bt gene expression in different strains of transgenic cry1C^＊ super japonica rice or different organs of the same strain; there was no relation in Bt gene expression among different organs; the Bt-mRNA and protein content of the same organ usually had consistent expression; the Bt gene expression of various organs had nothing to do with the Bt protein content of brown rice. [Conclusion] The Bt gene and its expression cassette can meet the requirement of cultivating japonica rice varieties against stem borers.

Bt水稻“克螟稻”稻米毒理性评价研究初报

经大鼠急性中毒试验确定 ,转Bt基因抗虫水稻稻米属无毒级。大鼠 90d喂养结果显示 ,各试验组大鼠体重、进食量和食物利用率与对照组均无显著差异。对大鼠肝、肾、胃、肠及睾丸进行组织切片检查 ,均未见病理性改变。大鼠脏体比、血象及血生化指标均在正常范围内 ,试验组与对照组无明显差异。

转Bt基因水稻表达的毒蛋白Cry1Ab在害虫及其捕食者体内的积累动态

在室内对转基因水稻KMD1中的Cry1Ab毒蛋白经食物链在几种主要害虫及其捕食性天敌体内的积累进行了研究。结果表明 :无论是水稻孕穗期还是成熟期 ,二化螟Chilosuppressalis连续取食KMD1或取食KMD136h后移至对照品种秀水 11上取食不同时间后 ,幼虫体内的Cry1Ab含量均随取食时间延长逐渐下降。稻眼蝶Mycalesisgotama幼虫连续取食KMD1或在KMD1上取食两天后移至秀水 11上继续取食不同时间 ,体内的Cry1Ab含量也都随取食时间延长而下降 ,但下降速度较二化螟更快。取食KMD1的二化螟和稻眼蝶幼虫的粪便中均检测到较高浓度的Cry1Ab ,对照组中均无Cry1Ab。取食KMD1的二化螟幼虫血淋巴中检测到Cry1Ab ,含量为 3 5ng g。取食KMD1的褐飞虱Nilaparvatalugens、稻蚜Sitobionavenae以及饲喂取食过KMD1的二化螟或稻眼蝶幼虫的拟水狼蛛Piratasubpiraticus体内都含有一定浓度的Cry1Ab ,其中 ,拟水狼蛛体内的CrylAb含量以饲喂取食KMD1稻眼蝶幼虫的含量最高 ,约为饲喂取食KMD1二化螟幼虫的 6 0倍。这些结果表明Cry1Ab可以沿水稻 害虫

Bt水稻“克螟稻”花粉对家蚕生长发育的影响

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。

Bt水稻杂交育种中转基因的遗传分析

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cry1Ab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。

转基因Bt水稻花粉在桑叶上的自然飘落浓度

实地 (杭州 )调查了桑稻混种地区水稻花粉飘落到桑叶上的浓度 ,同时对转 Bt基因水稻花粉向桑叶上飘飞程度进行了田间试验。 2 0个采样点的调查结果显示 ,超过一半 ( 5 4 .5 % )的桑叶检测到了水稻花粉 ,其浓度变化范围为 1 3.3～ 1 99.0粒 /cm2 ,平均浓度为 92 .9粒 /cm2 ,与 1 0 9.1粒 /cm2 (该浓度影响家蚕生长发育 )持平 ( t=0 .1 0 ,P>0 .5 )。不同桑稻距离 ( 0～ 1 0 m)的桑叶花粉浓度总体上是距离越近花粉浓度越高。将处于开花期的转基因 Bt水稻和非转基因水稻植株 (各 3株 )同时放入桑园中 ,然后观察水稻植株周边桑叶飘落上水稻花粉的浓度。两次试验采集的桑叶自然飘落的水稻花粉平均为 3.8粒 /cm2 ,这一结果证明水稻花粉可飘落到桑叶上 ,从而支持上述调查结果 ;试验同时证实转基因 Bt水稻和非转基因水稻植株的花粉飘飞能力没有差异。以上研究结果表明 ,Bt水稻在我国南方养蚕地区推广将可能对家蚕的生长发育造成负面影响。

Bt水稻对飞虱和叶蝉及其卵寄生蜂扩散规律的影响

研究了Bt水稻两个纯合品系TT9-3和TT9-4在田间对飞虱、叶蝉及其卵寄生蜂扩散规律的影响。结果表明:在Bt稻区与对照(IR72)区间相互扩散的飞虱有白背飞虱(Sogatellafurcifea)、褐飞虱(Nilaparvatalugens);叶蝉有黑尾叶蝉(Nephotetixcincticeps)、白翅叶蝉(Erythroneurasubrufa)和电光叶蝉(Deltpcephalusdorsalis);其中以白背飞虱和黑尾叶蝉为主。扩散数量叶蝉多于飞虱;扩散方向以Bt稻区向对照区扩散为主,其中TT9-3与IR72、TT9-4与IR72间白背飞虱与黑尾叶蝉的扩散量差异均达显著水平(P<0.05)。在Bt稻区与对照区间相互扩散的飞虱和叶蝉的卵寄生蜂有稻虱缨小蜂(Anagrusspp.)和叶蝉柄翅小蜂(Lynaenonlongicrus),数量上后者多于前者,扩散方向也以Bt稻区向对照区扩散为主。

转Bt基因水稻对二化螟绒茧蜂生物学特性的影响

用转Bt基因 (cry1Ab)水稻KMD1饲喂 3龄、4龄和 5龄二化螟Chilosuppressalis一定时间后 ,作为二化螟绒茧蜂Apanteleschilonis的寄主 ,研究了转基因水稻经寄主对绒茧蜂生物学特性的影响。结果发现 :KMD1处理后 ,三个龄期幼虫的寄生率都显著下降 ,其中 4龄和 5龄达极显著水平 ;3龄和 4龄上的结茧率显著低于对照 ;蜂蛹历期均短于对照 ,但仅 3龄差异显著 ;从 5龄幼虫所羽化的雄蜂寿命显著短于对照 ;蜂茧长显著短于对照 ;而对卵 +幼虫期、茧块茧数。

Bt水稻田不同斑块设计对田间节肢动物群落稳定性的影响

利用正交设计考察了B t水稻种植面积大小以及与非转基因水稻相间距离两个因素对稻田节肢动物群落稳定性的影响。结果表明:在转B t稻斑块与非转B t稻斑块共存格局下,尽管两因素对两个品种上节肢动物群落物种丰富度影响不大,但较大斑块面积对两个品种水稻而言均有利于其上节肢动物群落多样性的保持。同时还发现,两个因素对两个品种斑块上的节肢动物群落影响不尽相同,且起显著效应的时间分布也因品种而异。对转B t稻斑块上节肢动物群落显著影响集中在水稻生育期前期(7月份)和末期(9月份),以斑块面积因素相对而言对节肢动物群落稳定性影响更大,具体表现在其面积越大,多样性越高,优势集中度越大。而对于非转B t稻两个因素对其上节肢动物群落的影响几乎贯穿整个调查期,其中又以斑块相间距离因素的影响更大,距离越小,多样性越高,优势集中度越低。进一步品种间的比较发现,转B t稻对田间节肢动物群落影响显著,在靶标害虫危害盛期其上节肢动物群落明显比非转B t稻上的稳定,而在非靶标害虫危害为主的时期,转B t稻田上节肢动物群落均不如非转B t稻上的稳定,尤其在水稻移栽后初期表现显著。

Bt水稻中cry1Ab基因的遗传分析

用ＰＣＲ、ＧＵＳ染色和Ｗｅｓｔｅｒｎ点杂交技术检测了Ｂｔ水稻杂交后代群体，在３９４株ＧＵＳ阳性 株中，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ｃｒｙ１Ａｂ紧密连锁遗传与表达。ＧＵＳ组织染色和Ｓｏｕｔｈｅｒｎ杂交检测表明Ｂｔ水稻中的 ＣｒｙｌＡｂ基因呈单位点显性基因遗传，且在有性世代中能稳定传递。还发现，在ＢＣ１、ＢＣ１Ｆ２和粳粳 交Ｆ２群体中ＣｒｙｌＡｂ基因呈单位点显性基因遗传，而在籼粳交Ｆ２群体中偏离３∶１分离。