Effects of two kinds of transgenic poplar on protective enzymes system in the midgut of larvae of American white moth

The leaves of Bt (Bacillus thuringiensis) transgenic poplar (Populus nigra L.) and CpTI (Cowpea trypsin inhibitor) transgenic poplar ((P. tomentosa×P. bolleana)×P. Tomentosa) were taken to feed the 4th-5th-instar larvae of American white moth (Hyphantria cunea (Drury)) for determination of the activities of the protective enzyme system inside larvae’s body. The physiological and biochemical effects of the transgenic poplars on the larvae were studied. The results showed that the two kinds of transgenic poplars had similar effects on the protective enzyme system in the midgut of larvae. The activities of superoxide dismutase, catalase, and peroxidase in midgut of the larvae increased gradually, reached the highest value at a certain time, and then decreased suddenly. For the larvae that were fed with the leaves of Bt transgenic poplar, the peak value of superoxide dismutase and catalase presented at the time of 24-h feeding, while the peak of peroxidase took place at the time of 12-h feeding. The activities of these protective enzymes for the larvae that were fed with leaves of CpTI transgenic poplar peaked 12 h later than that of those fed with leaves of Bt transgenic poplar. The comparison of activities of the protective enzymes was also carried out between the larvae with different levels of intoxication. It was found that the activities of protective enzyme of the seriously intoxicant larvae were higher than that of the lightly intoxicant larvae. This difference was more obvious in the group treated with CpTI transgenic poplar.

Temporal and spatial changes in Bt toxin expression in Bt-transgenic poplar and insect resistance in field tests

Extensive planting of Bacillus thuringiensis （Bt）-transgenic plants economically benefits society; how-ever, the potential risk they pose is receiving increasing attention. This study used enzyme-linked immunosorbent assay and fluorescence quantitative PCR （RT-PCR） to monitor the temporal and spatial dynamics of the expression of Bt toxic protein in a forest of 6- to 8-year-old trees of transgenic insect-resistant poplar 741 for three consec- utive years. The enrichment, distribution, and degradation of Bt toxic protein and the influence of transgenic poplars on the targeted insect population, Hyphantria cunea, were investigated. The content of CrylAc toxic protein dynamically changed in transgenic poplar. During the annual growth cycle, the content initially increased, then decreased in the long and the short branches of the crown and in the root system, peaking in August. During the study, the protein did not accumulate overtime. The mRNA transcription of gene CrylAc was almost consistent with the level of the protein, but transcription peaked in July. In the transgenic and control forestland, microscale levels of the CrylAc toxic protein were detected from the soil, but increased accumulation was not observed with the planting year of transgenic poplar. Meanwhile, Bt was isolated and detected molecularly from the soil in the experimental forestland. A systematic investigation of the density of H. cunea in the experimental transgenic poplar forest indi- cated that transgenic Pb29 poplar could resist insects to a certain degree. At peak occurrence of the targeted insects, the density of H. cunea in the experimental forest was significantly lower than in the nontransgenic poplar forest.

Effects of transgenic poplar clone 741 with insect resistance on structure and diversity of arthropod community

Arthropod community structure and diversity were investigated in transgenic insect-resistant hybrid poplar 741 field trial plots, which had five isogenic clones with two genes （Bt. toxin [Cry IAc] and arrowhead proteinase inhibitor [API]） in comparison to control plots. Transgenic poplar clones were investigated according to their insect resistance （high and medium resistant clones）, being systematically probed into varying levels with feeding tests before. Investigations were carded out during three years （2002, 2003 and 2005）. The results indicated that among the basal species, transgenic plants in general had lower amounts of phytophagous insects, and an increased quantity of neutral arthropods such as saprophytic and strolling species. Among the top and intermediate species in transgenic variants, the quantity of predatory natural enemies was increased, while the amount of parasitoid ones was slightly reduced. The arthropod community characteristic indices were analyzed from different aspects in the three variants. It was discovered that, not only the characteristic indices of the whole arthropod community, but also the characteristic indices of the sub-communities （such as natural enemy, pest and neutral arthropods） and the functional groups categorized by their feeding patterns, showed a higher diversity and evenness and a lower dominancy concentration indices in the high-resistant and medium-resistant plots compared with the control. Transgenic poplar 741 showed improved ecological effects both in partial and total aspects. It could be concluded that composition and structure of the arthropod community in plots with transgenic insect-resistant poplar were turned to be more reasonable and itsdiversity and stability was enhanced.

Transgenic Poplar Plants for the Investigation of ABA-Dependent Salt and Drought Stress Adaptation in Trees

Important functions of the plant hormone abscisic acid (ABA) in stress reactions, growth and photosynthetic processes are extensively studied in the model plant Arabidopsis thaliana. This paper investigates the importance of Moco-sulphurase ABA3 and aldehyde oxidase (AO) on ABA-biosynthesis in Populus × canescens. ABA3 is essential for activation of the molybdenum enzymes AO and xanthine dehydrogenase (XDH). AO itself catalyzes the last step in ABA-biosynthesis. Generation of transgenic poplar plants altered in ABA3 and AO-activity using RNAi knock down and overexpression was performed. Whereas RNAi-AO plants show a specific loss of AO activity, the RNAi-ABA3 plants has a strongly reduced activity of both molybdenum enzymes: AO and XDH. Constructs of AO and ABA3-promoters fused to β-glucuronidase provide the basis to investigate transcriptional regulation of ABA-biosynthetic processes under stress conditions. Application of high salt concentrations and different drought stress intensities does change the endogenous AO or XDH neither on the side of transcription nor on protein activity. On phytohormone level however, water loss leads to increased ABA-amounts regardless of whether transgenic or wildtype plants are studied. Salt application resulted in higher ABA-levels in all analyzed plant lines. The down regulation of AO in the two different RNAi-plant lines strongly prevented a wildtype-like increase of ABA-levels. Whereas the WT plants accumulated up to 6000 ng ABA g-1 FW-1 after 16 h of salt stress exposure, plants of the RNAi lines revealed a markedly lower increase of only up to 2000 ng ABA g-1 FW-1. Opposing to these observations, ABA-levels increased during drought without any influence by the RNAi-effect. These results revealed that although stresses did not result in a visible increased AO-activity, ABA-production was influenced by AO and ABA3 at least under salinity.

Assessment of Rhizospheric Microorganisms of Transgenic Populus tomentosa with Cowpea Trypsin Inhibitor (CpTI) Gene

To have a preliminary insight into biosafety of genetically transformed hybrid triploid poplars （Populus tomentosa × P bolleana）× P. tomentosa with the cowpea trypsin inhibitor （CpTD gene, two layers of rhizospheric soil （from 0 to 20cm deep and from 20 to 40cm deep, respectively） were collected for microorganism culture, counting assay and PCR analysis to assess the potential impact of transgenic poplars on non-target microorganism population and transgene dispersal. When the same soil layer of suspension stock solution was diluted at both 1：1000 and 1：10000 rates, there were no significant differences in bacterium colony numbers between the inoculation plates of both transgenic and non-transgenic poplars. The uniform results were revealed for both soil layer suspension solutions of identical poplars at both dilution rates except for non-transgenic poplars at 1：10000 dilution rates from the same type of soil. No significant variation in morphology of both Gram-positive and Gram-negative bacteria was observed under the microscope. The potential transgene dispersal from root exudates or fallen leaves to non-target microbes was repudiated by PCR analysis, in which no CpTI gene specific DNA band was amplified for 15 sites of transgenic rhizospheric soil samples. It can be concluded that transgenic poplar with the CpTI gene has no severe impact on rhizospheric microorganisms and is tentatively safe to surrounding soil micro-ecosystem.

转抗虫基因107杨对根际和内源细菌群落的影响

为研究转抗虫基因对根际和内源细菌群落的影响,以转抗虫基因107杨田间对比试验林为对象,采用高通量测序技术对转基因株系和对照株系根际土壤细菌、树干韧皮部和根系内生细菌群落结构组成及多样性进行对比分析。结果显示:(1)土壤样品中有效序列平均在30000以上,杨树组织有效序列平均在300000以上。(2)转基因株系细菌物种组成与对照株系的极其相似,酸杆菌门与变形菌门为转基因和非转基因株系根际土壤的优势菌门,占比分别为16.6%和21.9%;蓝藻菌门和变形菌门为转基因和非转基因株系韧皮部的优势菌门,所占的比率分别为54.8%和59.2%;变形菌门为转基因和非转基因株系根部的优势菌门,所占的比率分别为40.7%和58.8%。(3)转基因和非转基因株系在细菌群落的丰富度和多样性上均没有表现出明显的差异,但不同部位之间差异显著。结果表明:转基因107杨没有对根际土壤和内生细菌造成显著影响,明确了转抗虫基因107杨不同部位细菌群落特征,为转抗虫基因107杨的生物安全评价提供了有益的参考。

杨树UGT198基因的生物信息学分析、基因克隆及功能初探

【目的】研究UGT198基因是否参与调控烟草抗虫性,为未来探索其参与调控杨树抗虫性奠定基础。【方法】前期通过分析公共转录组数据发现PtrUGT198基因在杨树响应虫害的转录组中高调表达,对其进行生物信息学及进化分析。以107杨为材料,克隆UGT198基因,构建过量表达载体,利用农杆菌介导法转化烟草获得过量表达转基因株系。通过棉铃虫饲虫试验初步验证UGT198基因是否参与调控抗虫性,分析UGT198基因在调控抗虫性中发挥的功能。【结果】PtrUGT198基因完整的ORF区为1440 bp,编码479个氨基酸,理论分子量为53.14 kDa,等电点为5.92,编码不稳定疏水性蛋白。PtrUGT198蛋白的二级结构α⁃螺旋占41.75%,β⁃折叠占14.61%,β⁃转角占7.10%,无规卷曲占6.53%。PtrUGT198蛋白不存在跨膜区,预测其不属于膜蛋白,为非分泌性蛋白,该蛋白的磷酸化修饰以丝氨酸为主。同源序列对比结果显示PtrUGT198蛋白的C末端存在高度保守的植物次生产物糖基转移酶(Plant secondary product glycosyltransferase,PSPG)基序。系统进化树分析发现,PtrUGT198蛋白与同属植物毛白杨、银白杨和胡杨亲缘关系最近,其次和模式植物烟草有较近的亲缘关系。成功克隆杨树UGT198基因,构建pNC⁃UGT198基因过量表达载体,并转化烟草获得过量表达转基因株系,进行饲虫实验,得到高表达量烟草相对抗虫。【结论】首次成功克隆了UGT198基因,并初步解析其调控抗虫性的功能,为深入了解UGT198基因调控杨树抗虫机理提供参考价值。

转BtCry1Ac基因107杨生长分析

以4个7年生转BtCry1Ac基因107杨无性系及其未转基因对照为材料,对河北深州市、枣强县和盐山县3个地点营建的对比试验林进行调查,对其生长进行分析,探究外源基因的转入对其生长产生的影响。结果表明,转基因株系间平均树高、胸径、材积无显著差异,但均显著低于未转基因对照。转基因株系在不同试验林生长量表现不同,枣强试验林与盐山、深州试验林在生长量方面均存在显著差异,盐山试验林表现最好,枣强试验林平均树高、胸径、材积仅为盐山的86.5%、84.8%、65.1%。多点联合方差分析表明,株系与地点之间互作明显。转基因株系与对照树高、胸径、材积方面,在速生期、年平均最大生长量等时期较为相近,生长模式基本相同。

Pulping performance of transgenic poplar with depressed Caffeoyl-CoA O-methyltransferase

This paper evaluated pulping performance of 3-year-old field-grown transgenic poplar (Populus tremula × Populus alba). The transgenic poplar with anti-sense CCoAOMT had an about 13% decreased lignin content, in which a slight increment was found in S/G ratio. Chemical analysis showed that the transgenic poplar had significantly less benezene-ethanol extractive than that of control wood, but no significant differences were found in contents of ash, cold water extractive, hot water extractive, 1% NaOH extractive, holocellulose, pentosans and cellulose. Fiber assay demonstrated that down-regulation of CCoAOMT expression improved the fiber quality in transgenic poplar. Kraft pulping showed that lower lignin in transgenic poplar led to remarkable improved pulp quality and increased pulp yield.

转Bt基因107杨根系分布特征

以2个转BtCry1Ac基因107杨株系及其未转基因对照为材料,研究转Bt基因107杨的根系分布特征。结果表明:1)垂直方向上,2个转基因株系与CK的总根系及各径级根长密度、表面积密度、体积密度以及生物量密度上均随土层深度的增加而显著降低,在0~30 cm土层中,根长密度、根表面积密度、根体积密度及生物量密度均达到最大值,且显著高于其他土层;2)水平方向0~150 cm, 2个转基因株系与CK的总根表面积密度、总生物量密度随着距树干水平距离的增加呈现出先减小后增大的趋势;不同径级根系表面积密度、根长密度在距树干0~30 cm处达到最大值;3)2个转基因株系总根长密度、根表面积密度、根体积密度和生物量密度均小于对照,对照与转基因株系存在显著性差异,而2个转基因株系间无显著性差异;4)3个株系在根系分布中均以细根为主,且转基因株系细根径级的根长密度、根表面积密度表现为对照大于转基因株系且存在显著性差异,对照和转基因株系中根与粗根根长密度、根表面积密度无显著性差异。

通过农杆菌介导法将兔防御素NP-1基因导入毛白杨(P.tomentosa)

采用叶盘法将兔防御素NP－1基因导入毛白杨后，经PCR分析、Southern杂交检测与筛选获得了转基因植株。并经体外抑菌实验表明，转NP－1基因毛白杨植株组织提取物对某些微生物的生长具有明显的抑制作用。

抗虫的转AaIT基因杨树的获得

通过根癌农杆菌叶盘法将构建在双元载体上的昆虫特异性神经蝎毒素AaIT基因转化至中国南方杨树N106(小叶杨×美洲黑杨,P.deltoides×P.simonii)中,共获得了62株再生植株。PCR分析及PCR产物Southernblotting的分析结果表明,AaIT基因整合在再生植株的基因组上。对部分转AaIT基因植株进行了杀虫实验,转基因植株A5对一龄舞毒蛾(Lymantriadispar)幼虫有明显的抗性,饲喂转基因杨树叶片的幼虫死亡率显著高于未转基因对照植株,其取食面积小,存活幼虫体重明显小于对照。ELISA分析证明了AaIT蛋白的表达。

转基因杨树的抗盐性分析

以转 1 磷酸甘露糖醇脱氢酶基因的八里庄杨为试材 ,对获得的杨树转化体进行了不同NaCl梯度的组织培养、水培和盆栽试验。抗盐试验中 ,转基因八里庄杨比对照的始分化天、分化率、芽头密度、苗高、生长势、生根率明显得到提高 ;主根数、侧根数、根长的发生数量均较多。结果表明在 4‰含盐量的基质上 ,转基因苗木比对照有更好的抗性。

转抗盐碱基因八里庄杨大田释放试验

对转抗盐碱基因 (mtl-D基因 )八里庄杨进行了大田释放造林试验 ,调查结果表明 :转基因杨比对照八里庄杨的造林成活率明显提高 ,在 0 .3%～ 0 .4 %土壤盐化范围内提高 0 .7倍 ;林木生长量显著增大 ,树体健壮 ,耐盐能力增强 ;经多点统计分析 ,转基因杨的田间耐盐极限为土壤含盐量 0 .4 3% ,可在中度盐碱地上正常生长。经大田试验林采样PCR检测 ,转化基因遗传性稳定 ,目前尚无基因丢失或沉默现象。

转CpTI基因杨树对美国白蛾幼虫中肠解毒酶及乙酰胆碱酯酶的影响

以转CpTI基因毛白杨回交杂种 [(Populustomentosa×Populusbolleana)×Populustomentosa]叶片饲喂 4～ 5龄美国白蛾 (HyphantriacuneaDrury)幼虫 ,对其中肠解毒酶和乙酰胆碱酯酶活性进行了测定。结果表明 ,酯酶、羧酸酯酶的活力受到明显抑制 ,且随时间的延长抑制强度增加 ,饲喂 48h后 ,上述两种酶的活力分别比对照降低 76 .42 %和 73.91% ;多功能氧化酶在饲喂的前 12h ,表现为受到抑制 ,最大抑制率为 35 .78% ,2 4h后酶活力反而高于对照 ;谷胱甘肽S -转移酶和乙酰胆碱酯酶活力受到抑制 ,而且两者表现极为相似 ,均在饲喂后 4h抑制作用最强 ,酶活力分别比对照降低 5 1.40 %和 40 .5 7% ,但在整个试验过程中 ,均没有表现出随时间加强的趋势。

Bt转基因杨树对杨树昆虫群落结构的影响

通过种植转基因杨树纯林和混交林 ,研究了Bt转基因杨树对杨树昆虫群落结构的影响。研究表明 1hm2的小面积的新疆转基因杨树和非转基因杨树之间群落结构和多样性差别不大 ;而对 6 7hm2 以上的转基因杨树纯林和 1∶1的转基因和非转基因杨树混交林中昆虫和蜘蛛群落结构的比较研究发现 ,Bt转基因杨树会改变其昆虫群落结构 ,在转基因纯林中杨叶蜂为优势种 ,而在混交林中杨扇舟蛾为优势种。为了避免其它非鳞翅目食叶害虫的危害 ,在转基因时应采用对非鳞翅目食叶害虫有抗性的种类或品系。转基因纯林的多样性和均匀性都较混交林高 ,有利于系统的稳定性 ,但转基因混交林中瓢虫的数量明显比转基因纯林高 ,分别为 0 2 1头·枝- 1 和 0 0 2 1头·枝 - 1 ,相差 1 0倍。蜘蛛的数量是纯林中较多 ,分别为 0 1 2 5头·枝 - 1 和 0 0 6 2 5头·枝 - 1 ,相差 1倍。转基因杨树对非目标昆虫和天敌的影响还需进行进一步详细的研究。转基因纯林和混交林的叶片被害率分别为 1 1 2 6 %和1 8 4 8% ,转基因纯林抑制目标食叶害虫的作用较好 ,但与混交林之间的差异不显著 ,两种林分的被害率都在不成灾的水平。

涝渍胁迫对转多基因库安托杨生长及生理性状的影响

以库安托杨转多基因株系(D5-9、D5-18、D5-19、D5-20、D5-21、D5-24、D5-26)和未转化株系(对照CK)为试材,研究不同水分处理(水涝胁迫、浸渍胁迫、正常供水)对其生长及生理性状的影响。结果表明:随着胁迫的加剧,各株系株高、根系生长和生物量累积呈下降趋势,而地径生长有所增加。浸渍、水涝胁迫下对照株系的净光合速率均最低,分别为11.99、10.37μmol.m-2.s-1,比正常供水分别减少了14.0%和25.6%;浸渍胁迫下D5-26的净光合速率最高,为13.95μmol.m-2.s-1;水涝胁迫下D5-19的净光合速率最高为12.01μmol.m-2.s-1。蒸腾速率、水分利用效率、气孔限制值、叶绿素a、叶绿素b和总叶绿素含量均下降,叶绿素a的降幅小于叶绿素b,叶绿素a/b值先降后升,而气孔导度和细胞间隙CO2浓度增大。PSⅡ的实际光化学效率、电子传递速率、潜在光化学活性和最大光化学效率均降低,初始荧光产量和最大荧光产量增加。涝渍胁迫对所有供试材料的生长及光合、叶绿素荧光等生理性状均有影响,但对转多基因株系的影响明显较对照小,且株系间存在差异。以生长、光合参数和叶绿素荧光参数为指标对供试材料进行综合评价,各株系的抗涝能力从高到低的排序为:D5-26>D5-19>D5-18>D5-21>D5-9>D5-20>D5-24>CK。

转基因欧洲黑杨中Bt基因表达特性的研究

利用PCR检测了转基因欧洲黑杨当代和杂交后代群体 ,外源Bt基因在 7年生的转基因欧洲黑杨 1 4个无性系中稳定存在 ,Bt基因在杂交后代中分离比例为 1∶1。转基因欧洲黑杨作母本进行杂交时有落花、落果现象 ,可能是基因改造影响了它们的某些生理功能。

转双抗虫基因三倍体毛白杨外源基因表达及性状相关性分析

用部分改造的BtCry I Ac基因与慈菇蛋白酶抑制剂（API）基因构建的双抗虫基因表达载体，通过农杆菌介导法转化了三倍体毛白杨Populus tomentosa Carr.，获得一批转双抗虫基因株系。对转基因株系的抗虫毒蛋白表达进行了ELISA和Westem Blot检测，同时用转基因株系叶片对杨扇舟蛾Clostera anochoreta Fabricius和舞毒蛾Lymantria dispar L．幼虫进行室内饲虫试验，并对各项外源基因表达指标进行了相关分析。结果表明：在检测的28个转基因株系中，对杨扇舟蛾高抗株系占总参试系号的41％，中抗系号占35．0％，低抗系号占24％，对舞毒蛾高抗株系占参试系号的70％。转基因植株可明显抑制存活幼虫的生长发育，且不同转基因株系饲养的幼虫发育存在显著差异。连续两年相关分析表明，不同转基因株系幼虫死亡率间存在极显著相关。转基因植株对舞毒蛾和杨扇舟蛾均表达出抗虫性，并存在极显著相关。ELISA检测结果表明，不同转基因株系Bt毒蛋白表达量存在差异，变化在0．0011％～0．0161％。转基因植株对害虫的杀虫效果与Bt杀虫蛋白的表达量存在显著相关，表明Bt毒蛋白在抗虫效果中占有重要地位。转基因植株对卡那霉素表现出一定的抗性，但与抗虫程度相关不明显，单纯用卡那霉素作为筛选手段并不能完全反映植株的真实抗虫效果。

盐胁迫对转AtNHX1基因杨树光合特性与叶绿体超微结构的影响

以欧美107杨(Populus×euramericana‘Neva’,Wt)和转拟南芥液泡膜Na+/H+逆向转运蛋白基因At-NHX1的欧美107杨新品系(Tr)幼苗为材料,研究了高低度盐胁迫对两品系幼苗光合色素含量、光合参数和叶绿体超微结构的影响,以阐明转AtNHX1基因杨树的耐盐性与其光合作用及叶绿体结构之间的关系。结果表明:(1)盐处理后,两品系叶片叶绿素含量、类胡萝卜素含量、净光合速率、蒸腾速率和气孔导度均下降,且高盐度处理下降幅度更大;同等盐度处理下,Tr品系叶片叶绿素含量、净光合速率和气孔导度的下降幅度显著低于Wt品系,且在高盐度处理间差异更大;两品系杨树叶片Pn下降的原因在低盐处理时以气孔限制为主,而在高盐下则是气孔限制和非气孔限制共同作用的结果。(2)盐胁迫对Tr品系叶片叶绿体超微结构的影响较轻,其在高盐下仍保持了较好的内部结构;盐胁迫Wt品系叶绿体则缩皱成球形,内部结构趋向简单,以至解体,脂质球显著增多。可见,盐胁迫导致杨树叶绿体结构破坏而引起叶绿体色素含量下降,最终降低其光合作用效率;同等盐度胁迫下,转At-NHX1基因品系叶片保持了较完整的叶绿体超微结构、更高的叶绿素含量,能维持较好的光合状态,从而表现出较高的耐盐能力。