摘要
杨树(Populus spp.)是全球重要的栽培树种,在我国的林业生产中也发挥着重要作用。转基因生物技术在杨树遗传育种领域的应用,大大加速了杨树优良新品种的选育过程,目前国内外已获得了抗虫、耐盐、改良木材品质等多方面性状改良的转基因杨树,显示出广阔的应用前景。在我国,转抗虫基因欧洲黑杨(P. nigra)、转双抗虫基因741杨等转基因杨树新品种已通过商品化许可。随着转基因林木的快速发展,由外源基因可能引起的转基因生态安全性问题也逐渐引起人们的关注。因此,对转基因林木进行生态安全性评价在理论研究和生产应用中都具有重要意义。但目前,转基因生态安全性评价研究的焦点多集中于作物,林业中相关研究还很少,而对转多个基因杨树的安全性评价研究更是尚未见报道。本研究利用基因枪转化法获得的转多基因库安托杨(P.×euramericana‘Guariento’)和转基因银腺杂种杨(P. alba×P. glandulosa)为材料,从抗生素标记基因NPTII蛋白表达量、目的基因的稳定性、转基因杨树基本生长情况、外源基因的水平转移、外源基因对目标昆虫、非目标昆虫和天敌种群结构和数量的影响以及外源基因对土壤微生物生态系统的影响这6个方面对以上2种转基因杨树幼龄林的生态安全性进行了初步评价。主要研究结果如下:
1、转基因杨树标记基因NPTII蛋白表达量ELISA检测。
选取温室扦插苗(转基因库安托杨15个株系及一个非转基因对照株系,转基因银腺杂种杨18个株系及一个非转基因对照株系)对其中的抗生素标记基因NPTII蛋白表达量进行ELISA检测。结果表明,含有不同数量外源基因的转基因株系间NPTII基因的蛋白表达量并无明显区别,其中库安托杨目的基因数分别为1、2、3、5(未检测出含有4个目的基因的株系)的转基因株系中NPTII基因的蛋白表达量分别为27.47、19.00、19.69、16.72(ng/g鲜叶重)。银腺杂种杨目的基因数分别为1、2、3、4、5的转基因株系中NPTII基因的蛋白表达量分别为2.44、3.51、3.82、7.54、5.60(ng/g鲜叶重)。因此转基因库安托杨和银腺杂种杨中NPTII基因的蛋白表达量未表现出随外源基因数量的增加而增加的趋势。
2、转基因杨树外源基因稳定性检测。
对中间试验2年生库安托杨3个转基因株系及银腺杂种杨3个转基因株系的目的基因进行PCR检测,电泳结果表明,所有转基因株系中均检测出目的基因PCR产物,而对照株系则无,证明目的基因仍稳定存在于转基因杨树的基因组中,可进一步进行生态安全性评价研究。
3、转基因杨树基本生长情况调查。
通过对2年生8个株系共800株转多基因库安托杨及其非转基因对照、转基因银腺杂种杨及其非转基因对照进行了高生长、径生长和死亡率的调查分析,发现转基因株系中在调查的各项指标中既有高于非转基因株系的,也有低于非转基因株系的,但是转基因银腺杂种杨各株系的平均径生长要高于非转基因株系,但是差异不显著,从而判断本试验地内并不是所有的转基因杨树都获得了比非转基因对照杨树更强的生长能力。
4、转基因杨树外源基因水平转移评价。
以试验林内转基因杨树根圈土壤微生物、昆虫和周边多种杂草基因组DNA为模板,对所有5种外源基因Vgb、SacB、BtCry3A、OC-1、JERF36和标记基因NPTII进行了PCR检测,结果表明,所有参试样品中均未能检测出目的基因PCR产物,说明本试验地中生长2年的转多基因库安托杨和转基因银腺杂种杨尚未发生外源基因向周边其它生物水平转移现象。
5、转基因杨树对目标、非目标昆虫及天敌数量的影响评价。
调查结果显示,在库安托杨林地内,转多基因库安托杨株系鞘翅目昆虫数量总体上要低于非转基因对照株系,表现出了一定的抗虫性,同时也发现转多基因库安托杨7个株系之间的鞘翅目昆虫发生量存在差异;转多基因库安托杨与非转基因对照林地内非目标昆虫的种类和数量随时间变化的趋势基本相同;转基因株系林内捕食性天敌数量高于对照,表明抗性株系可能有利于它们的生存。在银腺杂种杨林地内,转基因株系C013-5鞘翅目和鳞翅目发生量低于对照株系S13-0和转单价抗虫基因株系C13-5,说明转双价抗虫基因的转基因杨树有良好的抗虫效果;转基因与非转基因林地内非目标昆虫总数量变化趋势相似,无明显差异,转基因株系内天敌数量略高于对照。
6、转基因杨树对土壤微生物生态系统的影响评价。
以试验林中转基因杨树及其非转基因对照杨树共8个株系为材料,连续2年取其根圈土壤,采用稀释平板法对土样中的细菌、真菌、放线菌进行分离培养并计数。试验结果表明,试验地内三类土壤微生物数量随年份和月份表现出不同的增减趋势。方差分析与多重比较结果显示,库安托杨株系间土壤微生物数量无显著差异,而银腺杂种杨的个别株系间土壤微生物数量存在显著差异,但并未显示出与外源基因的种类和数量有相关性。并且有些转基因林地与非转基因林地间的土壤微生物数量也存在显著差异,但是否是由外源基因造成的影响还需要深入研究。同时对比不同年份的同一月份间三种土壤微生物数量,结果显示,大部分株系间存在极显著差异,但其变化无规律性,可能受自然条件影响所致,初步可以说明转基因杨树种植年限的增加并未对土壤微生物系统造成影响。
本研究首次对转不同数量及种类外源基因杨树进行了生态安全性初步评价,结果显示抗生素标记基因NPTII蛋白表达量未随目的基因数量增加而增加;目的基因均存在于杨树基因组中;本试验地内转基因杨树并未获得比非转基因对照杨树更强的生长能力;没有发现外源基因向其它物种的水平转移现象;对非目标昆虫和天敌尚未表现出明显的影响;也未对土壤微生物生态系统产生明显的影响。
The species of Populus spp. are important trees planted worldwide, and play crucial roles in forest industry in our country. The application of transgenic technology in poplar genetic breeding has been greatly accelerating the process of selection and cultivation of new poplar varieties. To date, many transgenic poplar trees with improved traits such as insect resistance, salt tolerance and modified wood properties have been obtained, showing a broad application in the future. In our country, several new poplar varieties, such as insect resistance transgenic P. nigra and transgenic hybrid 741 poplar have been permitted to be commercialized. With the rapid progress of research in transgenic trees, potential ecological risks caused by foreign genes are getting to be concerned. Thus, ecological risk assessment of transgenic trees has significant meanings and application. However, most of the current works on risks assessment are foused on crops, few work are carried out in research field of forest trees. In this study, transgenic P. alba×P. glandulosa and P.×euramericana‘Guariento’were used as materials to perform ecological risks assessment. Based on a 2-year-old field trail of transgenic poplar, preliminary evaluations were implementd on six aspects, including assay of the quantity of expressed protein of marker gene NPTII, stability of foreign genes, growth traits of transgenic trees, horizontal transfer of foreign genes, the effects of foreign genes on the community and density of target, non-target and parasite insects and on soil microorganisms ecosystems. The main results are described as following:
1. Protein expression level of marker gene NPTII using ELISA method. A total of 15 transgenic lines and 1 non-transgenic line of P.×euramericana‘Guariento’, 18 transgenic lines and 1 non-transgenic line of P. alba×P. glandulosa growing in greenhouse were used for this assay. There were no significant differences were observed in protein contents among different transgenic lines both in the two poplar species. In P.×euramericana‘Guariento’, the quantities of NPTII protein in lines with different number of foreign genes of 1, 2, 3 and 5 are 27.47, 0.19.00, 19.69, and 16.72 ng per gram fresh leaf tissues, respectively. In P. alba×P. glandulosa, the quantities of NPTII protein in lines with different number of foreign genes of 1, 2, 3, 4 and 5 are 2.44, 3.51, 3.82, 7.54 and 5.60 ng per gram fresh leaf tissues, respectively. The results suggested that the quantity of protein would not increase with increased number of transgenes.
2. Identification of genetic stability of foreign genes. Sets of PCR were performed on 3 lines of transgenic P.×euramericana‘Guariento’and 3 lines of transgenic P. alba×P. glandulosa. Results showed that all transgens were identified in these lines and not observed in control plants, suggesting that target genes were stable present in the genome of transgenic plants, and these trees were suitable to be assessed further.
3. Measurement of growth traits of transgenic poplar. Height, breast diameter and mortality were scored on 800 trees of 8 transgenic and non-transgenic lines of both 2-year-old poplar species. Higher and lower scores in different transgenic lines were observed compared with that of non-transgenic line. Among them, the average level of breast diameter in transgenic lines was higher than that of non-transgenic line in P. alba×P. glandulosa, but with no significant differeces. These results indicated that the expression of foreign genes conferred no advantage of growth ability to transgenic poplar compared with non-transgenic plants.
4. Assessment of horizontal transfer of foreign genes. Using genomic DNA from weed grasses, insects and soil microorganisms, series of PCR were conducted to amplify 5 transgenes Vgb, SacB, BtCry3A, OC-I and JERF36 and marker gene NPTII. No any target PCR product was observed in all samples analyzed. This result suggested that no horizontal transfer of transgenes occurred between transgenic poplar and other plant species, insects or microorganisms.
5. Assessment of the effects of transgenic poplar on target, non-target and parasite insects. In the stand of P.×euramericana‘Guariento’, the density of Lepidoptera in transgenic lines was lower than in non-transgenic line, and significant differences were observed among 7 transgenic lines. The developing trends of species and density of insects in transgenic and non-transgenic stands were the same with the change of time. The density of predator in transgenic lines was higher compared to that in control lines, which suggested that transgenic lines with resistance trait was favorable for the living of predator. In the stand of P. alba×P. glandulosa, the density of target insects of Coleoptera and Lepidoptera in C013-5 was lower compared with that of in control line S13-0, and also lower than that of in transgenic line C13-5 which has a single foreign gene, indicating that transgenic lines with two target genes had a higher resistance to insects. There was a similar character on the developing trend of the density of target insects in both transgenic and non-transgenic poplar stand. Furthermore, the density of parasite insects in transgenic poplar stand was slightly higher than that of non-transgenic plants.
6. Assessment of the effects of transgenic poplar on soil microbial ecosystems. Dilution-plate method was used to determine the quantities of main soil microbes of bacteria, fungi and actinomycetes in 8 poplar lines in different months of 2 successive years. The results showed different developing trends of three groups of microorganisms with different month and different years. In the stand of P.×euramericana‘Guariento’, there was no significant difference in the quantities of these three kinds of microbes in all lines, whereas significant different was found in several lines of P. alba×P. glandulosa stand, but did not show any relation to the type and number of foreign genes. Significant differences were also found in some lines between transgenic and non-transgenic stands, which need to be studied further whether foreign genes have any effects. The results from the same month in different years showed that there were significant differences in the quantity of microorganisms among most lines with no clear pattern, which might be caused by changes of natural environmental conditions. Our preliminary results indicated that the transgenic poplar growing in different years had no significant negative effects on soil microorganisms.
Our work is the first study on biosafety assessment of transgenic poplar with multiple transgenes with different functions. The results showed that the content of NPTII protein in poplar leave tissues did not increase with the increased number of foreign genes. Target genes were stable present in the poplar genome, and transgenic poplar did not showed advantage growth ability on non-transgenic plants. No horizontal gene transfer was found. No significant negative effects were found on non-target and predator insects, and on the soil microbial ecosystems.
引文
[1]董志峰,马荣才,彭于发.转基因植物中外源非目的基因片段的生物安全研究进展.植物学报, 2001, 43(7): 661-672
[2]全先庆,曹扬荣,赵彦修.转基因林木研究进展.中国生物工程杂志, 2003, 27(7): 47-51
[3]武芸,丁莉.转基因农作物的安全性及其发展前景.安徽农业科学, 2007, 35(7): 2096-2097
[4]苏晓华,张冰玉,黄烈健.转基因林木研究进展.林业科学研究, 2003, 16(1): 95-103
[5]钱迎倩,田彦.转基因植物的生态风险评价.植物生态学报, 1998, 22(4): 289-299
[6]康向阳,刘志民,李胜功.论转基因林木的潜在生态风险性.应用生态学报, 2004, 15(7): 1281-1284
[7] Gann PH, Ma J, Giovannucci E, et al. Lower prostate cancer risk in men with elevated plasma lycopene levels: results of a prospective analysis. Cancer Res, 1999, 59(6): 1225-1230
[8] Arab L, Steck S. Lycopene and cardiovascular disease. Am J Clin Nutr, 2000, 71(6): 1691-1695
[9] Matoba N, Doyama N, Yamada Y, et al. Design and production of genetically modified soybean protein with anti-hypertensive activity by incorporating potent analogue of ovokinin. FEBS Letters, 2001, 497(1): 50-54
[10] Paine JA, Shipton CA, Chaggar S, et al. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nat Biotechnol, 2005, 23(4): 482-487
[11] Huang J, Hu R, Rozelle S, et al. Insect-resistant GM rice in farmers’fields: assessing productivity and health effects in China. Science, 2005, 308(5722): 688-690
[12] Wang W, Vinocur B, Altman A. Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta, 2003, 218(1): 1-14
[13] Bevan MW, Flavell RB, Chilton MD. A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature, 1983, 304(5922): 184-187
[14] Herrera-Estrella L, Depicker A, Van Montagu M, et al. Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector. Nature, 1983, 303(5414): 209-213
[15] Murai N, Kemp JD, Sutton DW, et al. Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors. Science, 1983, 222(4623): 476-482
[16] Feldmann KA, Marks MD. Agrobacterium mediated transformation of germinating seeds of Arabidopsis thaliana: a non-tissue culture approach. Mol Gen Genet, 1987, 208(1-2): 1-9
[17] Kikkert JR. The Biolistic? PDS-1000/He device. Plant Cell Tiss Org Cult, 1993, 33(3): 221-226
[18] Sanford JC. The development of the biolistic process. In Vitro Cell Dev Biol Plant, 2000, 36(5): 303-308
[19] Joersbo M, Brunstedt J. Electroporation: mechanism and transient expression, stable transformation and biological effects in plant protoplasts. Physiol Plant, 1991, 81(2): 256-264
[20] Carozzi N, Koziel MG.Transgenic maize expressing a Bacillus thuringiensis insecticidal protein for control of European corn borer. In Advances in insect control: The role of transgenic plants (Eds), London, Taylor and Francis, 1997, 63-74
[21] Perlak FJ, Deaton RW, Armstrong TA, et al. Insect resistant cotton plants. Bio/Technology, 1990, 8(10): 939-943
[22] Perlak FJ, Stone TB, et al. Genetically improved potatoes: Protection from damage by Colorado potato beetle. Plant Mol. Biol, 1993, 22(2): 313-321
[23] James J,孙国凤. 2006年生物技术商业化及转基因作物的全球态势.生物技术通报, 2007, 2 :159-162
[24] van Frankenhuyzen K, Beardmore T. Current status and environmental impact of transgenic forest trees. Can J For Res , 2004, 34(6) :1163-1180
[25] FAO. Preliminary review of biotechnology in forestry, including genetic modification. 2005
[26] Gressel J. Tandem constructs: preventing the rise of superweeds. Trends Biotechnol, 1999, 17(9): 1013-1030
[27] Mack RN, Simberloff D, Lonsdale WM, et al. Biotic invasions: causes, epidemiology, global consequences, and control. Ecol Appl, 2000, 10(3): 689-710
[28] Kjellsson G, Simonsen V. Methods for risk assessment of transgenic plants 1: Competition, establishment and ecosystem effects. Birkhauser Verlag, 1994
[29] Dear BS, Sandral GA. Subterranean clover in NSW-identification and use. Agfact P2.5.16, 2nd edn. NSW Agriculture, Wagga Wagga, 2004
[30] Godfree RC, Woods MJ, Young AG, et al. Growth, fecundity and competitive ability of transgenic Trifolium subterraneum subsp. subterraneum cv. Leura expressing a sunflower seed albumin gene. Hereditas, 2004, 140(3): 229-244
[31] Halfhill MD, Sutherland JP, Moon HS, et al. Growth, productivity, and competitiveness of introgressed weedy Brassica rapa hybrids selected for the presence of Btcry1Ac and gfp transgenes. Mol Ecol, 2005, 14(10): 3177-3189
[32] Raybould AF, Gray AJ. Genetically modified crops and hybridization with wild relatives: A UK perspective. J Appl Ecol, 1993, 30(2): 199-219
[33] Grant V. The evolutionary process: A critical study of evolutionary theory. Columbia University Press, 1991
[34] Cox A L, Skipper J, Chen Y, et al. Identification of a peptide recognization by five melanoma-specific human cytotoxic T cell lines. Science, 1994, 264(5159):716-719
[35] Chevre A M, Eber E, Renard M. Transgenic rape and environmental hazards. Biofutur, 1997, 172(1997) :44-48
[36] Conner A J. 1997. Genetically engineering crops-environment and food safety issues. The Royal Society of New Zealand, Miscellaneous Series, 39:10-14
[37] Ford L B. 1998. Transgenic risks are not too low to be tested. Nature, 394(6695):715
[38] Wilkinson M J, Davenport I J, Charters Y M, et al. 2000. A direct regional scale estimate of transgenes movement from genetically modified oilseed rape to its wild progenitors. Mol Ecol, 9(7) :983-991
[39]王景平.转基因植物的环境安全性.安徽农业科学, 2007, 35(8): 2367-2369
[40]谢杰,余沛涛,王全喜.转基因植物的安全性问题及其对策.上海农业学报, 2006, 22(1): 80-84
[41] Darmency H, Vigouroux Y, Gestat De GarambéT, et al. Transgene escape in sugar beet production fields: data from six years farm scale monitoring. Environ Biosafety Res, 2007, 6(3): 197-206
[42] Abud S, Souza PI, Vianna GR, et al. Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil. Genet Mol Res, 2007, 6(2): 445-52
[43] Yuan QH, Shi L, Wang F, et al. Investigation of rice transgene flow in compass sectors by using male sterile line as a pollen detector. Theor Appl Genet, 2007, 115(4): 549-560
[44] Lawrence JG, Ochman H. Molecular archaeology of the Escherichia coli genome. Proc Natl Acad Sci USA, 1998, 95(16): 9413-9417
[45] Mel SF, Mekalanos JJ. Modulation of horizontal gene transfer in pathogenic bacteria by in vivo signals. Cell, 1996, 87(5): 795-798
[46] Bennett PM, Livesey CT, Nathwani D, et al. An assessment of the risks associated with the use of antibiotic resistance genes in genetically modified plants: report of the Workig Party of the British Society for Antimicrobial Chemotherapy. J Antimicro Chemother, 2004, 53(3): 418-431
[47]周琴,孙明,李林,等.苏云金芽胞杆菌标记重组菌株的构建于杀虫基因水平转移.应用生态学报, 2005, 16(1): 142-146
[48] Richter B, Smalla K. Screening of rhizosphere and soil bacteria for transformability. Environ Biosafety Res, 2007, 6(1-2): 91-99
[49] Mohr KI, Tebbe CC. Field study results on the probability and risk of a horizontal gene transfer from transgenic herbicide-resistant oilseed rape pollen to gut bacteria of bees. Appl Microbiol Biotechnol, 2007, 75(3): 573-582
[50] Paparini A, Romano-Spica V. Gene transfer and cauliflower mosaic virus promoter 35S activity in mammalian cells. J Environ Sci Health B, 2006, 41(4): 437-449
[51] Schubbert R, Hohlweg U, Renz D, et al. On the fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the foetus. Mol Gen Genet, 1998, 259(6): 569-576
[52] Pontiroli A, Simonet P, Frostegard A, et al. Fate of transgenic plant DNA in the environment. Environ Biosafety Res, 2007, 6(1-2): 15-35
[53] Netherwood T, Martín-Orúe SM, O’Donnell AG, et al. Assessing the survival of transgenic plant DNA in the human gastrointestinal tract. Nat Biotechnol, 2004, 22(2): 204-209
[54] Mazza R, Soave M, Morlacchini M, et al. Assessing the transfer of genetically modified DNA from feed to animal tissues. Transgenic Res, 2005, 14(5): 775-784
[55]赵志辉,杨立桃,艾晓杰,等.转codA基因的耐盐碱水稻对大鼠生理代谢的影响及外源基因的水平转移.农业生物技术学报, 2005, 13(3): 341-346
[56] Greene A, Allison R. Recombination between viral RNA and transgenic plant transcripts. Science, 1994, 236(5289): 1423-1425
[57] Falk B W, Bruening G. The risk of crop transgene new viruses and diseases? Science, 1994, 263(5289) :1395-1396
[58] Pruss G, Gex P, Shi XM, et al. Plant viral synergism: the potyviral gene encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell, 1997, 9(6): 859-868
[59] Voinner O, Pinto YM, Baulcombe DC. Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci USA, 1999, 96(24): 14147-14152
[60] Capote N, Pérez-Panadés J, MonzóC, et al. Assessment of the diversity and dynamics of Plum pox virus and aphid population in transgenic European plums under Mediterranean conditions. Transgenic Res, 2008, 17(3): 367-377
[61] Lommel SA, Xiong Z. Reconstitution of a functional red clover necrotic mosaic virus by recombinational rescue of the cell-to-cell movement gene expressed in a transgenic plant. J Cell Biochem, 1991, 15A: 151
[62] Borja M, Rubio T, Scholthof HB, et al. Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene. Mol Plant-Microbe Interact, 1999, 12(2): 153-162
[63] Lecoq H, Ravelonandro M, Wipf-Scheibel C, et al. Aphid transmission of a nonaphid-transmissible strain of Zucchini yellow mosaic potyvirus from transgenic plants expressing the capsid protein of Plum pox potyvirus. Mol Plant-Microbe Interact, 1993, 6: 403-406
[64]邓曙东,徐静,张青文,等.转Bt基因棉对非靶标害虫及害虫天敌种群动态的影响.昆虫学报, 2003, 46(1): 1-5
[65]柏立新,张龙娃,肖留斌,等.转Bt基因保铃棉对沿海棉区棉田节肢动物群落组成与多样性的影响.江苏农业科学, 2004, 20(4): 233-239
[66] Torres JB, Ruberson JR. Interactions of Bacillus thuringiensis Cry1Ac toxin in genetically engineered cotton with predatory beteropterans. Transgenic Res, 2008, 17(3): 345-54
[67]刘志诚,叶恭银,胡萃,等.转cry1Ab/cry1Ac基因籼稻对稻田节肢动物群落的影响.昆虫学报, 2003, 46(4): 454-464
[68]陈茂,叶恭银,胡萃,等.转Bt水稻对飞虱和叶蝉及其卵寄生蜂扩散规律的影响.浙江大学学报(农业与生命科学版), 2003, 29(1): 29-33
[69] Losey JE, Rayor LS, Carter ME. Transgenic pollen harms monarch larvae. Nature, 1999, 399(6733): 214
[70] McGaughey W H. Insect resistance to the biological insecticide Bacillus thuringiensis. Science, 1985, 229: 193-195
[71]郭同斌,嵇保中,诸葛强,等.转基因杨树对杨小舟蛾幼虫解毒酶活性的影响.林业科学, 2007, 43(5): 59-63
[72]高宝嘉,李川,刘军侠.转抗虫基因林木在害虫控制中的作用与合理利用途径.河北农业大学学报, 2003, 26 (4): 25-28
[73] Watkinson AR, Freckleton RP. Predict ions of biodiversity response to genetically modified herbicide tolerant crops. Science, 2000, 289: 1554-1557
[74] Kutlesa N J. Insecticidal activity of glufosinate through glufosinate depletion in a caterpillar. Pest Management Science, 2001, 57: 25-32
[75]张志罡,孙继英,付秀芹,等.转Bt基因水稻的生态安全性研究进展.中国稻米, 2007, 2 :1-4
[76]王洪兴,陈欣,唐建军,等.转Bt基因水稻秸秆降解对土壤微生物可培养类群的影响.生态学报2004, 24(1): 89-94
[77]芮玉奎. Bt棉与常规棉根际土壤Bt毒蛋白和植物激素变化动态.生物技术通讯, 2005, 16(5): 515-517
[78] Angles JS. Release of transgenic plants: biodiversity and population level consideration. Mol Ecol, 1994, 3: 45-50
[79] Lynch JM, Whipps JM.. Substrate flow in the rhizosphere. Plant Soil, 1990, 129: 1-10
[80] Saxena D, Stotzky G. Insecticidal toxin from Bacillus thuringiensis is released from roots of transgenic Bt corn in vitro and in situ. FEMS Microbiol Ecol, 2000, 33: 35-39
[81]孙彩霞,陈利军,武志杰. Bt毒蛋白在转基因棉花与土壤系统中的分布.应用生态学报, 2005, 16(9): 1765-1768
[82] Koskella J, Stotzky G. Microbial utilization of free and clay-bound insecticidal toxins from Bacillus thuringiensis and their retention of insecticidal activity after incubation with microbes. Appl Environ Microbio1, 1997, 63: 3561-3568
[83] Sims SR, Ream JE. Soil inactivation of the Bacillus thuringiensis subsp. Kurstaki CryIIA insecticidal protein with in transgenic cotton tissue: Laboratory microcosm and field studies. J Agr Food Chem, 1997, 45: 1502-1505
[84] Oger P, Petit A, Dessaux Y. Genetically engineered plants producing opines alter their biological environment. Nat Biotechnol, 1997, 15: 369-372
[85] Mansouri H, Petit A, Oger P, et al. Engineered rhizosphere: the trophic bias generated by opine-producing plants is independent of the opine type, the soil origin, and the plant species. Appl Environ Microbiol, 2002, 68: 2562-2566
[86] Glandorf DCM, Bakker PAHM, Loon LC. Influence of the production of antibacterial and antifungal proteins by transgenic plants on the saprophytic soil microflora. Acta Bot Neerl, 1997, 46(1): 85-104
[87] Hoffmann T, Golz C, Schieder O. Foreign DNA sequences are received by a wild-type strain of Aspergillus niger after co-culture with transgenic higher plants. Curr Genet, 1994, 27: 70-76
[88] Kremer RJ, Donald PA, Keaster AJ, et al. Herbicide impact on Fusarium spp. and soybean cyst nematode in glyphosate-tolerant soybean. In: Annual meeting abstracts. Madison, WI: ASA, CSSA, and SSSA, 2000, 257
[89] De Vries J, Harms K, Broer I, et al. The bacteriolytic activity in transgenic potatoes expressing a chimeric T4 lysozyme gene and the effect of T4 lysozyme on soil- and phytopathogenic bacteria. Syst Appl Microbiol, 1999, 22: 280-286
[90] Lottmann J, Heuer H, de Vries J, et al. Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community. FEMS Microbiol Ecol, 2000, 33: 41-49
[91] Lottmann J, Berg G. Phenotypic and genotypic characterization of antagonistic bacteria associated with roots of transgenic and non-transgenic potato plants. Microbiol Res, 2001, 156: 75-82
[92]徐晓宇,叶庆富,吴伟祥,等.转Bt基因“克螟稻”秸秆还田对稻田厌氧微生物种群和酶活性的影响.植物营养与肥料学报, 2004, 1: 63-67
[93] Donegan KK, Palm CJ, Fieland VJ, et al. Changes in levels, species and DNA fingerprints of soil microorganisms associated with cotton expressing the Bacillus thuringiensis var. kurstakiδ-endotoxin. Applied Soil Ecology, 1995, 2(2): 111-124
[94] Donegan KK, Seidler RJ, Fieland VJ, et al. Decomposition of genetically engineered tobacco under field conditions: persistence of the proteinase inhibitor I product and effects on soil microbial respiration and protozoa, nematode and microarthropod populations. J Appl Ecol, 1997, 34: 767-777
[95] Siciliano SD, Theoret CM, de Freitas JR, et al. Differences in the microbial communities associated with the roots of different cultivars of canola and wheat. Can J Microbiol, 1998, 44: 844-851
[96] Griffiths, BS, Geoghegan IE, Robertson WM. Testing genetically engineered potato, producing the lectins GNA and ConA, on non-target soil organisms and processes. J Appl Ecol, 2000, 37:159-170
[97] Boon T, Van Der B P. Human tumor antigens recognized by T lymphocytes. J Exp Med, 1996, 183: 725-729
[98] Tureci O, Sahin U, Pfreundschuh M. Serological analysis of human tumor antigens: molecular definition and implications. Mol Med Today, 1997, 3(8): 342-349
[99] Renkvist N, Castelli C, Robbins P, et al. A listing of human tumor antigens recognized by T cells. Cancer Immunl Inmunother, 2001, 50: 3-15
[100] Cook RJ, Thomshow LS, Weller DW, et al. Molecular mechanisms of defense by rhizobacteria against root disease. Proc Natl Acad Sci USA, 1995, 92: 4197-4201
[101] Smalla K, Gebhard F, Van Elsas JD, et al. Prevalence of nptII and Tn5 in kanamycin-resistant bacteria from different environments. FEMS Microbiol Ecol, 1993, 13: 47-58
[102] Kaiser J. Words (and axes) fly over transgenic trees. Science, 2001, 292(5514): 34-36
[103] DiFazio SP, Slavov GT, Burczyk J, et al. Gene flow from tree plantations and implication for transgenic risk assessment. Plantation Forest Biotechnology for the 21st Century, 2004: 405-422
[104] Williams CG, Ladeau SL, Oren R, et al. Modeling seed dispersal distances: implications for transgenic Pinus taeda. Ecological Applications, 2006, 16(1): 117-124
[105] Ellstrand NC. When transgenes wander, should we worry? Plant Ecol, 2001, 125 :1543-1545
[106] Wolf DE, Takebayashi N, Rieseberg LH. Predicting the risk of extinction through hybridization. Conserv Biol, 2001, 15 :1039-1053
[107]王明庥.中国森林可持续发展.南京林业大学学报, 2001, 1 (4 ) :1-3
[108] Fladung M, Nowitzki O, Ziegenhagen B, et al. Vegetative and generative dispersal capacity of field released transgenic aspen trees. Trees, 2003, 17: 412-416
[109]沈孝宙,钱迎倩,张树庸.基因工程树的现状、生态风险与对策.高技术通讯, 2002, 4 :100-105
[110]张真,王军辉,张建国. Bt转基因杨树对杨树昆虫群落结构的影响.林业科学, 2004, 40 (2) :84-89
[111]高宝嘉,张芳,侯东永.转基因741杨林节肢动物群落结构的研究.北京林业大学学报, 2003, 25 (1) :62-64
[112]高素红,毛富玲,王江柱.转双抗虫基因741杨节肢动物群落生态安全性评价-转基因741杨对节肢动物群落空间结构的影响.河北农业大学学报, 2005, 28 (3) :77-80
[113]高素红,高宝嘉,刘军侠,等.转基因741杨节肢动物群落主要害虫及天敌的动态变化.生态学报, 2006, 26(10): 3491-3498
[114]胡建军,张蕴哲,卢孟柱,等.欧洲黑杨转基因稳定性及对土壤微生物的影响.林业科学, 2004, 40 (5) :105-109
[115]胡海燕.转双抗虫基因741杨林土壤生物群落研究.河北农业大学硕士学位论文, 2004, 1-34
[116]刘军侠.转基因741杨的抗虫效应及生态安全性评价.东北林业大学博士学位论文, 2004, 1-83
[117] Pasonen H-L, Degefu Y, Brumós J, et al. Transgenic Betula pendula expressing sugar beet chitinase IV forms normal ectomycorrizae with Paxillus involutus in vitro. Scandinavian journal of Forest Research, 2005, 20(5): 385-392
[118] Ellstrand NC, Prentice HC, Hancock JF. Gene flow and introgression from domesticated plants into their wild relatives. Annu Rev Ecol Syst, 1999, 30 :539-563
[119] DiFazio S P. Progress Report-EPA Grant Number: U 91522, 2000
[120]闫新甫.转基因植物.北京:科学出版社, 2003
[121] Kjellesson G, Simonsen V, Ammanm K. Methods for risk assessment of transgenic plants, II: Pollination, gene transfer and popolation impacts. Basel: Brikhauser Verlag, 1997
[122]樊龙江,周雪平.转基因作物安全性争论与事实.北京:中国农业出版社, 2001
[123] Kjellesson D, Simonsson V. Methods for risk assessment of transgenic plant, I: Competition,establishment and ecosystem efforts. Basel: Brikhauser Verlag, 1994
[124]刘谦,朱鑫泉.生物安全.北京:科学出版社, 2001
[125]李尉民,岳宁,夏红民.转基因生物极其产品的风险与管理.生物技术通报, 2000, 16 (4) :41-44
[126]桑卫国,马克平,魏伟.国内外生物技术安全管理机制.生物多样性, 2000, 8 (4) :413-421
[127] Ramessar K, Peremarti A, Gómez-Galera S, et al. Biosafety and risk assessment framework for selectable marker genes in transgenic plants: a case of the science not supporting the politics. Transgenic Res, 2007, 16(3): 261-280
[128]王紫萱,易自力.卡那霉素在植物转基因中的应用及其抗性基因的生物安全性评价.中国生物工程杂志, 2003, 23 (6): 9-13
[129]司家钢.转基因植物中的卡那霉素抗性.生物技术通报, 1998, 1: 29-31
[130]徐茂军.转基因植物中卡那霉素抗性(Kanr)标记基因的生物安全性.生物学通报, 2001, 36 (2): 18-19
[131]崔林开,叶华智. 3种ELISA法检测Bt杀虫蛋白的比较研究.安徽农业科学, 2005, 33 (11): 2056-2057
[132]刘光明,苏文金.应用间接ELISA方法定量检测转基因抗虫玉米.无锡轻工大学学报, 2003, 22 (1):49-52
[133]严吉明,叶华智,伍光庆.转Bt抗虫基因植物中杀虫蛋白的酶联免疫检测技术研究Ⅲ.酶联免疫(ELISA)间接法检测Bt杀虫蛋白研究.四川农业大学学报, 2005, 23 (3):280-284
[134]李葱葱,刘娜,康岭生,等.转基因抗虫玉米Bt蛋白表达量的研究.玉米科学, 2006, 14 (3): 40-41
[135]王建革,苏晓华,纪丽丽,等.基因枪转多基因库安托杨的获得.科学通报, 2006, 51 (23): 2755-2760
[136] Baszczynski CL. A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues. Biochem Cell Biol, 1990, 68 (6): 983-987
[137] Nagel RJ, Manners JM, Birch RG. Evaluation of an ELISA assay for rapid detection and quantification of neomycin phosphotransferase II in transgenic plants. Plant Mol Biol Rep, 1992, 10 (3): 263-272
[138] Rogan GJ, Ream JE, Berberich, et al. Enzyme-linked immunosorbent assay for quantitation of neomycin phosphotransferase II in genetically modified cotton tissue extracts. J Agric Food chem, 1992, 40 (8): 1453-1458
[139] McKenzie MJ, Mett V, Jameson PE. Modified ELISA for the detection of neomycin phosphotransferaseⅡin transformed plant species. Plant Cell Rep, 2000, 19 (3): 286-289
[140] Anfossi L, Tozzi C, Giraudi G, et al. Evaluation of procedures for the extraction and purification of neomycin phosphotransferase II from a genetically modified Agrobacterium. Ann Chim, 2004, 94 (1-2): 93-99
[141] European Federation of Biotechnology. Antibiotic resistance markers in genetically modified (GM) crops, task group on public perceptions of biotechnology briefing paper 10, 2001
[142] Fuchs RL, Ream JE, Hammond BG, et al. Safety assessment of the neomycin phosphotransferase-II (NPTII) protein. Bio/Techno, 1993, 11: 1543-1547
[143] Gay P, Gillespie S. Antibiotic resistance markers in genetically modified plants: a risk to human health. Lancet Infect Dis, 2005, 5: 637-646
[144] Thomson J. Genetically modified food crops for improving agricultural proactic and their effects on human health. Trends Food Sci Techonl, 2003, 14(5): 210-228
[145]张冰玉,苏晓华,黄秦军,等.转果聚糖蔗糖转移酶基因银腺杂种杨的获得.林业科学, 2005, 4l(3): 48-53
[146]张冰玉,苏晓华,李义良,等.转抗鞘翅目害虫基因银腺杂种杨的获得及其抗虫性的初步研究.北京林业大学学报, 2006, 28 (2 ):102-105
[147] Halpin C, Barakate A, Askari BM, et al. Enabling technologies for manipulating multiple genes on complex pathways. Plant Mol Biol, 2001, 47: 295-310
[148] Kiran S, Pooja B-M, Trevor T. Genetic transformation technology: status and problems. In Vitro Cell Dev Biol. 2005, 41(2):102-112
[149] Kohli A, Twyman RM, Abranches R, et al. Transgene integration, organization and interaction in plants. Plant Mol Biol, 2003, 52(2): 247-258
[150] Brodersen P, Voinnet O. The diversity of RNA silencing pathways in plants. Trends Genet, 2006, 22(5): 268-280
[151] Garfinkel DJ, Simpson RB, Ream LW, et al. Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell, 1981, 27: 143-153
[152] De Wilde C, Van Houdt H, De Buck S, et al. Plants as bioreactors for protein production: avoiding the problem of transgene silencing. Plant Mol Biol, 2000, 43(2-3): 347-359
[153] Avesani L, Marconi G, Morandini F, et al. Stability of Potato virus X expression vectors is related to insert size: implications for replication models and risk assessment. Transgenic Res, 2007, 16(5): 587-597
[154]佘健明,蔡小宁,朱卫民,等.外源基因在不结球白菜转基因植株后代中的表达.江苏农业学报, 2002, 18(1): 33-36
[155]林良斌,管春云,周小云,等.外源基因在转基因抗虫油菜中的遗传行为.湖南农业大学学报(自然科学版), 2002, 28(2): 100-102
[156]姚方印,朱常香,李广贤,等. Bt水稻的抗虫鉴定及转基因的遗传分析. 2002, 35(2): 142-145
[157]刘允军,王国英. cry1A基因在转基因玉米中的遗传于表达.植物学报, 2003, 45(3): 253-256
[158] Neale DB, Ingvarsson PK. Population, quantitative and comparative genomics of adaptation in forest trees. Curr Opin Plant Bio, 2008, 11(2): 149-155
[159] Fredshavn JR, Poulsen GS. Growth behavior and competitive ability of transgenic crops. Field Crops Res, 1996, 45: 11-18
[160] Pe?a L, Séguin A. Recent advances in the genetic transformation of trees. Trends Biotechnol, 2001, 19(12): 500-506
[161] Daniell H, Dhingra A. Multigene engineering: dawn of an exciting new era in biotechnology. Curr Opin Biotechnol. 2002, 13(2): 136-141
[162] Czarny JC, Grichko VP, Glick BR. Genetic modulation of ethylene biosynthesis and signaling in plants. Biotechnol Adv, 2006, 24(4): 410-419
[163] Brown JR. Ancient horizontal gene transfer. Nat Rev Genet, 2003, 4(2): 121-132
[164] Choi IG, Kim SH. Global extent of horizontal gene transfer. Pro Natl Acad Sci USA, 2007, 104(11): 4489-4494
[165]李钧敏,金则新.一种高效可直接用于PCR分析的土壤总微生物DNA抽提方法.应用生态学报, 2006, 17 (11): 2107-2111
[166]印红,刘晓丽,王彦芳,等.一种改进的昆虫基因组DNA的提取方法.河北大学学报(自然科学版) . 2002, 22 (1): 80-82
[167] Velkov VV, Medvinsky AB, Sokolov MS, et al. Will transgenic plants adversely affect the environment? J Biosci, 2005, 30(4): 515-548
[168] Fladung M, Kaldorf M, Buscot F, et al. Untersuchungen zur Stabilit?t und Expressivit?t fremder Gene in Aspenklonen (Populus tremula und P. tremula×P. tremuloides) unter Freilandbedingungen. In: Schiemann J (ed) Biologische Sicherheitsforschung bei Freilandversuchen mit transgenen Organismen und anbaubegleitendes Monitoring. BEO (Projekttr?ger Biologie, Energie, Umwelt des BMBF), Braunschweig, 2000, 77-83
[169] Kaldorf M, Fladung M, Muhs HJ, et al. Interactions between mycorrhizal fungi and transgenic tress. In: Proceedings of the Workshop: Release of transgenic trees―present achievement, problems, future prospects. Federal Environment Agency Germany, Texte 99, 2000, 81-86
[170] Kaldorf M, Fladung M, Muhs HJ, et al. Mycorrhizal colonization of transgenic aspen in a field trial. Planta, 2002, 214: 653-660
[171] Nielsen KM, Bones AM, Smalla K, et al. Horizontal gene transfer from transgenic plants to terrestrial bacteria―a rare event? FEMS Microbiol Rev, 1998, 22: 79-103
[172] Mendelsohn M, Kough J, Vaituzis Z, et al. Are Bt crops safe? Nat Biotechnol, 2003, 21: 1003-1009
[173] Conner AJ, Glare TR, Nap JP. The release of genetically modified crops into the environment Part II. Overview of ecological risk assessment. Plant J, 2003, 33: 19-46
[174] Richardson AO, Palmer JD. Horizontal gene transfer in plants. J Exp Bot, 2007, 58(1): 1-9
[175] Adams KL, Qiu YL, Stoutemyer M, et al. Punctuated evolution of mitochondrial gene content: high and variable rates of mitochondrial gene loss and transfer to the nucleus during angiosperm evolution. Proc Natl Acad Sci USA, 2002, 99: 9905-9912
[176] Wilson FD, Flint HM, Deaton WR, et al. Resistance of cotton lines containing Bacillus thuringiensis toxin to pink bollworm and other insects. J Econ Entomol, 1992, 85(4): 1516-1521
[177] Rui YK, Yi GX, Zhao J, et al. Changes of Bt toxin in the rhizosphere of transgenic Bt cotton and its influence on soil functional bacteria. World Journal of Microbiology and Biotechnology, 2005, 21: 1279-1284
[178] Christopher B, Jeffrey S. Soil microbial communities associated with Bt and Non-Bt corn in three soils. J Environ Qual, 2004, 33: 832-836
[179]孙婷婷,胡宝忠.转基因植物及其安全性研究进展.黑龙江农业科学, 2007, 2 :72-74
[180] Crawley MJ, Brown SL, Hails RS. et a1. 2001. Biotechnology transgenic crops in natural habitats. Nature, 409 :682-683
[181] Prakash CS. The genetically modified crop debate in the context of agricultural evolution. Plant Physiol, 2001, 126: 8-15
[182]杨利峰,尹小敏,赵德明.农业转基因生物安全性.黑龙江畜牧兽医, 2007, 2 :1-3
[183] Richardson DM. Forestry trees as invasive aliens. Conserv Biol, 1998, 12 :18-26
[184] Stephen P, DiFazio, Gancho T, et al. Gene flow from tree plantations and implications for transgenic risk assessment. Plantation Forest Biotechnology for the 21st Century, 2004, 405-422