转基因杨树生态安全性评价初步研究
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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.
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
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