转Bt基因南林895杨的抗虫性检测与对土壤微生物影响的初步分析
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摘要
中国杨树的栽培居世界首位。但单一品种的过度利用导致杨树虫害愈演愈烈。利用基因工程技术培育抗虫杨树新品种已迫在眉睫。本文选择转抗虫基因(Bt、CpTI)南林895杨为试验材料,开展了分子检测、抗虫性测定及生物安全性初步评估,为选育抗虫转基因杨树新品种提供了依据。主要研究结果如下:
1.通过对基本培养基、激素组合、培养条件等筛选,改良了转基因南林895杨的组培繁殖体系。其中叶片诱导分化培养基为:MS+0.5mg/L 6-BA+0.002mg/LTDZ+0.1mg/L NAA,蔗糖30g/L,琼脂7.0g/L;芽增殖培养基:MS+0.2mg/L 6-BA+0.001mg/L TDZ+0.05mg/L NAA,蔗糖30g/L,琼脂7.0g/L;生根培养基:1/2MS+24g/L蔗糖。建立的组培体系提高了转基因杨树的组培繁殖效率:分化率由91%提高至100%,增值系数由11.6%提高至36.0%,为开展分子检测与抗虫转基因895杨生物学测定提供了基础;
2.由于林木生长周期长,为跟踪转基因895杨的遗传稳定性,对继代培养达4年的转Bt基因895杨11个株系B2、B17、B4、B1、B21、B16、B6、B8、B12、B3、B5等进行了PCR检测,分析结果表明这批转基因895杨仍携带有外源目的基因,为选育抗虫转基因杨新品种提供了依据;
3.本研究采用杨小舟蛾健康四龄幼虫进行田间试验转基因南林895杨10个株系B5、B7、B16、B2、B12、B4、B1、B17、B21、C4的抗虫性生物测定。结果表明,转基因植株表现出了不同程度的抗虫性。其中抗虫性较高的株系有B21、B1、B4、B17,幼虫平均校正死亡率(对照为0)分别达到25.6%、24.0%、15.0%、12.0%;转基因杨树叶片对幼虫的体重有显著抑制作用,其中B5、B4、C4、B21饲喂的幼虫平均相对体重均下降了50.0%以上;室内群体饲虫实验结果表明,B4、B1、B21植株对幼虫的化蛹率产生显著影响,化蛹率比对照下降30.0%以上,蛹重比对照下降33.3%以上B4、B1的羽化率均比对照下降了25.0%以上;
4.植物的生物安全性问题越来越受到人们的关注。为了解转Bt基因杨树对土壤微生物的影响,本文对5个转抗虫基因植株B21、B1、B17、B4、C4与对照进行了根际土壤微生物可培养类群的分析。初步结果表明转基因杨树根际土壤微生物中细菌、真菌、放线菌与对照相比没有显著差异。
The area of poplar planting in china is the biggest around the world.But overcrop of one kind of poplar brings on serious hazards carsed by pests.It has no time to delay for cultivating new insect resistance poplar by genetic engineering technology.In this paper, Populus×eramericana cv 'Nanlin895',with Bt gene or CpTI gene,were used as experiment materials.PCR analysis,larvae bioassay and priliminary biosafty assessment were carried on,which would supply a basis for breeding new excellent species of poplar.The main results described as follows:
1.Improvement of regenerate system of transgenic Populus×eramericana cv 'Nanlin895'with Bt gene or CpTI gene through the screening to the basic culture medium,the adjustment of hormone categories and concentration,election of the culture condition,the optimal medium for leaf differentiation was MS + 0.5mg/L 6-BA +0.002mg/L TDZ + 0.1 mg/L NAA.The optimal medium for bus inducement was MS + 0.2mg/L 6-BA +0.001mg/L TDZ+0.05mg/L NAA.The medium for rooting is 1/2 MS+24%sugar,which increased the ratio of poplar regeneration from 91%up to 100%,differentiation ratio from 11.6%up to 36.0%,and the system layed a solid foundation for PCR and insect bioassay;
2.The period of poplar growing is long.In order to trace the inheritance and segregation of transformants,tissue culture of 4 years was carried on.11 transgenic Populus×eramericana cv 'Nanlin895'with Bt gene:B2,B17,B4,B1,B21,B16,B6,B8,B12,B3,B5 etc.were detected with PCR analysis.The analysis result showed that foreign gene was still carried in the genome of poplar and this provided a solid foundation for cultivating new kinds of insect resistant poplar;
3.The larvae of Micromel alophatroglodyta(Graeser)were used in bioassay of 11 transgenic plant:B5,B7,B16,B2,B12,B4,B1,B17,B21,C4.The result of insect bioassay showed that all the transgenic plants were resistant to larvae at different levels.B4,B1,B17, B21 were confirmed extremely resistant to larvae,and the emending mortalitys were 25.6%, 24.0%,15.0%,12.0%(0%of CK).The leaf of transgenic poplar significantly restrained the weight of larvae,and the relative weight of insects feeding on B1,B4,B21,C4 descended more than 50.0%.And B1,B4,B21 remarkably produced negative influence on the larvae into chrysalis,the ratios descended more than 30.0%.Eclosion ratio of B4,B1,B7,B17,C4 decsended 25%at least;
4.Today biosafty is the focus of public concerns.In order to understand the effects of transgenic organisms on soil microbial flora,this paper analyzed the number of actinomyces, bacterium,epiphyte of 5 different transgenic 895 with Bt or CpTI gene:B21,B1,B17,B4, C4.The priliminary results indicated that the number of bacterium,epiphyte,actinomyces was not significantly different.
1.通过对基本培养基、激素组合、培养条件等筛选,改良了转基因南林895杨的组培繁殖体系。其中叶片诱导分化培养基为:MS+0.5mg/L 6-BA+0.002mg/LTDZ+0.1mg/L NAA,蔗糖30g/L,琼脂7.0g/L;芽增殖培养基:MS+0.2mg/L 6-BA+0.001mg/L TDZ+0.05mg/L NAA,蔗糖30g/L,琼脂7.0g/L;生根培养基:1/2MS+24g/L蔗糖。建立的组培体系提高了转基因杨树的组培繁殖效率:分化率由91%提高至100%,增值系数由11.6%提高至36.0%,为开展分子检测与抗虫转基因895杨生物学测定提供了基础;
2.由于林木生长周期长,为跟踪转基因895杨的遗传稳定性,对继代培养达4年的转Bt基因895杨11个株系B2、B17、B4、B1、B21、B16、B6、B8、B12、B3、B5等进行了PCR检测,分析结果表明这批转基因895杨仍携带有外源目的基因,为选育抗虫转基因杨新品种提供了依据;
3.本研究采用杨小舟蛾健康四龄幼虫进行田间试验转基因南林895杨10个株系B5、B7、B16、B2、B12、B4、B1、B17、B21、C4的抗虫性生物测定。结果表明,转基因植株表现出了不同程度的抗虫性。其中抗虫性较高的株系有B21、B1、B4、B17,幼虫平均校正死亡率(对照为0)分别达到25.6%、24.0%、15.0%、12.0%;转基因杨树叶片对幼虫的体重有显著抑制作用,其中B5、B4、C4、B21饲喂的幼虫平均相对体重均下降了50.0%以上;室内群体饲虫实验结果表明,B4、B1、B21植株对幼虫的化蛹率产生显著影响,化蛹率比对照下降30.0%以上,蛹重比对照下降33.3%以上B4、B1的羽化率均比对照下降了25.0%以上;
4.植物的生物安全性问题越来越受到人们的关注。为了解转Bt基因杨树对土壤微生物的影响,本文对5个转抗虫基因植株B21、B1、B17、B4、C4与对照进行了根际土壤微生物可培养类群的分析。初步结果表明转基因杨树根际土壤微生物中细菌、真菌、放线菌与对照相比没有显著差异。
The area of poplar planting in china is the biggest around the world.But overcrop of one kind of poplar brings on serious hazards carsed by pests.It has no time to delay for cultivating new insect resistance poplar by genetic engineering technology.In this paper, Populus×eramericana cv 'Nanlin895',with Bt gene or CpTI gene,were used as experiment materials.PCR analysis,larvae bioassay and priliminary biosafty assessment were carried on,which would supply a basis for breeding new excellent species of poplar.The main results described as follows:
1.Improvement of regenerate system of transgenic Populus×eramericana cv 'Nanlin895'with Bt gene or CpTI gene through the screening to the basic culture medium,the adjustment of hormone categories and concentration,election of the culture condition,the optimal medium for leaf differentiation was MS + 0.5mg/L 6-BA +0.002mg/L TDZ + 0.1 mg/L NAA.The optimal medium for bus inducement was MS + 0.2mg/L 6-BA +0.001mg/L TDZ+0.05mg/L NAA.The medium for rooting is 1/2 MS+24%sugar,which increased the ratio of poplar regeneration from 91%up to 100%,differentiation ratio from 11.6%up to 36.0%,and the system layed a solid foundation for PCR and insect bioassay;
2.The period of poplar growing is long.In order to trace the inheritance and segregation of transformants,tissue culture of 4 years was carried on.11 transgenic Populus×eramericana cv 'Nanlin895'with Bt gene:B2,B17,B4,B1,B21,B16,B6,B8,B12,B3,B5 etc.were detected with PCR analysis.The analysis result showed that foreign gene was still carried in the genome of poplar and this provided a solid foundation for cultivating new kinds of insect resistant poplar;
3.The larvae of Micromel alophatroglodyta(Graeser)were used in bioassay of 11 transgenic plant:B5,B7,B16,B2,B12,B4,B1,B17,B21,C4.The result of insect bioassay showed that all the transgenic plants were resistant to larvae at different levels.B4,B1,B17, B21 were confirmed extremely resistant to larvae,and the emending mortalitys were 25.6%, 24.0%,15.0%,12.0%(0%of CK).The leaf of transgenic poplar significantly restrained the weight of larvae,and the relative weight of insects feeding on B1,B4,B21,C4 descended more than 50.0%.And B1,B4,B21 remarkably produced negative influence on the larvae into chrysalis,the ratios descended more than 30.0%.Eclosion ratio of B4,B1,B7,B17,C4 decsended 25%at least;
4.Today biosafty is the focus of public concerns.In order to understand the effects of transgenic organisms on soil microbial flora,this paper analyzed the number of actinomyces, bacterium,epiphyte of 5 different transgenic 895 with Bt or CpTI gene:B21,B1,B17,B4, C4.The priliminary results indicated that the number of bacterium,epiphyte,actinomyces was not significantly different.
引文
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