北方防护林树种毛白杨抗虫基因遗传转化及其安全性评价
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摘要
毛白杨是我国北方一个常用的防护林树种,其特点是干形通直、材质好、生长快,但是也存在叶部虫害教严重、雌株春季飞絮等问题。本文利用败育毛白杨雄株-D为主要材料,在研究其雄性不育机制的基础上,利用转基因技术培育出抗虫新品种,并对环境安全等方面进行了评价。主要研究成果如下:
1、2004～2007年对败育毛白杨雄株-D花药外部形态及花药发育过程进行了观察研究。连续四年的花枝室内水培实验,均未发现花粉散出。花药发育的细胞组织学研究发现,花药后期干瘪不开裂、绒毡层解体延迟、大部分小孢子发育异常不能形成正常的花粉粒。
2、以败育毛白杨雄株-D去叶带芽茎段作为外植体,建立了其植株再生体系。采用组培苗叶片为外植体,建立了适合遗传操作的败育毛白杨雄株-D叶片再生体系。
3、通过抗生素临界浓度筛选实验,确定了卡那霉素的临界浓度为50mg/L。
4、通过农杆菌介导法将含有不同杀虫机制的Bt和API-A(重组质粒为pBtiA,农杆菌为LBA4404)转入败育毛白杨雄株-D。获得21株卡那霉素抗性植株。抗性植株的Bt和API-A基因PCR检测阳性率为90%。
5、对PCR检测阳性植株进行了抗虫生物检测。转基因败育毛白杨雄株-D对杨扇舟蛾和舞毒蛾幼虫具有明显的毒杀特性。筛选并获得4个高抗株系(编号为D-84、D-87、D-94、D-95)、3个中抗株系(编号为D-74、D-85、D-88)。各转基因株系(高抗株系和中抗株系系)对靶试昆虫的生长发育有明显抑制作用。生物测定的结果进一步证实了外源抗虫基因在植物基因组的整合和正常表达。其中D-84、D-88和D-95植物基因组中Bt基因的Southern杂交检测阳性。
6、为探讨转基因杨树的环境风险,对已批准环境释放的转基因741杨根部微生物进行了较为详细的研究。转基因741杨根部细菌的种类均高于对照未转基因741杨。转基因741杨根部真菌种类均低于对照为转基因741杨。
7、对从转基因741杨栽培地土壤中分离得到的860株次细菌(有重复)和120株次真菌(有重复)的总DNA进行PCR检测,在所有的被检测的细菌和真菌总DNA中均为发现Bt(含有35S启动子片段)以及NPTII基因片段。
P.tomentosa is the key shelter forest tree with nice biological characteristics.The sterile male P. tomentosa(NO.D) was used as materials of the insect-resistant genes transformation,its sterility and the environmental risk of transgenic poplar were studied.The purpose is to get new clone of sterile male P. tomentosa with the insect-resistance and environmental safety nature The results were shown as below.
1.The anther morphology and histology of the sterile male P.tomentosa was observed for 4 years, from 2004 to 2007.The results is that there is no pollination step during the process of anthers growth. The mature anther was not able to split.The tapetum cell delayed disintegration.The sporule couldn't grow normall.
2.Both shoots regeneration system and leaf regeneration systme were established by using its buds and leaf as explants seperately.
3.50mg/L of kanamycin was the selection limite concentration for pBtiA transformation of the sterile male P.tomentosa screened by agrobacterium-medium transformation methos.
4.21 Kan-resistant clones were obtained by agrobacterium-medium transformation method,they were identified by checking Bt gene and NPTⅡgene using PCR method,and 90%of kan-resistant clones were positive
5.Feeding Clostera anachoreta(Fabricius) and Lymantria dispar(Linnaeus) with the young tender leaves of transgenic sterile male P.tomentosa were carried out for checking the biological effects. Toxicity of transgenic sterile male P.tomentosa was significantly higher than that of normal sterile male P.tomentosa.According to the insect total death rate,four high insect-resistance clones(D--84、D-87、D-94、D-95) and three middling insect-resistance clones(D-74、D-85、D-88 ) were sieved.Both high and middle insect-resistant clones had the negative effects on the insects growth and their development. The southern blot results of 3 clones,D-84,D-88 and D-95,were positive.
6.The environmental risk of transgenic 741 poplar was studied.The species of the bacterium which were got from the rootage soil of the transgenic 741 poplus was much more than that of normal 741 poplar.The species of the fungi of the transgenic 741 poplus was less than that of normal 741 poplar.
7.Both of Bt gene and NPTⅡgene were not detected in 860 colonies of bacteria and 120 colonies of fungi isolated in the rhizosphere of transgenic 741 poplar by PCR method.
1、2004～2007年对败育毛白杨雄株-D花药外部形态及花药发育过程进行了观察研究。连续四年的花枝室内水培实验,均未发现花粉散出。花药发育的细胞组织学研究发现,花药后期干瘪不开裂、绒毡层解体延迟、大部分小孢子发育异常不能形成正常的花粉粒。
2、以败育毛白杨雄株-D去叶带芽茎段作为外植体,建立了其植株再生体系。采用组培苗叶片为外植体,建立了适合遗传操作的败育毛白杨雄株-D叶片再生体系。
3、通过抗生素临界浓度筛选实验,确定了卡那霉素的临界浓度为50mg/L。
4、通过农杆菌介导法将含有不同杀虫机制的Bt和API-A(重组质粒为pBtiA,农杆菌为LBA4404)转入败育毛白杨雄株-D。获得21株卡那霉素抗性植株。抗性植株的Bt和API-A基因PCR检测阳性率为90%。
5、对PCR检测阳性植株进行了抗虫生物检测。转基因败育毛白杨雄株-D对杨扇舟蛾和舞毒蛾幼虫具有明显的毒杀特性。筛选并获得4个高抗株系(编号为D-84、D-87、D-94、D-95)、3个中抗株系(编号为D-74、D-85、D-88)。各转基因株系(高抗株系和中抗株系系)对靶试昆虫的生长发育有明显抑制作用。生物测定的结果进一步证实了外源抗虫基因在植物基因组的整合和正常表达。其中D-84、D-88和D-95植物基因组中Bt基因的Southern杂交检测阳性。
6、为探讨转基因杨树的环境风险,对已批准环境释放的转基因741杨根部微生物进行了较为详细的研究。转基因741杨根部细菌的种类均高于对照未转基因741杨。转基因741杨根部真菌种类均低于对照为转基因741杨。
7、对从转基因741杨栽培地土壤中分离得到的860株次细菌(有重复)和120株次真菌(有重复)的总DNA进行PCR检测,在所有的被检测的细菌和真菌总DNA中均为发现Bt(含有35S启动子片段)以及NPTII基因片段。
P.tomentosa is the key shelter forest tree with nice biological characteristics.The sterile male P. tomentosa(NO.D) was used as materials of the insect-resistant genes transformation,its sterility and the environmental risk of transgenic poplar were studied.The purpose is to get new clone of sterile male P. tomentosa with the insect-resistance and environmental safety nature The results were shown as below.
1.The anther morphology and histology of the sterile male P.tomentosa was observed for 4 years, from 2004 to 2007.The results is that there is no pollination step during the process of anthers growth. The mature anther was not able to split.The tapetum cell delayed disintegration.The sporule couldn't grow normall.
2.Both shoots regeneration system and leaf regeneration systme were established by using its buds and leaf as explants seperately.
3.50mg/L of kanamycin was the selection limite concentration for pBtiA transformation of the sterile male P.tomentosa screened by agrobacterium-medium transformation methos.
4.21 Kan-resistant clones were obtained by agrobacterium-medium transformation method,they were identified by checking Bt gene and NPTⅡgene using PCR method,and 90%of kan-resistant clones were positive
5.Feeding Clostera anachoreta(Fabricius) and Lymantria dispar(Linnaeus) with the young tender leaves of transgenic sterile male P.tomentosa were carried out for checking the biological effects. Toxicity of transgenic sterile male P.tomentosa was significantly higher than that of normal sterile male P.tomentosa.According to the insect total death rate,four high insect-resistance clones(D--84、D-87、D-94、D-95) and three middling insect-resistance clones(D-74、D-85、D-88 ) were sieved.Both high and middle insect-resistant clones had the negative effects on the insects growth and their development. The southern blot results of 3 clones,D-84,D-88 and D-95,were positive.
6.The environmental risk of transgenic 741 poplar was studied.The species of the bacterium which were got from the rootage soil of the transgenic 741 poplus was much more than that of normal 741 poplar.The species of the fungi of the transgenic 741 poplus was less than that of normal 741 poplar.
7.Both of Bt gene and NPTⅡgene were not detected in 860 colonies of bacteria and 120 colonies of fungi isolated in the rhizosphere of transgenic 741 poplar by PCR method.
引文
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