利用基因拆分技术限控转基因飘流的研究
摘要
随着转基因作物的大面积商业化种植，转基因飘流带来的环境安全性问题成为人们关注的热点和焦点问题之一。为了打消公众的疑虑，最好的办法是从根本上防止基因飘流的发生。当前普遍应用的物理隔离只能减少转基因飘流，并不能从根本上杜绝转基因飘流，需要探索研究新的限控转基因飘流的生物学措施。鉴于此，本研究以抗草甘膦除草剂的G2-aroA基因为目的基因，以烟草和水稻为试材，分析G2-aroA基因拆分后重新组装成完整有功能蛋白的效率，探讨基因拆分技术限控转基因飘流的效果，以期建立基因拆分技术限控转基因飘流的技术平台。具体试验结果如下：
1.烟草中进行基因拆分技术限控转基因飘流的效果分析
1）将单独含有完整G_2-aroA、G_2-aroA的N端（EPSPSn-In）或G2-aroA的C端（IC-EPSPSC）基因的转基因烟草T_0代种子接种到含有100mg/L卡那霉素的MS0培养基上，分析Kan~R与KanS的分离比，初步确定单独含有EPSPSn-In的N-33、单独含有IC-EPSPSC的C-11和含有完整G2-aroA基因的G2-24为单拷贝插入。利用Southern杂交和荧光定量PCR对其进行分析，进一步确定了转基因烟草N-33、C-11和G2-24为单拷贝插入。
2）连续2代筛选，获得了转基因烟草N-33和C-11的纯合系，并通过有性杂交获得了同时含有2个基因片段的转基因烟草杂交种N-33×C-11。
3）利用Tail-PCR的方法获得了N-33、C-11的侧翼序列，初步分析二者插入到了不同染色体上。另外，杂交种N-33×C-11自交，获得的自交种进行卡那抗性筛选，共分析3096株，其中非卡那抗性苗为188株，所占比例约为6.07%，符合两个基因片段位于不同染色体上时的孟德尔遗传规律，进一步确定拆分后的EPSPSn-In和Ic-EPSPSc基因片段位于非同源染色体上。
4）对不同类型的转基因烟草进行草甘膦抗性分析，结果表明非转基因烟草NC89以及单独含有1个基因片段的转基因烟草N-33和C-11均不能正常生长，杂交种N-33×C-11与G2-24生长正常，而且N-33×C-11种子100%能抗草甘膦。RT-PCR分析发现杂交种N-33×C-11中只能扩增出两个基因片段，不能扩增出完整基因。Western杂交和质谱分析证明杂交种N-33×C-11中含有完整的EPSPS蛋白。上述结果表明G2-aroA基因拆分后，在intein介导下形成了完整有功能的EPSPS蛋白，且在植株水平上的重新组装效率可达100%，能够满足实际生产的需要。
5）在人工授粉的情况下，将N-33×C-11与受体NC89回交，回交种进行草甘膦抗性分析，共筛选2812株，得到草甘膦抗性植株664个，抗性植株所占比例为23.61%，符合两个基因片段位于不同染色体上时，基因飘流频率为25%的假设。表明基因拆分技术至少能降低75%的基因飘流频率，即使在100%异交率情况下，基因飘流频率也只有23.61%。
2.水稻中建立基因拆分技术限控转基因飘流的技术体系
1)构建了用于水稻遗传转化的高效表达载体13UEI（含有EPSPSn-In）、13UIC（含有Ic-EPSPSc）和13UG2（含有完整的G2-aroA），并通过农杆菌介导法导入水稻品种中花11中，分别获得转基因水稻En（含有EPSPSn-In融合基因片段）15株，Ec （含有Ic-EPSPSc融合基因片段）14株，G_2（含有完整G_2-aroA基因）14株。
2)通过Southern blot分析确定En-1、En-3、En-12、En-19、En-31、En-33、En-34、Ec-2、Ec-5、Ec-8、G_2-6、G_2-7、G_2-10为单拷贝插入。
3)利用Tail-PCR方法对单拷贝的转基因水稻进行了侧翼序列克隆，并进行染色体定位分析，确定了En-1、En-3、En-19和En-31中外源基因分别插入到2号、11号、1号和7号染色体上，Ec-2、Ec-5和Ec-8中外源基因分别插入到4号、3号和9号染色体上。
4)通过荧光定量PCR方法对外源基因在转基因水稻中的表达量进行了分析，表明外源基因在Ec中的表达为Ec-2＞Ec-5＞Ec-8，外源基因在En中的表达情况为En-12＞En-3＞En-1＞En-34＞En-33＞En-31。
综上所述，本研究在烟草上建立了利用基因拆分技术限控转基因飘流的技术平台。同时对在水稻中利用基因拆分技术限控转基因飘流进行了技术储备。在水稻实际应用中，可以将目的基因拆分成两个片段，其中一个片段导入恢复系中，另一个片段导入保持系中。这样，只有杂交种中才具有完整有功能的目的蛋白，而亲本中的基因片段不具有功能，不会对环境产生影响，从而为在杂交稻上采用转基因技术铺平道路，并对保持我国杂交稻研究和生产在国际上的领先地位，进一步促进我国转基因水稻的商业化具有重要意义。
With the rapid development and commercialization of GMC (Genetically Modified Crop), thepotential environmental problem caused by transgene flow from GMC to related plants has become oneof the focuses of public concern. The best strategy for promoting public acceptance of transgenicproducts is to eliminate transgene flow fundamentally. Existing physical technologies such as distanceisolation, florescence isolation or physical barrier can only reduce the frequency of transgene flow to acertain extent. So, it is important to develop new biological strategy with more satisfied efficiency oncontrolling transgene flow. In this study, the G2-aroA gene, conferring resistance to herbicideglyphosate, was used to study the transgene split technology and the reassembling efficiency of theprotein via intein-mediated protein-splicing and to explore the effect of gene split technology oncontrolling transgene flow in tobacco and rice, in the hope of establishing a technology platform tocontrol transgene flow.
The main results obtained are as follows:
1. Gene split technology to controlling transgene flow on tobacco::
1） Seeds of T_0generation of transgenic tobacco, which harboring the integrated G2-aroA(EPSPS),EPSPSn-In and IC-EPSPSC, respectively, were generated on kanamycin medium to analyzethe segregation ratio of KanR/KanS. It was indicated that transgenic plants N-33、C-11andG2-24were integrated with single copy of foreign gene according to the segregation ratio ofKanR/KanSand reconfirmed through southern blot and quantitative Real-time PCR study.
2） Through self-polination and screening with kanamycin of transgenic tobacco plants,homozygous line of N-33and C-11were obtained. F1seeds of N-33×C-11were also obtainedby hybridization.
3） The flanking sequence of inserted genes in transgenic tobacco plants N-33and C-11werecloned with Tail-PCR and it was preliminary indicated that integration of foreign genes ofEPSPSn-In and Ic-EPSPSc were occurred at different chromosomes. The KanR/KanSsegregation ratio of self-pollinated seeds from N-33×C-11was analyzed. Among3096seedlings tested,1880seedlings were KanRand the resistant proportion was60.7%.According to the basic rules of genetics, we further confirmed that the two fusion genesEPSPSn-In and Ic-EPSPSc were inserted into two non-homologous chromosomes.
4） The glyphosate resistance of different types of transgenic tobacco was analyzed. It wasindicated that both N-33and C-11as well as the non-transgenic tobacco NC89weresusceptible to glyphosate, while100%seedlings from hybrid N-33×C-11harboring bothEPSPSn-In and Ic-EPSPSc showed the same resistance to glyphosate as the positive controlG2-24. RT-PCR analysis showed that each of the two gene fragments can be amplified fromhybrid N-33×C-11, but the complete gene can’t be found in hybrid. It was proved through Western blot and mass spectral analysis that the hybrid N-33×C-11plants contain thecomplete EPSPS protein. The above results showed that the two fragments of G2-aroA genecan be complemented and EPSPS activity can be recovered by intein mediated proteincomplementation, with a assembly efficiency up to100%in plant level, which can meet theneeds of practical application.
5）The glyphosate resistance of the seedlings from back crossing generation of hybrid N-33×C-11and non-transgenic control NC89was analyzed. Among2812seedlings totally tested,664seedlings or23.61%of the total seedlings showed resistance to glyphosate, which fit to thehypothesis that the transgenic flow rate is25%when the two fragments were integrated intotwo non-homologous chromosomes. The results suggest that the transgene split technologycan at least reduce the gene flow frequence of75%.
2. Constructing a gene split technology system for controlling transgene flow in rice
1）Plant expression vectors13UG2,13UEI and13UIC, harboring the integrated G2-aroA(EPSPS),EPSPSn-In and IC-EPSPSC, respectively, were constructed and transformed into rice byagrobacterium-mediated gene transfer and14,15,14transgenic rice plants were obtainedrespectively.
2）Results from Southern-blot analysis indicated that transgenic rice plants En-1、En-3、En-12、En-19、En-31、En-33、En-34、Ec-2、Ec-5、Ec-8、G2-6、G2-7、G2-10were integrated withone copy of target gene.
3）The flank sequence of transgenic rice were cloned by Tail-PCR, and chromosome localizationanalysis showed that the foreign genes in transgenic rice En-1, En-3, En-19, En-31, Ec-2,Ec-5and Ec-8were localized in chromosome2,11,1,7,4,3and9respectively.
4）The quantity of foreign protein expressed in transgenic rice was indentified via Real-timequantitative PCR. The foreign protein expressing level from high to low is Ec-2, Ec-5andEc-8respectively for Ec line, while in the En line, the order is En-12, En-3, En-1, En-34,En-33and En-31.
In summary, our study on tobacco has established a technological platform to control trangene flowby using gene split technology and laid the foundation for the same study in transgenic rice. In thepractical application in rice, the target gene can be split into two fragments. One fragment may beintroduced into the restore line, while another introduced into the maintainer line. There for, only thehybrids can produce complete and functional target protein, and the parents with only one fragment hasno function and will not impact on the environment. Thus it paving the way for using trangenictechnology on hybrid rice, and to maintain China’s leading position in hybrid rice research andproduction. It will has significant benefits for commercialization of transgenic rice in the future.
1.烟草中进行基因拆分技术限控转基因飘流的效果分析
1）将单独含有完整G_2-aroA、G_2-aroA的N端（EPSPSn-In）或G2-aroA的C端（IC-EPSPSC）基因的转基因烟草T_0代种子接种到含有100mg/L卡那霉素的MS0培养基上，分析Kan~R与KanS的分离比，初步确定单独含有EPSPSn-In的N-33、单独含有IC-EPSPSC的C-11和含有完整G2-aroA基因的G2-24为单拷贝插入。利用Southern杂交和荧光定量PCR对其进行分析，进一步确定了转基因烟草N-33、C-11和G2-24为单拷贝插入。
2）连续2代筛选，获得了转基因烟草N-33和C-11的纯合系，并通过有性杂交获得了同时含有2个基因片段的转基因烟草杂交种N-33×C-11。
3）利用Tail-PCR的方法获得了N-33、C-11的侧翼序列，初步分析二者插入到了不同染色体上。另外，杂交种N-33×C-11自交，获得的自交种进行卡那抗性筛选，共分析3096株，其中非卡那抗性苗为188株，所占比例约为6.07%，符合两个基因片段位于不同染色体上时的孟德尔遗传规律，进一步确定拆分后的EPSPSn-In和Ic-EPSPSc基因片段位于非同源染色体上。
4）对不同类型的转基因烟草进行草甘膦抗性分析，结果表明非转基因烟草NC89以及单独含有1个基因片段的转基因烟草N-33和C-11均不能正常生长，杂交种N-33×C-11与G2-24生长正常，而且N-33×C-11种子100%能抗草甘膦。RT-PCR分析发现杂交种N-33×C-11中只能扩增出两个基因片段，不能扩增出完整基因。Western杂交和质谱分析证明杂交种N-33×C-11中含有完整的EPSPS蛋白。上述结果表明G2-aroA基因拆分后，在intein介导下形成了完整有功能的EPSPS蛋白，且在植株水平上的重新组装效率可达100%，能够满足实际生产的需要。
5）在人工授粉的情况下，将N-33×C-11与受体NC89回交，回交种进行草甘膦抗性分析，共筛选2812株，得到草甘膦抗性植株664个，抗性植株所占比例为23.61%，符合两个基因片段位于不同染色体上时，基因飘流频率为25%的假设。表明基因拆分技术至少能降低75%的基因飘流频率，即使在100%异交率情况下，基因飘流频率也只有23.61%。
2.水稻中建立基因拆分技术限控转基因飘流的技术体系
1)构建了用于水稻遗传转化的高效表达载体13UEI（含有EPSPSn-In）、13UIC（含有Ic-EPSPSc）和13UG2（含有完整的G2-aroA），并通过农杆菌介导法导入水稻品种中花11中，分别获得转基因水稻En（含有EPSPSn-In融合基因片段）15株，Ec （含有Ic-EPSPSc融合基因片段）14株，G_2（含有完整G_2-aroA基因）14株。
2)通过Southern blot分析确定En-1、En-3、En-12、En-19、En-31、En-33、En-34、Ec-2、Ec-5、Ec-8、G_2-6、G_2-7、G_2-10为单拷贝插入。
3)利用Tail-PCR方法对单拷贝的转基因水稻进行了侧翼序列克隆，并进行染色体定位分析，确定了En-1、En-3、En-19和En-31中外源基因分别插入到2号、11号、1号和7号染色体上，Ec-2、Ec-5和Ec-8中外源基因分别插入到4号、3号和9号染色体上。
4)通过荧光定量PCR方法对外源基因在转基因水稻中的表达量进行了分析，表明外源基因在Ec中的表达为Ec-2＞Ec-5＞Ec-8，外源基因在En中的表达情况为En-12＞En-3＞En-1＞En-34＞En-33＞En-31。
综上所述，本研究在烟草上建立了利用基因拆分技术限控转基因飘流的技术平台。同时对在水稻中利用基因拆分技术限控转基因飘流进行了技术储备。在水稻实际应用中，可以将目的基因拆分成两个片段，其中一个片段导入恢复系中，另一个片段导入保持系中。这样，只有杂交种中才具有完整有功能的目的蛋白，而亲本中的基因片段不具有功能，不会对环境产生影响，从而为在杂交稻上采用转基因技术铺平道路，并对保持我国杂交稻研究和生产在国际上的领先地位，进一步促进我国转基因水稻的商业化具有重要意义。
With the rapid development and commercialization of GMC (Genetically Modified Crop), thepotential environmental problem caused by transgene flow from GMC to related plants has become oneof the focuses of public concern. The best strategy for promoting public acceptance of transgenicproducts is to eliminate transgene flow fundamentally. Existing physical technologies such as distanceisolation, florescence isolation or physical barrier can only reduce the frequency of transgene flow to acertain extent. So, it is important to develop new biological strategy with more satisfied efficiency oncontrolling transgene flow. In this study, the G2-aroA gene, conferring resistance to herbicideglyphosate, was used to study the transgene split technology and the reassembling efficiency of theprotein via intein-mediated protein-splicing and to explore the effect of gene split technology oncontrolling transgene flow in tobacco and rice, in the hope of establishing a technology platform tocontrol transgene flow.
The main results obtained are as follows:
1. Gene split technology to controlling transgene flow on tobacco::
1） Seeds of T_0generation of transgenic tobacco, which harboring the integrated G2-aroA(EPSPS),EPSPSn-In and IC-EPSPSC, respectively, were generated on kanamycin medium to analyzethe segregation ratio of KanR/KanS. It was indicated that transgenic plants N-33、C-11andG2-24were integrated with single copy of foreign gene according to the segregation ratio ofKanR/KanSand reconfirmed through southern blot and quantitative Real-time PCR study.
2） Through self-polination and screening with kanamycin of transgenic tobacco plants,homozygous line of N-33and C-11were obtained. F1seeds of N-33×C-11were also obtainedby hybridization.
3） The flanking sequence of inserted genes in transgenic tobacco plants N-33and C-11werecloned with Tail-PCR and it was preliminary indicated that integration of foreign genes ofEPSPSn-In and Ic-EPSPSc were occurred at different chromosomes. The KanR/KanSsegregation ratio of self-pollinated seeds from N-33×C-11was analyzed. Among3096seedlings tested,1880seedlings were KanRand the resistant proportion was60.7%.According to the basic rules of genetics, we further confirmed that the two fusion genesEPSPSn-In and Ic-EPSPSc were inserted into two non-homologous chromosomes.
4） The glyphosate resistance of different types of transgenic tobacco was analyzed. It wasindicated that both N-33and C-11as well as the non-transgenic tobacco NC89weresusceptible to glyphosate, while100%seedlings from hybrid N-33×C-11harboring bothEPSPSn-In and Ic-EPSPSc showed the same resistance to glyphosate as the positive controlG2-24. RT-PCR analysis showed that each of the two gene fragments can be amplified fromhybrid N-33×C-11, but the complete gene can’t be found in hybrid. It was proved through Western blot and mass spectral analysis that the hybrid N-33×C-11plants contain thecomplete EPSPS protein. The above results showed that the two fragments of G2-aroA genecan be complemented and EPSPS activity can be recovered by intein mediated proteincomplementation, with a assembly efficiency up to100%in plant level, which can meet theneeds of practical application.
5）The glyphosate resistance of the seedlings from back crossing generation of hybrid N-33×C-11and non-transgenic control NC89was analyzed. Among2812seedlings totally tested,664seedlings or23.61%of the total seedlings showed resistance to glyphosate, which fit to thehypothesis that the transgenic flow rate is25%when the two fragments were integrated intotwo non-homologous chromosomes. The results suggest that the transgene split technologycan at least reduce the gene flow frequence of75%.
2. Constructing a gene split technology system for controlling transgene flow in rice
1）Plant expression vectors13UG2,13UEI and13UIC, harboring the integrated G2-aroA(EPSPS),EPSPSn-In and IC-EPSPSC, respectively, were constructed and transformed into rice byagrobacterium-mediated gene transfer and14,15,14transgenic rice plants were obtainedrespectively.
2）Results from Southern-blot analysis indicated that transgenic rice plants En-1、En-3、En-12、En-19、En-31、En-33、En-34、Ec-2、Ec-5、Ec-8、G2-6、G2-7、G2-10were integrated withone copy of target gene.
3）The flank sequence of transgenic rice were cloned by Tail-PCR, and chromosome localizationanalysis showed that the foreign genes in transgenic rice En-1, En-3, En-19, En-31, Ec-2,Ec-5and Ec-8were localized in chromosome2,11,1,7,4,3and9respectively.
4）The quantity of foreign protein expressed in transgenic rice was indentified via Real-timequantitative PCR. The foreign protein expressing level from high to low is Ec-2, Ec-5andEc-8respectively for Ec line, while in the En line, the order is En-12, En-3, En-1, En-34,En-33and En-31.
In summary, our study on tobacco has established a technological platform to control trangene flowby using gene split technology and laid the foundation for the same study in transgenic rice. In thepractical application in rice, the target gene can be split into two fragments. One fragment may beintroduced into the restore line, while another introduced into the maintainer line. There for, only thehybrids can produce complete and functional target protein, and the parents with only one fragment hasno function and will not impact on the environment. Thus it paving the way for using trangenictechnology on hybrid rice, and to maintain China’s leading position in hybrid rice research andproduction. It will has significant benefits for commercialization of transgenic rice in the future.
引文
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