植物基因安全转化体系关键技术的研究
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摘要
转基因植物安全性问题一直是各界关注的问题,本研究利用分子生物学技术优化改造传统的植物表达载体,以获得无选择标记基因、无目的基因和无目的蛋白的转基因产品,从而减轻转基因植物带来的安全性问题。
利用PCR方法扩增玉米乙酰苯胺类化合物诱导启动子(In5-2启动子),并与GUS基因相连构建植物表达载体,转化烟草获得转基因烟草植株。在不同诱导时间和不同佐剂浓度条件下,对转基因烟草的GUS活性进行分析。结果表明,诱导剂的存在与GUS基因的表达呈正相关,且随着诱导时间的延长和佐剂浓度的增加,GUS活性均呈增长的趋势。利用PCR技术获得6个不同长度的玉米In5-2启动子5′端缺失片段,分别克隆到植物表达载体p1301中构建一系列植物表达载体,转化烟草获得转基因植株。GUS活性定量检测结果显示,玉米In5-2启动子的核心功能区可能位于ATG上游-1520至-1431bp之间。利用DNAMAN软件分析,在-1108至-1079bp和-891至-864bp之间存在着两个茎环结构,推测茎环结合蛋白位点可能位于-1079至-897bp之间,且ATG上游-388至-86bp之间可能存在乙酰苯胺类化合物诱导元件。
构建诱导型Cre/lox基因删除系统,并在转基因烟草中进行验证。即在诱导剂的处理下,烟草愈伤组织中发生重组反应,基因组中的选择标记基因被删除。在该系统中,乙酰苯胺诱导启动子(In5-2)控制Cre基因的表达。对转基因烟草植株进行分子检测的结果表明,不论标记基因是否被删除,目的基因(gus)均整合到烟草基因组中;在48株To代转基因烟草植株中,有45株是无选择标记基因(hpt)的。该系统只有一个载体,克服了双载体重组系统的缺点。
设计了一个“3-free”系统,用于生产无选择标记基因、无目的基因及其蛋白的转基因植物产品。该系统由三部分组成,即R/RS无标记基因转化体系、绿色组织特异性基因表达系统、诱导型Cre/lox基因删除体系。以转Cry1Ac基因烟草为模型对该系统进行了验证。结果显示,选择标记基因在组织培养阶段被删除了;目的基因在除草剂安全剂田间喷施后是可以被删除的,其删除效率为20%;且由于目的基因的删除,目的蛋白在转基因烟草的叶和根中逐渐降解;在种子中没有检测到目的蛋白。该系统为解决转基因植物生物安全性问题提供了一个新的方向。
利用PCR方法克隆了苏云金芽孢杆菌毒蛋白水解酶基因(nprA),并根据植物密码子偏好性对其进行改造,进行全基因人工合成,命名为mnprA基因。分别将两个基因连入植物表达载体p1301构建植物表达载体,并转化Cry1AC转基因烟草,希望nprA(或mnprA)基因的表达产物在转基因烟草中能将Cry1Ac蛋白水解。然而,我们没有能够得到突变植株,说明nprA基因可能是一个非专一性水解酶基因。
The biosafety of transgenic plants was always a key problem in the world. We transformated the tranditional plant expressed vector by biotechnology, to obtain transgenic product without selectable marker gene, target gene and target peotein.The DNA fragment of chloroacetanilide-induced promoter (In5-2) was amplified by PCR method from maize genome and fused with GUS gene to construct the expression vector pCGUS for tobacco transformation. The results showed that the GUS gene was expressed under chemical induction in different adjuvant concentration and inducing time, in which the GUS activity increased correlatively with the chemical concentration and decreased when the inducing time was elongated. Maize In5-2 promoter was induced by herbicide-safener. The boundaries required for maximal promoter expression were defined by 5' deletion analysis of the maize In5-2 promoter fragments coupled to a GUS reporter gene. The expression patterns of these chimeric gene constructs were evaluated in transgenic Nicotiana tabacum. Analysis of the 5' deletion constructs showed the regulation region of its basic transcription was mainly within - 1520 to - 1431 bp upstream of ATG Two stem-loop structures were within - 1108 to - 1079 and - 891 to - 864 bp relative to ATG It suggests that their protein binding site is between - 1079 to - 897 bp relative to ATG. In addition, cis-acting elements responsive to chloroacetanilide could be mainly located within the range of - 388 to - 86 bp upstream of ATGA new inducible Cre/lox system was constructed in transgenic tobacco plants. The inducer-treatment of tobacco callus mediates an excision event in which the selectable marker gene and Cre gene between two lox sites were deleted. A chloroacetanilide-induced promoter (In5-2) was used to control the expression of Cre gene in this system. Molecular analysis of transgenic tobacco plants showed the interest gene, gus, was integrated into the genome whether removing has been successful, and forty-five out of forty-eight T0 plants were transgenic tobacco without the marker gene, hpt. This system uses a single vector to circumvent the flaw of other dual recombinase vector systems.A new transgenic plant product that has not marker gene, target gene and target protein was obtained. It is derived from a 3-free system, the novel system included R/RS marker-free system, green tissue- specific expression system and chemical-inducible Cre/lox target gene excision system. The system was tested with the transgenic CrylAc tobacco. And the results showed that the selectable marker gene was deleted in medium culture, the target gene was moved from plant genome after induced by spraying of herbicide safener, the target protein in the leaf and root was degraded. This system could provide a new way to resolve the problem of biosafety in transgenic plants.The non-transgenic nprA (neutral protease A) gene was amplified by PCR, and it was modified according to plant code bias. The modified nprA gene was artificially synthesized, and the synthesized nprA gene was renamed mnprA gene. The two genes were cloned into plant expressed vector pl301 to transform the transgneic CrylAc tobacco. While no mutant plant was obtained. Maybe because the
nprA protein was not specific proteinase.
利用PCR方法扩增玉米乙酰苯胺类化合物诱导启动子(In5-2启动子),并与GUS基因相连构建植物表达载体,转化烟草获得转基因烟草植株。在不同诱导时间和不同佐剂浓度条件下,对转基因烟草的GUS活性进行分析。结果表明,诱导剂的存在与GUS基因的表达呈正相关,且随着诱导时间的延长和佐剂浓度的增加,GUS活性均呈增长的趋势。利用PCR技术获得6个不同长度的玉米In5-2启动子5′端缺失片段,分别克隆到植物表达载体p1301中构建一系列植物表达载体,转化烟草获得转基因植株。GUS活性定量检测结果显示,玉米In5-2启动子的核心功能区可能位于ATG上游-1520至-1431bp之间。利用DNAMAN软件分析,在-1108至-1079bp和-891至-864bp之间存在着两个茎环结构,推测茎环结合蛋白位点可能位于-1079至-897bp之间,且ATG上游-388至-86bp之间可能存在乙酰苯胺类化合物诱导元件。
构建诱导型Cre/lox基因删除系统,并在转基因烟草中进行验证。即在诱导剂的处理下,烟草愈伤组织中发生重组反应,基因组中的选择标记基因被删除。在该系统中,乙酰苯胺诱导启动子(In5-2)控制Cre基因的表达。对转基因烟草植株进行分子检测的结果表明,不论标记基因是否被删除,目的基因(gus)均整合到烟草基因组中;在48株To代转基因烟草植株中,有45株是无选择标记基因(hpt)的。该系统只有一个载体,克服了双载体重组系统的缺点。
设计了一个“3-free”系统,用于生产无选择标记基因、无目的基因及其蛋白的转基因植物产品。该系统由三部分组成,即R/RS无标记基因转化体系、绿色组织特异性基因表达系统、诱导型Cre/lox基因删除体系。以转Cry1Ac基因烟草为模型对该系统进行了验证。结果显示,选择标记基因在组织培养阶段被删除了;目的基因在除草剂安全剂田间喷施后是可以被删除的,其删除效率为20%;且由于目的基因的删除,目的蛋白在转基因烟草的叶和根中逐渐降解;在种子中没有检测到目的蛋白。该系统为解决转基因植物生物安全性问题提供了一个新的方向。
利用PCR方法克隆了苏云金芽孢杆菌毒蛋白水解酶基因(nprA),并根据植物密码子偏好性对其进行改造,进行全基因人工合成,命名为mnprA基因。分别将两个基因连入植物表达载体p1301构建植物表达载体,并转化Cry1AC转基因烟草,希望nprA(或mnprA)基因的表达产物在转基因烟草中能将Cry1Ac蛋白水解。然而,我们没有能够得到突变植株,说明nprA基因可能是一个非专一性水解酶基因。
The biosafety of transgenic plants was always a key problem in the world. We transformated the tranditional plant expressed vector by biotechnology, to obtain transgenic product without selectable marker gene, target gene and target peotein.The DNA fragment of chloroacetanilide-induced promoter (In5-2) was amplified by PCR method from maize genome and fused with GUS gene to construct the expression vector pCGUS for tobacco transformation. The results showed that the GUS gene was expressed under chemical induction in different adjuvant concentration and inducing time, in which the GUS activity increased correlatively with the chemical concentration and decreased when the inducing time was elongated. Maize In5-2 promoter was induced by herbicide-safener. The boundaries required for maximal promoter expression were defined by 5' deletion analysis of the maize In5-2 promoter fragments coupled to a GUS reporter gene. The expression patterns of these chimeric gene constructs were evaluated in transgenic Nicotiana tabacum. Analysis of the 5' deletion constructs showed the regulation region of its basic transcription was mainly within - 1520 to - 1431 bp upstream of ATG Two stem-loop structures were within - 1108 to - 1079 and - 891 to - 864 bp relative to ATG It suggests that their protein binding site is between - 1079 to - 897 bp relative to ATG. In addition, cis-acting elements responsive to chloroacetanilide could be mainly located within the range of - 388 to - 86 bp upstream of ATGA new inducible Cre/lox system was constructed in transgenic tobacco plants. The inducer-treatment of tobacco callus mediates an excision event in which the selectable marker gene and Cre gene between two lox sites were deleted. A chloroacetanilide-induced promoter (In5-2) was used to control the expression of Cre gene in this system. Molecular analysis of transgenic tobacco plants showed the interest gene, gus, was integrated into the genome whether removing has been successful, and forty-five out of forty-eight T0 plants were transgenic tobacco without the marker gene, hpt. This system uses a single vector to circumvent the flaw of other dual recombinase vector systems.A new transgenic plant product that has not marker gene, target gene and target protein was obtained. It is derived from a 3-free system, the novel system included R/RS marker-free system, green tissue- specific expression system and chemical-inducible Cre/lox target gene excision system. The system was tested with the transgenic CrylAc tobacco. And the results showed that the selectable marker gene was deleted in medium culture, the target gene was moved from plant genome after induced by spraying of herbicide safener, the target protein in the leaf and root was degraded. This system could provide a new way to resolve the problem of biosafety in transgenic plants.The non-transgenic nprA (neutral protease A) gene was amplified by PCR, and it was modified according to plant code bias. The modified nprA gene was artificially synthesized, and the synthesized nprA gene was renamed mnprA gene. The two genes were cloned into plant expressed vector pl301 to transform the transgneic CrylAc tobacco. While no mutant plant was obtained. Maybe because the
nprA protein was not specific proteinase.
引文
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