苜蓿体细胞胚作为生物反应器表达外源蛋白的研究
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摘要
苜蓿（Medicago Sativa L.）是世界上分布最广泛的一种豆科多年生牧草，素有“牧草之王”的美誉。随着分子生物学的发展，传统农业正在向分子农业转变。植物生物反应器作为一种大规模重组蛋白的生产系统，已广泛运用于工业、农业尤其是生命科学以及医学制造领域。
与微生物发酵、动物细胞和转基因动物等生产系统相比，苜蓿作为一种生物反应器,为人类提供了一个更加安全和廉价的生产体系。它具有操作方便、便于大面积推广等许多潜在的优势。利用转基因苜蓿作为植物生物反应器生产工业酶制剂、疫苗、药用蛋白等的操作路线与现在的苜蓿转基因技术路线相同，即目的基因的克隆→载体构建→苜蓿外植体的转化→转基因植株再生；只需在得到转基因植株以后，再从转基因植株中进行疫苗、酶制剂、药用蛋白等的下一步提取工作。论文针对外源蛋白在转基因苜蓿植物组织中表达量低、转基因苜蓿的种质保存困难的问题进行研究。
本论文选用农杆菌株系GV3101，含gus/npt II的双相载体pCAMBIA2301和pBin9，以N4.4.2作为转化受体进行转化,在卡那霉素筛选压下，经过一系列的诱导培养，形成转基因植株。然后，通过GUS组织化学定位分析转基因植株中的GUS表达，并进一步通过PCR分子检测。经分析检测确定为阳性植株后，对转基因植株进行体细胞胚诱导，通过GUS荧光定量分析不同组织中的GUS表达，评价外源GUS在苜蓿体细胞胚中的积累。同时，对获得的7个转基因苜蓿株系进行体细胞胚的诱导，随后利用外源ABA和6种干燥剂处理来获得转基因苜蓿人工种子，考察不同株系转基因苜蓿人工种子的萌发率和遗传特性。以上研究为苜蓿体细胞胚作为植物反应器生产外源有用蛋白和转基因人工种质保存提供理论依据。主要结论如下：
1.进一步完善了苜蓿转化体系。以N4.4.2为转化受体，感染的子叶期苜蓿体细胞胚在75mg/L卡那霉素筛选压下，经过一系列诱导培养，最终获得转基因植株。然后，通过GUS组织化学定位分析检测转基因植株不同组织器官中的GUS表达，并进一步通过PCR和Southern杂交确定转基因的整合和转化率。结果证实：苜蓿次级体细胞胚的遗传转化法相对于叶柄、叶子和上胚轴等为外植体的常规的转化方法，缩短了苜蓿转化的时间；次级体细胞胚作为外植体的遗传转化方法大大提高了次级体细胞胚的诱导频率；它还提供了一个导入多基因到苜蓿植株中的方法。同时，这种方法也适用于别的转化效率低的一些苜蓿品种，拓宽了苜蓿遗传转化方法。
2.证明了苜蓿体细胞胚是最佳的外源蛋白表达材料，为苜蓿体细胞胚作为生物反应器生产外源蛋白提供理论依据。以N4.4.2紫花苜蓿品种叶柄为材料，进行农杆菌GV3101介导的苜蓿遗传转化。农杆菌株系GV3101包括双向载体pBin9。这个载体含有2个CaMV35s启动子调控下的gus/npt II融合基因。感染的叶柄外植体置于不同的转化培养基中培养，形成了适合于农杆菌介导的转基因体系。获得的转基因植株，采用组织化学定位分析GUS表达，进一步的PCR方法检测其中11个抗性株系，并对表现为阳性的转基因植株的不同组织器官进行GUS荧光定量分析。结果表明：GUS外源蛋白表达量在体细胞胚中明显高于根、茎和叶，是根、茎和叶的3~6倍；而在根、茎和叶中GUS表达量相似，没有明显的差异。
3.本试验建立了使用干燥的转基因苜蓿体细胞胚作为种质保藏的模式体系。将转基因苜蓿植株进行体细胞胚的诱导，随后,子叶期的体细胞胚在含有10mM ABA的BOi2Y培养基中培养14d，随后，体细胞胚依次放在相对湿度恒定的饱和溶液中：硫酸钾饱和溶液（87%）、碳酸钠饱和溶液（87%）、氯化钠饱和溶液（75%）、硝酸铵饱和溶液（63%）、四水硝酸钙饱和溶液（50%）、二水碳酸钾饱和溶液（43%）6种干燥剂中缓慢干燥，获得干化的转基因体细胞胚。干化的体细胞在MSO培养基中萌发，获得转基因苜蓿植株。结果表明：干化的体细胞胚和未干化的体细胞胚具有相似的萌发率，恢复的植株在形态上相似。经过Southern杂交分析遗传变异特性，源自干化的和未干燥的苜蓿体细胞胚恢复的植株具有相同的整合位点，研究证实了干燥的体细胞胚可以用于转基因苜蓿种质保藏的可行性。
4.35s CaMV是一种强启动子，通常用于转基因的组成型表达调控。在苜蓿转化试验中还发现，使用2个35s CaMV启动子调控GUS表达时，明显高于单个35s CaMV启动子调控的GUS表达。为了获得高水平的外源蛋白表达，可以使用双强35s CaMV启动子的载体。
Alfalfa (Medicago sativa L.) is important, perennial and leguminous forage and is widelydistributed in the world with the reputation of “King Forage”. With the development ofmolecular biology, the traditional farming is gradually transiting to the molecular farming. Theplant bioreactor has been extensively used as the scale production system of recombinantprotein in field of industry and agriculture, especially in life science and medicinalmanufacture.
Alfalfa as a bioreactor provided a more safe and cheap production system of recombinantprotein comparing with which used the microbe fermentation, animal cell or transgenicanimal as bioreactor. Alfalfa production system has more potential superiority such as easyoperation and easy spread to large area. The application of transgenic alfalfa as a plantbioreactor has the similar technical route with the operation process of current transgenicalfalfa in the production of vaccine, industry enzyme preparation and pharmaceutical protein.That is to say: Clone of interest gene→construction of vector→alfalfa transformation in vitro→Regeneration of transgenic alfalfa; Then we may extract the vaccine, enzyme preparation,pharmaceutical protein from these transgenic plants. This paper focused on the issue of lowexogenous protein expression in transgenic plants and difficult storage of transgenic alfalfagermplasm.
The Agrobacterium strain GV3101contained binary vector pCAMBIA2301and pBin19which had gus gene as reporter and nptⅡ gene as selectable marker. N4.4.2genotype(Medicago sativa) as donor plants was induced through series of medium under75mg/Lkanamycin selection. And we obtained the transgenic alfalfa plants. Then, GUS expression indifferent tissue of transgenic alfalfa was tested by GUS histochemical analysis. Further, thestable integration and transformation efficiency were tested by polymerase chain reaction. Toevaluate the accumulation of the exogenous GUS in somatic embryos, the somatic embryoswere induced from transgenic plantlets after the positive transgenic alfalfa plantlets wereconfirmed by GUS stain and polymerase chain reaction. And we compare the expression ofGUS in different transgenic alfalfa tissue (roots, stems, leaves and somatic embryos) by GUSfluorescence assay. At the same time, somatic embryos were induced from3transgenic alfalfa lines. Subsequently, the artificial transgenic alfalfa seeds were obtained by exogenousABA and6series of desiccators. The germination rate and genetic characteristics oftransgenic artificial alfalfa seeds was investigated. These studies provide the theoretical basisfor alfalfa somatic embryos used as bioreactor to produce exogenous pharmaceutical proteinand for artificial transgenic alfalfa used as germplasm storage. The followings are the majorconclusions:
1. The alfalfa transformation system was further improved. N4.4.2alfalfa genotype wasused as donor plants. The infected cotyledonary-stage embryos were induced through seriesof medium under75mg/L kanamycin selection. And we obtained the transgenic alfalfa plants.Then, GUS expression in different tissue of transgenic alfalfa was tested by GUShistochemical analysis. Further, the stable integration and transformation efficiency weretested by polymerase chain reaction and Southern blot hybridization. The result showed thatthe protocol of genetic transformation via secondary somatic embryos can shorten alfalfatransformation time than the protocol of genetic transformation via explants of petiole, leavesand hypocotyls and so on. The protocol significantly improved the induce frequency ofsecondary somatic embryos and provide a method of transferring multigene to alfalfa plants.At the same time, the protocol is also suited to those alfalfa genotypes which have lowtransformation efficiency to extend the method of genetic alfalfa transformation.
2. Alfalfa somatic embryos are the optimal materials of exogenous protein expressionwhich provides theoretical basis for alfalfa somatic embryos used as plant bioreactor toproduce recombinant protein. In this paper, petiole explants from N4.4.2alfalfa genotype astransformation materials were induced by Agrobacterium tumefaciens strain GV3101whichharbors the plasmid pBin9. pBin9vector contains gus/npt II fusion gene Both genes weredriven by double35s CaMV35S promoter. Infected petiole explants were cultured in differentmedia and the alfalfa tissue culture and transgenic system was developed. We tested11resistant Kanamycin alfalfa lines selected from the recovered transgenic plantlets by GUSstain and PCR. Moreover, we tested GUS activity expression in different tissue in transgenicalfalfa by GUS fluorometric measure in positive plantlets. The result showed that the amountof exogenous protein GUS expression in somatic embryos is significantly higher than in othertissue (roots, stems and leaves). The amount in somatic embryos is3~6folds than in roots,stems and leaves. However, the amount of GUS expression was no significant difference inroots, stems and leaves. Alfalfa somatic embryos can accumulate higher exogenous protein.
3. In this paper, we set up a germplasm preservation model system from desiccationsomatic embryos of transgenic alfalfa. The somatic embryos were induced original from thetransgenic pants and the cotyledonary-stage somatic embryos were cultured on a maturation medium supplemented with10mM abscisic acid for14d. Then somatic embryos weredesiccated in a series of desiccators K2SO4(RH87%), Na2CO3(RH87%), NaCl (RH75%), NH4NO3(RH63%), Ca (NO3)2.4H2O (RH51%), and K2CO3.2H2O (RH43%) over1week to slowly dry.Finally, the desiccation somatic embryos treated by the MSO medium were germinated totransgenic plantlets. Our result showed that the germination rate of transgenic somaticembryos is74.3%after desiccation. The recovery transgenic alfalfa posterity from desiccationsomatic embryos could express stable GUS. In addition, the plants produced were similarmorphology. Moreover, southern hybridization has confirmed that integration of the gene inrecovery plant was consistent in three different alfalfa lines. So these findings have proved thefeasibility of using desiccation somatic embryos as germplasm preservation method fortransgenic plants.
4.35s CaMV is a constitutive, strong promoter and is used for induce of constitutiveexpression in the transgenic plants. In this paper, the result show that GUS expression ishigher by double35s CaMV promoter than single one. To obtain higher exogenous proteinexpression, The vector with double35s CaMV promoter can be possible.
与微生物发酵、动物细胞和转基因动物等生产系统相比，苜蓿作为一种生物反应器,为人类提供了一个更加安全和廉价的生产体系。它具有操作方便、便于大面积推广等许多潜在的优势。利用转基因苜蓿作为植物生物反应器生产工业酶制剂、疫苗、药用蛋白等的操作路线与现在的苜蓿转基因技术路线相同，即目的基因的克隆→载体构建→苜蓿外植体的转化→转基因植株再生；只需在得到转基因植株以后，再从转基因植株中进行疫苗、酶制剂、药用蛋白等的下一步提取工作。论文针对外源蛋白在转基因苜蓿植物组织中表达量低、转基因苜蓿的种质保存困难的问题进行研究。
本论文选用农杆菌株系GV3101，含gus/npt II的双相载体pCAMBIA2301和pBin9，以N4.4.2作为转化受体进行转化,在卡那霉素筛选压下，经过一系列的诱导培养，形成转基因植株。然后，通过GUS组织化学定位分析转基因植株中的GUS表达，并进一步通过PCR分子检测。经分析检测确定为阳性植株后，对转基因植株进行体细胞胚诱导，通过GUS荧光定量分析不同组织中的GUS表达，评价外源GUS在苜蓿体细胞胚中的积累。同时，对获得的7个转基因苜蓿株系进行体细胞胚的诱导，随后利用外源ABA和6种干燥剂处理来获得转基因苜蓿人工种子，考察不同株系转基因苜蓿人工种子的萌发率和遗传特性。以上研究为苜蓿体细胞胚作为植物反应器生产外源有用蛋白和转基因人工种质保存提供理论依据。主要结论如下：
1.进一步完善了苜蓿转化体系。以N4.4.2为转化受体，感染的子叶期苜蓿体细胞胚在75mg/L卡那霉素筛选压下，经过一系列诱导培养，最终获得转基因植株。然后，通过GUS组织化学定位分析检测转基因植株不同组织器官中的GUS表达，并进一步通过PCR和Southern杂交确定转基因的整合和转化率。结果证实：苜蓿次级体细胞胚的遗传转化法相对于叶柄、叶子和上胚轴等为外植体的常规的转化方法，缩短了苜蓿转化的时间；次级体细胞胚作为外植体的遗传转化方法大大提高了次级体细胞胚的诱导频率；它还提供了一个导入多基因到苜蓿植株中的方法。同时，这种方法也适用于别的转化效率低的一些苜蓿品种，拓宽了苜蓿遗传转化方法。
2.证明了苜蓿体细胞胚是最佳的外源蛋白表达材料，为苜蓿体细胞胚作为生物反应器生产外源蛋白提供理论依据。以N4.4.2紫花苜蓿品种叶柄为材料，进行农杆菌GV3101介导的苜蓿遗传转化。农杆菌株系GV3101包括双向载体pBin9。这个载体含有2个CaMV35s启动子调控下的gus/npt II融合基因。感染的叶柄外植体置于不同的转化培养基中培养，形成了适合于农杆菌介导的转基因体系。获得的转基因植株，采用组织化学定位分析GUS表达，进一步的PCR方法检测其中11个抗性株系，并对表现为阳性的转基因植株的不同组织器官进行GUS荧光定量分析。结果表明：GUS外源蛋白表达量在体细胞胚中明显高于根、茎和叶，是根、茎和叶的3~6倍；而在根、茎和叶中GUS表达量相似，没有明显的差异。
3.本试验建立了使用干燥的转基因苜蓿体细胞胚作为种质保藏的模式体系。将转基因苜蓿植株进行体细胞胚的诱导，随后,子叶期的体细胞胚在含有10mM ABA的BOi2Y培养基中培养14d，随后，体细胞胚依次放在相对湿度恒定的饱和溶液中：硫酸钾饱和溶液（87%）、碳酸钠饱和溶液（87%）、氯化钠饱和溶液（75%）、硝酸铵饱和溶液（63%）、四水硝酸钙饱和溶液（50%）、二水碳酸钾饱和溶液（43%）6种干燥剂中缓慢干燥，获得干化的转基因体细胞胚。干化的体细胞在MSO培养基中萌发，获得转基因苜蓿植株。结果表明：干化的体细胞胚和未干化的体细胞胚具有相似的萌发率，恢复的植株在形态上相似。经过Southern杂交分析遗传变异特性，源自干化的和未干燥的苜蓿体细胞胚恢复的植株具有相同的整合位点，研究证实了干燥的体细胞胚可以用于转基因苜蓿种质保藏的可行性。
4.35s CaMV是一种强启动子，通常用于转基因的组成型表达调控。在苜蓿转化试验中还发现，使用2个35s CaMV启动子调控GUS表达时，明显高于单个35s CaMV启动子调控的GUS表达。为了获得高水平的外源蛋白表达，可以使用双强35s CaMV启动子的载体。
Alfalfa (Medicago sativa L.) is important, perennial and leguminous forage and is widelydistributed in the world with the reputation of “King Forage”. With the development ofmolecular biology, the traditional farming is gradually transiting to the molecular farming. Theplant bioreactor has been extensively used as the scale production system of recombinantprotein in field of industry and agriculture, especially in life science and medicinalmanufacture.
Alfalfa as a bioreactor provided a more safe and cheap production system of recombinantprotein comparing with which used the microbe fermentation, animal cell or transgenicanimal as bioreactor. Alfalfa production system has more potential superiority such as easyoperation and easy spread to large area. The application of transgenic alfalfa as a plantbioreactor has the similar technical route with the operation process of current transgenicalfalfa in the production of vaccine, industry enzyme preparation and pharmaceutical protein.That is to say: Clone of interest gene→construction of vector→alfalfa transformation in vitro→Regeneration of transgenic alfalfa; Then we may extract the vaccine, enzyme preparation,pharmaceutical protein from these transgenic plants. This paper focused on the issue of lowexogenous protein expression in transgenic plants and difficult storage of transgenic alfalfagermplasm.
The Agrobacterium strain GV3101contained binary vector pCAMBIA2301and pBin19which had gus gene as reporter and nptⅡ gene as selectable marker. N4.4.2genotype(Medicago sativa) as donor plants was induced through series of medium under75mg/Lkanamycin selection. And we obtained the transgenic alfalfa plants. Then, GUS expression indifferent tissue of transgenic alfalfa was tested by GUS histochemical analysis. Further, thestable integration and transformation efficiency were tested by polymerase chain reaction. Toevaluate the accumulation of the exogenous GUS in somatic embryos, the somatic embryoswere induced from transgenic plantlets after the positive transgenic alfalfa plantlets wereconfirmed by GUS stain and polymerase chain reaction. And we compare the expression ofGUS in different transgenic alfalfa tissue (roots, stems, leaves and somatic embryos) by GUSfluorescence assay. At the same time, somatic embryos were induced from3transgenic alfalfa lines. Subsequently, the artificial transgenic alfalfa seeds were obtained by exogenousABA and6series of desiccators. The germination rate and genetic characteristics oftransgenic artificial alfalfa seeds was investigated. These studies provide the theoretical basisfor alfalfa somatic embryos used as bioreactor to produce exogenous pharmaceutical proteinand for artificial transgenic alfalfa used as germplasm storage. The followings are the majorconclusions:
1. The alfalfa transformation system was further improved. N4.4.2alfalfa genotype wasused as donor plants. The infected cotyledonary-stage embryos were induced through seriesof medium under75mg/L kanamycin selection. And we obtained the transgenic alfalfa plants.Then, GUS expression in different tissue of transgenic alfalfa was tested by GUShistochemical analysis. Further, the stable integration and transformation efficiency weretested by polymerase chain reaction and Southern blot hybridization. The result showed thatthe protocol of genetic transformation via secondary somatic embryos can shorten alfalfatransformation time than the protocol of genetic transformation via explants of petiole, leavesand hypocotyls and so on. The protocol significantly improved the induce frequency ofsecondary somatic embryos and provide a method of transferring multigene to alfalfa plants.At the same time, the protocol is also suited to those alfalfa genotypes which have lowtransformation efficiency to extend the method of genetic alfalfa transformation.
2. Alfalfa somatic embryos are the optimal materials of exogenous protein expressionwhich provides theoretical basis for alfalfa somatic embryos used as plant bioreactor toproduce recombinant protein. In this paper, petiole explants from N4.4.2alfalfa genotype astransformation materials were induced by Agrobacterium tumefaciens strain GV3101whichharbors the plasmid pBin9. pBin9vector contains gus/npt II fusion gene Both genes weredriven by double35s CaMV35S promoter. Infected petiole explants were cultured in differentmedia and the alfalfa tissue culture and transgenic system was developed. We tested11resistant Kanamycin alfalfa lines selected from the recovered transgenic plantlets by GUSstain and PCR. Moreover, we tested GUS activity expression in different tissue in transgenicalfalfa by GUS fluorometric measure in positive plantlets. The result showed that the amountof exogenous protein GUS expression in somatic embryos is significantly higher than in othertissue (roots, stems and leaves). The amount in somatic embryos is3~6folds than in roots,stems and leaves. However, the amount of GUS expression was no significant difference inroots, stems and leaves. Alfalfa somatic embryos can accumulate higher exogenous protein.
3. In this paper, we set up a germplasm preservation model system from desiccationsomatic embryos of transgenic alfalfa. The somatic embryos were induced original from thetransgenic pants and the cotyledonary-stage somatic embryos were cultured on a maturation medium supplemented with10mM abscisic acid for14d. Then somatic embryos weredesiccated in a series of desiccators K2SO4(RH87%), Na2CO3(RH87%), NaCl (RH75%), NH4NO3(RH63%), Ca (NO3)2.4H2O (RH51%), and K2CO3.2H2O (RH43%) over1week to slowly dry.Finally, the desiccation somatic embryos treated by the MSO medium were germinated totransgenic plantlets. Our result showed that the germination rate of transgenic somaticembryos is74.3%after desiccation. The recovery transgenic alfalfa posterity from desiccationsomatic embryos could express stable GUS. In addition, the plants produced were similarmorphology. Moreover, southern hybridization has confirmed that integration of the gene inrecovery plant was consistent in three different alfalfa lines. So these findings have proved thefeasibility of using desiccation somatic embryos as germplasm preservation method fortransgenic plants.
4.35s CaMV is a constitutive, strong promoter and is used for induce of constitutiveexpression in the transgenic plants. In this paper, the result show that GUS expression ishigher by double35s CaMV promoter than single one. To obtain higher exogenous proteinexpression, The vector with double35s CaMV promoter can be possible.
引文
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