抗逆转录因子基因的功能验证与转基因百脉根的获得
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摘要
百脉根(Lotus corniculatus L.)别名牛角花,是豆科百脉根属多年生牧草,原产于欧亚温暖地区,目前在世界各地广泛种植。作为优良饲草,百脉根营养含量居豆科牧草的首位,成熟收种后,蛋白质含量可达17.4%。含皂素低,适口性好,耐牧性强,不会引起家畜膨胀病,为一般豆科牧草所不及。同时,百脉根也是建立人工草地、补播改良草场和建立人工放牧地的不可多得的牧草品种。近年从加拿大引进的品种里奥(Leo),抗寒性强,但其耐旱耐盐特性较差,难以适于国内极端气候日益频发的环境。为解决这一难题,本研究对本实验室从沙生植物胡杨(Populus euphratica)和铃铛刺(Halimodendron halodendron (Pall.)Voss.)中克隆的四个抗逆转录因子基因进行了功能分析,从中筛选了2个高抗基因PeDREB2a和HhERF2,构建单、双价以生物安全标记基因pmi为选择标记的植物表达载体,转化百脉根Leo品种,以期提高其在抗旱耐盐特性方面的能力,为我国畜牧业增添新的优良牧草种质资源。主要研究结果如下:
1.本研究以实验室从沙生植物胡杨和铃铛刺中克隆的PeDREB2a、PeDREB2b、HhDREB2和HhERF2四个抗逆相关转录因子基因为基础,构建了分别含有PeDREB2a和PeDREB2b基因以诱导型启动子rd29A为启动子的植物表达载体:rd29A::PeDREB2a和rd29A:: PeDREB2b(rd29A::HhDREB2和rd29A::HhERF2实验室已构建);
2.将四个转录因子基因分别转化模式植物拟南芥,通过潮霉素(Hyg)多代筛选获得纯合系转基因拟南芥植株分别为:PeDREB2a(20株)、PeDREB2b(18株)、HhDREB2(23株)和HhERF2(27株)。研究中对转基因拟南芥萌发期、幼苗期和生长期进行干旱控水和盐胁迫梯度实验,通过发芽率、含水量、可溶性糖、丙二醛(MDA)等抗逆指标检测,验证了四个转录因子在植物不同时期均提高了转基因拟南芥对干旱和盐胁迫的耐受力和适应性;
3.结合四种转基因拟南芥各种指标结果的比较,发现PeDREB2a和HhERF2基因在植物抗逆调控中起到明显的调控作用。因此,本研究在实验室已构载体pCAMBIA1302-pmi的基础上,构建了pmi-rd29A::PeDREB2a、pmi-rd29A:: HhERF2和pmi-PeDREB2a::HhERF2三种单、双价安全型植物表达载体,为安全型抗旱耐盐百脉根转基因植株的获得打下基础;
4.将以上构建的三种单、双价植物表达载体,通过农杆菌介导法转化百脉根子叶,对通过甘露糖筛选的抗性植株进行PCR和Southern-blotting检测,结果表明,目的基因均已整合入牧草百脉根的基因组中。现已获得转pmi-rd29A::PeDREB2a、pmi-rd29A:: HhERF2和pmi-PeDREB2a::HhERF2百脉根植株7株、12株和5株(其他转化事件还在检测当中)。干旱盐碱胁迫处理,初步结果表明,转录因子基因PeDREB2a和HhERF2明显改善了百脉根的抗逆特性,尤其双价转基因百脉根抗逆效果明显。
Lotus corniculatus L. (Birdsfoot), a member of the perennial Leguminosae native to warm regions of Eurasia, is non-bloating for cattle and sheep that have the potential to become a major crop in temperate forage-producing regions of the world. The Lotus has a high nutritive proportion rank first in legume forages. It has a protein content of as high as 17.4% even after seed harvest. It also has low saponin content, palatability and strong regrowth habits advantages. At the same time, the Lotus as a pioneer grass is often used in establishing managed grasslands and artificial sowing. Leo, one of Lotus breeds introduced from Canada, is more tolerant to environmental extremes of cold, but less tolerant to salt and drought that influenced popularization in China. In order to solve the puzzle, we identify two highly resistant genes PeDREB2a and HhERF2 through functional analysis of the four transcriptional factor genes cloning from Populus euphratica and Halimodendron halodendron, respectively. PeDREB2a and HhERF2 are two factors with synogestic effects involved in plant resistance. Based on this fact, a series of plant expression vectors either harboring PeDREB2a or HhERF2 or both were constructed with non-resistance marker gene pmi and transferred into the Lotus genome to expect to improve salt endurance and drought resistance and adding fine varieties of herbage to Animal Husbandry Industry of China. The major work includes:
1. By genetic recombination technology, the transcription factors, PeDREB2a, PeDREB2b, HhDREB2 and HhERF genes, cloned from Populus euphratica and Halimodendron halodendron, were amplified by PCR. Based on the vector pCAMBIA1302, two plant expression vectors rd29A::PeDREB2a and rd29A:: PeDREB2b were constructed under the controlling of rd29A promoter. (rd29A::HhDREB2 and rd29A::HhERF2 had already constructed by other person and conserved in our laboratory).
2. The transcription factor genes were transferred into Arabidopsis. Under the selection of Hyg, 20,18, 23 and 27 pure transgenic lines of Arabidopsis were obtained for PeDREB2a, PeDREB2b, HhDREB2 and HhERF2 genes,respectively. These transgenic lines were further evaluated according to their germination rate, moisture content, soluble sugar and MDA of Arabidopsis induced by dehydration and high salt within germinating, seedling stage and leaf growth periods.The results thus obtained suggested that the introduction of these four genes into the Arabidopsis genomes can improve drought and salt resistance of the transgenic Arabidopsis at the different growth stages.
3. The results further showed that the introduction of PeDREB2 and HhERF2 genes enhanced drought and salt tolerance of transgenic Arabidopsis significantly. Three plant expression vectors pmi-rd29A::PeDREB2a, pmi-rd29A::HhERF2, pmi-PeDREB2a::HhERF were further constructed basing on pCAMBIA1302-PMI, for transformation of the genes into Lotus corniculatus L.
4. The constructed vectors were tranfered into the legume plant Lotus corniculatus“Leo”via Agrobacterium tumefaciens. PCR of the shoots thus obtained indicated that shoots had been transformed. Southern-blotting of selected regenerated plants further confirmed integration of the target genes into the Lotus genome. 7,12, and 5 transgenic lines of Lotus were obtained transfored by pmi-rd29A::PeDREB2a, pmi-rd29A::HhERF2, pmi-PeDREB2a::HhERF, respectively(Other transformed material detections are pending). Preliminary study of the transgenic regenerated plants under stress conditions showed that the transformed genes improved extreme environment tolerance of transgenic Lotus, especially the plant transformed with pCAMBIA1302-pmi-PeDREB2a::HhERF2.
1.本研究以实验室从沙生植物胡杨和铃铛刺中克隆的PeDREB2a、PeDREB2b、HhDREB2和HhERF2四个抗逆相关转录因子基因为基础,构建了分别含有PeDREB2a和PeDREB2b基因以诱导型启动子rd29A为启动子的植物表达载体:rd29A::PeDREB2a和rd29A:: PeDREB2b(rd29A::HhDREB2和rd29A::HhERF2实验室已构建);
2.将四个转录因子基因分别转化模式植物拟南芥,通过潮霉素(Hyg)多代筛选获得纯合系转基因拟南芥植株分别为:PeDREB2a(20株)、PeDREB2b(18株)、HhDREB2(23株)和HhERF2(27株)。研究中对转基因拟南芥萌发期、幼苗期和生长期进行干旱控水和盐胁迫梯度实验,通过发芽率、含水量、可溶性糖、丙二醛(MDA)等抗逆指标检测,验证了四个转录因子在植物不同时期均提高了转基因拟南芥对干旱和盐胁迫的耐受力和适应性;
3.结合四种转基因拟南芥各种指标结果的比较,发现PeDREB2a和HhERF2基因在植物抗逆调控中起到明显的调控作用。因此,本研究在实验室已构载体pCAMBIA1302-pmi的基础上,构建了pmi-rd29A::PeDREB2a、pmi-rd29A:: HhERF2和pmi-PeDREB2a::HhERF2三种单、双价安全型植物表达载体,为安全型抗旱耐盐百脉根转基因植株的获得打下基础;
4.将以上构建的三种单、双价植物表达载体,通过农杆菌介导法转化百脉根子叶,对通过甘露糖筛选的抗性植株进行PCR和Southern-blotting检测,结果表明,目的基因均已整合入牧草百脉根的基因组中。现已获得转pmi-rd29A::PeDREB2a、pmi-rd29A:: HhERF2和pmi-PeDREB2a::HhERF2百脉根植株7株、12株和5株(其他转化事件还在检测当中)。干旱盐碱胁迫处理,初步结果表明,转录因子基因PeDREB2a和HhERF2明显改善了百脉根的抗逆特性,尤其双价转基因百脉根抗逆效果明显。
Lotus corniculatus L. (Birdsfoot), a member of the perennial Leguminosae native to warm regions of Eurasia, is non-bloating for cattle and sheep that have the potential to become a major crop in temperate forage-producing regions of the world. The Lotus has a high nutritive proportion rank first in legume forages. It has a protein content of as high as 17.4% even after seed harvest. It also has low saponin content, palatability and strong regrowth habits advantages. At the same time, the Lotus as a pioneer grass is often used in establishing managed grasslands and artificial sowing. Leo, one of Lotus breeds introduced from Canada, is more tolerant to environmental extremes of cold, but less tolerant to salt and drought that influenced popularization in China. In order to solve the puzzle, we identify two highly resistant genes PeDREB2a and HhERF2 through functional analysis of the four transcriptional factor genes cloning from Populus euphratica and Halimodendron halodendron, respectively. PeDREB2a and HhERF2 are two factors with synogestic effects involved in plant resistance. Based on this fact, a series of plant expression vectors either harboring PeDREB2a or HhERF2 or both were constructed with non-resistance marker gene pmi and transferred into the Lotus genome to expect to improve salt endurance and drought resistance and adding fine varieties of herbage to Animal Husbandry Industry of China. The major work includes:
1. By genetic recombination technology, the transcription factors, PeDREB2a, PeDREB2b, HhDREB2 and HhERF genes, cloned from Populus euphratica and Halimodendron halodendron, were amplified by PCR. Based on the vector pCAMBIA1302, two plant expression vectors rd29A::PeDREB2a and rd29A:: PeDREB2b were constructed under the controlling of rd29A promoter. (rd29A::HhDREB2 and rd29A::HhERF2 had already constructed by other person and conserved in our laboratory).
2. The transcription factor genes were transferred into Arabidopsis. Under the selection of Hyg, 20,18, 23 and 27 pure transgenic lines of Arabidopsis were obtained for PeDREB2a, PeDREB2b, HhDREB2 and HhERF2 genes,respectively. These transgenic lines were further evaluated according to their germination rate, moisture content, soluble sugar and MDA of Arabidopsis induced by dehydration and high salt within germinating, seedling stage and leaf growth periods.The results thus obtained suggested that the introduction of these four genes into the Arabidopsis genomes can improve drought and salt resistance of the transgenic Arabidopsis at the different growth stages.
3. The results further showed that the introduction of PeDREB2 and HhERF2 genes enhanced drought and salt tolerance of transgenic Arabidopsis significantly. Three plant expression vectors pmi-rd29A::PeDREB2a, pmi-rd29A::HhERF2, pmi-PeDREB2a::HhERF were further constructed basing on pCAMBIA1302-PMI, for transformation of the genes into Lotus corniculatus L.
4. The constructed vectors were tranfered into the legume plant Lotus corniculatus“Leo”via Agrobacterium tumefaciens. PCR of the shoots thus obtained indicated that shoots had been transformed. Southern-blotting of selected regenerated plants further confirmed integration of the target genes into the Lotus genome. 7,12, and 5 transgenic lines of Lotus were obtained transfored by pmi-rd29A::PeDREB2a, pmi-rd29A::HhERF2, pmi-PeDREB2a::HhERF, respectively(Other transformed material detections are pending). Preliminary study of the transgenic regenerated plants under stress conditions showed that the transformed genes improved extreme environment tolerance of transgenic Lotus, especially the plant transformed with pCAMBIA1302-pmi-PeDREB2a::HhERF2.
引文
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