GmDREB1基因转化苜蓿及三种转基因苜蓿种子耐盐生理
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摘要
高盐是限制牧草生产的重要环境因素。我国的1亿公顷耕地中有667万公顷盐渍化土壤,另有0.3亿公顷盐碱荒地,其中吉林省西部地区是我国重要的牧草产区,也是我国土地盐碱化最严重的地区之一。近几十年来,由于气候变化和高强度的人类活动的影响,该区土地盐碱化面积不断扩展、程度不断加剧。相关资料显示,盐碱灾害使得吉林省西部某些地区豆科牧草几乎绝迹,而盐生植物却迅速发展。
紫花苜蓿是重要的豆科牧草,其自身具中度耐盐性,更兼应用植物基因工程技术方法对紫花苜蓿进行特异性遗传改良,使其适应盐碱化的土地,对改良吉林省西部植被现状以及合理开发利用盐碱土地有着重要的现实意义。
本研究主要分以下两个部分的实验:
第一部分实验以含有pBIGmDREB1质粒(含目的基因GmDREB1)的根癌农杆菌LBA4404感染公农1号紫花苜蓿的初生真叶,并通过一系列植物组织培养操作,获得含有GmDREB1基因的耐盐性紫花苜蓿。经卡那霉素初步筛选和PCR检测,得到阳性转GmDREB1基因植株119株;对其中4个PCR阳性株系进行Southern Blot杂交显影检测,进一步证明目的基因已经成功转入苜蓿基因组中。同时,我们对转SsNHX1基因、转SsNHX1-PDH45基因双元载体基因和转GmDREB1基因的阳性成活植株进行田间移栽,并对转SsNHX1基因植株进行了扩繁。最终得到SsNHX1转基因阳性植株116株,SsNHX1-PDH45转基因阳性植株112株和转GmDREB1基因阳性植株75株,并获得三种转基因植株的T1代种子。
第二部分实验以三种转基因苜蓿种子(转SsNHX1基因、转SsNHX1-PDH45双元基因和转GmDREB1基因)和公农1号紫花苜蓿种子为材料,研究了不同基因型对紫花苜蓿种子在5个不同盐浓度梯度(0mM, 100mM, 200mM, 300mM, 400mM)胁迫下发芽及幼苗素质的影响。结果表明,在200-400mM浓度范围内,转基因苜蓿种子的发芽势、发芽率、发芽指数、活力指数、根重和鲜重等指标均显著高于非转基因植株(p<0.05),而三种转基因种子之间并未出现显著差异。最终得出结论:三种外源基因在转基因苜蓿种子发芽期间进行了表达,转基因植株与对照相比表现出了显著的耐盐性。
本研究通过将GmDREB1基因转入苜蓿,经PCR和Southern Blot检测得到转基因阳性植株;并将本实验室所获得的三种转基因苜蓿进行田间移栽和扩繁,最终获得大量的转基因苜蓿T1代种子。另外,T1代种子的发芽试验证实了三种转基因植株的耐盐性与对照相比有着显著的耐盐性。这为进一步为转基因苜蓿大面积种植与盐碱地的可行性提供了理论依据。
High salinity is one of the most important environmental limiting factors of forage growth and productivity. There is 100 million ha of arable land in China, of which 6.67 million ha is saline land, and another 0.3 million hectares of saline-alkali wasteland. Grassland in the western of Jilin Province is an important forage producing area, but also one of the most serious saline-alkali lands in China. In recent decades, with the impact of climate change and increasement of intensive human activities, the area of saline-alkali soil and the degree of saline-alkali have grown constantly. Relevant data shows that leguminous grass has almost completely disappeared in some of this region, while the halophytes have been developing rapidly.
Alfalfa (Medicago sativa L.), an important leguminous forage plant, has a certain degree of salt tolerance. It is a highly significant way to improve vegetation of Western Jilin, develop and utilize saline-alkali land resources rationally by using plant genetic engineering technique to make specific genetic improvement of alfalfa and to adapt saline-alkali conditions.
This study mainly consists of two parts as follows:
Part one: The first true leaves of Alfalfa as recipient were infected by Agrobacterium tumefaciens strain LBA4404 which was transformed by pBI121 with GmDREB1 gene, Callus inducted from transgenic leaves were used for plant tissue culture operation with antibiotics (Kan) resistance including differentiation, rooting, and seedlings. Then PCR were used to analyze transgenic alfalfa plants, and the results showed that 119 individual plants were positive. Three kinds of transgenic plants harboring SsNHX1 gene, SsNHX1-PDH45 double gene and GmDREB1 gene were transplanted on Stud-farm, Changling Country, Jilin Province on May 1st, 2009. Among them, the transgenic plants harboring SsNHX1 gene were propagated by cutting and layering methods. In conclusion, 116 transformants with SsNHX1 gene, 112 transformants with SsNHX1-PDH45 double gene and 75 transformants with GmDREB1 gene were obtained, and the seeds were harvested.
Part two: Seeds from three transgenic alfalfa transformants with SsNHX1 gene, SsNHX1-PDH45 double gene and transformants with GmDREB1 gene, and Gongnong-1 alfalfa seeds were used to investigate the effects of germination and growth under different gradient of NaCl conditions (0mM, 100mM, 200mM, 300mM and 400mM). By statistical analysis, results showed that the germinating potentiality, germinating rate, germination index, vigor index, root natural weight and nature fresh weight in three transgenic alfalfa seeds were significantly higher than these in Gongnong-1 seeds(p<0.05) with the concentration at 200-400 mM. The final conclusion was that: The integration and expression of stress-resistant genes gave salt resistance of transgenic alfalfa.
This research transferred GmDREB1 gene into alfalfa tested by PCR and transgenic plants were obtained. Three transgenic alfalfa were transplanted in field and propagated by cutting and layering. Mass of T1 seeds from transformants were obtained. Salt tolerance assays of three transgenic alfalfa T1 seeds and Gongnong-1 seeds demonstrated that the salt tolerance of transgenic alfalfa T1 seed had an obvious progress compared with the control. It provides the theoretical basis for further research on feasibility of planting in large areas of saline-alkaline land.
紫花苜蓿是重要的豆科牧草,其自身具中度耐盐性,更兼应用植物基因工程技术方法对紫花苜蓿进行特异性遗传改良,使其适应盐碱化的土地,对改良吉林省西部植被现状以及合理开发利用盐碱土地有着重要的现实意义。
本研究主要分以下两个部分的实验:
第一部分实验以含有pBIGmDREB1质粒(含目的基因GmDREB1)的根癌农杆菌LBA4404感染公农1号紫花苜蓿的初生真叶,并通过一系列植物组织培养操作,获得含有GmDREB1基因的耐盐性紫花苜蓿。经卡那霉素初步筛选和PCR检测,得到阳性转GmDREB1基因植株119株;对其中4个PCR阳性株系进行Southern Blot杂交显影检测,进一步证明目的基因已经成功转入苜蓿基因组中。同时,我们对转SsNHX1基因、转SsNHX1-PDH45基因双元载体基因和转GmDREB1基因的阳性成活植株进行田间移栽,并对转SsNHX1基因植株进行了扩繁。最终得到SsNHX1转基因阳性植株116株,SsNHX1-PDH45转基因阳性植株112株和转GmDREB1基因阳性植株75株,并获得三种转基因植株的T1代种子。
第二部分实验以三种转基因苜蓿种子(转SsNHX1基因、转SsNHX1-PDH45双元基因和转GmDREB1基因)和公农1号紫花苜蓿种子为材料,研究了不同基因型对紫花苜蓿种子在5个不同盐浓度梯度(0mM, 100mM, 200mM, 300mM, 400mM)胁迫下发芽及幼苗素质的影响。结果表明,在200-400mM浓度范围内,转基因苜蓿种子的发芽势、发芽率、发芽指数、活力指数、根重和鲜重等指标均显著高于非转基因植株(p<0.05),而三种转基因种子之间并未出现显著差异。最终得出结论:三种外源基因在转基因苜蓿种子发芽期间进行了表达,转基因植株与对照相比表现出了显著的耐盐性。
本研究通过将GmDREB1基因转入苜蓿,经PCR和Southern Blot检测得到转基因阳性植株;并将本实验室所获得的三种转基因苜蓿进行田间移栽和扩繁,最终获得大量的转基因苜蓿T1代种子。另外,T1代种子的发芽试验证实了三种转基因植株的耐盐性与对照相比有着显著的耐盐性。这为进一步为转基因苜蓿大面积种植与盐碱地的可行性提供了理论依据。
High salinity is one of the most important environmental limiting factors of forage growth and productivity. There is 100 million ha of arable land in China, of which 6.67 million ha is saline land, and another 0.3 million hectares of saline-alkali wasteland. Grassland in the western of Jilin Province is an important forage producing area, but also one of the most serious saline-alkali lands in China. In recent decades, with the impact of climate change and increasement of intensive human activities, the area of saline-alkali soil and the degree of saline-alkali have grown constantly. Relevant data shows that leguminous grass has almost completely disappeared in some of this region, while the halophytes have been developing rapidly.
Alfalfa (Medicago sativa L.), an important leguminous forage plant, has a certain degree of salt tolerance. It is a highly significant way to improve vegetation of Western Jilin, develop and utilize saline-alkali land resources rationally by using plant genetic engineering technique to make specific genetic improvement of alfalfa and to adapt saline-alkali conditions.
This study mainly consists of two parts as follows:
Part one: The first true leaves of Alfalfa as recipient were infected by Agrobacterium tumefaciens strain LBA4404 which was transformed by pBI121 with GmDREB1 gene, Callus inducted from transgenic leaves were used for plant tissue culture operation with antibiotics (Kan) resistance including differentiation, rooting, and seedlings. Then PCR were used to analyze transgenic alfalfa plants, and the results showed that 119 individual plants were positive. Three kinds of transgenic plants harboring SsNHX1 gene, SsNHX1-PDH45 double gene and GmDREB1 gene were transplanted on Stud-farm, Changling Country, Jilin Province on May 1st, 2009. Among them, the transgenic plants harboring SsNHX1 gene were propagated by cutting and layering methods. In conclusion, 116 transformants with SsNHX1 gene, 112 transformants with SsNHX1-PDH45 double gene and 75 transformants with GmDREB1 gene were obtained, and the seeds were harvested.
Part two: Seeds from three transgenic alfalfa transformants with SsNHX1 gene, SsNHX1-PDH45 double gene and transformants with GmDREB1 gene, and Gongnong-1 alfalfa seeds were used to investigate the effects of germination and growth under different gradient of NaCl conditions (0mM, 100mM, 200mM, 300mM and 400mM). By statistical analysis, results showed that the germinating potentiality, germinating rate, germination index, vigor index, root natural weight and nature fresh weight in three transgenic alfalfa seeds were significantly higher than these in Gongnong-1 seeds(p<0.05) with the concentration at 200-400 mM. The final conclusion was that: The integration and expression of stress-resistant genes gave salt resistance of transgenic alfalfa.
This research transferred GmDREB1 gene into alfalfa tested by PCR and transgenic plants were obtained. Three transgenic alfalfa were transplanted in field and propagated by cutting and layering. Mass of T1 seeds from transformants were obtained. Salt tolerance assays of three transgenic alfalfa T1 seeds and Gongnong-1 seeds demonstrated that the salt tolerance of transgenic alfalfa T1 seed had an obvious progress compared with the control. It provides the theoretical basis for further research on feasibility of planting in large areas of saline-alkaline land.
引文
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