海藻糖合酶基因遗传转化烟草的研究
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摘要
干旱是我国农业最主要的自然灾害。它影响了植物的生长发育，造成作物严重减产，加剧了全球性的粮食危机。在中国，有将近1／3的土地处于干旱和半干旱状态，7％以上耕地处于干旱和盐碱之中。随着土地沙漠化的加剧，以及淡水资源的日益短缺，这一数字还在不断上升。因此培育抗旱新品种是利用干旱地的一条经济而有效的途径。
海藻糖在生物体内扮演极其特殊的角色，它具有保护细胞膜和蛋白质结构的特殊功能，使生物体具有较强的抗旱、抗寒的能力，且对蛋白质制品、食品具有特殊的保护作用。
本研究利用担子菌灰树花海藻糖合酶基因(Tsase)遗传转化烟草，以进一步验证该基因的功能。首先将Tsase基因取代植物表达载体pBBB-ETR的ETR基因构建植物表达载体pBBBT，然后利用“三亲交配”法导入超毒性农杆菌菌株EHA105中，接着通过农杆菌介导法遗传转化烟草，经过连续的PPT抗性筛选，获得20株抗性植株。抗性植株经PPT体外抗性初筛，获得3株阳性植株；经PCR检测，其中的2株呈阳性反应；接下来的Southern杂交检测结果仍呈阳性，证明海藻糖合酶基因已整合到烟草基因组中。通过HPLC／ELSD法测得这两株转基因烟草海藻糖含量分别为2.126mg／g FW和2.556mg／g FW，而对照则检测不出海藻糖。与对照相比转基因烟草在形态上发生了明显的改变：叶片颜色变深绿；叶片增厚；主叶脉变粗壮等。田间抗旱试验结果证明转基因烟草的抗旱性得到了增强。这为植物抗旱基因工程的研究打下了良好的基础，同时也为利用植物生物反应器生产海藻糖奠定了基础。
Drought has been one of the greatest natural disaster threatening agriculture in our country . It affects the growth of plants and sharply reduces the yield. In china, about one-third of the land is in the drought and half-drought state and above 7% of the arable is threatened by drought and salinization. However the number is still increasing with the desertification of land and the lack of fresh water. So, it is an economical and efficient approach for utilizing the drought-stress land better to cultivate a new variety with drought-resistance trait.
Trehalose plays a special role in the organism.. It endows the trehalose-producing organisms with the ability to resist drought and freeze because it can protect the structures of the protein and the membrane. Besides, it also has the special protective function on protein products and food.
In our experiments, we transformed the Grifola frondos ( bacidiomycete ) trehalose synthase gene (Tsase) into tobacco to make further study to verify the function of the gene. First, a plant expression vector, pBBBT was constructed by inserting Tsase into pBBB-ETR to substitute the ETR gene within the plant expression vector. Then, it was introduced into the super-poison Agrobacterium tumerfaciens EHA105 through the "Triparental mating" method. Twenty regenerated tobacco plantlets were obtained with selection medium under the PPT selective pressure. Three plantlets were selected positive via vitro studies with PPT. We used the method of PCR to detect the trehalose synthase gene in them and there were two PCR-positive plants in the three. Southern blotting analysis suggested that the trehalose synthase gene had been integrated into the genome of the two PCR-positive regenerated plants. Here we used a method of HPLC/ELSD to analyse the content from leaf extract of the two transformants and CK. There were 2.126 mg/gFW and 2.556 mg/gFW trehalose in these two
transformants, respectively, whilst no trehalose was detected in CK. In comparison with CK, obvious morphological variation were observed in the transgenic tobaccos. Their leaves became greener and thicker, chief veins turned bolder and thicker than those of CK. In addition, drought-stress trials in field proved that the capability of drought-resistance of the transgenic tobaccos were improved. All this have made a firm foundation for the gene engineering research of drought-resistance in plant and will be useful in producing trehalose by plant bioreactors.
海藻糖在生物体内扮演极其特殊的角色，它具有保护细胞膜和蛋白质结构的特殊功能，使生物体具有较强的抗旱、抗寒的能力，且对蛋白质制品、食品具有特殊的保护作用。
本研究利用担子菌灰树花海藻糖合酶基因(Tsase)遗传转化烟草，以进一步验证该基因的功能。首先将Tsase基因取代植物表达载体pBBB-ETR的ETR基因构建植物表达载体pBBBT，然后利用“三亲交配”法导入超毒性农杆菌菌株EHA105中，接着通过农杆菌介导法遗传转化烟草，经过连续的PPT抗性筛选，获得20株抗性植株。抗性植株经PPT体外抗性初筛，获得3株阳性植株；经PCR检测，其中的2株呈阳性反应；接下来的Southern杂交检测结果仍呈阳性，证明海藻糖合酶基因已整合到烟草基因组中。通过HPLC／ELSD法测得这两株转基因烟草海藻糖含量分别为2.126mg／g FW和2.556mg／g FW，而对照则检测不出海藻糖。与对照相比转基因烟草在形态上发生了明显的改变：叶片颜色变深绿；叶片增厚；主叶脉变粗壮等。田间抗旱试验结果证明转基因烟草的抗旱性得到了增强。这为植物抗旱基因工程的研究打下了良好的基础，同时也为利用植物生物反应器生产海藻糖奠定了基础。
Drought has been one of the greatest natural disaster threatening agriculture in our country . It affects the growth of plants and sharply reduces the yield. In china, about one-third of the land is in the drought and half-drought state and above 7% of the arable is threatened by drought and salinization. However the number is still increasing with the desertification of land and the lack of fresh water. So, it is an economical and efficient approach for utilizing the drought-stress land better to cultivate a new variety with drought-resistance trait.
Trehalose plays a special role in the organism.. It endows the trehalose-producing organisms with the ability to resist drought and freeze because it can protect the structures of the protein and the membrane. Besides, it also has the special protective function on protein products and food.
In our experiments, we transformed the Grifola frondos ( bacidiomycete ) trehalose synthase gene (Tsase) into tobacco to make further study to verify the function of the gene. First, a plant expression vector, pBBBT was constructed by inserting Tsase into pBBB-ETR to substitute the ETR gene within the plant expression vector. Then, it was introduced into the super-poison Agrobacterium tumerfaciens EHA105 through the "Triparental mating" method. Twenty regenerated tobacco plantlets were obtained with selection medium under the PPT selective pressure. Three plantlets were selected positive via vitro studies with PPT. We used the method of PCR to detect the trehalose synthase gene in them and there were two PCR-positive plants in the three. Southern blotting analysis suggested that the trehalose synthase gene had been integrated into the genome of the two PCR-positive regenerated plants. Here we used a method of HPLC/ELSD to analyse the content from leaf extract of the two transformants and CK. There were 2.126 mg/gFW and 2.556 mg/gFW trehalose in these two
transformants, respectively, whilst no trehalose was detected in CK. In comparison with CK, obvious morphological variation were observed in the transgenic tobaccos. Their leaves became greener and thicker, chief veins turned bolder and thicker than those of CK. In addition, drought-stress trials in field proved that the capability of drought-resistance of the transgenic tobaccos were improved. All this have made a firm foundation for the gene engineering research of drought-resistance in plant and will be useful in producing trehalose by plant bioreactors.
引文
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