农杆菌介导bdDREB2基因转化紫花苜蓿的研究
摘要
紫花苜蓿(Medicago sativa)是世界上栽培最早、分布最广、也是最为重要的一种多年生优质豆科牧草,由于其具有产量高、适口性好、蛋白质含量高等优点,素有“牧草之王”的美誉。近年来,紫花苜蓿在我国东北、华北以及西北等地被广泛种植,且其种植规模具有逐年扩大的趋势。近年来,随着气候条件的恶化,干旱和水资源的匮乏成为了限制我过农业发展的重要因素,同时也限制了紫花苜蓿的种植及畜牧业的发展。近年来通过基因改良技术改造植物品种,成为生物工程研究的一个热点。
为培育出具有抗旱能力的紫花苜蓿品种,本研究选取由中国农业科学院畜牧兽医研究所牧草育种研究室通过常规育种技术育成的紫花苜蓿品种“中苜一号”作为基因转化受体,分别选用胚根、下胚轴及子叶作为转化受体,进行组织培养学系统研究,寻找相对分化率较高的外植体和适合于该品种的培养基搭配,测定了最佳菌液工作浓度、侵染时间、共培养时间等要素。并利用农杆菌介导的方法,将从野牛草(Buchloe dactyloides(Nutt.) Engelm.)中克隆得到的抗逆基因BdDREB2导入紫花苜蓿“中苜一号”中。获得如下实验结果:
1、胚根、下胚轴和子叶愈伤率均较高,但胚根和下胚轴不易分化出胚状体,子叶相对愈伤率低于下胚轴,但分化率较高。适合作为转化受体。最适合培养基搭配为:UM培养基为最佳诱导愈伤培养基和胚状体分化培养基;1/2MS为最佳诱导生根培养基。
2、农杆菌菌液最佳工作浓度为OD600≈0.6;侵染时间为10 min;共培养时间为3d。
3、最佳转化体系为:诱导愈伤培养基: UM+2,4-D 2 mg/L+KT 0.25 mg/L+Cef 500 mg/L+Kan 75 mg/L最佳分化培养基: UM+KT 2 mg/L+Cef 500 mg/L+Kan 75 mg/L最佳生根培养基: 1/2MS
4、获得8个转化株系,通过PCR分子鉴定,获得4株阳性苗。阳性率为50%。RT-PCR检测结果表明目的基因已经整合到苜蓿基因组中;Southern blot分析表明,BdDREB2基因已经稳定整合到转基因植株的基因组中。
As“the king of forages”,Medicago sativa is the earliest cultivation,the most extensive distribution and the most important perennial quality legumes which is high yield, good palatability and protein-rich. Recently, Medicago sativa is cultivated in north China area widely and the cultivation scale has the trendency of expanding gradually year. As the environment of China turn worse, the water resourse turn less. The soil salinization of part areas’land is serious. These greatly restrict the planting of alfalfa and the development of animal husbandry. Improving the soil has many shortcomings, for example, tremendous cost of soil and little effect. But these years,improveing plant cultivar by genetic engineering technology becomes a hot spot of biological engineering research.
In this study, the gene transformation acceptor selected is the alfalfa cultivar Medicago sativa L.“zhongmu NO.1”, which comes from the conventional big field breeding technology by the CASI. I selected the radicle, the hypocotyls and the cotylecdons as the explant, conducted the tissue culture systematic research, seeked a high differentiation rate explant and the basic culture medium which suit for this variety, and determined the best concentration of bacilli, the best infection time and the co-culture time. Addition to these, the BdDREB2 (Buchloe dactyloides Dehydration Responsive Element Binding protein 2)gene which was cloned from Buchloe dactyloides (Nutt.) Engelm was introduced into the genome of the alfalfa (Zhongmu No.1) by using Agrobacterium tumefaciens-mediated transformation. The main results were as following:
1. The radicle, the hypocotyls and the cotylecdons callus rate are all higher, but the radicle and the hypocotyls are hard to differentiated embryo. The callus rate of cotylecdon is lower than the hypocotyl’s callus rate, but its differentiation rate is higher. It is indicated that cotylecdon is suitable as explant. UM medium is the best embryogenic callus induction medium and the best embryo induction medium and 1/2MS medium is the best root development medium.
2. The best concentration of bacilli is OD600≈0.6; the best infection time is 10 minutes; and the co-culture time is 3 days.
3. The best transformation system is as following.
Embryogenic callus induction medium: UM+2,4-D 2mg/L+KT 0.25mg/L+Cef 500mg/L+Kan 75mg/L The best embryo induction medium: UM+KT 2mg/L+Cef 500mg/L+Kan 75mg/L The best root development medium: 1/2MS
4. Finally eight transformant was achieved. PCR analysis indicated that four of the eight transformant were transgenic plants. The positive rate is 50%. RT-PCR results showed that the geneof BdDREB2 is transcribed into mRNA in positive transgenic alfalfa. Stable integration of the BdDREB2 gene into the genome of transgenic plants was confirmed by Southern blot analyses.
为培育出具有抗旱能力的紫花苜蓿品种,本研究选取由中国农业科学院畜牧兽医研究所牧草育种研究室通过常规育种技术育成的紫花苜蓿品种“中苜一号”作为基因转化受体,分别选用胚根、下胚轴及子叶作为转化受体,进行组织培养学系统研究,寻找相对分化率较高的外植体和适合于该品种的培养基搭配,测定了最佳菌液工作浓度、侵染时间、共培养时间等要素。并利用农杆菌介导的方法,将从野牛草(Buchloe dactyloides(Nutt.) Engelm.)中克隆得到的抗逆基因BdDREB2导入紫花苜蓿“中苜一号”中。获得如下实验结果:
1、胚根、下胚轴和子叶愈伤率均较高,但胚根和下胚轴不易分化出胚状体,子叶相对愈伤率低于下胚轴,但分化率较高。适合作为转化受体。最适合培养基搭配为:UM培养基为最佳诱导愈伤培养基和胚状体分化培养基;1/2MS为最佳诱导生根培养基。
2、农杆菌菌液最佳工作浓度为OD600≈0.6;侵染时间为10 min;共培养时间为3d。
3、最佳转化体系为:诱导愈伤培养基: UM+2,4-D 2 mg/L+KT 0.25 mg/L+Cef 500 mg/L+Kan 75 mg/L最佳分化培养基: UM+KT 2 mg/L+Cef 500 mg/L+Kan 75 mg/L最佳生根培养基: 1/2MS
4、获得8个转化株系,通过PCR分子鉴定,获得4株阳性苗。阳性率为50%。RT-PCR检测结果表明目的基因已经整合到苜蓿基因组中;Southern blot分析表明,BdDREB2基因已经稳定整合到转基因植株的基因组中。
As“the king of forages”,Medicago sativa is the earliest cultivation,the most extensive distribution and the most important perennial quality legumes which is high yield, good palatability and protein-rich. Recently, Medicago sativa is cultivated in north China area widely and the cultivation scale has the trendency of expanding gradually year. As the environment of China turn worse, the water resourse turn less. The soil salinization of part areas’land is serious. These greatly restrict the planting of alfalfa and the development of animal husbandry. Improving the soil has many shortcomings, for example, tremendous cost of soil and little effect. But these years,improveing plant cultivar by genetic engineering technology becomes a hot spot of biological engineering research.
In this study, the gene transformation acceptor selected is the alfalfa cultivar Medicago sativa L.“zhongmu NO.1”, which comes from the conventional big field breeding technology by the CASI. I selected the radicle, the hypocotyls and the cotylecdons as the explant, conducted the tissue culture systematic research, seeked a high differentiation rate explant and the basic culture medium which suit for this variety, and determined the best concentration of bacilli, the best infection time and the co-culture time. Addition to these, the BdDREB2 (Buchloe dactyloides Dehydration Responsive Element Binding protein 2)gene which was cloned from Buchloe dactyloides (Nutt.) Engelm was introduced into the genome of the alfalfa (Zhongmu No.1) by using Agrobacterium tumefaciens-mediated transformation. The main results were as following:
1. The radicle, the hypocotyls and the cotylecdons callus rate are all higher, but the radicle and the hypocotyls are hard to differentiated embryo. The callus rate of cotylecdon is lower than the hypocotyl’s callus rate, but its differentiation rate is higher. It is indicated that cotylecdon is suitable as explant. UM medium is the best embryogenic callus induction medium and the best embryo induction medium and 1/2MS medium is the best root development medium.
2. The best concentration of bacilli is OD600≈0.6; the best infection time is 10 minutes; and the co-culture time is 3 days.
3. The best transformation system is as following.
Embryogenic callus induction medium: UM+2,4-D 2mg/L+KT 0.25mg/L+Cef 500mg/L+Kan 75mg/L The best embryo induction medium: UM+KT 2mg/L+Cef 500mg/L+Kan 75mg/L The best root development medium: 1/2MS
4. Finally eight transformant was achieved. PCR analysis indicated that four of the eight transformant were transgenic plants. The positive rate is 50%. RT-PCR results showed that the geneof BdDREB2 is transcribed into mRNA in positive transgenic alfalfa. Stable integration of the BdDREB2 gene into the genome of transgenic plants was confirmed by Southern blot analyses.
引文
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