大豆规模化转基因体系构建
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摘要
大豆是现代农业十分重要的农作物之一,富含蛋白质、植物油和多种对人体有益的成分,是人类食品中主要的植物蛋白来源,同时也是重要的工业原料和牲畜饲料。随着科技的发展和人们生活水平的提高,大豆的产量逐渐开始无法满足人们的生活需求,而传统的育种方法又无法有效解决这一矛盾。于是自1988年开始,越来越多的研究人员开始利用基因工程技术,培育具有优良性状的大豆品种,并取得了一定的成就。但大豆的基因转化一直存在着转化率低、重复性差等问题。因此,建立高效、稳定的大豆转化技术体系仍是目前国内外学者研究的热点之一。本实验采取目前最常用的农杆菌介导的转化方法,以多个大豆品种的子叶节外植体为受体材料,进行了大豆遗传转化实验。并且针对转化过程中的重要步骤实施了一系列优化措施,旨在通过提高农杆菌侵染效率、丛生芽的诱导效率等,进一步提高大豆再生效率和转化效率,建立一种稳定性好、转化率高的大豆转化技术体系,为今后的大豆分子育种和基因功能研究打下良好的基础。
本实验主要研究成果如下:
1、高再生能力的大豆基因型筛选。实验采用含PBI121质粒的农杆菌EHA105和GV3101侵染7个大豆品种的子叶节外植体,发现不同大豆基因型之间再生效率差异明显,并从中筛选出中黄10号、文丰7号两个再生效率较高的大豆品种。
2、降低了组织培养过程中的褐化现象。通过对共培培养基进行半胱氨酸(L-cy)浓度的梯度试验,发现L-cy浓度为400 mg?L-1时,能有效地抑制子叶节外植体的组织褐化现象,提高了外植体的成活效率。
3、降低了残留农杆菌污染。本实验通过优化共培后的除菌方法,配合除菌剂的使用,大大降低了残留农杆菌污染的比率,有效地解决了农杆菌介导转化体系容易造成侵染后的农杆菌残留污染的现象,保证了转化实验的顺利进行。
4、提高了丛生芽的诱导效率。通过在恢复期去掉子叶节外植体第一组新芽的方法,大大提高了外植体诱发的丛生芽数量,从而提高了再生效率;并以此降低了丛生芽发育的顶端优势,提高了筛选效率。
5、Thidiazuron(TDZ)的活化效果。TDZ是一种高效的细胞分裂素,本实验在丛生芽诱导阶段添加了适量的TDZ,结果发现诱导的活性细胞数量得到了大幅度增加。
6、生根培养基的优化。通过对多品种的生根诱导实验,确定了生根诱导高效稳定的生根培养基配比及IBA的最适浓度1.0mg?L-1,并且选出了容易发根的大豆品种中黄10号和文丰7号。
7、优化炼苗移栽步骤。本实验对再生植株的炼苗移栽步骤作了一系列优化,解决了幼嫩再生植株无法快速适应外界环境的问题,提高了移栽成活效率,为之后的检测与研究创造了条件。
8、转化植株的获得。通过对7个品种的大豆进行优化转化实验,最终获得了43株含有转化外源基因的转基因再生植株,平均转化效率为1.76%,效率最高的EHA105侵染大豆中黄10转化实验中,最终转化效率达到4.61%,高于目前大豆转化的平均水平。
Soybean is one of the most important crops, which is rich in protein, vegetable oil and a variety of useful physiological chemical compositions of human. Soybean is not only a major human food source of vegetable protein, is also an important materials for industry and animal feed. With the technology and people's living standards improve, soybean production began to not content the needs of people's lives. Traditional breeding methods and can not effectively solve the problem of soybean’s gap between supply and demand. So that, more and more researchers have tried to overcome these problems use genetic engineering technology since 1988, and has achieved some success. But, compare with other plants, the transformation frequency of soybean is still low relative, and the repeatability is not good. Actually a few of cases of transgenic soybeans were successful due to the low transformation rate.Establish an efficient and stable system for soybean transformation technology is still one of the hot topic for the scientists.
In this study,we use Agrobacterium-mediated transformation method for transforming 7 soybeans, the cotyledon-node explant was uesd for the transgenic receptor. In order to improve transformation efficiency, this experiment was conducted to compare the efficiency of several factors during transformation. This will lay a good foundation for the gene function research and molecular breeding of soybean.
The main results of this research as follows
1. 7 soybean genotypes were used for transformation.The Agrobacterium tumefaciens strain was EHAl05 and GV3101,which carried binary vector PBI121.The result in this research had shown that regeneration frequency was higher of“zhonghuang10”and“wenfeng7”.
2. To reduce the browning effect,we added L-cy in the co-cultivation medium.The result indicate that though the experiments for concentration gradient of L-cy,the suitable L-cy cencentration is 400mg/L for the zhonghuang13 soybean.The browning rate has dropped by more than 90%。
3. Reduce the level of residual Agrobacterium contamination.We design of three methods to wash the cotyledon-node explant,after the co-cultivation,and finally chose one methods that the lever of residual Agrobacterium was lowest.
4. Improve the efficiency of shoot induction.We chose some of cotyledon-node explant that just shooting after the co-cultivation,cut off the new shoot,and then transfer them to a new medium.The result indicate that the efficiency of shoot induction was most increased.
5. We add Thidiazuron(TDZ) in the shoot induction medium.And found TDZ is more effective than 6-BA at activated cell development.
6. Duing the root regeneration stage,We had determined the optimal concentration of IBA,and found that the genotypes has some infulence on the rooting ability.
7. We also did some research on transplanting, and improve the efficiency of the survival of transplanted.It is made some good provide for the molecular detection.
8. Through the soybean transformation experiments,we finally obtained 43 transgenic plants,increases in transformation efficiency to an average of 1.7%, which the most is 4.61%. Conversion efficiency is higher than the current average level of soybean transformation.
本实验主要研究成果如下:
1、高再生能力的大豆基因型筛选。实验采用含PBI121质粒的农杆菌EHA105和GV3101侵染7个大豆品种的子叶节外植体,发现不同大豆基因型之间再生效率差异明显,并从中筛选出中黄10号、文丰7号两个再生效率较高的大豆品种。
2、降低了组织培养过程中的褐化现象。通过对共培培养基进行半胱氨酸(L-cy)浓度的梯度试验,发现L-cy浓度为400 mg?L-1时,能有效地抑制子叶节外植体的组织褐化现象,提高了外植体的成活效率。
3、降低了残留农杆菌污染。本实验通过优化共培后的除菌方法,配合除菌剂的使用,大大降低了残留农杆菌污染的比率,有效地解决了农杆菌介导转化体系容易造成侵染后的农杆菌残留污染的现象,保证了转化实验的顺利进行。
4、提高了丛生芽的诱导效率。通过在恢复期去掉子叶节外植体第一组新芽的方法,大大提高了外植体诱发的丛生芽数量,从而提高了再生效率;并以此降低了丛生芽发育的顶端优势,提高了筛选效率。
5、Thidiazuron(TDZ)的活化效果。TDZ是一种高效的细胞分裂素,本实验在丛生芽诱导阶段添加了适量的TDZ,结果发现诱导的活性细胞数量得到了大幅度增加。
6、生根培养基的优化。通过对多品种的生根诱导实验,确定了生根诱导高效稳定的生根培养基配比及IBA的最适浓度1.0mg?L-1,并且选出了容易发根的大豆品种中黄10号和文丰7号。
7、优化炼苗移栽步骤。本实验对再生植株的炼苗移栽步骤作了一系列优化,解决了幼嫩再生植株无法快速适应外界环境的问题,提高了移栽成活效率,为之后的检测与研究创造了条件。
8、转化植株的获得。通过对7个品种的大豆进行优化转化实验,最终获得了43株含有转化外源基因的转基因再生植株,平均转化效率为1.76%,效率最高的EHA105侵染大豆中黄10转化实验中,最终转化效率达到4.61%,高于目前大豆转化的平均水平。
Soybean is one of the most important crops, which is rich in protein, vegetable oil and a variety of useful physiological chemical compositions of human. Soybean is not only a major human food source of vegetable protein, is also an important materials for industry and animal feed. With the technology and people's living standards improve, soybean production began to not content the needs of people's lives. Traditional breeding methods and can not effectively solve the problem of soybean’s gap between supply and demand. So that, more and more researchers have tried to overcome these problems use genetic engineering technology since 1988, and has achieved some success. But, compare with other plants, the transformation frequency of soybean is still low relative, and the repeatability is not good. Actually a few of cases of transgenic soybeans were successful due to the low transformation rate.Establish an efficient and stable system for soybean transformation technology is still one of the hot topic for the scientists.
In this study,we use Agrobacterium-mediated transformation method for transforming 7 soybeans, the cotyledon-node explant was uesd for the transgenic receptor. In order to improve transformation efficiency, this experiment was conducted to compare the efficiency of several factors during transformation. This will lay a good foundation for the gene function research and molecular breeding of soybean.
The main results of this research as follows
1. 7 soybean genotypes were used for transformation.The Agrobacterium tumefaciens strain was EHAl05 and GV3101,which carried binary vector PBI121.The result in this research had shown that regeneration frequency was higher of“zhonghuang10”and“wenfeng7”.
2. To reduce the browning effect,we added L-cy in the co-cultivation medium.The result indicate that though the experiments for concentration gradient of L-cy,the suitable L-cy cencentration is 400mg/L for the zhonghuang13 soybean.The browning rate has dropped by more than 90%。
3. Reduce the level of residual Agrobacterium contamination.We design of three methods to wash the cotyledon-node explant,after the co-cultivation,and finally chose one methods that the lever of residual Agrobacterium was lowest.
4. Improve the efficiency of shoot induction.We chose some of cotyledon-node explant that just shooting after the co-cultivation,cut off the new shoot,and then transfer them to a new medium.The result indicate that the efficiency of shoot induction was most increased.
5. We add Thidiazuron(TDZ) in the shoot induction medium.And found TDZ is more effective than 6-BA at activated cell development.
6. Duing the root regeneration stage,We had determined the optimal concentration of IBA,and found that the genotypes has some infulence on the rooting ability.
7. We also did some research on transplanting, and improve the efficiency of the survival of transplanted.It is made some good provide for the molecular detection.
8. Through the soybean transformation experiments,we finally obtained 43 transgenic plants,increases in transformation efficiency to an average of 1.7%, which the most is 4.61%. Conversion efficiency is higher than the current average level of soybean transformation.
引文
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