抗草甘膦转基因大豆生物测定方法的研究
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摘要
转基因大豆的种植,极大地提高了农田除草效率,取得了巨大的经济效益和社会效益。但是随着研究的不断深入,转基因大豆的生态安全性、抗性基因的流向和由此引发的食品安全性问题则引起了越来越多的争议,甚至在一定程度上阻碍了转基因技术的发展。因此,建立完善的转基因大豆检测方法对于我国的大豆育种研究、环境安全性和食品安全性等有重要的意义。
目前关于抗草甘膦转基因大豆的检测方法主要集中在分子检测法和田间(温室)整株鉴定法。前者由于仪器、试剂要求精确,成本较高,专一性强,且只是针对靶标基因进行检测,并不能验证靶标基因的表达与否;后者虽然直观准确,但所需时间比较长、工作量大,且易受季节影响。
本研究针对目前的的研究现状,利用除草剂生物测定技术,构建了抗草甘膦转基因大豆生物生化测定方法;并且对检测指标、该方法适用的不同培养条件、温度等进行了深入的研究,同时辅以田间表型鉴定和分子检测手段对方法进行了验证。
主要研究结果如下:
一、抗草甘膦转基因大豆生物测定方法的建立
1、在26℃、持续黑暗培养72 h,以大豆萌发过程中草甘膦对下胚轴抑制率为检测指标,建立了检测转基因大豆的生物测定方法。研究结果表明:转基因大豆下胚轴抑制中浓度为1620.86-2269.33mg a. i./L;非转基因大豆的下胚轴抑制中浓度为78.04-97.43 mg a. i./L,二者有显著性差异。因此,为方便快速,在各自95%置信区间内,确定快速甄别剂量为100 mg a. i./L,或者2000 mg a. i./L,在此甄别剂量下可以有效区分转基因大豆和非转基因大豆。
2、依据生物测定的方法,建立了以莽草酸积累量为指标的生物化学检测方法:即在100 mg a. i./L(非转基因大豆的抑制中浓度)下,非转基因大豆的莽草酸积累量约为1237.7 ug/g FW,转基因大豆的莽草酸积累量只有64.61ug/g FW,二者之间有显著性差异,可以有效地鉴别转基因大豆和非转基因大豆。
3、利用生物和生化测定方法,对样本龙抗630、绥10-6047进行了检测。结果表明:龙抗630和绥10-6047为转基因大豆,确定EPSPS基因已经成功转入大豆体内,并且获得表达。田间表型鉴定表明龙抗630、绥10-6047为高抗草甘膦大豆,同时分子检测结果表明两份大豆为阳性。三种检测结果相互印证,证明生物生化测定方法可行。
二、室内生物测定方法建立的生理生化机理研究
1、受到草甘膦胁迫时,莽草酸途径是主要的靶标途径。转基因大豆因其转入了EPSPS基因,表现了此生理途径不受影响,莽草酸的积累量较小,从而能够在较高浓度的草甘膦水培条件下正常的萌发生长,而非转基因大豆则没有此能力。
2、受到草甘膦胁迫时,转基因大豆和非转基因大豆的GSTS活性有显著性差异,表明GSTS参与了大豆体内的解毒代谢作用,是大豆产生抗性的“解毒代谢层次”的重要原因。后期随着浓度的增加和时间的延长活性逐渐减弱表明其解毒代谢能力是有限的。
3、在抵抗草甘膦的胁迫过程中,转基因大豆和非转基因大豆体内SOD、POD活性虽然在最初有上升,但二者之间并无显著性差异,表明这种机制其并不是抗性产生主要原因。可能是由于大豆受到“除草剂逆境和水分逆境”的胁迫而诱发了自由基和活性氧等有害物质的产生,进而引起了SOD、POD活性的增加,是一种本能自卫反映,起到一个协同抗性的作用。
三、检测方法的在杂交育种中的应用
运用生物测定方法较为快捷的检测了7个杂交后代品系,结果表明其大豆品种抗性程度明显比杂交亲本提高20-30倍,鉴定为转基因抗草甘膦大豆,表明EPSPS基因转入成功,与分子检测结果相符。
The cultivation and promotion of t Genetically Modified soybean increased the efficiency of weeding observably,obtained great economic benefit。As researching deeply, the ecological security of Genetically Modified soybean.the flow of resistance genes and food security by alien genes in transgenic herbicide resistants crops has caused more and more controversies and inhibited its developing. So it has important significance to establish a perfect detect GM soybean method for China's soybean breeding research, environmental security and food safety.
Currently the method to detect Roundup Ready soybeans mainly focused on molecular detection and field (greenhouse) whole plant identification method. As the former method,the majority of the research institutes can not carry out because of the precision of equipment, reagents ,high cost or high specificity and also only for target genes were detected, and can not verify whether the expression of target genes which although visually accurate; The latter method equired a long time, heavy workload, and vulnerability to seasonal effects even though intuitive and accurate.
In this study,we constructed the biological and biochemical method to detect Roundup Ready soybeans combining with herbicide biometric technology for the current status of research. We discussed the detection index, the different culture conditions and temperature which is the method applicable and verified it by field testing of phenotypic and molecular mean.
The main results are as follows:
ⅠThe establish of biological method to detection Roundup Ready soybeans
1. we took for soybean hypocotyl inhibition rate as an indicator to establish the biological method of detection Roundup Ready soybeans at 26℃, continuous cultured 72 h in darkness. In the method , the hypocotyl inhibition LD50 of GM soybean was1620.86-2269.33mg a. i./L while Non-GM soybean was 78.04-97.43 mg a. i./L and it has 20 times between the two soybeans. So we defined the discrimination dose as 100 mg a.i /L or 2000 mg a.i /L whlile GM soybean and Non-GM soybean can effectively distinguish in the two doses.
2. we established the biochemical method of detection Roundup Ready soybeans for the biological method. In the concentration of 100 mg a.i /L, shikimic acid accumulation of Non-GM soybean was 1237.7 mg a.i /L and GM soybean was 175.10 mg a.i. /L. There was a significant difference between the two soybeans and can effectively identify GM soybean and Non-GM soybean.
3.We detected the samples of Longkang630 and Suinong10-6047 based on the biological, biochemical method。The results showed that Longkang630 and Suinong10-6047 was GM soybean and EPSPS gene was transferred into soybean which obtained the resistannce. Field testing of phenotypic showed that Longkang630 and Suinong10-6047 was Roundup Ready soybean and the two samples was identified as positive by molecular mean. Three kinds of test results confirmed each other and proved biological and biochemical determination viablely creditable.
Ⅱ.The determination of physiological and biochemical mechanism to resistance in the biological and biochemical method
1. The shikimic acid is the main way of targe pathway with the stress of glyphosate. Because of a EPSPS gene into GM soybean,which can germination and growth normally and the shikimic acid accumulation was smaller and not affected thus to in a higher concentration of glyphosate in water culture conditions , while non-transgenic soybeans do not have this capability.
2.The GSTS activity has significant difference between GM soybean and Non-GM soybean.with the stress of glyphosate,which shown that GSTS involved in the detoxification metabolism of soybean and the metabolizable ability of GSTs is the other important reason of herbicide resistance to glyphosate . Late activity was gradually reduced with the concentration increase and time extension and shown that the detoxification capacity is limited.
3.Although the SOD, POD activity rised in the first in the process of glyphosate Stress between the GM soybean and Non-GM soybean in vivo, but both no significant difference between them and shown that this mechanism is not the main reason of their resistance. Maybe the free radicals and active oxygen and other harmful substances was induced by the herbicide stress and water stress ,which caused the SOD, POD activity increased,. It was an instinct of self-defense, played a synergistic resistance Role.
Ⅲ.Application of biological, biochemical method in the Hybridization
We detected the the offspring 06-698,1568, HF1, HF9, Sui BC1F6006, in as J9331, H04-7-11 efficiently by the method,which shown that the resistance extent to glyphosate significantly higher due to EPSPS gene transfer into successful and increased 20-30 times than their than hybrid parents。So we identified them as Roundup Ready soybeans and the results are consistent with the molecule detection.
目前关于抗草甘膦转基因大豆的检测方法主要集中在分子检测法和田间(温室)整株鉴定法。前者由于仪器、试剂要求精确,成本较高,专一性强,且只是针对靶标基因进行检测,并不能验证靶标基因的表达与否;后者虽然直观准确,但所需时间比较长、工作量大,且易受季节影响。
本研究针对目前的的研究现状,利用除草剂生物测定技术,构建了抗草甘膦转基因大豆生物生化测定方法;并且对检测指标、该方法适用的不同培养条件、温度等进行了深入的研究,同时辅以田间表型鉴定和分子检测手段对方法进行了验证。
主要研究结果如下:
一、抗草甘膦转基因大豆生物测定方法的建立
1、在26℃、持续黑暗培养72 h,以大豆萌发过程中草甘膦对下胚轴抑制率为检测指标,建立了检测转基因大豆的生物测定方法。研究结果表明:转基因大豆下胚轴抑制中浓度为1620.86-2269.33mg a. i./L;非转基因大豆的下胚轴抑制中浓度为78.04-97.43 mg a. i./L,二者有显著性差异。因此,为方便快速,在各自95%置信区间内,确定快速甄别剂量为100 mg a. i./L,或者2000 mg a. i./L,在此甄别剂量下可以有效区分转基因大豆和非转基因大豆。
2、依据生物测定的方法,建立了以莽草酸积累量为指标的生物化学检测方法:即在100 mg a. i./L(非转基因大豆的抑制中浓度)下,非转基因大豆的莽草酸积累量约为1237.7 ug/g FW,转基因大豆的莽草酸积累量只有64.61ug/g FW,二者之间有显著性差异,可以有效地鉴别转基因大豆和非转基因大豆。
3、利用生物和生化测定方法,对样本龙抗630、绥10-6047进行了检测。结果表明:龙抗630和绥10-6047为转基因大豆,确定EPSPS基因已经成功转入大豆体内,并且获得表达。田间表型鉴定表明龙抗630、绥10-6047为高抗草甘膦大豆,同时分子检测结果表明两份大豆为阳性。三种检测结果相互印证,证明生物生化测定方法可行。
二、室内生物测定方法建立的生理生化机理研究
1、受到草甘膦胁迫时,莽草酸途径是主要的靶标途径。转基因大豆因其转入了EPSPS基因,表现了此生理途径不受影响,莽草酸的积累量较小,从而能够在较高浓度的草甘膦水培条件下正常的萌发生长,而非转基因大豆则没有此能力。
2、受到草甘膦胁迫时,转基因大豆和非转基因大豆的GSTS活性有显著性差异,表明GSTS参与了大豆体内的解毒代谢作用,是大豆产生抗性的“解毒代谢层次”的重要原因。后期随着浓度的增加和时间的延长活性逐渐减弱表明其解毒代谢能力是有限的。
3、在抵抗草甘膦的胁迫过程中,转基因大豆和非转基因大豆体内SOD、POD活性虽然在最初有上升,但二者之间并无显著性差异,表明这种机制其并不是抗性产生主要原因。可能是由于大豆受到“除草剂逆境和水分逆境”的胁迫而诱发了自由基和活性氧等有害物质的产生,进而引起了SOD、POD活性的增加,是一种本能自卫反映,起到一个协同抗性的作用。
三、检测方法的在杂交育种中的应用
运用生物测定方法较为快捷的检测了7个杂交后代品系,结果表明其大豆品种抗性程度明显比杂交亲本提高20-30倍,鉴定为转基因抗草甘膦大豆,表明EPSPS基因转入成功,与分子检测结果相符。
The cultivation and promotion of t Genetically Modified soybean increased the efficiency of weeding observably,obtained great economic benefit。As researching deeply, the ecological security of Genetically Modified soybean.the flow of resistance genes and food security by alien genes in transgenic herbicide resistants crops has caused more and more controversies and inhibited its developing. So it has important significance to establish a perfect detect GM soybean method for China's soybean breeding research, environmental security and food safety.
Currently the method to detect Roundup Ready soybeans mainly focused on molecular detection and field (greenhouse) whole plant identification method. As the former method,the majority of the research institutes can not carry out because of the precision of equipment, reagents ,high cost or high specificity and also only for target genes were detected, and can not verify whether the expression of target genes which although visually accurate; The latter method equired a long time, heavy workload, and vulnerability to seasonal effects even though intuitive and accurate.
In this study,we constructed the biological and biochemical method to detect Roundup Ready soybeans combining with herbicide biometric technology for the current status of research. We discussed the detection index, the different culture conditions and temperature which is the method applicable and verified it by field testing of phenotypic and molecular mean.
The main results are as follows:
ⅠThe establish of biological method to detection Roundup Ready soybeans
1. we took for soybean hypocotyl inhibition rate as an indicator to establish the biological method of detection Roundup Ready soybeans at 26℃, continuous cultured 72 h in darkness. In the method , the hypocotyl inhibition LD50 of GM soybean was1620.86-2269.33mg a. i./L while Non-GM soybean was 78.04-97.43 mg a. i./L and it has 20 times between the two soybeans. So we defined the discrimination dose as 100 mg a.i /L or 2000 mg a.i /L whlile GM soybean and Non-GM soybean can effectively distinguish in the two doses.
2. we established the biochemical method of detection Roundup Ready soybeans for the biological method. In the concentration of 100 mg a.i /L, shikimic acid accumulation of Non-GM soybean was 1237.7 mg a.i /L and GM soybean was 175.10 mg a.i. /L. There was a significant difference between the two soybeans and can effectively identify GM soybean and Non-GM soybean.
3.We detected the samples of Longkang630 and Suinong10-6047 based on the biological, biochemical method。The results showed that Longkang630 and Suinong10-6047 was GM soybean and EPSPS gene was transferred into soybean which obtained the resistannce. Field testing of phenotypic showed that Longkang630 and Suinong10-6047 was Roundup Ready soybean and the two samples was identified as positive by molecular mean. Three kinds of test results confirmed each other and proved biological and biochemical determination viablely creditable.
Ⅱ.The determination of physiological and biochemical mechanism to resistance in the biological and biochemical method
1. The shikimic acid is the main way of targe pathway with the stress of glyphosate. Because of a EPSPS gene into GM soybean,which can germination and growth normally and the shikimic acid accumulation was smaller and not affected thus to in a higher concentration of glyphosate in water culture conditions , while non-transgenic soybeans do not have this capability.
2.The GSTS activity has significant difference between GM soybean and Non-GM soybean.with the stress of glyphosate,which shown that GSTS involved in the detoxification metabolism of soybean and the metabolizable ability of GSTs is the other important reason of herbicide resistance to glyphosate . Late activity was gradually reduced with the concentration increase and time extension and shown that the detoxification capacity is limited.
3.Although the SOD, POD activity rised in the first in the process of glyphosate Stress between the GM soybean and Non-GM soybean in vivo, but both no significant difference between them and shown that this mechanism is not the main reason of their resistance. Maybe the free radicals and active oxygen and other harmful substances was induced by the herbicide stress and water stress ,which caused the SOD, POD activity increased,. It was an instinct of self-defense, played a synergistic resistance Role.
Ⅲ.Application of biological, biochemical method in the Hybridization
We detected the the offspring 06-698,1568, HF1, HF9, Sui BC1F6006, in as J9331, H04-7-11 efficiently by the method,which shown that the resistance extent to glyphosate significantly higher due to EPSPS gene transfer into successful and increased 20-30 times than their than hybrid parents。So we identified them as Roundup Ready soybeans and the results are consistent with the molecule detection.
引文
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