抗除草剂草甘膦基因导入玉米自交系的研究
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摘要
本研究以玉米优良自交系综31为受体材料，用PDS1000/He基因枪将抗除草剂草甘膦基因(EroA)导入其中，对玉米愈伤筛选不同次数的筛选效果和继代不同次数的愈伤在不同培养基上的分化做了初步的研究，并对T_0代和T_1代进行了分子鉴定，还对玉米田间自然状态杂草发生和自交系综31及杂草在田间水平上对草甘膦的耐受度进行了调查，确定了筛选时期和筛选浓度，并在此基础上对T_1代植株进行了除草剂田间抗性鉴定。主要结果如下：
采用五点取样法，在田间随机取样，每个样点0.2平方米，调查了玉米田间的杂草种类及其发生规律。调查结果显示，在科学园玉米田间，有11种杂草，涉及六个科属。
对玉米的生育期和杂草发生情况对比结果显示，在当年春季玉米三四叶期时杂草发生达到峰值。草甘膦在玉米三叶一心时对玉米和杂草的喷施效果表明，1.2g/l的浓度时对杂草的防治效果良好，对玉米接近致死浓度，所以确定筛选时期选择在玉米三叶一心时期，浓度为1.2g/l。
将Ubiquitin启动子构建的抗草甘膦基因导入优良玉米自交系综31的愈伤，对愈伤筛选次数效果的研究结果表明，筛选两次与筛选多次没有差异，并且筛选两次愈伤组织在分化能力和幼苗的出苗率上都衰退的不是很严重，所以筛选两次效果较好。
不同时期的愈伤用11种不同的分化培养基进行分化，结果显示各个继代时间的愈伤都在F培养基上分化情况较好，但苗较弱。
将Ubiquitin启动子构建的抗草甘膦基因导入优良玉米自交系综31的幼胚或愈伤组织，得到456个结实的再生植株。PCR扩增结果显示其中153株扩增出特异目标条带，其中134株为结实植株。PCR-Southern检测结果与PER相同。部分样品Southern检测结果也说明外源基因已整合到玉米基因组中。部分样品的RT-PCR结果说明外源基因在转基因植株中能够转录。
将T_0代的12640多粒种子种植于国家玉米改良中心海南南繁基地，于五叶期叶面喷1.2g/l的草甘膦，结果剩下十株。其中八株死亡未散粉，两株回交结实。
实验室内盆育部分种子T_1小苗，进行分子检测，PCR，Sourthern结果显示部分外源基因已传到T1代中。
This study mainly dealts with the transfer of glyphosate-resistance gene (EroA) into the elite maize inbred line of Zong 31 by using gene gun, the molecular identification of TO , TI generations and the field identification of T1. In addition, the field identification was based on the investigation result of glyphosate tolerance of Zong 3land weeds and the law of the weeds naturally occurrence in the field. The main results were as follows:
l.The kinds and the occurrence peak of weeds were investigated by five-spot-sampling method. There were 11 kinds of weeds in the maize trial field.
2.The development comparison between weeds and the maize verified that the weeds occurrence peak was in the 3-leaf and 4-leaf period of Zong 31 .The tolerance to glyphosate of Zong 31 and weeds in the 3-leaf period indicated that it was belter to prevente and cure weeds when the glyphosate concentration was 1.2g/l,which was nearly the lethal concentration of Zong 31 in the 3-leaf period.
3 The effect of different culture mediums on the different callus, which were different in the times of subculture, was investigated.The result of the "F" culture medium is better than others.
4.The glyphosate resistant gene, RA was transferred into Zong 31. Only 456 generated plants were gotten seeds, because of bad weather of continuous high temperature in greenhouse. Results from PCR, PCR-Southern, Southern blot and RT-PCR showed that the glyphosate resistant genes were integrated into the maize genome and could be transcripted in 153 TO plants, in which there were!34 plants were gotten seeds.
5.The effect of selective time was investigated in the callus, which showed that about one month was better. Because the callus in the condition kept stronger differentiation abilities but the most of control callus died and brownized, which was advantage for the plantlet.
6.The plants of Tj generation and inbred Zong 31 were spraied with glyphosate of 1.2g/J on leave surfaces in the 5-leaf period. Due to the typhoon and the other factors' effect, ten plant were remain, and only get two ears.
7.The results of PCR, PCR-Southern and Southern blot of the plant potted in the room also confirmed that the glyphosate resistant gene was transmitted to Tl generation.
采用五点取样法，在田间随机取样，每个样点0.2平方米，调查了玉米田间的杂草种类及其发生规律。调查结果显示，在科学园玉米田间，有11种杂草，涉及六个科属。
对玉米的生育期和杂草发生情况对比结果显示，在当年春季玉米三四叶期时杂草发生达到峰值。草甘膦在玉米三叶一心时对玉米和杂草的喷施效果表明，1.2g/l的浓度时对杂草的防治效果良好，对玉米接近致死浓度，所以确定筛选时期选择在玉米三叶一心时期，浓度为1.2g/l。
将Ubiquitin启动子构建的抗草甘膦基因导入优良玉米自交系综31的愈伤，对愈伤筛选次数效果的研究结果表明，筛选两次与筛选多次没有差异，并且筛选两次愈伤组织在分化能力和幼苗的出苗率上都衰退的不是很严重，所以筛选两次效果较好。
不同时期的愈伤用11种不同的分化培养基进行分化，结果显示各个继代时间的愈伤都在F培养基上分化情况较好，但苗较弱。
将Ubiquitin启动子构建的抗草甘膦基因导入优良玉米自交系综31的幼胚或愈伤组织，得到456个结实的再生植株。PCR扩增结果显示其中153株扩增出特异目标条带，其中134株为结实植株。PCR-Southern检测结果与PER相同。部分样品Southern检测结果也说明外源基因已整合到玉米基因组中。部分样品的RT-PCR结果说明外源基因在转基因植株中能够转录。
将T_0代的12640多粒种子种植于国家玉米改良中心海南南繁基地，于五叶期叶面喷1.2g/l的草甘膦，结果剩下十株。其中八株死亡未散粉，两株回交结实。
实验室内盆育部分种子T_1小苗，进行分子检测，PCR，Sourthern结果显示部分外源基因已传到T1代中。
This study mainly dealts with the transfer of glyphosate-resistance gene (EroA) into the elite maize inbred line of Zong 31 by using gene gun, the molecular identification of TO , TI generations and the field identification of T1. In addition, the field identification was based on the investigation result of glyphosate tolerance of Zong 3land weeds and the law of the weeds naturally occurrence in the field. The main results were as follows:
l.The kinds and the occurrence peak of weeds were investigated by five-spot-sampling method. There were 11 kinds of weeds in the maize trial field.
2.The development comparison between weeds and the maize verified that the weeds occurrence peak was in the 3-leaf and 4-leaf period of Zong 31 .The tolerance to glyphosate of Zong 31 and weeds in the 3-leaf period indicated that it was belter to prevente and cure weeds when the glyphosate concentration was 1.2g/l,which was nearly the lethal concentration of Zong 31 in the 3-leaf period.
3 The effect of different culture mediums on the different callus, which were different in the times of subculture, was investigated.The result of the "F" culture medium is better than others.
4.The glyphosate resistant gene, RA was transferred into Zong 31. Only 456 generated plants were gotten seeds, because of bad weather of continuous high temperature in greenhouse. Results from PCR, PCR-Southern, Southern blot and RT-PCR showed that the glyphosate resistant genes were integrated into the maize genome and could be transcripted in 153 TO plants, in which there were!34 plants were gotten seeds.
5.The effect of selective time was investigated in the callus, which showed that about one month was better. Because the callus in the condition kept stronger differentiation abilities but the most of control callus died and brownized, which was advantage for the plantlet.
6.The plants of Tj generation and inbred Zong 31 were spraied with glyphosate of 1.2g/J on leave surfaces in the 5-leaf period. Due to the typhoon and the other factors' effect, ten plant were remain, and only get two ears.
7.The results of PCR, PCR-Southern and Southern blot of the plant potted in the room also confirmed that the glyphosate resistant gene was transmitted to Tl generation.
引文
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