巴西固氮螺菌Yu62在小麦中的定殖以及对小麦的促生作用
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摘要
利用增强型绿色荧光蛋白EGFP基因作为报告基因对巴西固氮螺菌Yu62进行标记。首先将质粒pEGFP-C1中EGFP基因克隆至原核表达载体pVK-100上,构建重组质粒pVK-EGFP,此质粒中含有固氮螺菌复制子,可在巴西固氮螺菌Yu62中稳定复制。然后利用电转化法将重组质粒pVK-EGFP转入巴西固氮螺菌Yu62中,通过聚丙烯酰胺凝胶电泳对绿色荧光蛋白表达情况进行检测,结果表明EGFP基因在巴西固氮螺菌Yu62中能稳定表达,获得EGFP标记的菌株。
在限菌条件下,用标记菌株侵染小麦(小偃107)露白种子,取接菌1、3、5、7、30天后的小麦幼苗,经75%乙醇和1%次氯酸钠表面消毒后置于含四环素的YMA培养基上贴板培养,进行固氮菌的分离以及固氮菌在小麦中的初步定位。培养48小时后于波长为365nm紫外灯观测,结果表明,随着接菌时间的推移,小麦发荧光部位由根尖生长点逐步向上蔓延,直至茎部和叶部。另取接菌相同天数的小麦制作切片,用荧光显微镜观测固氮菌在小麦内的定殖规律。结果显示,在接菌后的第1天,标记菌株主要定殖根表面。在接菌后的第3天,标记菌株进入到小麦根的皮层,主要位于细胞间隙。在接菌后的第5天,标记菌进入内皮层,少数菌株进入到维管束。在接菌后的第7天,大量菌株进入到维管束,导管内有少量标记菌的侵入。在接菌后的第30天发现在茎、叶中有标记菌定殖,结果表明固氮菌可以由根部向地上部位迁移。
在开放条件下,将挑选出籽粒饱满的小麦(小偃107)分为两组,其中一组以巴西固氮螺菌Yu62作为菌肥,与小麦(小偃107)作拌种试验,拌种方法为:将培养至OD值约为0.6的巴西固氮螺菌菌液浸泡小麦种子,浸泡时间为2h,浸泡后将接菌小麦和不做任何处理的对照小麦种于田间,种植时沟宽0.2m,每行小麦均匀种植,保证接菌小麦和对照在数量上基本一致。在接菌五个月后对小麦观测,结果表明接菌小麦在株高以及叶片颜色上比对照小麦更高、更绿。在接菌七个月后对小麦观测,结果表明接菌小麦在株高、分蘖数以及根系发达程度上比对照小麦都有较大优势。从中可以得到以下结论:巴西固氮螺菌作为禾本科植物内生固氮菌对植物有明显的促生作用。
Using of EGFP gene as a reporter gene of Azospirillum brasilenseYu62 tagged. First, the EGFP gene was amplified by using PCR amplification methods and cloned to the prokaryotic expression vector pVK-100 to construct the recombinant plasmid pVK-EGFP, the plasmid containing Azospirillum replicon can be copied stablity. The recombinant plasmid was transformed into Azospirillum brasilense Yu62 by electroporation. Detection of the expression of green fluorescent protein through polyacrylamide gel electrophoresis, the result showed that the EGFP gene can be expressed stablity in Azospirillum brasilenseYu62 .In restricted bacteria conditions, using of marker strains inoculated wheat (Xiaoyan 107) seedlings, after 1,3,5,7,30 days, disinfection from wheat seedlings with 1% sodium hypochlorite and 75% ethanol at the surface, and then, placed in YMA medium containing tetracycline to culture for the separation of Azospirillum and initial position in wheat. After cultured for 48 hours, observation under the UV-light of 365nm wavelength, the results showed that the fluorescent position from rootstalk growth point spread upward gradually until the stem and leaf with the change in inoculation time. Checking the wheat which inoculated the same number of days to make slices and observe the colonization law of Azospirillum in wheat by Fluorescence microscope. The results showed that marked strains mainly colonizing the root surface after inoculation the one day, After inoculation three days, marked strains into the cortex of wheat roots, mainly located in cell space. After inoculation five days, marker bacteria have entered the inner cortex, a small amount of strain into the vascular bundle. After inoculation seven days, a large number of strains into the vascular bundle, a small amount of strain into the catheter. After inoculation thirty days, marked bacteria colonization in the stems and leaves of wheat, The results showed that Azospirillum can migrate from the roots to the ground site.
In open conditions, the big plump grains of wheat (Xiaoyan 107) were selected and then divided into two groups, as fertilizer, Azospirillum brasilenseYu62 was mixed with one group. The main method: when the Azospirillum brasilenseYu62 was cultured to OD value of approximately 0.6, mixed it with wheat seed soaking about 2h. After soaking, planting the inoculation and other wheat without any treatment in the fileds, with the groove width 0.2 m, uniform the plants per line, and Ensure that inoculation and the control is consistent in the quantity. In the fifth month after inoculation, the observation results show that the inoculation plants not only grow higher than the control plants, but also have more green leaves. In the seventh month after inoculation, the observation results show that the inoculation plants have large advandage in the height, tiller and the level of root developed compared to the control. All these lead us conclusion that as a grass endophytic diazotrophs, Azospirillum brasilenseYu62 has obvious growth-promoting effect on the plant.
在限菌条件下,用标记菌株侵染小麦(小偃107)露白种子,取接菌1、3、5、7、30天后的小麦幼苗,经75%乙醇和1%次氯酸钠表面消毒后置于含四环素的YMA培养基上贴板培养,进行固氮菌的分离以及固氮菌在小麦中的初步定位。培养48小时后于波长为365nm紫外灯观测,结果表明,随着接菌时间的推移,小麦发荧光部位由根尖生长点逐步向上蔓延,直至茎部和叶部。另取接菌相同天数的小麦制作切片,用荧光显微镜观测固氮菌在小麦内的定殖规律。结果显示,在接菌后的第1天,标记菌株主要定殖根表面。在接菌后的第3天,标记菌株进入到小麦根的皮层,主要位于细胞间隙。在接菌后的第5天,标记菌进入内皮层,少数菌株进入到维管束。在接菌后的第7天,大量菌株进入到维管束,导管内有少量标记菌的侵入。在接菌后的第30天发现在茎、叶中有标记菌定殖,结果表明固氮菌可以由根部向地上部位迁移。
在开放条件下,将挑选出籽粒饱满的小麦(小偃107)分为两组,其中一组以巴西固氮螺菌Yu62作为菌肥,与小麦(小偃107)作拌种试验,拌种方法为:将培养至OD值约为0.6的巴西固氮螺菌菌液浸泡小麦种子,浸泡时间为2h,浸泡后将接菌小麦和不做任何处理的对照小麦种于田间,种植时沟宽0.2m,每行小麦均匀种植,保证接菌小麦和对照在数量上基本一致。在接菌五个月后对小麦观测,结果表明接菌小麦在株高以及叶片颜色上比对照小麦更高、更绿。在接菌七个月后对小麦观测,结果表明接菌小麦在株高、分蘖数以及根系发达程度上比对照小麦都有较大优势。从中可以得到以下结论:巴西固氮螺菌作为禾本科植物内生固氮菌对植物有明显的促生作用。
Using of EGFP gene as a reporter gene of Azospirillum brasilenseYu62 tagged. First, the EGFP gene was amplified by using PCR amplification methods and cloned to the prokaryotic expression vector pVK-100 to construct the recombinant plasmid pVK-EGFP, the plasmid containing Azospirillum replicon can be copied stablity. The recombinant plasmid was transformed into Azospirillum brasilense Yu62 by electroporation. Detection of the expression of green fluorescent protein through polyacrylamide gel electrophoresis, the result showed that the EGFP gene can be expressed stablity in Azospirillum brasilenseYu62 .In restricted bacteria conditions, using of marker strains inoculated wheat (Xiaoyan 107) seedlings, after 1,3,5,7,30 days, disinfection from wheat seedlings with 1% sodium hypochlorite and 75% ethanol at the surface, and then, placed in YMA medium containing tetracycline to culture for the separation of Azospirillum and initial position in wheat. After cultured for 48 hours, observation under the UV-light of 365nm wavelength, the results showed that the fluorescent position from rootstalk growth point spread upward gradually until the stem and leaf with the change in inoculation time. Checking the wheat which inoculated the same number of days to make slices and observe the colonization law of Azospirillum in wheat by Fluorescence microscope. The results showed that marked strains mainly colonizing the root surface after inoculation the one day, After inoculation three days, marked strains into the cortex of wheat roots, mainly located in cell space. After inoculation five days, marker bacteria have entered the inner cortex, a small amount of strain into the vascular bundle. After inoculation seven days, a large number of strains into the vascular bundle, a small amount of strain into the catheter. After inoculation thirty days, marked bacteria colonization in the stems and leaves of wheat, The results showed that Azospirillum can migrate from the roots to the ground site.
In open conditions, the big plump grains of wheat (Xiaoyan 107) were selected and then divided into two groups, as fertilizer, Azospirillum brasilenseYu62 was mixed with one group. The main method: when the Azospirillum brasilenseYu62 was cultured to OD value of approximately 0.6, mixed it with wheat seed soaking about 2h. After soaking, planting the inoculation and other wheat without any treatment in the fileds, with the groove width 0.2 m, uniform the plants per line, and Ensure that inoculation and the control is consistent in the quantity. In the fifth month after inoculation, the observation results show that the inoculation plants not only grow higher than the control plants, but also have more green leaves. In the seventh month after inoculation, the observation results show that the inoculation plants have large advandage in the height, tiller and the level of root developed compared to the control. All these lead us conclusion that as a grass endophytic diazotrophs, Azospirillum brasilenseYu62 has obvious growth-promoting effect on the plant.
引文
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姜伯乐,赵宇,刘建元,苏莉莉,刘娇,王爱勤,周琼,李素丽,陆凤花,蒋建荣,唐纪良.含有增强
型绿色荧光蛋白报告子的载体系统的构建[J].广西农业生物科学, 2008,(04)
李久蒂,安千里.联合固氮研究进展[J] .植物学通报,1997 ,14(3) :14 - 21.
林敏,尤崇杓.根际联合固氮作用的研究进展[J] .植物生理通讯,1992 ,28(5) :323 - 329.
田宏,张德罡,姚拓.土壤联合固氮菌的研究进展[J] .草原与草坪,2004 ,105 , (2) :3 - 7.
温晓芳,黄俊生.细菌的群体感应及其信号分子[J] .华南热带农业大学报,2005 ,11(1) :31 - 35.
汪恒英,周守标,常志州,等.绿色荧光蛋白(GFP)研究进展[J] .生物技术,2004 ,14 (3) :70 - 73.
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