高粱幼苗水分胁迫诱导表达差异cDNA的研究
摘要
干旱是能够引起植物发生水分胁迫的一种最重要的环境因子,它极大限度地制约着植物生长和作物产量。改善和提高植物的抗旱性研究是植物抗逆性研究的重要内容。随着分子生物学技术的发展,通过基因工程改良植物的抗旱能力被广大科研工作者认为是一种快捷、有效的方法。
差异显示反转录聚合酶链式反应,简称DDRT-PCR。因其操作简单、快速、灵敏性高、易重复等优点,自1992年由Liang和Pardee发明以来,已成为分子生物学领域一种强有力的工具,被广泛地用来分离差异表达的基因。
在当前的研究中,我们采用了mRNA差异显示技术和银染技术,对经过用19.5%(ψ=-0.7MPa)PEG-6000溶液处理不同时间而诱导表达的高粱基因进行分离,共得到差异表达的cDNA片段53条。方法是:用19.5%(ψ=-0.7MPa)PEG-6000溶液来处理高粱幼苗,用去离子水培育幼苗做对照,提取的总RNA经反转录后进行PCR扩增,扩增的PCR产物最后经6%的变性聚丙烯酰胺凝胶电泳分离,银染染色后可观察到清晰的条带。所回收的53条差异带,其中包括6个完全诱导表达片段,42个上调表达片段和5个下调表达片段。经Reverse Northern.验证后,得到阳性表达的cDNA片段13个。当用单个随机引物对上述13个片段进行再次扩增时,全部表现为阴性。得到的13个阳性cDNA片段克隆并测序,通过与GenBank中的EST数据库比对,结果发现,10个cDNA片段与已知序列有较高的同源性,3个为未知基因序列。其中,受水分胁迫处理诱导表达信号增强的cDNA片段T_(11)A—AP_3与损伤的二色高粱叶片cDNA克隆同源性100%,与氧化胁迫的二色高粱叶片和根cDNA克隆同源性100%,与酸碱处理的二色高粱根cDNA克隆同源性100%,与水杨酸处理的二色高粱苗期cDNA克隆同源性100%;T_(11)G—AP_3-2与氧化胁迫的二色高粱叶片和根cDNA克隆同源性100%;T_(11)G—AP_4与脱落酸、铁缺乏和盐胁迫诱导的二色高粱苗期cDNA克隆同源性100%。另外7个受水分胁迫处理诱导表达的cDNA片段序列与水杨酸处理的二色高粱苗期cDNA、损伤的二色高粱叶片cDNA、氧化胁迫的二色高粱叶片和根cDNA、酸碱处理的二色高粱根cDNA、盐胁迫诱导的二色高粱苗期cDNA、干旱胁迫诱导的二色高粱开花前cDNA、干旱胁迫诱导的二色高粱开花后cDNA、二色高粱光生长cDNA、铁缺乏诱导的二色高粱苗期cDNA同源性高达95%以上。另外3个差异片段与已知序列同源性非常低,推测可能为新的基因序列,其序列全长及其功能鉴定有待于进一步的工作。
Drought is one of the most important environmental factors that cause osmotic stress and dramatically limit plant growth and crop productivity. It's an important branch of plant research on stress resistance to enhance or improve the capability of drought and water deficit—tolerance in plant. With the advance of molecular biological technique, bioengineering becomes a rapid efficient method of improving plant resistance to drought, water deficit, on the viewpoint of many researchers.Differential display reverse transcription polymerase chain reaction (DDRT-PCR), one of the most widely employed techniques to identify differential gene expression, has become a powerful tool in the area of molecular genetics since 1992 when the technique was invented by Liang and Pardee. The technique is simple, time-saving, sensitive and reproducible.In present research, we adopted both mRNA differential display and silver staining techniques to isolate grain sorghum cDNA induced by 19.5%(-0.7MPa)PEG-6000, total 53 differential expression cDNA fragments were obtained. Sorghum seedling in 19.5% PEG-6000 (polyethylene glycol, MW=6000, V= -0.7MPa) solution was used as treatment sample, and sorghum seedling in dH_2O as control sample. Differential expression of mRNA between treatment sample and control sample was analysed by DDRT-PCR. The PCR amplification products were separated by 6% denaturing polyacrylamide gels. Distinct bands were visualized by silver staining. A total of 53 differential expression cDNA fragments had been detected, including 6 absolute expression fragments induced by water stress, 42 enhanced expression fragments, 5 inhibited expression fragments. After further certified with reverse northern dot blot, thirteen fragments are proved to be positive. When amplified by single arbitrary primer, all of the above fragments appeared negative. These fragments were cloned in the pGEM?-T Easy Vector . Through sequencing and then querying EST database of GenBank, we can find that ten of them have high homologous sequences while the other 3 have poor. Of these ten fragments, T_11A—AP_3 was 100% homologous to wounded leaves Sorghum bicolor cDNA clone . Oxidatively-stressed leaves and roots Sorghum bicolor cDNA clone. Acid- and alkaline-treated roots Sorghum bicolor cDNA clone and Salicylic acid-treated seedlings Sorghum bicolor cDNA clone. T_11G—AP_3-2 was 100% homologous to Oxidatively-stressed leaves and roots Sorghum bicolor cDNA clone. T_11G—AP_4 was 100% homologous to Abscisic acid-treated seedlings Sorghum bicolor cDNA clone. Iron-deficient seedlings Sorghum bicolor cDNA clone and Salt-stressed seedlings Sorghum bicolor cDNA clone. Other seven fragments were more than 95% homologous to the Sorghum bicolor cDNA clone induced by Abscisic acid-treated 、 wounded 、 Oxidatively-stressed 、 Acid- and alkaline-treated、 Salt-stressed、 Drought-stressed before flowering、 Light Grown and Iron-deficient. The others there were low homologous to EST library and were supposed novel genes, whose functions need further study. Therefor, it most probably be a novel gene, and there is still much work to do for its full length and its function identification.
差异显示反转录聚合酶链式反应,简称DDRT-PCR。因其操作简单、快速、灵敏性高、易重复等优点,自1992年由Liang和Pardee发明以来,已成为分子生物学领域一种强有力的工具,被广泛地用来分离差异表达的基因。
在当前的研究中,我们采用了mRNA差异显示技术和银染技术,对经过用19.5%(ψ=-0.7MPa)PEG-6000溶液处理不同时间而诱导表达的高粱基因进行分离,共得到差异表达的cDNA片段53条。方法是:用19.5%(ψ=-0.7MPa)PEG-6000溶液来处理高粱幼苗,用去离子水培育幼苗做对照,提取的总RNA经反转录后进行PCR扩增,扩增的PCR产物最后经6%的变性聚丙烯酰胺凝胶电泳分离,银染染色后可观察到清晰的条带。所回收的53条差异带,其中包括6个完全诱导表达片段,42个上调表达片段和5个下调表达片段。经Reverse Northern.验证后,得到阳性表达的cDNA片段13个。当用单个随机引物对上述13个片段进行再次扩增时,全部表现为阴性。得到的13个阳性cDNA片段克隆并测序,通过与GenBank中的EST数据库比对,结果发现,10个cDNA片段与已知序列有较高的同源性,3个为未知基因序列。其中,受水分胁迫处理诱导表达信号增强的cDNA片段T_(11)A—AP_3与损伤的二色高粱叶片cDNA克隆同源性100%,与氧化胁迫的二色高粱叶片和根cDNA克隆同源性100%,与酸碱处理的二色高粱根cDNA克隆同源性100%,与水杨酸处理的二色高粱苗期cDNA克隆同源性100%;T_(11)G—AP_3-2与氧化胁迫的二色高粱叶片和根cDNA克隆同源性100%;T_(11)G—AP_4与脱落酸、铁缺乏和盐胁迫诱导的二色高粱苗期cDNA克隆同源性100%。另外7个受水分胁迫处理诱导表达的cDNA片段序列与水杨酸处理的二色高粱苗期cDNA、损伤的二色高粱叶片cDNA、氧化胁迫的二色高粱叶片和根cDNA、酸碱处理的二色高粱根cDNA、盐胁迫诱导的二色高粱苗期cDNA、干旱胁迫诱导的二色高粱开花前cDNA、干旱胁迫诱导的二色高粱开花后cDNA、二色高粱光生长cDNA、铁缺乏诱导的二色高粱苗期cDNA同源性高达95%以上。另外3个差异片段与已知序列同源性非常低,推测可能为新的基因序列,其序列全长及其功能鉴定有待于进一步的工作。
Drought is one of the most important environmental factors that cause osmotic stress and dramatically limit plant growth and crop productivity. It's an important branch of plant research on stress resistance to enhance or improve the capability of drought and water deficit—tolerance in plant. With the advance of molecular biological technique, bioengineering becomes a rapid efficient method of improving plant resistance to drought, water deficit, on the viewpoint of many researchers.Differential display reverse transcription polymerase chain reaction (DDRT-PCR), one of the most widely employed techniques to identify differential gene expression, has become a powerful tool in the area of molecular genetics since 1992 when the technique was invented by Liang and Pardee. The technique is simple, time-saving, sensitive and reproducible.In present research, we adopted both mRNA differential display and silver staining techniques to isolate grain sorghum cDNA induced by 19.5%(-0.7MPa)PEG-6000, total 53 differential expression cDNA fragments were obtained. Sorghum seedling in 19.5% PEG-6000 (polyethylene glycol, MW=6000, V= -0.7MPa) solution was used as treatment sample, and sorghum seedling in dH_2O as control sample. Differential expression of mRNA between treatment sample and control sample was analysed by DDRT-PCR. The PCR amplification products were separated by 6% denaturing polyacrylamide gels. Distinct bands were visualized by silver staining. A total of 53 differential expression cDNA fragments had been detected, including 6 absolute expression fragments induced by water stress, 42 enhanced expression fragments, 5 inhibited expression fragments. After further certified with reverse northern dot blot, thirteen fragments are proved to be positive. When amplified by single arbitrary primer, all of the above fragments appeared negative. These fragments were cloned in the pGEM?-T Easy Vector . Through sequencing and then querying EST database of GenBank, we can find that ten of them have high homologous sequences while the other 3 have poor. Of these ten fragments, T_11A—AP_3 was 100% homologous to wounded leaves Sorghum bicolor cDNA clone . Oxidatively-stressed leaves and roots Sorghum bicolor cDNA clone. Acid- and alkaline-treated roots Sorghum bicolor cDNA clone and Salicylic acid-treated seedlings Sorghum bicolor cDNA clone. T_11G—AP_3-2 was 100% homologous to Oxidatively-stressed leaves and roots Sorghum bicolor cDNA clone. T_11G—AP_4 was 100% homologous to Abscisic acid-treated seedlings Sorghum bicolor cDNA clone. Iron-deficient seedlings Sorghum bicolor cDNA clone and Salt-stressed seedlings Sorghum bicolor cDNA clone. Other seven fragments were more than 95% homologous to the Sorghum bicolor cDNA clone induced by Abscisic acid-treated 、 wounded 、 Oxidatively-stressed 、 Acid- and alkaline-treated、 Salt-stressed、 Drought-stressed before flowering、 Light Grown and Iron-deficient. The others there were low homologous to EST library and were supposed novel genes, whose functions need further study. Therefor, it most probably be a novel gene, and there is still much work to do for its full length and its function identification.
引文
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