疫霉菌诱导的南瓜SSH文库构建及表达序列标签分析
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摘要
南瓜是葫芦科南瓜属一年生经济作物,其经济价值和药用价值均十分显著。随着对外贸易的发展和南瓜籽及南瓜系列产品的开发,南瓜种植面积不断扩大,为农民带来了巨大的经济效益。在有的年份和地块由于南瓜疫病的流行,籽用南瓜的产区受疫病危害,引起的死秧、烂瓜现象严重,重症年份部分地区甚至绝产,因此,疫病已成为南瓜生产的限制因子,生产上迫切需要解决疫病危害的问题。然而,只有深入研究南瓜的抗病机制以及南瓜与病原菌的互作关系,才能从根本上解决这个问题。本研究运用抑制差减杂交(SSH)技术构建cDNA文库,应用EST技术并结合生物信息学技术,通过对南瓜抗疫病相关基因的表达分析,从基因水平来研究南瓜抗病机制。为今后的南瓜抗疫病遗传育种奠定理论基础。
本试验构建了一个南瓜接种疫霉菌后6,12,24,36,48,72小时的混合SSHcDNA文库,通过反向Northern杂交、质粒双酶切、菌液PCR进行初步筛选后,对其中60个阳性克隆进行单向测序获得55个质量较好的EST序列。与GenBank数据库进行BLASTx和BLASTn比较分析,共有52条EST具有蛋白编码功能,占全部分析EST的94.5%,3条EST找不到任何同源序列,推测可能是一类新的基因,或变异度较高的cDNA非编码区。与52条EST同源的推测蛋白质序列来自黄瓜、西瓜、拟南芥、水稻、甘蓝型油菜、甘蓝、马铃薯、葡萄、玉米、烟草、高粱、甜菜、菜豆等多个物种。
通过对获得的EST序列进行分析,推测钙调蛋白激酶、茉莉酸等信号传导系统可能参与了南瓜抗疫病防卫反应过程中的信号传导,但各信号传导间的相互作用机制还不清楚。抗病相关转录因子包括锌指蛋白、MYC转录因子、G-box结合蛋白,它们可能在南瓜抗病防卫反应基因的表达调控中起到重要作用。
在本研究中,有两条EST与马铃薯和拟南芥的抗病基因类似物有部分同源性,应该是参与南瓜抗疫病反应的重要基因。另外在所获得的EST中存在部分非生物胁迫诱导蛋白如热休克蛋白、盐诱导蛋白等。说明生物机体对外界的胁迫反应,存在某种协同机制。
在获得的EST中还包括一些RNA加工、蛋白翻译与加工、光合作用、能量代谢基因,说明这些基础功能基因也与南瓜抗病机制相关。
Pumpkin is annual economic crop. And it is a plant genus of the family cucurbitaceae. It has very important economic value and medicine value. As the foreign trade and the serial products about pumpkin developed, cultivated area of pumpkin keeps expanding. And because of that, farmers gained more economic returns. But pumpkin blight caused huge loss in some years and some areas. So pumpkin loemia is one of the limiting factors about pumpkin production. And it is a troublesome question need to be deal with. At first ,we need to know the resistance mechanism and the mechanism of action between pumpkin and pathogenic bacterium. In this study, a suppression subtractive hybridization (SSH) cDNA library was first constructed. Then using expression sequence tags (ESTs) and bioinformatics to analyze the expression of related gene resisted pumpkin loemia. To study the resistance mechanism about pumpkin at molecule level. And the study may help disease resistance breeding.
In this study, SSHcDNA library was detected by reserve northern blot, PCR, double digestion on plasmid. And then 60 cDNA positive clones were randomly selected and sequenced. 55 ESTs with high quality were obtained. Similarity analysis was based on BLASTx and BLASTn softwares in GenBank. 52 of the ESTs were identified encoding putative proteins. 3 of the ESTs were found no significant similarity and they may represent new genes or high variant non-coding regions of cDNAs. The best matched protein sequences with those encoded by the 52 ESTs were from so many plant species, such as Cucumis sativus, Citrullus lanatus, Arabidopsis thaliana, Oryza sativa, Brassica napus, Brassica oleracea, Solanum tuberosum, Vitis vinifera, Zea mays, Nicotiana tobacum, Sorghum vulgare, Beta vulgaris, Phaseolus vulgaris and so on.
After EST analysis, we supposed that calcium-mediated signal transduction system and jasminate acid signal transduction system play roles in pumpkin’s resistance to blight. But the interactions among various signal transduction pathways are not clear. Transcription factor related resistance contains zinc finger protein, MYC transcription factor, G-box binding protein. They may play important roles in expression and regulation of gene related pumpkin’s resistance.
In this study, we found two resistance gene analog (RGA). They should be to participate pumpkin’s resistance, and play a very important part in it. So this two genes need to be studied to know the resistance mechanism. In addition, in the ESTs obtained, there are some ESTs for abiotic stress-induced protein, such as heat shock protein, salt induced protein and so on. This result suggests that a possible common mechanism exist during the stress reaction of plants towards different environments.
These ESTs contained other aspects, such as RNA processing, protein interpretation and processing, photosynthesis, energy metabolism. These genes should be related to pumpkin resistance mechanism.
本试验构建了一个南瓜接种疫霉菌后6,12,24,36,48,72小时的混合SSHcDNA文库,通过反向Northern杂交、质粒双酶切、菌液PCR进行初步筛选后,对其中60个阳性克隆进行单向测序获得55个质量较好的EST序列。与GenBank数据库进行BLASTx和BLASTn比较分析,共有52条EST具有蛋白编码功能,占全部分析EST的94.5%,3条EST找不到任何同源序列,推测可能是一类新的基因,或变异度较高的cDNA非编码区。与52条EST同源的推测蛋白质序列来自黄瓜、西瓜、拟南芥、水稻、甘蓝型油菜、甘蓝、马铃薯、葡萄、玉米、烟草、高粱、甜菜、菜豆等多个物种。
通过对获得的EST序列进行分析,推测钙调蛋白激酶、茉莉酸等信号传导系统可能参与了南瓜抗疫病防卫反应过程中的信号传导,但各信号传导间的相互作用机制还不清楚。抗病相关转录因子包括锌指蛋白、MYC转录因子、G-box结合蛋白,它们可能在南瓜抗病防卫反应基因的表达调控中起到重要作用。
在本研究中,有两条EST与马铃薯和拟南芥的抗病基因类似物有部分同源性,应该是参与南瓜抗疫病反应的重要基因。另外在所获得的EST中存在部分非生物胁迫诱导蛋白如热休克蛋白、盐诱导蛋白等。说明生物机体对外界的胁迫反应,存在某种协同机制。
在获得的EST中还包括一些RNA加工、蛋白翻译与加工、光合作用、能量代谢基因,说明这些基础功能基因也与南瓜抗病机制相关。
Pumpkin is annual economic crop. And it is a plant genus of the family cucurbitaceae. It has very important economic value and medicine value. As the foreign trade and the serial products about pumpkin developed, cultivated area of pumpkin keeps expanding. And because of that, farmers gained more economic returns. But pumpkin blight caused huge loss in some years and some areas. So pumpkin loemia is one of the limiting factors about pumpkin production. And it is a troublesome question need to be deal with. At first ,we need to know the resistance mechanism and the mechanism of action between pumpkin and pathogenic bacterium. In this study, a suppression subtractive hybridization (SSH) cDNA library was first constructed. Then using expression sequence tags (ESTs) and bioinformatics to analyze the expression of related gene resisted pumpkin loemia. To study the resistance mechanism about pumpkin at molecule level. And the study may help disease resistance breeding.
In this study, SSHcDNA library was detected by reserve northern blot, PCR, double digestion on plasmid. And then 60 cDNA positive clones were randomly selected and sequenced. 55 ESTs with high quality were obtained. Similarity analysis was based on BLASTx and BLASTn softwares in GenBank. 52 of the ESTs were identified encoding putative proteins. 3 of the ESTs were found no significant similarity and they may represent new genes or high variant non-coding regions of cDNAs. The best matched protein sequences with those encoded by the 52 ESTs were from so many plant species, such as Cucumis sativus, Citrullus lanatus, Arabidopsis thaliana, Oryza sativa, Brassica napus, Brassica oleracea, Solanum tuberosum, Vitis vinifera, Zea mays, Nicotiana tobacum, Sorghum vulgare, Beta vulgaris, Phaseolus vulgaris and so on.
After EST analysis, we supposed that calcium-mediated signal transduction system and jasminate acid signal transduction system play roles in pumpkin’s resistance to blight. But the interactions among various signal transduction pathways are not clear. Transcription factor related resistance contains zinc finger protein, MYC transcription factor, G-box binding protein. They may play important roles in expression and regulation of gene related pumpkin’s resistance.
In this study, we found two resistance gene analog (RGA). They should be to participate pumpkin’s resistance, and play a very important part in it. So this two genes need to be studied to know the resistance mechanism. In addition, in the ESTs obtained, there are some ESTs for abiotic stress-induced protein, such as heat shock protein, salt induced protein and so on. This result suggests that a possible common mechanism exist during the stress reaction of plants towards different environments.
These ESTs contained other aspects, such as RNA processing, protein interpretation and processing, photosynthesis, energy metabolism. These genes should be related to pumpkin resistance mechanism.
引文
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