黑腐病诱导的甘蓝SSH文库构建及ESTs分析
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摘要
结球甘蓝(Brassica oleracea var. capitata)简称甘蓝。因耐寒、适应性强、易贮耐运、产量高、品质好等特点,现在我国各地普遍栽培。随着生产上大面积栽培,甘蓝的病虫害危害日益严重,引起甘蓝产量和品质的严重下降。甘蓝黑腐病是由野油菜黄单胞菌引起的一种世界性病害,是甘蓝栽培生产的主要限制因子。本研究采用了抑制差减杂交(SSH)技术构建SSH文库,应用EST技术与生物信息学分析技术,对甘蓝抗黑腐病相关基因进行分析,从基因水平上研究甘蓝抗病机制。为今后甘蓝抗黑腐病遗传育种奠定理论基础。
本研究采用抗黑腐病的甘蓝自交系“A21”为试材,以接种黑腐病菌后6h、8h、12h、24h后的材料为试验方(Tester)和不接菌的材料为驱动方(Driver)构建SSH文库。通过反向Northern杂交、菌液PCR进行筛选后,对其中92个阳性克隆进行单向测序获得87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83条EST具有蛋白编码功能,占全部分析EST的95.4%。3条ESTS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83条EST同源的推测蛋白质序列来自拟南芥、甜瓜、豇豆、小麦、水稻、葡萄、黄瓜、玉米、烟草、甘蓝、亚麻等物种。
通过对获得的ESTS序列进行分析,发现了一些与信号传导有关的蛋白如serine/threonine kinase丝/苏氨酸激酶,MAP3K alpha protein kinase。一些与初级代谢有关的酶如peptidylprolyl isomerase肽基脯氨酰异构酶,malate dehydrogenase苹果酸脱氢酶等。putative rubisco activase核酮糖二磷酸羟化酶和叶绿素chlorophyll a/b-binding proteina/b结合蛋白等是与光合作用有关的蛋白。在本研究中,有8条ESTs与甘蓝和拟南芥的抗病基因类似物有部分同源性,应该是参与甘蓝抗黑腐病反应的重要基因。另外在所获得的ESTS中存在部分非生物胁迫诱导蛋白如heat shock protein 20 (hsp20)热休克蛋白,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ESTS中还包括一些次生代谢、抗病和防御基因、蛋白质合成、能量代谢等基因,推测这些基础功能基因与甘蓝抗病机制相关。
The cabbage (Brassica oleracea var. capitata), which was also called head cabbage or heading cabbage .Because of hardy, adaptable and easy to transport storage-resistant, high yield, good quality and so on .It was a common vegetable crop in China. With the production of large-scale cultivation The cabbage arised increasingly serious pests and diseases. In recent years, Black rot caused by Xanthomonas campestris pv. Campestris (Xcc), was one of the most serious diseases of the cabbage causing serious reduction in yield and quality. In this reserch, a suppression subtractive hybridization (SSH) cDNA library was constructed. After analysis of reserve northern blot, PCR, double digestion on plasmid. and then 92 cDNA positive clones were randomly selected and sequenced. 87 ESTs with high quality were obtained. Similarity analysis was based on BLASTx and BLASTn softwares in GenBank. 83 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. 83of the ESTs were different similarity with some protein sequences and genes which were from so many plant species,such as Arabidopsis thaliana,Citrullus lanatus Triticum aestivum L.Oryza sativa,Phaseolus vulgaris, Vitis vinifera,Cucumis sativus,Zea mays,Brassica oleracea,Nicotiana tobacum,Linumusitatissimum and so on.By the ESTs function classification found:The genes were related with primery metabolism,energy metabolism, protein synthesis, disease/defense, and single. Transduction secondary metabolism.
After ESTS analysis,we found some Signal transduction-related protein such as serine/threonine kinase、MAP3K alpha protein kinase and Some enzymes of primary metabolism such as peptidylprolyl isomerase malate dehydrogenase. putative rubisco activase chlorophyll a/b-binding protein are photosynthesis-related proteins.
In this experiment we found 8 ESTs of Cabbage and Arabidopsis resistance gene analogues and some homology and play a very important part in it .In addition, in the ESTs obtained, there are some ESTs for abiotic stress-induced protein, such as heat shock protein, This result suggested that a possible common mechanism exists during the stress reaction of plants towards different environments.
These ESTs contained other aspects, such as Secondary metabolism, disease resistance and defense genes, protein synthesis, energy metabolism genes, These genes should be related to cabbage resistance mechanism.
本研究采用抗黑腐病的甘蓝自交系“A21”为试材,以接种黑腐病菌后6h、8h、12h、24h后的材料为试验方(Tester)和不接菌的材料为驱动方(Driver)构建SSH文库。通过反向Northern杂交、菌液PCR进行筛选后,对其中92个阳性克隆进行单向测序获得87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83条EST具有蛋白编码功能,占全部分析EST的95.4%。3条ESTS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83条EST同源的推测蛋白质序列来自拟南芥、甜瓜、豇豆、小麦、水稻、葡萄、黄瓜、玉米、烟草、甘蓝、亚麻等物种。
通过对获得的ESTS序列进行分析,发现了一些与信号传导有关的蛋白如serine/threonine kinase丝/苏氨酸激酶,MAP3K alpha protein kinase。一些与初级代谢有关的酶如peptidylprolyl isomerase肽基脯氨酰异构酶,malate dehydrogenase苹果酸脱氢酶等。putative rubisco activase核酮糖二磷酸羟化酶和叶绿素chlorophyll a/b-binding proteina/b结合蛋白等是与光合作用有关的蛋白。在本研究中,有8条ESTs与甘蓝和拟南芥的抗病基因类似物有部分同源性,应该是参与甘蓝抗黑腐病反应的重要基因。另外在所获得的ESTS中存在部分非生物胁迫诱导蛋白如heat shock protein 20 (hsp20)热休克蛋白,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ESTS中还包括一些次生代谢、抗病和防御基因、蛋白质合成、能量代谢等基因,推测这些基础功能基因与甘蓝抗病机制相关。
The cabbage (Brassica oleracea var. capitata), which was also called head cabbage or heading cabbage .Because of hardy, adaptable and easy to transport storage-resistant, high yield, good quality and so on .It was a common vegetable crop in China. With the production of large-scale cultivation The cabbage arised increasingly serious pests and diseases. In recent years, Black rot caused by Xanthomonas campestris pv. Campestris (Xcc), was one of the most serious diseases of the cabbage causing serious reduction in yield and quality. In this reserch, a suppression subtractive hybridization (SSH) cDNA library was constructed. After analysis of reserve northern blot, PCR, double digestion on plasmid. and then 92 cDNA positive clones were randomly selected and sequenced. 87 ESTs with high quality were obtained. Similarity analysis was based on BLASTx and BLASTn softwares in GenBank. 83 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. 83of the ESTs were different similarity with some protein sequences and genes which were from so many plant species,such as Arabidopsis thaliana,Citrullus lanatus Triticum aestivum L.Oryza sativa,Phaseolus vulgaris, Vitis vinifera,Cucumis sativus,Zea mays,Brassica oleracea,Nicotiana tobacum,Linumusitatissimum and so on.By the ESTs function classification found:The genes were related with primery metabolism,energy metabolism, protein synthesis, disease/defense, and single. Transduction secondary metabolism.
After ESTS analysis,we found some Signal transduction-related protein such as serine/threonine kinase、MAP3K alpha protein kinase and Some enzymes of primary metabolism such as peptidylprolyl isomerase malate dehydrogenase. putative rubisco activase chlorophyll a/b-binding protein are photosynthesis-related proteins.
In this experiment we found 8 ESTs of Cabbage and Arabidopsis resistance gene analogues and some homology and play a very important part in it .In addition, in the ESTs obtained, there are some ESTs for abiotic stress-induced protein, such as heat shock protein, This result suggested that a possible common mechanism exists during the stress reaction of plants towards different environments.
These ESTs contained other aspects, such as Secondary metabolism, disease resistance and defense genes, protein synthesis, energy metabolism genes, These genes should be related to cabbage resistance mechanism.
引文
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