斯氏线虫抗逆性幼虫cDNA文库和差减文库的建立与基因表达分析
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摘要
昆虫病原线虫是一类新型的生物杀虫剂,在害虫治理中具有广阔的应用前景。抗逆性幼虫是昆虫病原线虫发育过程中的一个特殊阶段,它是唯一能在自然环境中存活的虫态,其口、食道和肛门封闭,发育停止,具有较强的抵抗外界不良环境的能力。了解抗逆性幼虫的基因表达对探讨抗逆性机制以及抗逆性代谢机理具有十分重要的意义,进而为昆虫病原线虫的生产与应用奠定理论基础。
本文构建了芜菁夜蛾斯氏线虫Steinernema feltiae G26品系抗逆性幼虫的cDNA文库,对部分片段进行了测序和同源性分析,并用抑制差减杂交技术构建了其抗逆性幼虫和三龄发育幼虫的差减cDNA文库。
1芜菁夜蛾线虫抗逆性幼虫cDNA文库构建与基因表达分析
通过提取S. feltiae G26品系抗逆性幼虫的总RNA,采用LD-PCR(Long-Distance PCR)法合成了双链cDNA,成功构建了cDNA文库,得到原始文库的库容为3.7×108cuf/ml。随机挑取118个克隆,测序获得100条序列,这些序列按照蛋白质功能分为热激蛋白(heat shock protein),核糖体大小亚基蛋白(ribosomal protein )、延伸因子(translation elongation factor 1-alpha)等。
2芜菁夜蛾线虫正常发育三龄幼虫和抗逆性幼虫差减文库构建
分别以抗逆性线虫cDNA为tester,以发育三龄幼虫的cDNA为driver,采用抑制性减法杂交构建了差减文库,共获得1628个克隆。通过反向Northern杂交,将其中38个阳性克隆送出测序,最终得到20条序列。利用BLAST在GenBank数据库中对获得的cDNA序列进行相似性比对分析,除去重复序列后,共得到10个上升表达差异基因。这些基因所编码的蛋白质中有3个功能已知序列,其中一个为半胱氨酸蛋白酶抑制剂,推测与线虫的抗逆性代谢机理有关;有7个基因在数据库中找到同源性序列,但功能尚不清楚。
本研究结果可为阐明抗逆性幼虫形成与发育机制以及抗逆代谢机理等提供重要理论依据,进而为调控其形成与发育奠定基础,最终达到降低昆虫病原线虫生产成本,提高其防治效果的目的。
Entomopathogenic nematodes are a new type of bioinsecticide. It has broad application prospects in the pest management. Infective juveniles(IJs) are a special development stage of entomopathogenic nematodes. It is the only stage that survived in the natural environment, and its mouth, esophagus and anus close to stop the development. It has strong capacity to resist adverse environment. The genes espression profile of the IJs provided insight into the understanding of resistance mechanism as well as metabolic mechanism of the resistance, which may lay the theoretical basis for the nematodes production and application.
A cDNA library from IJs of Steinernema feltiae G26 strains was constructed and the partial fragments were sequenced and analysed. A suppression subtractive hybridization (SSH) library from IJs and their 3rd developmental satge juvenile was constructed with suppression subtractive hybridization technique.
1 Construction and sequence analysis of IJs of S. feltiae G26 strains cDNA library
LD-PCR(Long-Distance PCR)method was used to construct the cDNA library successfully from the IJs of S. feltiae G26 strains total RNA, and the original titer for 3.7×10~8cuf/ml was obtained. 100 sequences were acquired from the selected 118 clones randomly by sequencing, including heat shock protein, ribosomal protein genes, translation elongation factor 1-alpha genes etc.
2 Construction of IJs and their 3rd instar normal larvae of S. feltiae G26 strains SSH library
A SSH library was constructed by suppression subtractive hybridization, using cDNA from IJs as tester and cDNA from 3rd developmental satge juvenile as driver. The SSH library was totally composed of 1628 recombinant clones, and 38 of them were sent for sequencing after Dig detection. Finally we got 20 sequences. After similarity analysis based on BLASTx and BLASTn in GenBank, the redundant cDNAs were removed, and 10 differentially expressed genes were obtained. Three genes of them were known, and one was Cysteine Protease Inhibitor family member (cpi-2) that was related to the resistance of metabolic mechanism. Seven of them were found similar to some of sequence in the database, but the functions were still not clear.
This research provided theoretical foundation for revealing the resistance mechanism, development mechanism and resistance metabolic mechanism of IJs, moreover, it laid the foundation for controlling formation and development of IJs. Ultimately, costs for entomopathogenic nematodes production were reduced and the its control efficancy was enhanced.
本文构建了芜菁夜蛾斯氏线虫Steinernema feltiae G26品系抗逆性幼虫的cDNA文库,对部分片段进行了测序和同源性分析,并用抑制差减杂交技术构建了其抗逆性幼虫和三龄发育幼虫的差减cDNA文库。
1芜菁夜蛾线虫抗逆性幼虫cDNA文库构建与基因表达分析
通过提取S. feltiae G26品系抗逆性幼虫的总RNA,采用LD-PCR(Long-Distance PCR)法合成了双链cDNA,成功构建了cDNA文库,得到原始文库的库容为3.7×108cuf/ml。随机挑取118个克隆,测序获得100条序列,这些序列按照蛋白质功能分为热激蛋白(heat shock protein),核糖体大小亚基蛋白(ribosomal protein )、延伸因子(translation elongation factor 1-alpha)等。
2芜菁夜蛾线虫正常发育三龄幼虫和抗逆性幼虫差减文库构建
分别以抗逆性线虫cDNA为tester,以发育三龄幼虫的cDNA为driver,采用抑制性减法杂交构建了差减文库,共获得1628个克隆。通过反向Northern杂交,将其中38个阳性克隆送出测序,最终得到20条序列。利用BLAST在GenBank数据库中对获得的cDNA序列进行相似性比对分析,除去重复序列后,共得到10个上升表达差异基因。这些基因所编码的蛋白质中有3个功能已知序列,其中一个为半胱氨酸蛋白酶抑制剂,推测与线虫的抗逆性代谢机理有关;有7个基因在数据库中找到同源性序列,但功能尚不清楚。
本研究结果可为阐明抗逆性幼虫形成与发育机制以及抗逆代谢机理等提供重要理论依据,进而为调控其形成与发育奠定基础,最终达到降低昆虫病原线虫生产成本,提高其防治效果的目的。
Entomopathogenic nematodes are a new type of bioinsecticide. It has broad application prospects in the pest management. Infective juveniles(IJs) are a special development stage of entomopathogenic nematodes. It is the only stage that survived in the natural environment, and its mouth, esophagus and anus close to stop the development. It has strong capacity to resist adverse environment. The genes espression profile of the IJs provided insight into the understanding of resistance mechanism as well as metabolic mechanism of the resistance, which may lay the theoretical basis for the nematodes production and application.
A cDNA library from IJs of Steinernema feltiae G26 strains was constructed and the partial fragments were sequenced and analysed. A suppression subtractive hybridization (SSH) library from IJs and their 3rd developmental satge juvenile was constructed with suppression subtractive hybridization technique.
1 Construction and sequence analysis of IJs of S. feltiae G26 strains cDNA library
LD-PCR(Long-Distance PCR)method was used to construct the cDNA library successfully from the IJs of S. feltiae G26 strains total RNA, and the original titer for 3.7×10~8cuf/ml was obtained. 100 sequences were acquired from the selected 118 clones randomly by sequencing, including heat shock protein, ribosomal protein genes, translation elongation factor 1-alpha genes etc.
2 Construction of IJs and their 3rd instar normal larvae of S. feltiae G26 strains SSH library
A SSH library was constructed by suppression subtractive hybridization, using cDNA from IJs as tester and cDNA from 3rd developmental satge juvenile as driver. The SSH library was totally composed of 1628 recombinant clones, and 38 of them were sent for sequencing after Dig detection. Finally we got 20 sequences. After similarity analysis based on BLASTx and BLASTn in GenBank, the redundant cDNAs were removed, and 10 differentially expressed genes were obtained. Three genes of them were known, and one was Cysteine Protease Inhibitor family member (cpi-2) that was related to the resistance of metabolic mechanism. Seven of them were found similar to some of sequence in the database, but the functions were still not clear.
This research provided theoretical foundation for revealing the resistance mechanism, development mechanism and resistance metabolic mechanism of IJs, moreover, it laid the foundation for controlling formation and development of IJs. Ultimately, costs for entomopathogenic nematodes production were reduced and the its control efficancy was enhanced.
引文
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