松材线虫乙酰胆碱酯酶基因Bx-ace-2克隆和序列分析及RNAi研究
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摘要
松材线虫是松树萎蔫病(Pine wilt disease)的病原,通过介体天牛传播病害,自1982年在江苏南京首次发现后,目前松材线虫已经蔓延至我国14个省(自治区、直辖市)的192个县级行政区,使我国松林资源与生态平衡遭到严重破坏,经济损失巨大。目前对松材线虫的研究主要集中在松材线虫的生物学、分类鉴定、松材线虫病发生条件和防治方法等方面,而关于松材线虫致病性的分子机理知之甚少。
乙酰胆碱酯酶(acetylcholinesterase, AChE)是生物神经传导中的一种关键性酶,除了神经学功能外,还可能涉及细胞凋亡和寄生线虫细胞发育、行为调控及侵染致病等多个生理进程。松材线虫AChE基因克隆的相关研究尚未见报道,本论文通过反转录PCR (RT-PCR)和cDNA末端快速扩增(RACE)技术获得了松材线虫AChE基因Bx-ace-2的cDNA全长,研究了该基因的内含子及外显子的排列结构；通过Southern杂交技术检测了该基因在基因组中的拷贝数；通过氨基酸序列比对分析,构建了乙酰胆碱酯酶基因的进化树；利用RNAi技术对克隆的Bx-ace-2进行了松材线虫体外干涉的初步研究。研究结果为今后进一步开展对松材线虫乙酰胆碱酯酶基因功能分析,从而推进对松材线虫致病分子机制的理解打下基础。
1松材线虫乙酰胆碱酯酶基因Bx-ace-2克隆和分析
克隆的Bx-ace-2 cDNA全长为2010 bp,有1878 bp的开放阅读框(ORF),推测其编码的蛋白有625个氨基酸；Bx-ace-2基因组克隆长为2192bp,含有7个内含子,Southern杂交实验表明该基因在松材线虫基因组中以单拷贝存在。氨基酸序列比对和同源性分析显示,Bx-ace-2编码的乙酰胆碱酯酶与已报道的秀丽小杆线虫ACE-1、ACE-3和ACE-4型乙酰胆碱酯酶的相似性在28%-31%之间,而与秀丽小杆线虫ACE-2及甘薯茎线虫、南方根结线虫、大豆胞囊线虫、马铃薯白线虫和胎生网尾线虫的ACE-2型乙酰胆碱酯酶的氨基酸序列相似性在44%-52%之间；构建的系统进化树也显示了松材线虫乙酰胆碱酯酶与其它线虫的ACE-2型乙酰胆碱酯酶具有相对更近的遗传距离,因此推测该松材线虫乙酰胆碱酯酶也属于ACE-2型。
2松材线虫乙酰胆碱酯酶基因Bx-ace-2的RNAi研究
研究了松材线虫吞咽FITC过程,浸泡10 h后松材线虫吞咽明显,24 h后线虫全身荧光信号最强。以Bx-ace-2 cDNA为模板,体外转录合成dsRNA,对经过不同dsRNA浸泡液处理24小时后的松材线虫进行RT-PCR检测显示,ace dsRNA浸泡处理后的松材线虫Bx-ace-2的转录水平明显降低,而经gfp dsRNA浸泡处理后的松材线虫没有检测到Bx-ace-2的转录水平变化。对处理后线虫的表型观察显示,ace dsRNA浸泡处理对松材线虫的活力没有明显影响,同时未发现线虫的贲门收缩的差异；浸泡液中加入脂质体对RNA干涉作用的影响不明显；松材线虫的灰葡萄孢接种实验显示经ace处理的松材线虫繁殖力明显低于对照。
Pinewood nematode (PWN), Bursaphelenchus xylophilus, and its vector insects, cerambycid beetles belonging to the genus Monochamus have been found associated with pine wilt disease. B. xylophilus represents a great threat to forestry industry. Since 1982 the first discovery of PWN was reported on Pinus thunbergii in Nanjing, more than 20 years continuous spread of PWN has crossed 192 countries of 14 provinces of China. Millions of trees were killed and the forest landscape and ecology were faced with a devastating threat.
Despite the damage caused by B. xylophilus throughout the world, little is clearly known about the molecular mechanisms underlying nematode pathogenicity. The major studies that have been carried out to date have focused on the biology, morphology, disease occurring condition and control strategies rather than examining mechanisms of pathogenicity. Understanding the interaction between B. xylophilus and it host plants is essential for designing disease control programs that are based on the structure and function of proteins encoded by nematode parasitism genes. Therefore, identifying candidate genes related to pathogenicity or parasitism of the nematode will provide breeding strategies to develop pine varieties with broad and durable resistance and contribute greatly to the efficient management of the pine wilt disease.
Acetylcholinesterase (AChE) plays a key role in neurotransmission. AChE terminates the cholinergic synapses to assure the neuro signal transmission, consequently regulate the muscle movement. AChE is also found may having other functions, such as apoptosis potential markers and regulators or secretions involving in cell development, behavior regulation and pathogenesis. AChE has been extensively studied in C. elegans in which four molecular classes have been found and encoded by four different genes ace-1 to ace-4. No AChE gene was reported from B. xylophilus, therefore the following works was carried out:
1. Molecular cloning and characterization of a new acetylcholinesterase gene Bx-ace-2 from B. xylophilus
A new gene, Bx-ace-2, encoding an acetylcholinesterase (AChE), was cloned from B. xylophilus by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full length cDNA is 2010 bp with an open reading frame (ORF) of 1878bp which was deduced encoding a protein with 625 amino acids. The length of genomic clone of Bx-ace-2 is 2192 bp which contained seven introns. Southern blot analysis demonstrated Bx-ace-2 is probably a signal copy gene. The deduced protein sequence encoded by Bx-ace-2 from B. xylophilus shares 28%～31% similarities with those of ACE-1, ACE-3 and ACE-4 from Caenorhabditis elegans. However, it shares 44%～52% similarities with ACE-2 from C. elegans, Ditylenchus destructor, Meloidogyne incognita, Heterodera glycines, Globodera pallida and Dictyocaulus viviparous. The phylogenetic analysis of AChEs demonstrated that the protein encoded by Bx-ace-2 was clustered into a same group with ACE-2 type of acetylcholinesterases from the aforementioned nematodes. Therefore, it was deduced that the protein encoded by Bx-ace-2 is belonged to ACE-2 type of acetylcholinesterase. ace-2 is the only AChE expressed in the C. elegans pharyngeo-intestinal valve which could be implicated involving into the action of feeding for nematode. More works need to be carried out for the gene expression and function analysis of Bx-ace-2.
2. RNAi of Bx-ace-2 in B. xylophilus
The uptake of Fluorescein isothiocyanate (FITC) by B. xylophilus was observed in soaking solution. After 10 h soaking, the FITC uptake by nematodes was clearly viewed under light microscopy. Fluorescent signals were appeared in full body with strong light after 24 h soaking. dsRNA was synthsized by in vitro transcription from cDNA of Bx-ace-2 which was a template. Reverse Transcription PCR (RT-PCR) analyses were conducted after B. xylophilus was soaked in different dsRNA solutions. The result relvealed that the transcription levels of Bx-ace-2 were reduced obviously when nematodes were exposed to ace dsRNA, no changes was observed when exposed to gfp dsRNA and blank control. No difference was showed in mortality of nematodes after soaked in different dsRNA solutions. Treatment of nematodes uptaking ace dsRNA resulted in reduction in ability to propagation. Adding lipofectin to the dsRNA-nematode mixture did not produce clear promotion to RNAi.for Bx-ace-2 in B. xylophilus.
乙酰胆碱酯酶(acetylcholinesterase, AChE)是生物神经传导中的一种关键性酶,除了神经学功能外,还可能涉及细胞凋亡和寄生线虫细胞发育、行为调控及侵染致病等多个生理进程。松材线虫AChE基因克隆的相关研究尚未见报道,本论文通过反转录PCR (RT-PCR)和cDNA末端快速扩增(RACE)技术获得了松材线虫AChE基因Bx-ace-2的cDNA全长,研究了该基因的内含子及外显子的排列结构；通过Southern杂交技术检测了该基因在基因组中的拷贝数；通过氨基酸序列比对分析,构建了乙酰胆碱酯酶基因的进化树；利用RNAi技术对克隆的Bx-ace-2进行了松材线虫体外干涉的初步研究。研究结果为今后进一步开展对松材线虫乙酰胆碱酯酶基因功能分析,从而推进对松材线虫致病分子机制的理解打下基础。
1松材线虫乙酰胆碱酯酶基因Bx-ace-2克隆和分析
克隆的Bx-ace-2 cDNA全长为2010 bp,有1878 bp的开放阅读框(ORF),推测其编码的蛋白有625个氨基酸；Bx-ace-2基因组克隆长为2192bp,含有7个内含子,Southern杂交实验表明该基因在松材线虫基因组中以单拷贝存在。氨基酸序列比对和同源性分析显示,Bx-ace-2编码的乙酰胆碱酯酶与已报道的秀丽小杆线虫ACE-1、ACE-3和ACE-4型乙酰胆碱酯酶的相似性在28%-31%之间,而与秀丽小杆线虫ACE-2及甘薯茎线虫、南方根结线虫、大豆胞囊线虫、马铃薯白线虫和胎生网尾线虫的ACE-2型乙酰胆碱酯酶的氨基酸序列相似性在44%-52%之间；构建的系统进化树也显示了松材线虫乙酰胆碱酯酶与其它线虫的ACE-2型乙酰胆碱酯酶具有相对更近的遗传距离,因此推测该松材线虫乙酰胆碱酯酶也属于ACE-2型。
2松材线虫乙酰胆碱酯酶基因Bx-ace-2的RNAi研究
研究了松材线虫吞咽FITC过程,浸泡10 h后松材线虫吞咽明显,24 h后线虫全身荧光信号最强。以Bx-ace-2 cDNA为模板,体外转录合成dsRNA,对经过不同dsRNA浸泡液处理24小时后的松材线虫进行RT-PCR检测显示,ace dsRNA浸泡处理后的松材线虫Bx-ace-2的转录水平明显降低,而经gfp dsRNA浸泡处理后的松材线虫没有检测到Bx-ace-2的转录水平变化。对处理后线虫的表型观察显示,ace dsRNA浸泡处理对松材线虫的活力没有明显影响,同时未发现线虫的贲门收缩的差异；浸泡液中加入脂质体对RNA干涉作用的影响不明显；松材线虫的灰葡萄孢接种实验显示经ace处理的松材线虫繁殖力明显低于对照。
Pinewood nematode (PWN), Bursaphelenchus xylophilus, and its vector insects, cerambycid beetles belonging to the genus Monochamus have been found associated with pine wilt disease. B. xylophilus represents a great threat to forestry industry. Since 1982 the first discovery of PWN was reported on Pinus thunbergii in Nanjing, more than 20 years continuous spread of PWN has crossed 192 countries of 14 provinces of China. Millions of trees were killed and the forest landscape and ecology were faced with a devastating threat.
Despite the damage caused by B. xylophilus throughout the world, little is clearly known about the molecular mechanisms underlying nematode pathogenicity. The major studies that have been carried out to date have focused on the biology, morphology, disease occurring condition and control strategies rather than examining mechanisms of pathogenicity. Understanding the interaction between B. xylophilus and it host plants is essential for designing disease control programs that are based on the structure and function of proteins encoded by nematode parasitism genes. Therefore, identifying candidate genes related to pathogenicity or parasitism of the nematode will provide breeding strategies to develop pine varieties with broad and durable resistance and contribute greatly to the efficient management of the pine wilt disease.
Acetylcholinesterase (AChE) plays a key role in neurotransmission. AChE terminates the cholinergic synapses to assure the neuro signal transmission, consequently regulate the muscle movement. AChE is also found may having other functions, such as apoptosis potential markers and regulators or secretions involving in cell development, behavior regulation and pathogenesis. AChE has been extensively studied in C. elegans in which four molecular classes have been found and encoded by four different genes ace-1 to ace-4. No AChE gene was reported from B. xylophilus, therefore the following works was carried out:
1. Molecular cloning and characterization of a new acetylcholinesterase gene Bx-ace-2 from B. xylophilus
A new gene, Bx-ace-2, encoding an acetylcholinesterase (AChE), was cloned from B. xylophilus by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full length cDNA is 2010 bp with an open reading frame (ORF) of 1878bp which was deduced encoding a protein with 625 amino acids. The length of genomic clone of Bx-ace-2 is 2192 bp which contained seven introns. Southern blot analysis demonstrated Bx-ace-2 is probably a signal copy gene. The deduced protein sequence encoded by Bx-ace-2 from B. xylophilus shares 28%～31% similarities with those of ACE-1, ACE-3 and ACE-4 from Caenorhabditis elegans. However, it shares 44%～52% similarities with ACE-2 from C. elegans, Ditylenchus destructor, Meloidogyne incognita, Heterodera glycines, Globodera pallida and Dictyocaulus viviparous. The phylogenetic analysis of AChEs demonstrated that the protein encoded by Bx-ace-2 was clustered into a same group with ACE-2 type of acetylcholinesterases from the aforementioned nematodes. Therefore, it was deduced that the protein encoded by Bx-ace-2 is belonged to ACE-2 type of acetylcholinesterase. ace-2 is the only AChE expressed in the C. elegans pharyngeo-intestinal valve which could be implicated involving into the action of feeding for nematode. More works need to be carried out for the gene expression and function analysis of Bx-ace-2.
2. RNAi of Bx-ace-2 in B. xylophilus
The uptake of Fluorescein isothiocyanate (FITC) by B. xylophilus was observed in soaking solution. After 10 h soaking, the FITC uptake by nematodes was clearly viewed under light microscopy. Fluorescent signals were appeared in full body with strong light after 24 h soaking. dsRNA was synthsized by in vitro transcription from cDNA of Bx-ace-2 which was a template. Reverse Transcription PCR (RT-PCR) analyses were conducted after B. xylophilus was soaked in different dsRNA solutions. The result relvealed that the transcription levels of Bx-ace-2 were reduced obviously when nematodes were exposed to ace dsRNA, no changes was observed when exposed to gfp dsRNA and blank control. No difference was showed in mortality of nematodes after soaked in different dsRNA solutions. Treatment of nematodes uptaking ace dsRNA resulted in reduction in ability to propagation. Adding lipofectin to the dsRNA-nematode mixture did not produce clear promotion to RNAi.for Bx-ace-2 in B. xylophilus.
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