马尾松钙调素基因的克隆及响应松材线虫侵染的表达特征

Cloning and Expression Characteristics of Pinus massoniana pmCaM Response to Bursaphelenchus xylophilus Infection

松材线虫病是由松材线虫(Bursaphelenchus xylophilus)侵染引起的严重病害,引起重大的经济和生态损失。钙调素(CaM)是真核生物中Ca2+主要传导蛋白,本研究采用RT-PCR方法,首次从马尾松中克隆获得CaM,命名为pmCaM,并检测了其响应松材线虫侵染的表达特征。序列分析表明:pmCaM完整的开放阅读框核苷酸序列为450bp,编码149个氨基酸的蛋白质;该蛋白含有4个EF-hand结构域,具有钙调素的典型特征,与其它植物的CaM蛋白均有较高同源性。实时荧光定量PCR分析显示:接种松材线虫后30 180 min时间段,马尾松苗根、茎、叶器官中的pmCaM均下调表达,但不同器官间显著下调表达的时间点存在差异;同时,不同器官pmCaM表达量变化均存在特异的随时间发展的波动特征,其中发现,根茎pmCaM在松材线虫接种45 min时均处于表达量高峰。本研究结果显示pmCaM表达响应了松材线虫侵染,揭示pmCaM可能参与了调控松材线虫-马尾松互作早期的钙信号响应。

Pine wilt disease （PWD） caused by the pine wood nematode （PWN）, Bursaphelenchus xylophilus, is believed to be the most destructive disease of pine species, causing significant economic and ecological losses around the world. Calmodulin （CaM） is a major calcium-binding messenger protein expressed in all eukaryotic cells. A cDNA sequence of CaM was cloned from Masson pine （P. massoniana） by reverse transcription polymerase chain reaction （RT-PCR） methods, and named as pmCaM. And the pmCaM expression characteristics response to pine wood nematode infection was checked by real-time fluorescent quantitative PCR. The sequence analysis showed that the pmCaM consisted of an opening reading frame of 450 bp, encoded a protein of 149 aa containing four EF-hand domains and having the typical features of calmodulin. The comparison analysis showed that pmCaM shared high homology with CaMs from other plants. Real-time PCR analysis showed that 30 - 180 min post-PWD inoculation, pm- CaM genes in root, stem and leaf of pine seedling were all down-regulated expression, but the times for significantly down-regulated expression of pmCaM were difference among different organs; specifiC fluctuate characteristics of pro- CaM expression amount along with time development were appeared in root, stem and leaf, and the pmCaM expression amount peaked in root and stem 45 min p0st-PWD inoculation. This study showed that the expression ofpmCaM was response to PWD infection, and inferred that the pmCaM in pine may play an important role in the regulation of calcium signal response early interaction between Dine wood nematode with Masson pine.