Molecular characterization and functional analysis of four β-1,4-endoglucanases from the root-lesion nematode Pratylenchus vulnus
Molecular characterization and functional analysis of four β-1,4-endoglucanases from the root-lesion nematode Pratylenchus vulnus
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DOI：
10.1111/ppa.12222
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发表时间：
2014-12-01
期刊：
影响因子：
2.7
通讯作者：
De Luca, F.
中科院分区：
文献类型：
--
作者：
Fanelli, E.;Troccoli, A.;De Luca, F.
本研究报道了四种不同的内切葡聚糖酶的根病变线虫Pratylenchus vulnus的分离和特性。其中两个，Pv-eng-1和Pv-eng-2的基因结构被完全确定，Pv-eng-3和Pv-eng-5被部分测序。原位杂交检测Pv-eng-1、Pv-eng-2和Pv-eng-5的空间表达。Pv-eng-1和Pv-eng-2转录本位于腹下食管腺，而Pv-eng-5转录本位于肠中。实时RT-PCR结果表明，四个内切葡聚糖酶中的三个在成年男性和女性中具有最高的转录水平，从而表明成年人也寄生在P. vulnus。用Pv-eng-1的双链RNA（dsRNA）处理混合期的创伤疟原虫，以研究基因沉默（RNAi）的效果。通过靶向碳水化合物结合模块（CBM）的dsRNA沉默Pv-eng-1导致转录物水平的显著降低（88-98%），表明创伤毕赤酵母对RNAi敏感。此外，在5周的时间内，沉默P.vulnus显示出在胡萝卜微盘上的线虫繁殖减少（54%）。这些结果表明，Pv-eng-1的沉默可能会导致线虫定位和侵入根的能力降低，因此，建立和繁殖。
This study reports on the isolation and characterization of four different endoglucanases in the root-lesion nematode Pratylenchus vulnus. The gene structures of two of these, Pv-eng-1 and Pv-eng-2, were fully determined, and Pv-eng-3 and Pv-eng-5 were partially sequenced. Spatial expression of Pv-eng-1, Pv-eng-2 and Pv-eng-5 was examined by in situ hybridization. Pv-eng-1 and Pv-eng-2 transcripts were localized in the subventral oesophageal glands, whilst the Pv-eng-5 transcript was localized in the intestine. Real-time RT-PCR showed that three of the four endoglucanases had the highest transcriptional level in adult males and females, thus demonstrating that adults are also parasitic in P.vulnus. Mixed stages of P.vulnus were treated with double-stranded RNA (dsRNA) of Pv-eng-1 in order to study the effect of gene silencing (RNAi). Silencing Pv-eng-1 by dsRNA targeting of the carbohydrate-binding module (CBM) resulted in a significant reduction (88-98%) of the transcript level, suggesting that P.vulnus is susceptible to RNAi. Furthermore, silencing P.vulnus showed a reduction (54%) in nematode reproduction on carrot minidiscs over a 5week period. These results suggest that silencing of Pv-eng-1 may result in reduction of the ability of the nematode to locate and invade roots and, therefore, to establish and reproduce.