低剂量杀线虫剂对马铃薯腐烂茎线虫三个热激蛋白70 mRNA表达的影响
Effects of low-dose nematicides on expression of three HSP70 mRNA in Ditylenchus destructor

为了解马铃薯腐烂茎线虫Ditylenchus destructor对低剂量杀线虫剂胁迫的适应机制,采用RT-PCR和RACE技术克隆了3个热激蛋白70基因Dd-hsp70-A(KF792307)、Dd-hsp70-C(JQ422276)和Dd-hsp70-F(JQ422277),并利用实时荧光定量PCR分别检测了33 ℃热激及低剂量(10、1、0.1、0.01和0.001 μg/mL)杀线虫剂阿维菌素、丙溴磷和涕灭威处理对3个Dd-hsp70基因mRNA表达量的影响。结果表明,3个Dd-hsp70基因在翻译区均含有内含子,编码的氨基酸序列均包含了完整的保守结构域和3段保守的HSP70家族签名序列,且在氨基酸C末端具有保守的亚细胞定位基序。33 ℃热激3、6 h及0.1 μg/mL涕灭威浸渍处理24 h,马铃薯腐烂茎线虫Dd-hsp70-A基因mRNA表达量分别比对照显著上调1.865、4.127和1.634倍;其它处理的mRNA表达量与对照差异均不显著。证实了Dd-hsp70-A、Dd-hsp70-C和Dd-hsp70-F基因均为组成型,其中Dd-hsp70-A基因的响应在植物线虫应对低剂量杀线虫剂胁迫过程中具有重要作用。
To understand the molecular mechanism of adaptability to low-dose nematicides stress in Ditylenchus destructor, three hsp70 genes, Dd-hsp70-A (accession no.KF792307), Dd-hsp70-C (accession no. JQ422276) and Dd-hsp70-F (accession no. JQ422277), were cloned from D.destructor using RT-PCR and RACE techniques. The mRNA expression of three hsp70 genes in D.destructor exposed to heat shock and low-doses (10, 1, 0.1, 0.01, 0.001 μg/mL) of three kinds of nematicides, abamectin, profenofos and aldicarb, were analyzed using real-time quantitative PCR. Amino acids translation and sequence analysis indicated that introns existed in the translated region of the three Dd-hsp70 genes, and complete conserved domains, three signature sequences of HSP70 family, signal sequences of subcellular location at the carboxyl (C)-terminal were found in amino acid sequences of the three Dd-HSP70s. The real-time PCR results indicated that the expression level of Dd-hsp70-A was significantly upregulated by 1.865, 4.127 and 1.634 folds after exposed to 33 ℃ for 3, 6 h and 0.1μg/mL aldicarb for 24 h, respectively, compared to the control. No significant difference was observed in the expression of the three hsp70 genes after treatment with other temperatures of heat-shock and other doses of nematicides. The results suggested that Dd-hsp70-A, Dd-hsp70-C and Dd-hsp70-F are constitutively expressed genes, of which the expression of Dd-hsp70-A in D.destructor might play an important role in response to low-dose nematicide stress.