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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RTPCR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0.001μg/m L)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0.1μg/m L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。
In order to understand the adaptive mechanism of Ditylenchus destructor to low dose nematicide stress, three heat shock protein 70 genes, Dd-hsp70-A (KF792307), Dd-hsp70-C (JQ422276), were cloned by RTPCR and RACE techniques. And Dd-hsp70-F (JQ422277) respectively. Heat shock and low dose (10, 1, 0.1, 0.01 and 0.001 μg / mL) nematicides abamectin at 33 ℃ were detected by real- Effects of Bromophos and Aldicarb Treatment on the mRNA Expression of Three Dd-hsp70 Genes. The results showed that all three Dd-hsp70 genes contained introns in the translation region. The encoded amino acid sequences all contained a complete conserved domain and three conserved HSP70 family signature sequences with conserved subcellular C-terminal amino acids Positioning motifs. The expression of Dd-hsp70-A gene in D. destructor was significantly increased by 1.865, 4.127 and 1.634 folds compared with the control at heat shock of 33 ℃ for 3 and 6 h and 0.1 μg / mL of aldicarb immersed for 24 h, respectively. The mRNA expression level was not significantly different from the control. It was confirmed that the Dd-hsp70-A, Dd-hsp70-C and Dd-hsp70-F genes are all constitutive. The Dd-hsp70-A gene response plays an important role in plant nematode response to low dose nematicide stress.