南方根结线虫乳酸脱氢酶基因克隆及其沉默效应分析

【目的】对南方根结线虫乳酸脱氢酶基因（mildh）进行克隆分析，并探索mildh沉默对南方根结线虫（Meloidogyne incognita）病害的抑制作用。【方法】在前期研究中，利用生物信息学方法在线虫全基因组中预测了一些线虫RNA干扰（RNAi）表型基因。本研究以预测的线虫ldh设计特异引物克隆南方根结线虫中mildh。对克隆到的mildh序列进行了特征分析后，利用病毒诱导的基因沉默（VIGS）技术，将其导入番茄植株，并接种南方根结线虫，研究mildh基因沉默对根结线虫根结数量影响。【结果】克隆到的mildh包含1个完整的编码框序列，编码315个氨基酸。该基因与预测到的mildh核苷酸序列一致性高达94.9%，所编码的氨基酸一致性高达95.5%。构建烟草脆裂病毒（Tobacco rattle virus，TRV）介导的沉默载体pTV-mildhi，并用该沉默载体对番茄植株进行处理后，接种南方根结线虫。60 d后统计发现，pTV-mildhi处理的番茄植株上根结数分别比空载体对照减少48.6%，比清水对照降低48.4%。【结论】mildh基因沉默对南方根结线虫具有显著的抑制作用，也说明mildh可能参与根结线虫的致病性,该研究对线虫的病理学研究及线虫病害防控研究提供了新的理论基础。

Molecular Clone and Its RNA Interference Effect Analysis of Lactate Dehydrogenase Gene in Meloidogyne incognita

【Objective】The objective of this study is to clone and analyze the lactate dehydrogenase gene in Meloidogyne incognita (mildh), and to determine the inhibitory effect of mildh silencing on the diseases caused by Meloidogyne spp..【Method】In the preliminary studies, the number of RNA interference (RNAi) phenotype genes of nematodes in their genomes were predicted by bioinformatics methods. In this study, using special primers designed based on the predicted nematode ldh, the ldh in M. incognita (mildh) were cloned which was transferred into tomato plantlets by virus-induced gene silence (VIGS) technique after analyzing the sequence characters to investigate the influence of the mildh silencing on the amounts of root knot on the tomato plantlet inoculated with M. incognita.【Result】The cloned mildh contained a complete coding sequence that coded 315 amino acids. The identity between the cloned mildh and the predicted mildh was as high as 94.9% in nucleotide sequence and 95.5% in the deduced amino acid sequence. The silencing vector pTV-mildhi, mediated by Tobacco rattle virus (TRV), was constructed. The tomato plantlets were inoculated with M. incognita after treated by pTV-mildhi. Sixty days later, statistical analysis showed that the amounts of root knot on tomato plantlets of which the mildh was silenced decreased by 48.6% compared to that of the empty vector control, by 48.4% compared to that of the water control. 【Conclusion】The study revealed the mildh silencing had a significant inhibitory effect on root-knot nematode disease caused by M. incognita. It also showed that mildh genes may be involved in the pathogenesis of nematode, which provided a theoretical basis to the research of nematode pathology and to the control of nematode disease.