Abstract: 【Objective】Soybean cyst nematode (Heterodera glycines) is a devastating disease all over the world. The expanded protein (expansin) secreted by stylet plays an important role in the parasitism of H. glycines. The objective of this study is to identify the expansin gene from H. glycines, understand its structure, tissue localization and the expression characteristics at different developmental stages, so as to lay a foundation for further clarifying the parasitic and pathogenic mechanism of H. glycines. 【Method】According to the conserved sequence of the reported expansin gene, upstream and downstream degenerate primers were designed and the EST fragment of expansin gene from the 2nd stage juveniles of H. glycines was cloned. According to the sequence of EST fragment, RACE specific primers were designed. After amplified and sequenced by RACE technique, the sequence was compared and spliced by DNAstar 7.1 and DNAman software. The full length of expansin gene cDNA of H. glycines was obtained. CLC sequence viewer 6 was used for open reading frame search, protein translation and sequence alignment. On-line software SignalP 3.0 Server and TMHMM were used to predict protein precursor signal peptide and transmembrane domain, and GSDS was used to analyze the genome structure. Using PHYML and MEGA 5.0 software maximum likelihood method, the obtained genes were compared with other nematode expansin genes to construct phylogenetic tree. Southern hybridization was used to analyze the number of the Hg-exp-1 copies in the genome. The expression sites of two genes were confirmed by in situ hybridization. The cDNAs of eggs, pre-parasitic 2nd stage juvenile, parasitic 2nd stage juvenile, parasitic 3rd stage juvenile, parasitic 4th stage juvenile and females were extracted as templates, the developmental expression characteristics were analyzed by semi quantitative PCR. According to the sequence of Hg-exp-1, primers were designed to amplify and synthesize dsRNA. Soybean plantlets were inoculated with 2nd stage juvenile after immersion for 24 h. RNA interference in vitro method was used to identify the function of Hg-exp-1. 【Result】The full-length cDNAs of two expansin genes named Hg-exp-1 and Hg-exp-2 were successfully cloned from the 2nd stage juveniles of H. glycines, with a length of 1 047 and 1 037 bp, and the peptides with length of 288 and 295 amino acids were encoded. Both of the two predicted proteins contained a signal peptide in N-terminal and had no transmembrane domain, indicating that they were secretory proteins. Sequence alignment showed that the HG-EXP-1 sequence of H. glycines was highly consistent with GR-EXPB1 (CAC83611) and GR-EXPB2 (CAC84564) in Globodera rostochiensis and DA-EXPB1 (ADJ57307) in Ditylenchus africanus. Southern blot analysis showed the expansin genes might exist in H. glycines in multi-copy mode or members of a small multi-gene family. In situ hybridization analyses showed that the transcripts of them accumulated exclusively in the subventral oesophageal gland cells of H. glycines. The results of Hg-exp-1 interference in vitro showed that the transcriptional level of the target gene was down-regulated in nematode treated with dsRNA for 24 h. After Hg-exp-1 was silenced, the number of 2nd stage juveniles infected in soybean root and females decreased by 38.3% and 43.4% than the control, respectively.【Conclusion】 two expansin genes were successfully isolated and identified from H. glycines, and their important role in the early parasitic process of H. glycines was also clarified.

Key words: Heterodera glycines, expansin, developmental expression, RNA interference