关键词: 福寿螺, 广州管圆线虫, G型溶菌酶

Abstract:

Objective To evaluate the effect of Angiostrongylus cantonensis infection on the expression of G-type lysozyme in snail Pomacea canaliculate. Methods Primers were designed based on the DNA sequence coding for G-type lysozyme of P. canaliculate and the coding DNA was amplified by PCR with the primers from the snail tissue. The basic bioinformatics of P. canaliculate G-type lysozyme was analyzed using ExPASy, signalP 4.1, TMHMM2.0, Smart, MEGA7. P. canaliculate snails were infected with A. cantanensis by incubating with rat feces containing A. cantonensis first-stage larva for 24 h. The tissues of hepatopancrea, kidney, intestine, cephalopod and gill were collected from 3-5 P. canaliculate snails after being infected for 0, 1, 10, 20 and 30 days. The same number of uninfected snails were collected as negative control. Total RNA was extracted from these tissues and cDNA was synthesized by reverse transcription. Fluorescence quantitative PCR (qPCR) was used to analyze the tissue gene expression level of G-type lysozyme in P. canaliculata after being infected with A. cantonensis at different time points. Results The DNA coding for G-type lysozyme of P. canaliculata was amplified by PCR with the size of about 800 bp. The sequencing results showed that the coding DNA for G-type lysozyme of P. canaliculata was 828 bp encoding for an open reading frame of 275 amino acids. The P. canaliculate G-type lysozyme is a stable hydrophobic protein with a signal peptide at first 1-19 amino acids, mainly playing a role in the extracellular activity on the cell membrane. A SH3b domain is located between 18-85 amino acids. The phylogenetic analysis indicated that P. canaliculata G-type lysozyme is genetically close to the homologue in Physella acuta with 79% sequence identity. It clusters with homologues in other mollusks such as Argopecten irradians. qPCR results showed that the transcription level of G-type lysozyme in the hepatopancreatic tissues was significantly higher in female snail (2 238 ± 158) than in the male (685 ± 27) (P < 0.05), also significantly higher than that in other 4 tissues of the snail (P < 0.01). After being infected with A. cantonensis, the relative expression levels of G-type lysozyme in the tissues of hepatopanpancreas, kidney, intestine, gill and cephalopod all decreased to a certain extent compared with the tissues in un-infected snails. The relative expression levels of G-type lysozyme mRNA in gill 1, 10, 20 and 30 d after infection were 0.002 ± 0.002, 0.012 ± 0.050, 0.001 ± 0.000, and 0.004 ± 0.003, respectively, that were significantly lower than that in the un-infected snail (P < 0.01). The relative expression levels of G-type lysozyme in kidney were significantly lower than control only at 10 d and 20 d after infection (P < 0.05). The relative expression levels of G-type lysozyme was significantly lower than control in hepatopancreatic tissues at 1, 10 and 20 d after infection (P < 0.05). The relative expression levels of G-type lysozyme in the intestine at 1, 10, 20 and 30 d were 0.280 ± 0.040, 0.070 ± 0.030, 0.039 ± 0.002, and 0.120 ± 0.050, respectively, significantly lower than that in control group (P < 0.05). Similarly, the relative expression levels in cephalopod tissues were 0.280 ± 0.150, 0.150 ± 0.008, 0.031 ± 0.011, and 0.120 ± 0.030, significantly lower than that in control group (P < 0.05). Conclusion After being infected with A. cantonensis, the expression of G-type lysozyme gene in the tissues of gills, intestines and cephalopods of P. canaliculate was decreased for 1-30 d, decreased in hepatopancreas for 1-20 d and in kidneys for 10 d and 20 d after infection.

Key words: Pomacea canaliculata, Angiostrongylus cantonensis, G-type lysozyme