福寿螺感染广州管圆线虫后G型溶菌酶基因差异表达分析

Effect of Angiostrongylus cantonensis infection on the expression of G-type lysozyme in Pomacea canaliculata

目的分析感染广州管圆线虫后福寿螺G型溶菌酶基因的组织表达特征。方法根据福寿螺G型溶菌酶基因序列,设计特异性引物, PCR扩增后进行测序,利用在线软件ExPASy、 signalP 4.1、 TMHMM2.0、Smart、 MEGA7对其序列的基本生物学信息进行分析,并构建系统进化树。取含有广州管圆线虫Ⅰ期幼虫的大鼠粪便喂食福寿螺,感染后24 h统一饲养,分别于感染后1、 10、 20、 30 d各取3~5只福寿螺采集肝胰腺、肾脏、肠道、头足和鳃组织,未感染的福寿螺为阴性对照组。提取各个感染时期福寿螺各组织总RNA,逆转录为cDNA,荧光定量PCR分析福寿螺G型溶菌酶mRNA的组织表达特征,设定对照组福寿螺各组织的G型溶菌酶m RNA的相对表达量为1.0±0.0。结果PCR扩增福寿螺G型溶菌酶基因片段,长度约为800 bp。测序结果显示,为一段828 bp的完整开放读码框,编码275个氨基酸。生物信息学分析结果显示, G型溶菌酶基因编码产物为稳定的疏水性蛋白,在1~19位存在1个信号肽,主要在细胞外包括细胞壁发挥作用,在18~85位氨基酸处存在1个SH3b结构域。系统进化树分析显示,福寿螺G型溶菌酶与尖膀胱螺的亲缘关系最近,序列一致性达79%;其次与海湾扇贝等其他软体动物较近,并聚类在一个大的分支上。荧光定量PCR结果显示,对照组雌性成年螺肝胰腺组织中, G型溶菌酶mRNA的拷贝数为2 238±158,高于雄性的685±27 (P <0.05)。福寿螺感染广州管圆线虫后,肝胰腺、肾脏、肠道、鳃和头足部的G型溶菌酶基因相对表达量,与对照组相比均有不同程度的下降(P <0.01)。感染后1、 10、 20、 30 d鳃中G型溶菌酶mRNA的相对表达量分别为0.002±0.002、 0.012±0.050、 0.001±0.000、 0.004±0.003,均低于对照组(P <0.01);肾脏组织中10 d和20 d的相对表达量低于对照组(P <0.05);肝胰腺组织中1、 10和20 d的相对表达量低于对照组(P <0.05);感染后1、 10、 20、 30 d肠道组织中的相对表达量分别为0.280±0.040、 0.070±0.030、 0.039±0.002、 0.120±0.050,均低于对照组(P <0.05),头足部组织中的相对表达量分别为0.280±0.150、 0.150±0.008、 0.031±0.011、 0.120±0.030,均低于对照组(P <0.05)。结论福寿螺感染广州管圆线虫后, G型溶菌酶基因在鳃、肠、头足组织中各个感染时期表达均下调,肝胰腺组织中感染后1、 10、 20 d表达下调,肾脏组织中感染后10 d、 20 d表达下调。

Objective To evaluate the effect of Angiostrongylus cantonensis infection on the expression of Gtype 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 SH3 b 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 were0.280 ± 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.