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目的鉴定血根碱致钉螺肝脏差异表达的基因。方法浓度梯度法测定血根碱浸杀钉螺的半数致死浓度(LC50);以80%LC50血根碱浸泡钉螺,分离血根碱组和清水组活钉螺肝脏;提取mRNA,逆转录为cDNA,进行抑制消减杂交;巢式PCR扩增差异cDNA,克隆至pMD-18T载体,PCR和测序鉴定,NCBI中blastx比对分析表达序列标签(expressed sequence tag,EST)。结果血根碱浸杀钉螺LC50为7.5 mg/L。获得的EST序列大小在150~450 bp,经blastx比对,表达上调的基因有α-4(VI)胶原链、α-5(VI)胶原链、40 S核糖体蛋白S19、假定蛋白(WP_009787 197.1)、kelch样蛋白、颗粒体样蛋白;表达下调的基因有β-微管蛋白、α-淀粉酶1、大亚基核糖体蛋白L23e、几丁质酶-1、捷蛋白-3、假定蛋白(EKC34 262.1)、线粒体乙醛脱氢酶、肽聚糖识别蛋白、真核转录延长因子1A、铁蛋白、去整合素金属蛋白酶、溶菌酶1、1,3(4)-β-葡聚糖酶1、血蓝蛋白α-4D亚基,这些基因编码蛋白的功能涉及物质转运、蛋白合成、增殖诱导、营养、抗氧化、抗炎、呼吸、信号转导等。结论血根碱处理导致钉螺肝脏基因表达发生改变。
Objective To identify the genes that are differentially expressed in the liver of cauline snail. Methods Concentration gradient method was used to determine the half lethal concentration (LC50) of dipyridamole. The somatic stem of Solanum nigrum was isolated by soaking snails at 80% LC50, and the mRNA was extracted and reverse transcribed into cDNA. The amplified cDNA was amplified by nested PCR, cloned into pMD-18T vector, identified by PCR and sequencing, and expressed sequence tag (EST) by blastx analysis in NCBI. Results Sanguinarine snail LC50 was 7.5 mg / L. The size of the EST sequence obtained was 150-450 bp and the genes that were upregulated by blastx were α-4 (VI) collagen chain, α-5 (VI) collagen chain, 40 S ribosomal protein S19, putative protein (WP_009787 197.1), kelch-like protein and granulocyte-like protein. The down-regulated genes are β-tubulin, α-amylase 1, big subunit ribosomal protein L23e, chitinase-1, Protein (EKC34 262.1), mitochondrial aldehyde dehydrogenase, peptidoglycan recognition protein, eukaryotic transcription elongation factor 1A, ferritin, desintegrin metalloproteinase, lysozyme 1,1,3 (4) Carbohydrase 1, hemocyanin α-4D subunit. The function of these genes encoding proteins involved in substance transport, protein synthesis, proliferation induction, nutrition, anti-oxidation, anti-inflammatory, respiration, signal transduction. Conclusion Sanguinarine treatment results in altered gene expression in liver.