凡纳滨对虾围食膜蛋白基因全长cDNA的克隆及序列分析

摘要

In order to study the peritrophin function of Litopenaeus vannamei related to IHHNV invasion,the full-length cDNA library from intestinal tissue of Litopenaeus vannamei was constructed by switching mechanism at 5' end of the RNA transcript technique ( SMART) , a full-length cDNA of peritrophin was obtained by gene splicing and its characterization was analyzed. The capacity of constructed library was up to 1.05 × 106 pfu/mL, and its recombinant coefficient was 95% . 1 600 clones selected randomly were sequenced,l 531 available sequences with inserted length 3:450 bp were obtained after removing vector. One cDNA of peritrophin gene sequence of 1 002 bp was obtained from the clones, its protein encodes 303 amino acids. The nucleotide sequences of Litopenaeus vannamei have an overall similarity of 80% to peritrophin 1 , 2 precursor and peritrophin 3 precursor of Penaeus monodon. Expression of peritrophin gene in different tissue was analyzed by semi-quantitative reverse transcription PCR ( RT-PCR) , the result showed that the expression of peritrophin transcript in myocardium and gastric was higher, the expression was lower in hepatopancreas, intestines and muscle, the minimal expression was in gills, but not in eyes; Expression was obviously weakened in heart of IHHNV injected Litopenaeus vannamei, expression was lower in hepatopancreas, eyestalk, muscle and gills tissues, there was almost no expression in intestines and stomach. These results suggest that peritrophin of Litopenaeus vannamei was involved in prevention of IHHNV invasion.%为研究凡纳滨对虾围食膜蛋白在IHHNV感染虾体过程中的作用,实验采用SMART技术构建了凡纳滨对虾肠道组织的全长cDNA文库,电子拼接获得了对虾围食膜蛋白基因全长cDNA序列,并进行了功能分析.全长cDNA文库的库容达1.05×106 pfu/mL,重组率为95％.随机挑选1 600个克隆进行测序,去除载体后得到插入长度≥450 bp的有效序列1 531条,根据同源片段长度不小于100 bp,90％同源性的原则对这些序列归并unigene,得到unigene 399条.从所测克隆中得到一个围食膜蛋白基因,其cDNA序列长度为1 002 bp,编码由303个氨基酸组成的蛋白,基因序列比对发现该序列与斑节对虾卵巢围食膜蛋白1和2、围食膜蛋白前体3,短钩对虾围食膜蛋白1、2,以及中国对虾围食膜蛋白编码序列的同源性较高,均达到80％以上.通过半定量RT-PCR对该基因在不同组织的表达分析结果表明该基因在抗感染对虾传染性皮下及造血组织坏死病毒( IHHNV)的对虾的心脏和胃中高表达,在肝脏、肠和肌肉中表达较低,在鳃中表达最弱,在眼中不表达；该基因在感染IHHNV病毒的对虾的心脏表达明显减弱,在肝脏、眼、肌肉和鳃中表达较低,在肠道和胃中几乎不表达,说明该基因参与对虾抵御病原体侵入过程.