虾夷马粪海胆NLR家族基因片段的克隆及表达特征分析

Cloning of partial sequence and expression of NLR family in sea urchin Strongylocentrotus intermedius

采用同源克隆和半定量RT-PCR方法,对虾夷马粪海胆Strongylocentrotus intermedius NLR家族基因片段进行了克隆和组织表达特征分析。将紫球海胆S.purpuratus的9组NLR家族聚类的编码区序列进行比对后,根据保守区域设计引物扩增虾夷马粪海胆中相应的基因,通过测序获得了9组不同NLR聚类基因片段,每组各10个测序结果,通过基因序列及氨基酸序列比对分析,发现存在长度及序列结果上的差异,这说明每组NLR家族聚类都存在多个家族成员。同时选取虾夷马粪海胆的不同组织提取RNA,反转录后使用同样的引物进行半定量RT-PCR分析,结果表明,NLR基因在虾夷马粪海胆的管足、肠、围口膜、体腔细胞、性腺中存在高效表达,且在围口膜及性腺中表达量最高,表明NLR基因在虾夷马粪海胆中普遍表达,并存在表达差异。研究表明,NLR基因在海胆免疫过程中起着重要作用。

In this study, homologous cloning and semi-quantitative RT-PCR technologies were used to clone the fragments of NLR genes and to detect their expression patterns in tissues of sea urchin Strongylocentrotus intermedi-us. The primers were designed according to the homology alignment of NLR genes from purple sea urchin S. purpu-ratus, and 9 fragment groups of NLR genes were obtained from the sea urchin, by sequencing of 10 clones for each group. Then bioinformatic analysis of these genes was conducted by the method of Blast, finding the diversity of cDNA sequences and amino acid sequences of these NLR genes. Also, the RNA was extracted from different tissues in the sea urchin, and the expression of NLR genes was analyzed by semi-quantitative PCR in different tissues of the sea urchin after reverse transcription. The results showed that 9 fragment groups of NLR genes were all ex-pressed in coelomocytes （ CL ） , membrana peristomialis （ WK ） , intestines （ GT ） , tube-feet （ GZ ） , and gonad （ GD） at a high level, the maximum in membrana peristomialis and gonad, indicating that NLR gene family plays an important role in the congenital immune system of the sea urchin, with different expression. The findings will provide the foundation for further study of NLR genes in the sea urchin.