新芋螺毒素基因的克隆及其遗传进化分析

Gene Cloning of Novel Conotoxins and Phylogenetic Analysis

全世界有约800种芋螺,每种芋螺产生多达2 000种的肽类毒素,这些毒素可以作用于电压门控离子通道(Na+,K+,Ca2+)、配体门控离子通道(n ACh Rs,5-HT3R,NMDAR)、G蛋白偶联受体(神经降压素和血管加压素)和神经递质转运蛋白。虽然已有大量的芋螺毒素通过毒液分离、c DNA克隆和转录组测序获得,但已发现的芋螺毒素不足其总量的0.5%。A-超家族中α-芋螺毒素基因结构包含了一个内含子和被该内含子分开的两个外显子,成熟肽具有标准的4个半胱氨酸骨架(CC-C-C)。本研究利用具有保守性的α-芋螺毒素基因内含子序列,采用多个PCR退火温度,从海南产疣缟芋螺中克隆到了1个新的具有6个半胱氨酸骨架(CC-C-C-CC)的M-超家族芋螺毒素基因和1个含有5个半胱氨酸新颖骨架(CC-C-C-C)的未知新家族芋螺毒素,并对它们的基因结构、成熟肽序列,以及与其他M-超家族芋螺毒素的遗传进化关系进行了深入分析。首次证实保守的α-芋螺毒素基因内含子序列可能存在于其他超家族中。

There are around 800 species of cone snails living in the world tropical ocean. Venom of each cone snail species consists of up to 2 000 conopeptides or conotoxins, which are targeting voltage-gated ion channels （Na^＋, K^＋, Ca^2＋）, ligand-gated ion channels （nAChRs, 5-HT3R, NMDAR）, G-protein-coupled recep- tors （neurotensin, vasopressin）, and neurotransmitter transporters （NET）. However, the number of conotoxins identified is less than 0.5% of the total conotoxins. The genomic a-conotoxin precursor genes of A-gene su- perfamily contain a long intron inserted in their pro-region and divided their encoding region into two exons. The α-conotoxins consist of standard four-Cys framework （CC-C-C）. Here, the C. lividus genomic DNA was used as a template for gene cloning of novel non-α-conotoxins using the conserved intron of α-conotoxins by PCR at multiple annealing temperatures. Two novel conotoxin genes, Lv Ⅲ-M01 ＆ LvXXⅦ-M02, were cloned from genomic DNA of C. lividus native to Hainan, China, which do not belong to classic α-conotoxins. Lv Ⅲ-MO1 encodes a new member of M-superfamily with six-Cys framework （C C-C-C-C C）. And LvXXⅦ-M02 encodes a new conotoxin of unknown gene superfamily with five-Cys framework（CC-C-C-C）, which is a new cys pattern. Gene structure, sequence characteristics and phynolgeny of the two novel conotoxins were ana-lyzed. It is the first time showing that the conserved intron sequence of α-conotoxins exists in other gene su-perfamily.