亚洲蝮属蛇毒蛋白组种间变异的系统发育信号与食性选择作用

Snake venom represents a critical innovation in advanced snake, allowing the transition from mechanical to chemical means of subduing, killing and digesting prey. Venom toxins evolved diversely from proteins with normal physiological functions, and are thought to have an important role in adapting to the different types of food ingested by advanced snake. It has been proved that snake venom has evolved across the entire advanced snake radiation at molecular level and, as such, the venom composition and function may still retain some information on the evolutionary history of these snakes. The variation in venom composition and function may covary with phylogenetic relationship of advanced snake and the positive selection imposed by their diet, but it still needs to be demonstrated by more examples. Here, we will detect the interspecific variation and its phylogenetic signal in composition and function of venoms from seven species (and subspecies) of the genus Gloydius found in China, using the point-scaled pit viper (Protobothrops mucrosquamatus) as an outgroup species. Furthermore, we will use phylogenetic independent contrast to determine associations of venom composition and function with diet. Questions addressed in this study include: (1) whether the impact of phylogenetic relationship and diet selection on variation of venom composition and function differ between taxa, and between venom components? and (2) whether the variation pattern of phylogenetic signal of different venom components reported for species of the genera Sistrurus and Agksitrodon can be also detected in snakes of the genus Gloydius. We will also evaluate the magnitude of diet selection indirectly using the ratio calculated from the toxin encoding genes under positive selection. Our study may overcome the shortcomings associated with the relevant hypotheses explaining variation in snake venom based on either phylogenetic relationship or diet adaption. The comprehensieve analysis of venom proteome and transcriptome of snakes conducted in this study may be helpful in understanding the differences in snakebite symptoms caused by species of the genus Gloydius found in China, and may have implications on the improvement of antivenom preparation and the development of the active ingredients with potential medical value.

蛇毒作为新蛇类一个重要的革新，可使其由机械捕杀猎物向化学捕杀并辅助消化猎物的手段转变；蛇毒组成与功能的多样性对新蛇类适应不同的食物类型具有重要作用。分子水平的研究表明，蛇毒进化贯穿了整个新蛇类的辐射过程，可能仍保留了与物种进化历史相关的信息。由此推测，蛇毒的变异可能是谱系关系和食性结构共同作用的结果，但这一推测仍有待于通过更多的实例研究给予检验。申请人拟以原矛头蝮为外群，检测国内亚洲蝮属7种（亚种）毒蛇的蛇毒蛋白组成与功能的种间变异及其系统发育信号，并结合谱系独立比较来探讨：(1)谱系关系和食性选择作用对蛇毒变异的影响是否存在类群间、组分间差异？(2)蛇毒不同组分系统发育信号强度的变化格局是否具有普遍性？利用转录组信息分析毒素编码基因受到的正选择作用，间接评估食性选择作用对蛇毒组成变异的影响强度。本项目有望克服以往谱系相关论或食性适应论在单方面解释蛇毒变异机制时的不足。

本项目通过检测亚洲蝮属主要毒蛇粗毒蛋白组与毒腺转录组构成特征及蛇毒酶活性与毒性，探讨蛇毒组成与功能的种间差异是否与物种间系统发育关系和食性结构相关。研究发现：（1）亚洲蝮属毒蛇粗毒全蛋白组由9-13种毒素家族构成，毒素的种类构成与相对含量存在显著的种间差异，其中缓激肽增强肽与利钠肽（BPP/CNP）、蛇毒金属蛋白酶（SVMP）、L-氨基酸氧化酶（LAAO）、C型类凝集素（CTL）、磷脂酶A2（PLA2）、丝氨酸蛋白酶（SVSP）和富半胱氨酸分泌蛋白（CRISP）是主要的毒素家族；（2）六种毒蛇毒腺转录组中毒素转录本主要归属24个家族，毒素种类构成的种间差异较小，但相对含量存在显著的种间差异，其中蛇毒金属蛋白酶、磷脂酶A2、C型类凝集素、缓激肽增强肽/C型利钠肽、丝氨酸蛋白酶、L-氨基酸氧化酶、神经生长因子、血管内皮生长因子和富半胱氨酸分泌蛋白为主要成分；（3）部分毒素受到明显的正选择作用，但仍有部分毒素并未受到选择作用，主要毒素家族在翻译水平与转录水平呈现显著的正相关；（4）蛇毒的酶活性与致死毒性存在显著的种间差异，但各检测指标间不呈现特定的规律性；（5）蛇毒组成、酶活性和毒性的种间差异与食性结构相关，但r值相对较低，蛇毒酶活性和毒性与种间遗传距离显著相关，蛇毒组成与种间遗传之间也虽不存在相关性，但与显著性临界值的差距较小；（6）短期内投饲不同食物，不影响高丰度蛋白家族在不同处理组蛇毒中的存在与否，但对蛇毒不同蛋白家族的相对含量存在影响，且高丰度蛇毒组分含量的变化较低丰度组分相对稳定。研究结果表明，亚洲蝮属毒蛇蛇毒蛋白组构成与功能特征受系统发育关系与食性结构的影响，部分毒素组分受自然选择正选择驱动显著。