ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISMS OF THE 5-HT1A GENE WITH DIFFERENT AGGRESSION PHENOTYPE IN SEBASTES SCHLEGELII
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摘要:
以许氏平鲉(Sebastes schlegelii)幼鱼为研究对象, 旨在探究5-HT1A受体基因在攻击行为调控中的作用。首先, 通过注射8-OH-DPAT (特异性5-HT1A受体激动剂)对许氏平鲉幼鱼的攻击行为进行了分析, 评估了受体对幼鱼攻击行为的影响; 其次, 对不同攻击表型的许氏平鲉幼鱼进行了5-HT1A受体基因的多态性位点筛选, 以期获得与许氏平鲉攻击行为显著相关的潜在SNP标记。结果显示: (1) 8-OH-DPAT处理组幼鱼攻击行为的频率和时间显著低于对照组(P<0.05), 但攻击潜伏期显著高于对照组(P<0.05); (2)在5-HT1Aα启动子区域中发现了7个与攻击表型差异显著相关的多态性位点(P<0.05), 分别为SNP 1236、SNP 1245、SNP 1260、SNP 1301、SNP 1302、SNP 1309和SNP 1330位点; 在5-HT1Aβ启动子区域中发现了2个与攻击表型差异显著相关的多态性位点(P<0.05), 分别为SNP 892和SNP 1147位点, 但在5-HT1A基因CDS编码区并未发现与攻击差异表型显著相关的多态性位点(P>0.05)。综上所述, 研究结果证明了5-HT1A受体的激活能够显著抑制许氏平鲉的攻击行为, 筛选了与攻击性差异个体相关的5-HT1A受体基因SNP位点。
Abstract:We used juvenile black rockfish (Sebastes schlegelii) to investigate the impact of the 5-HT1A receptor gene on aggression control in this study. The administration of 5-HT1A receptor agonist, 8-OH-DPAT, was utilized to evaluate its impact on aggression among juvenile black rockfish. Subsequently, different aggression phenotypes within the juvenile groups were scrutinized for genetic variations in the 5-HT1A receptor gene, aiming to identify potential SNP markers correlated with aggression. Our findings showed that fish treated with 8-OH-DPAT exhibited significantly reduction in the frequency and duration of aggressive behavior compared to the control group (P<0.05). The latency period for aggression was significantly higher in the treated group (P<0.05). We discovered seven significant polymorphic sites linked to attack phenotypes in the 5-HT1Aα promoter region (P<0.05): SNP 1236, SNP 1245, SNP 1260, SNP 1301, SNP 1302, SNP 1309, and SNP 1330. Additionally, two polymorphic sites were significantly associated with attack phenotypes in the 5-HT1Aβ promoter region (P<0.05): SNP 892 and SNP 1147. However, no significant polymorphic sites were identified in the coding region of the 5-HT1A gene in relation to attack phenotypes (P>0.05). In summary, the results demonstrate that activating the 5-HT1A receptor effectively inhibits aggressive behavior in black rockfish. Furthermore, we also identified SNP loci within the 5-HT1A receptor gene that are associated with different aggression profiles.
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攻击行为是鱼类在面对外界压力时所表现出的自身生理反应, 具有获取有限资源的适应性特征, 因此在鱼类生态中具备重要地位[1]。尽管个体会随着环境条件调整自身行为, 但在鱼类自然种群中, 攻击性仍然表现出稳定、一致的个体差异, 即一些个体表现出更强的攻击倾向[2—4]。这种攻击行为的差异在进化过程中呈现一定的保守性, 并且表现出高度的遗传率, 这表明鱼类攻击性差异可能受遗传突变的影响[5, 6], 但这种一致性表型差异的遗传基础尚未明确查明。
5-羟色胺(5-hydroxy tryptamine, 5-HT)是调控鱼类攻击行为的重要单胺类神经递质, 在信号转导中与不同功能的5-HT受体(5-hydroxytryptamine receptor)在突触前膜和突触后膜上结合以发挥生理作用。目前, 已经发现17种5-HT受体, 包括5-HT1A、5-HT1B、5-HT1D、5-HT2A和5-HT2B等。其中, 5-HT1A受体属于G蛋白偶联受体(GPCR)超家族, 其对5-HT的亲和性显著高于其他受体[7]。在硬骨鱼类中, 5-HT1A受体存在两种同源基因, 分别为5-HT1Aα和5-HT1Aβ, 这两种同源基因与人类的5-HT1A受体基因同源性较高[8], 且存在相似的结构域[9]。5-HT1A受体基因可以通过与典型信号通路偶联来激活第二信使, 调节5-HT的释放, 进而影响攻击行为[10, 11]。对斑马鱼(Danio rerio)[5]和伯氏妊丽鱼(Astatotilapia burtoni)[12]攻击行为的研究结果表明, 低攻击个体的5-HT1A受体基因表达显著高于高攻击个体。8-OH-DPAT是一种选择性的5-HT1A受体激动剂, 通过激活5-HT1A受体来产生效应, 能够显著抑制高攻击个体的攻击行为[13], 但对低攻击个体攻击行为没有显著影响[14], 表明5-HT1A受体基因可能是导致鱼类攻击行为表型分化的潜在候选基因之一。
单核苷酸多态性(Single nucleotide polymorphisms, SNPs)作为DNA序列中最丰富的多态性变异类型, 具有多态性高、覆盖密度大和遗传稳定性强等特点, SNP分为基因编码区和非编码区单碱基突变两种形式, 它们可以通过影响基因编码区的氨基酸序列或非编码区的基因表达水平影响个体的生理、代谢过程和发育调节, 从而导致个体间的表型差异。因此, SNP在遗传研究中具有重要地位, 是鱼类遗传研究的首选标记[15]。近年来, SNP分子标记技术已被广泛应用于与水产动物生长性状[16, 17]、抗病性状[18]和种群保护[19]等相关的研究中。
许氏平鲉(Sebastes schlegelii)因其肉质细嫩, 味道鲜美备受欢迎, 成为我国北方重要的经济鱼种[20]。在许氏平鲉养殖中, 常出现攻击行为, 甚至同类残食, 致使幼鱼成活率下降, 给产业带来严重损失[21, 22]。降低许氏平鲉的攻击行为已经是养殖业急需解决的一个问题, 鉴于5-HT1A受体基因在鱼类攻击行为中的重要调控作用, 本研究在划分攻击行为表型差异群体基础上, 对许氏平鲉幼鱼的5-HT1A受体基因进行SNP筛选, 以期为许氏平鲉低攻击品种分子标记辅助选择育种提供参考。
1. 材料与方法
1.1 实验条件
实验用许氏平鲉幼鱼[体长为(6.53±0.46) cm, 体重为(7.72±0.92) g]为当地商业孵化场于同一种群中获得的有相同遗传背景的幼鱼, 暂养于设施渔业教育部重点实验室。暂养期间保持水温为(20.0±1.0)℃, 盐度为31, 维持自然光周期, 以体重的2%进行投喂(粗蛋白质≥44%, 粗脂肪≥5%), 投喂时间为8:30、12:30和16:30。
1.2 实验设置
实验在规格为21 cm×14 cm×17.5 cm的玻璃水箱中进行, 水深10 cm, 有效水体为2.94 L。玻璃水箱侧面覆盖不透明白色贴纸以减少外界环境影响。将水箱用白色不透明PVC板分成2个大小相等的隔间, 每个隔间配有1个气石, 使水中的溶解氧保持在6 mg/L以上。为排除大小差异对个体攻击性的影响, 对幼鱼的体长体重进行严格筛选, 保证每组幼鱼体长差异<0.1 cm, 体重差异<0.6 g。在实验系统顶部架设摄像机(HIK-DS-2CD3T35D-I5, 海康威视)进行行为录制, 视频采样率为25帧/秒。
8-OH-DPAT注射实验 实验选取40尾许氏平鲉幼鱼, 设置8-OH-DPAT (Sigma-Aldrich, 上海)处理组(0.9%生理盐水稀释, 浓度为1.54 mg/mL)和0.9%生理盐水对照组, 每个处理组10组重复。幼鱼在实验系统中适应3d后, 使用Dosys式连续分液注射器(Socorex 174, 瑞士)对处理组和对照组的每尾幼鱼分别腹腔注射5 μL的8-OH-DPAT溶液和生理盐水。在注射完成后, 将幼鱼放回实验系统适应60min, 随后移除隔板, 让两尾鱼开始自由接触, 进行10min的视频录制和行为监测。
攻击行为表型差异群体划分 实验选取60尾许氏平鲉幼鱼, 将幼鱼配对后, 使用MS-222麻醉, 采用手术带线缝合针将不同颜色的塑料圆片(d=5 mm)固定在幼鱼的背鳍前端进行个体标记识别。标记后立即放回水箱中, 1min内恢复正常运动。该过程没有引起幼鱼的任何出血或行为异常等现象。标记的幼鱼在实验系统中适应3d后, 移除隔板, 让两尾鱼开始自由接触, 进行10min的视频录制和行为监测, 通过颜色标记识别攻击差异个体, 准确识别后放置隔板, 将两尾鱼分开, 以便后续样品采集。
1.3 行为分析和样品采集
在实验结束后, 使用Noldus EthoVision XT (版本12.0; Noldus信息技术公司)的动态减影法(Dynamic Ethogram)跟踪和分析幼鱼的运动和行为。分析数据采用全事件观测法进行复核, 确保结果的准确性。攻击行为定义为高攻击个体对低攻击个体做出的追逐、咬的过程, 视频分别记录了幼鱼的攻击频率、攻击持续时间和第一次攻击潜伏期。
在高攻击和低攻击个体用MS-222麻醉后, 置于冰盘上解剖取全脑和1/5背鳍、胸鳍和尾鳍的混合样本。脑组织样品立即放入装有1 mL RNA wait保存液的冻存管中, 样品浸润后置4℃冰箱过夜, 使RNA wait完全渗入到组织中, 后转移到–20℃冰箱长期保存, 用于RNA的提取。鱼鳍混合样本放入冻存管中, 立即浸入液氮中, 30min后转移至–80℃冰箱长期保存, 用于DNA的提取。
1.4 攻击行为表型差异个体5-HT1A基因SNP筛选
按照天根生化科技有限公司(北京)的基因组DNA提取试剂盒许氏平鲉鱼鳍总DNA, 按照艾科瑞生物工程有限公司(湖南)总RNA提取试剂盒提取幼鱼脑组织中的RNA, 按照1%琼脂糖凝胶电泳检测DNA和RNA的完整性, 并用核酸微量定量仪(莫纳生物, 江苏)检测DNA和RNA浓度, 保证A260/ A280在1.8—2.0。根据Fast Quant cDNA第一链合成试剂盒(艾科瑞, 湖南), 反转录合成cDNA。
根据许氏平鲉5-HT1A基因序列信息, 对5-HT1Aα和5-HT1Aβ基因启动子区及编码区进行扩增。利用Primer Premier 5.0进行引物设计(引物信息见表 1), 将引物序列送至生工生物工程股份有限公司(上海)进行合成。以许氏平鲉攻击差异群体基因组的cDNA和DNA为模板, 对5-HT1Aα和5-HT1Aβ基因进行扩增, 反应体系(50 μL)包括: 模板2 μL, 5-HT1A-F/R各1 μL, Pro Taq Master Mix 25 μL, ddH2O 21 μL, PCR反应程序为: 94℃预变性30s; 98℃变性10s, 退火复性30s (退火温度见表 1), 72℃延伸1min, 共进行35个扩增循环, 最后72℃延伸2min, PCR产物经1%琼脂糖凝胶电泳检测, 合格PCR产物送诺禾致源科技股份有限公司(北京)进行测序。
表 1 5-HT1A基因编码区和启动子引物序列Table 1. The primer sequences of the coding region and promoter region of the 5-HT1A gene基因
Gene引物名称
Primer name引物序列
Primer sequence产物长度
Product length (bp)退火温度
Annealing temperature (℃)5-HT1Aα Ssc_10010295-CDS GTAACTCCGATGGACCCG 985 55 ATATGATGGGGTTGAGGAGA Ssc_10010295-Promoter AATAGTGTCACATTGTCCATCTTT 1280 57 GTGTTGTAGTTGAGCGTTGACA 5-HT1Aβ Ssc_10008317-CDS CAATTTGGCCTCAGTTCGA 1014 57 AGCCCAGCCAGTTGATGAC Ssc_10008317-Promoter AAGTCAATTCCACAATAGTTCC 1443 53 GAAAACTCGAGGTGTTACTTGA 1.5 统计分析
统计计算均使用SPSS 26.0进行, 采用单因素方差分析(One-way ANOVA)方法对行为数据进行分析。利用SnapGene软件分别对攻击表型差异群体的5-HT1Aα和5-HT1Aβ扩增产物编码区和启动子区的测序结果进行比对, 结合峰图, 筛选出突变频率大于或等于10%的位点作为与攻击表型相关的潜在SNP位点。对启动子区域, 本实验利用http://bioinfo.life.hust.edu.cn/AnimalTFDB/#!/tfbs_predict在线软件进行转录因子结合位点的预测, 筛选P<0.0001的转录因子结合位点, 按照得分情况对启动子区域SNP和转录因子结合位点进行对应。对筛选出的与攻击表型相关的编码区和启动子区的潜在SNP进行卡方检验, 当P<0.05时, 表明筛选出的SNP与攻击表型相关。
2. 结果
2.1 5-HT1A受体对许氏平鲉幼鱼攻击行为的影响
图 1所示, 8-OH-DPAT处理组的第一次攻击潜伏期显著高于对照组(P<0.05), 攻击时间和频率显著低于对照组(P<0.05)。
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图 1 不同处理对许氏平鲉幼鱼攻击行为的影响*P<0.05表示差异显著; **P<0.01表示差异极显著Figure 1. The effects of different treatment groups on aggression behavior in S. schlegelii*P<0.05 indicates significant differences; **P<0.01 indicates extremely significant differences2.2 5-HT1A受体基因编码区SNP的筛选
本研究对60个高、低攻击差异个体的5-HT1Aα和5-HT1Aβ基因进行检测, 在编码区初步筛选到5个可能与攻击性表型相关的SNP位点, 所有位点均为碱基转换(C/T、T/C、G/A、A/C和A/G), 其中在5-HT1Aα编码区筛选到3个可能的SNP位点, 5-HT1Aβ编码区筛选到2个可能的SNP位点(表 2), 分别占5-HT1Aα和5-HT1Aβ编码序列总碱基的0.24%和0.16%。5个SNP位点中只有SNP 214位点改变所编码的氨基酸, 其天门冬氨酸变为组氨酸, 为非同义突变, 其他均为同义突变。
表 2 5-HT1A基因编码区SNP位点基因分型结果Table 2. The coding region SNP genotyping results of 5-HT1A gene基因
GeneSNP 位点
SNP site位点碱基
Site base高攻击(基因型/
突变频率)
High-aggression
(Genotype/
Mutation
frequency)低攻击(基因型/
突变频率)
Low-aggression
(Genotype/
Mutation
frequency)5-HT1Aα SNP 234 C T 17/13 T 13/17 SNP 942 T C 8/22 C 10/20 SNP 999 G G 0/30 A 2/28 5-HT1Aβ SNP 214 A C 2/22 A 0/30 SNP 1122 A G 2/22 A 0/30 2.3 5-HT1A受体基因编码区SNP与许氏平鲉幼鱼攻击行为相关性研究
通过核苷酸比对和测序峰图分析, 对5-HT1Aα的3个SNP位点和5-HT1Aβ的2个SNP位点进行分型[以5-HT1Aα基因的SNP 999 (G/A)的测序峰图为例, 图 2]。通过攻击表型与突变位点的关联分析表明, 非同义突变SNP 214位点对基因功能影响不显著(P=0.096)。
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图 2 许氏平鲉幼鱼5-HT1Aα基因测序峰图分析a. 5-HT1Aα基因SNP 999 (G/A)测序峰图; b. 高攻击个体5-HT1Aα基因SNP 999 (G/A)测序峰图; c. 低攻击个体5-HT1Aα基因SNP 999 (G/A)测序峰图Figure 2. The sequencing chromatograms of 5-HT1Aα gene of S. schlegeliia. The sequencing chromatograms of 5-HT1Aα gene SNP 999 (G/A); b. The sequencing chromatograms of 5-HT1Aα gene SNP 999 (G/A) of High-aggression; The sequencing chromatograms of 5-HT1Aα gene SNP 999 (G/A) of Low-aggression2.4 5-HT1A受体基因启动子区SNP的筛选
许氏平鲉攻击表型差异个体的5-HT1Aα和5-HT1Aβ启动子区域SNP筛选结果显示: 在许氏平鲉启动子区域存在34个与攻击性表型差异可能有关的SNP位点, 其中5-HT1Aα启动子区域共有16个(表 3), 5-HT1Aβ启动子区域共有18个(表 4)。
表 3 5-HT1Aα启动子SNP位点基因分型结果Table 3. The promoter SNP genotyping results of 5-HT1AαSNP位点
SNP site位点碱基
Site base高攻击(基因型/
突变频率)
High-aggression
(Genotype/Mutation
frequency)低攻击(基因型/
突变频率)
Low-aggression
(Genotype/Mutation
frequency)SNP 987 G A 4/26 A 6/24 SNP 1235 C C 0/30 T 2/28 SNP 1236 T T 0/30 C 4/26 SNP 1245 T T 0/30 G 5/25 SNP 1255 A A 0/30 C 3/27 SNP 1260 T T 0/30 A 4/26 SNP 1291 T G 6/24 G 2/28 SNP 1301 C G 5/25 C 0/30 SNP 1302 G C 3/27 A 8/22 SNP 1309 T C 4/26 T 0/30 SNP 1315 G A 6/24 A 7/23 SNP 1330 C T 5/25 C 0/30 SNP 1333 C T 4/26 T 2/28 SNP 1435 C T 7/23 T 10/20 SNP 1542 T C 8/22 C 15/15 SNP 1682 G C 12/18 C 13/17 表 4 5-HT1Aβ启动子SNP位点基因分型结果Table 4. The promoter SNP genotyping results of 5-HT1Aβ geneSNP位点
SNP site位点碱基
Site base高攻击(基因型/
突变频率)
High-aggression
(Genotype/Mutation
frequency)低攻击(基因型/
突变频率)
Low-aggression
(Genotype/Mutation
frequency)SNP 813 T A 2/28 T 0/30 SNP 892 G A 6/24 G 0/30 SNP 898 G A 3/27 G 0/30 SNP 1030 G A 2/28 A 3/27 SNP 1033 G A 8/22 A 6/24 SNP 1050 C A 4/26 C 0/30 SNP 1147 T T 0/30 C 3/27 SNP 1198 G A 10/20 A 7/23 SNP 1290 G A 2/28 G 0/30 SNP 1373 C T 9/21 T 8/22 SNP 1388 G A 3/27 G 0/30 SNP 1401 C T 11/19 T 6/24 SNP 1474 C T 3/27 T 3/27 SNP 1571 C T 11/19 T 8/22 SNP 1582 C T 11/19 T 13/17 SNP 1680 T G 3/27 T 0/30 SNP 1756 G A 4/26 G 0/30 SNP 1821 G A 2/28 G 0/30 通过对转录因子结合位点的预测, 筛选得到与许氏平鲉攻击差异个体5-HT1Aα和5-HT1Aβ启动子SNP相对应的转录因子结合位点。在5-HT1Aα启动子中所有筛选到的SNP位点均与转录因子结合位点对应(表 5), 5-HT1Aβ启动子中有15个SNP位点与转录因子结合位点对应(表 6)。
表 5 5-HT1Aα启动子SNP位点中转录因子结合位点预测Table 5. 5-HT1Aα promoter SNP genotyping resultsSNP位点
SNP site转录因子
Transcription factorScore P-value 序列
SequenceSNP 987 FLI1 4.409 <0.0001 CCTCATCCTTCCTCCCTACAGT SNP 1235 CREB1 10.506 <0.0001 TTTTTTTCTCTT SNP 1236 SNP 1245 TRIM28 10.026 <0.0001 TTTTTTCTCTTTAAGGTCTTTT SNP 1255 TRIM63 10.348 <0.0001 CTTTTTTTCACTTAT SNP 1260 SNP 1291 PITX2 10.939 <0.0001 TTGGCTTAAA SNP 1301 E2F1 11.571 <0.0001 CGAAAAGT SNP 1302 SNP 1309 PXR:RXR 10.727 <0.0001 AAAGTTCA SNP 1315 TBX21 10.957 <0.0001 TTAAAAGTGATAAAG SNP 1330 NOTCH1 11.105 <0.0001 AAACCACTT SNP 1333 SNP 1435 CBFB 11.184 <0.0001 CACCCCACAGAA SNP 1542 ONECUT3 9.959 <0.0001 AGTATAGATTTTCT SNP 1682 VDR 9.743 <0.0001 CAATTCAGTGGGTCCA 表 6 5-HT1Aβ启动子SNP位点中转录因子结合位点预测Table 6. 5-HT1Aβ promoter SNP genotyping resultsSNP位点
SNP site转录因子
Transcription factorScore P-value 序列
SequenceSNP 813 FOXM1 11.130 <0.0001 AAAATAAATAAA SNP 892 TP53 –9.056 <0.0001 GAAAAAGAAAAAAGAAATGAAA SNP 898 FLI1 –14.590 <0.0001 AAAAAGAAAAAAGAAATG SNP 1030 PAX3 11.493 <0.0001 ACCAAAACTAATAGAA SNP 1147 POU2F3 10.713 <0.0001 TTATTTGCTAATTAGT SNP 1198 NOBOX 10.491 <0.0001 CAGATCAATTAGCTTTG SNP 1290 GTF2A1:
GTF2A211.606 <0.0001 TATTAAAGCGGG SNP 1373 SETDB1 7.053 <0.0001 CACAGCCCGTATTCAGAAACTC SNP 1401 ZBTB33 8.316 <0.0001 CGTCAGGATATCTG SNP 1474 HOXA9 10.020 <0.0001 TTGTAAATCTGTCC SNP 1571 CIITA 10.258 <0.0001 CCTAGCAACGCAATTC SNP 1582 CTCF 10.474 <0.0001 GTTGCAATTCTA SNP 1680 SOX17 6.744 <0.0001 GGAAAATAAAGTTTTTTT SNP 1756 POU6F1 10.699 <0.0001 CCTGAAAATGAGCATGA SNP 1821 ZNF384 11.307 <0.0001 ACAAAAAACAGC 2.5 5-HT1A受体基因启动子区SNP与许氏平鲉幼鱼攻击行为相关性分析
攻击表型与启动子SNP的关联研究结果发现, 5-HT1Aα启动子区域中共筛选到7个位点与许氏平鲉攻击表型差异显著相关(P<0.05), 分别为SNP 1236 (T/C)、SNP 1245 (T/G)、SNP 1260 (T/A)、SNP 1301 (C/G)、SNP 1302 (G/C)、SNP 1309 (T/C)和SNP 1330 (C/T)位点, 其他位点与许氏平鲉攻击表型差异相关性不显著(P>0.05; 表 7), 筛选到5-HT1Aβ启动子区域中SNP 892 (G/A)和SNP 1147 (T/C)位点与攻击性表型差异显著相关(P<0.05), 其他位点均不显著(P>0.05; 表 8)。
表 7 5-HT1Aα启动子的卡方检验结果Table 7. Chi-square test results of 5-HT1Aα promoterSNP位点
SNP site基因型
Genotypeχ² P SNP 987 G>A 0.403 0.526 SNP 1235 C>T 2.001 0.157 SNP 1236 T>C 4.138 0.042 SNP 1245 T>G 5.261 0.022 SNP 1255 A>C 3.052 0.081 SNP 1260 T>A 4.138 0.042 SNP 1291 T>G 2.439 0.118 SNP 1301 C>G 5.606 0.018 SNP 1302 G>C 3.252 0.071 SNP 1302 G>A 7.629 0.006 SNP 1309 T>C 4.409 0.036 SNP 1315 G>A 0.061 0.805 SNP 1330 C>T 5.606 0.018 SNP 1333 C>T 0.809 0.368 SNP 1435 C>T 0.601 0.438 SNP 1542 T>C 3.015 0.083 SNP 1682 G>C 0.024 0.877 表 8 5-HT1Aβ启动子的卡方检验结果Table 8. Chi-square test results of 5-HT1Aβ promoterSNP位点
SNP site基因型
Genotypeχ² P SNP 813 T>A 1.564 0.211 SNP 892 G>A 5.128 0.024 SNP 898 G>A 2.397 0.122 SNP 1030 G>A 0.668 0.414 SNP 1147 T>C 4.261 0.039 SNP 1198 G>A 0.03 0.862 SNP 1290 G>A 1.564 0.211 SNP 1373 C>T 0.188 0.665 SNP 1401 C>T 0.608 0.436 SNP 1474 C>T 0.142 0.706 SNP 1571 C>T 0.007 0.933 SNP 1582 C>T 2.457 0.117 SNP 1680 T>G 2.397 0.122 SNP 1756 G>A 3.267 0.071 SNP 1821 G>A 1.564 0.211 3. 讨论
研究表明5-HT1A受体在调控鱼类攻击行为方面起到重要作用, 攻击性差异个体5-HT1A受体敏感性不同[23]。在本研究中, 8-OH-DPAT能显著降低高攻击性许氏平鲉幼鱼的攻击强度, 增加攻击潜伏期, 这与Antunes等[24]的研究结果一致, 表明5-HT1A受体的激活可显著抑制许氏平鲉高攻击个体的攻击性。值得注意的是, 8-OH-DPAT在不同物种体内的代谢率不同。在啮齿动物中, 其作用时间较短, 代谢较快, 半衰期约为8min[25]。在本研究中, 注射实验的连续监测结果显示, 注射8-OH-DPAT的1h后, 许氏平鲉攻击行为反应出现显著降低。在对红鳍东方鲀(Takifugu rubripes)攻击行为的研究结果中同样发现, 8-OH-DPAT处理55—65min后, 幼鱼的攻击行为反应变化显著[26]。本文结果证实此时间对许氏平鲉幼鱼的攻击反应同样有效, 表明鱼类中8-OH-DPAT药物代谢时间较长, 半衰期较长。
单核苷酸多态是真核生物基因组中经常出现的遗传突变, 其与某些确切的表型相关联。然而, 对5-HT1A受体基因SNP位点筛选及其与攻击行为关联的研究以灵长类和啮齿类动物较多, 关于鱼类的相关研究尚未见报道[27—29]。本研究从许氏平鲉5-HT1Aα和5-HT1Aβ基因编码区序列上分别筛选到3个和2个SNP位点, 分别占5-HT1Aα和5-HT1Aβ编码序列总碱基的0.24%和0.16%, 相比于ND2、IPS-1等基因编码区序列的突变率[30, 31], 许氏平鲉5-HT1A基因编码区序列多态性较低。其中SNP 214位点改变了氨基酸的种类, 属于非同义突变。非同义突变通常会导致蛋白质结构与活性的变化, 甚至导致蛋白质翻译提前终止, 从而发生一系列生物学效应并对动物机体产生影响。但在本研究中, 通过对SNP 214位点与攻击行为关联性分析发现, SNP 214位点对攻击行为差异群体的影响不显著。这可能是因为5-HT1A受体基因具有7个疏水跨膜结构域的共同基序, 硬骨鱼类中的5-HT1A基因序列同源性较高[8], 跨膜区域(TM)的氨基酸在许多物种之间高度保守[32]。
通常位于非编码区的SNP位点对蛋白质无直接影响, 对个体的表型也没有显著影响。但本研究发现5-HT1Aα启动子区域中有7个SNP位点与许氏平鲉攻击表型差异显著相关, 5-HT1Aβ启动子区域中有2个SNP位点与许氏平鲉攻击表型差异显著相关, 相较于编码区, 启动子区筛选到的SNP位点更多。在对团头鲂(Megalobrama amblycephala)、黄颡鱼(Pelteobagrus fulvidraco)等开展的SNP位点的研究中也发现, SNP位点位于启动子区域的频率大于编码区域[33, 34]。这可能是因为启动子是基因转录起始所必需的顺式作用元件之一[35]。启动子区域可能存在较多的转录因子结合位点, 调控多个下游基因的表达[36]。先前的研究表明, 启动子区域的SNP位点可以通过改变与转录因子的结合来激活或抑制基因表达, 从而导致表型的改变[37]。在本研究筛选到的9个SNP位点中, 携带T等位基因个体多为高攻击个体, 在之前的研究中, T等位基因通常被认为是攻击行为的风险等位基因[38]。本研究结果中T等位基因突变整合了5种转录因子结合位点, 分别是CREB1、TRIM28、TRIM63、NOTCH1和TP53。其中, CREB (cAMP效应元件结合蛋白)是cAMP-PKA-CREB-BDNF通路中的一个交汇点。5-HT1A受体与Gi/Go蛋白偶联, 直接或间接介导CREB转录因子的分子磷酸化, 磷酸化的CREB可能通过激活转录辅激活物(CRTC1)的表达, 从而影响攻击性表型的变化[39]。NOTCH信号对调节细胞分化、增殖和细胞存活具有重要作用[40]。它可能通过抑制树突的延伸和分支来抑制5-HT1A受体的表达[41]。然而, 关于TRIM28、TRIM63和TP53介导5-HT1A受体表达进而影响攻击行为的机制尚不清楚, 需要进一步的研究来验证。此外, 许氏平鲉在5-HT1Aα基因上筛选到的SNP位点数量高于5-HT1Aβ基因, 表明5-HT1Aα基因多态性高于5-HT1Aβ基因, 这可能导致5-HT1Aα受体表达高于5-HT1Aβ, 使5-HT1Aα受体表达区域更加广泛, 而5-HT1Aβ表达受限[9]。因此, 位于启动子区域的SNP可能通过影响启动子与转录位点的结合来调控5-HT1A基因的表达, 进而影响鱼类攻击行为反应。
4. 结论
本研究通过证明了5-HT1A受体的激活可以显著降低许氏平鲉攻击行为, 从许氏平鲉5-HT1Aα基因非编码区筛选到7个与攻击性表型差异显著相关的多态性位点, 在5-HT1Aβ基因非编码区筛选到2个, 但未在编码区发现与攻击差异显著相关的多态性位点。该结果可为许氏平鲉标记辅助选育低攻击品种提供参考。
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图 1 不同处理对许氏平鲉幼鱼攻击行为的影响
*P<0.05表示差异显著; **P<0.01表示差异极显著
Figure 1. The effects of different treatment groups on aggression behavior in S. schlegelii
*P<0.05 indicates significant differences; **P<0.01 indicates extremely significant differences
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图 2 许氏平鲉幼鱼5-HT1Aα基因测序峰图分析
a. 5-HT1Aα基因SNP 999 (G/A)测序峰图; b. 高攻击个体5-HT1Aα基因SNP 999 (G/A)测序峰图; c. 低攻击个体5-HT1Aα基因SNP 999 (G/A)测序峰图
Figure 2. The sequencing chromatograms of 5-HT1Aα gene of S. schlegelii
a. The sequencing chromatograms of 5-HT1Aα gene SNP 999 (G/A); b. The sequencing chromatograms of 5-HT1Aα gene SNP 999 (G/A) of High-aggression; The sequencing chromatograms of 5-HT1Aα gene SNP 999 (G/A) of Low-aggression
表 1 5-HT1A基因编码区和启动子引物序列
Table 1 The primer sequences of the coding region and promoter region of the 5-HT1A gene
基因
Gene引物名称
Primer name引物序列
Primer sequence产物长度
Product length (bp)退火温度
Annealing temperature (℃)5-HT1Aα Ssc_10010295-CDS GTAACTCCGATGGACCCG 985 55 ATATGATGGGGTTGAGGAGA Ssc_10010295-Promoter AATAGTGTCACATTGTCCATCTTT 1280 57 GTGTTGTAGTTGAGCGTTGACA 5-HT1Aβ Ssc_10008317-CDS CAATTTGGCCTCAGTTCGA 1014 57 AGCCCAGCCAGTTGATGAC Ssc_10008317-Promoter AAGTCAATTCCACAATAGTTCC 1443 53 GAAAACTCGAGGTGTTACTTGA 
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表 2 5-HT1A基因编码区SNP位点基因分型结果
Table 2 The coding region SNP genotyping results of 5-HT1A gene
基因
GeneSNP 位点
SNP site位点碱基
Site base高攻击(基因型/
突变频率)
High-aggression
(Genotype/
Mutation
frequency)低攻击(基因型/
突变频率)
Low-aggression
(Genotype/
Mutation
frequency)5-HT1Aα SNP 234 C T 17/13 T 13/17 SNP 942 T C 8/22 C 10/20 SNP 999 G G 0/30 A 2/28 5-HT1Aβ SNP 214 A C 2/22 A 0/30 SNP 1122 A G 2/22 A 0/30
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表 3 5-HT1Aα启动子SNP位点基因分型结果
Table 3 The promoter SNP genotyping results of 5-HT1Aα
SNP位点
SNP site位点碱基
Site base高攻击(基因型/
突变频率)
High-aggression
(Genotype/Mutation
frequency)低攻击(基因型/
突变频率)
Low-aggression
(Genotype/Mutation
frequency)SNP 987 G A 4/26 A 6/24 SNP 1235 C C 0/30 T 2/28 SNP 1236 T T 0/30 C 4/26 SNP 1245 T T 0/30 G 5/25 SNP 1255 A A 0/30 C 3/27 SNP 1260 T T 0/30 A 4/26 SNP 1291 T G 6/24 G 2/28 SNP 1301 C G 5/25 C 0/30 SNP 1302 G C 3/27 A 8/22 SNP 1309 T C 4/26 T 0/30 SNP 1315 G A 6/24 A 7/23 SNP 1330 C T 5/25 C 0/30 SNP 1333 C T 4/26 T 2/28 SNP 1435 C T 7/23 T 10/20 SNP 1542 T C 8/22 C 15/15 SNP 1682 G C 12/18 C 13/17
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表 4 5-HT1Aβ启动子SNP位点基因分型结果
Table 4 The promoter SNP genotyping results of 5-HT1Aβ gene
SNP位点
SNP site位点碱基
Site base高攻击(基因型/
突变频率)
High-aggression
(Genotype/Mutation
frequency)低攻击(基因型/
突变频率)
Low-aggression
(Genotype/Mutation
frequency)SNP 813 T A 2/28 T 0/30 SNP 892 G A 6/24 G 0/30 SNP 898 G A 3/27 G 0/30 SNP 1030 G A 2/28 A 3/27 SNP 1033 G A 8/22 A 6/24 SNP 1050 C A 4/26 C 0/30 SNP 1147 T T 0/30 C 3/27 SNP 1198 G A 10/20 A 7/23 SNP 1290 G A 2/28 G 0/30 SNP 1373 C T 9/21 T 8/22 SNP 1388 G A 3/27 G 0/30 SNP 1401 C T 11/19 T 6/24 SNP 1474 C T 3/27 T 3/27 SNP 1571 C T 11/19 T 8/22 SNP 1582 C T 11/19 T 13/17 SNP 1680 T G 3/27 T 0/30 SNP 1756 G A 4/26 G 0/30 SNP 1821 G A 2/28 G 0/30
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表 5 5-HT1Aα启动子SNP位点中转录因子结合位点预测
Table 5 5-HT1Aα promoter SNP genotyping results
SNP位点
SNP site转录因子
Transcription factorScore P-value 序列
SequenceSNP 987 FLI1 4.409 <0.0001 CCTCATCCTTCCTCCCTACAGT SNP 1235 CREB1 10.506 <0.0001 TTTTTTTCTCTT SNP 1236 SNP 1245 TRIM28 10.026 <0.0001 TTTTTTCTCTTTAAGGTCTTTT SNP 1255 TRIM63 10.348 <0.0001 CTTTTTTTCACTTAT SNP 1260 SNP 1291 PITX2 10.939 <0.0001 TTGGCTTAAA SNP 1301 E2F1 11.571 <0.0001 CGAAAAGT SNP 1302 SNP 1309 PXR:RXR 10.727 <0.0001 AAAGTTCA SNP 1315 TBX21 10.957 <0.0001 TTAAAAGTGATAAAG SNP 1330 NOTCH1 11.105 <0.0001 AAACCACTT SNP 1333 SNP 1435 CBFB 11.184 <0.0001 CACCCCACAGAA SNP 1542 ONECUT3 9.959 <0.0001 AGTATAGATTTTCT SNP 1682 VDR 9.743 <0.0001 CAATTCAGTGGGTCCA
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表 6 5-HT1Aβ启动子SNP位点中转录因子结合位点预测
Table 6 5-HT1Aβ promoter SNP genotyping results
SNP位点
SNP site转录因子
Transcription factorScore P-value 序列
SequenceSNP 813 FOXM1 11.130 <0.0001 AAAATAAATAAA SNP 892 TP53 –9.056 <0.0001 GAAAAAGAAAAAAGAAATGAAA SNP 898 FLI1 –14.590 <0.0001 AAAAAGAAAAAAGAAATG SNP 1030 PAX3 11.493 <0.0001 ACCAAAACTAATAGAA SNP 1147 POU2F3 10.713 <0.0001 TTATTTGCTAATTAGT SNP 1198 NOBOX 10.491 <0.0001 CAGATCAATTAGCTTTG SNP 1290 GTF2A1:
GTF2A211.606 <0.0001 TATTAAAGCGGG SNP 1373 SETDB1 7.053 <0.0001 CACAGCCCGTATTCAGAAACTC SNP 1401 ZBTB33 8.316 <0.0001 CGTCAGGATATCTG SNP 1474 HOXA9 10.020 <0.0001 TTGTAAATCTGTCC SNP 1571 CIITA 10.258 <0.0001 CCTAGCAACGCAATTC SNP 1582 CTCF 10.474 <0.0001 GTTGCAATTCTA SNP 1680 SOX17 6.744 <0.0001 GGAAAATAAAGTTTTTTT SNP 1756 POU6F1 10.699 <0.0001 CCTGAAAATGAGCATGA SNP 1821 ZNF384 11.307 <0.0001 ACAAAAAACAGC
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表 7 5-HT1Aα启动子的卡方检验结果
Table 7 Chi-square test results of 5-HT1Aα promoter
SNP位点
SNP site基因型
Genotypeχ² P SNP 987 G>A 0.403 0.526 SNP 1235 C>T 2.001 0.157 SNP 1236 T>C 4.138 0.042 SNP 1245 T>G 5.261 0.022 SNP 1255 A>C 3.052 0.081 SNP 1260 T>A 4.138 0.042 SNP 1291 T>G 2.439 0.118 SNP 1301 C>G 5.606 0.018 SNP 1302 G>C 3.252 0.071 SNP 1302 G>A 7.629 0.006 SNP 1309 T>C 4.409 0.036 SNP 1315 G>A 0.061 0.805 SNP 1330 C>T 5.606 0.018 SNP 1333 C>T 0.809 0.368 SNP 1435 C>T 0.601 0.438 SNP 1542 T>C 3.015 0.083 SNP 1682 G>C 0.024 0.877
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表 8 5-HT1Aβ启动子的卡方检验结果
Table 8 Chi-square test results of 5-HT1Aβ promoter
SNP位点
SNP site基因型
Genotypeχ² P SNP 813 T>A 1.564 0.211 SNP 892 G>A 5.128 0.024 SNP 898 G>A 2.397 0.122 SNP 1030 G>A 0.668 0.414 SNP 1147 T>C 4.261 0.039 SNP 1198 G>A 0.03 0.862 SNP 1290 G>A 1.564 0.211 SNP 1373 C>T 0.188 0.665 SNP 1401 C>T 0.608 0.436 SNP 1474 C>T 0.142 0.706 SNP 1571 C>T 0.007 0.933 SNP 1582 C>T 2.457 0.117 SNP 1680 T>G 2.397 0.122 SNP 1756 G>A 3.267 0.071 SNP 1821 G>A 1.564 0.211
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