徐欣玉 刘洁 夏慧敏 陈丽婷 李鹏飞 肖俊 陆颖
摘要:【目的】比較低温胁迫下野生罗非鱼和养殖吉富罗非鱼的基因表达模式,揭示野生罗非鱼低温应答的特有分子调控机制,为后续开展野生罗非鱼耐冷亲本大范围筛选打下基础。【方法】挑选规格相近的野生罗非鱼和养殖吉富罗非鱼进行梯度降温试验,水温先以1 ℃/12 h的速度从26 ℃降至14 ℃,并保持288 h(12 d),于26 ℃、20 ℃及14 ℃保持0、120和288 h等5个时间点分别解剖罗非鱼采集脑组织样品,构建cDNA文库后以Illumina HiSeq×Ten测序平台进行双端测序及比较转录组分析,并采用实时荧光定量PCR对具有重要功能的差异表达基因(DEGs)进行表达验证。【结果】野生罗非鱼在11 ℃时开始出现死亡个体,但在8 ℃时仍有50.0%的存活个体,说明野生罗非鱼较养殖吉富罗非鱼具有更强的低温耐受能力。在14 ℃的长时间低温胁迫过程中,养殖吉富罗非鱼脑组织中表达显著上调的差异表达基因数量约是野生罗非鱼的10倍,即养殖吉富罗非鱼的应激反应远比野生罗非鱼强烈。KEGG信号通路富集分析结果显示,养殖吉富罗非鱼和野生罗非鱼在14 ℃保持120和288 h的上调差异表达基因均富集到核糖体发生、内质网蛋白加工及剪接体信号等信号通路上,野生罗非鱼的上调差异表达基因还额外富集到核苷酸切除修复、N-糖基化生物合成和DNA复制信号等通路上。与养殖吉富罗非鱼相比,在野生罗非鱼中发现577个特有的差异表达基因,主要富集在NOD受体信号通路、凋亡和内吞等通路上,且表现为NOD受体信号通路被启动,而细胞凋亡受抑制。在野生罗非鱼中,参与NOD受体信号通路的关键功能基因(Nemo、NFκB、p38、JNK和IL-1β)在长时间低温胁迫中均维持在一个较高的表达水平,而内吞途径关键基因的表达上升倍数也明显高于养殖吉富罗非鱼。【结论】野生罗非鱼通过避免过度的应激反应和维持低水平代谢的细胞稳定以减轻低温胁迫对机体的损伤,并持续启动NOD受体信号通路及内吞途径以维持其免疫能力,而具有较养殖罗非鱼更强的低温耐受力。
关键词: 罗非鱼;脑组织;低温胁迫;差异表达基因(DEGs);转录组测序
中图分类号: S917.4;S965.125 文献标志码: A 文章编号:2095-1191(2022)03-0704-10
Transcriptome sequencing analysis of the brain tissues responding to low temperature stress in wild tilapia
XU Xin-yu LIU Jie XIA Hui-min CHEN Li-ting LI Peng-fei XIAO Jun LU Ying
(1Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources (Shanghai Ocean University), Shanghai 201306, China; 2Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, (Shanghai Ocean University),Shanghai 201306, China; 3Guangxi Academy of Fishery Sciences/Guangxi Key
Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture, Nanning, Guangxi 530021, China; 4Guangxi Academy of Sciences/Guangxi Engineering Research Center for Fishery Major Diseases Control and Efficient
Healthy Breeding Industrial Technology, Nanning, Guangxi 530007, China)
Abstract:【Objective】To investigate regulatory mechanism of the responses to low temperature stress in wild tilapia, comparing to that of the cultured tilapia, so as to contribute to screen cold tolerant parents from wild tilapia in the future.【Method】A wild and a cultured tilapia populations with similar sizes were selected for a gradient low temperature stress test that the water temperature was decreased from 26 ℃ to 14 ℃ at the rate of 1 ℃/12 h and kept at 14 ℃ for 288 h (12 d). The brain tissues were collected at 26 ℃, 20 ℃ and 14 ℃ for 0, 120 and 288 h during this period, respectively, and were dissected. The prepared cDNA libraries were sequenced using Illumina HiSeq×Ten platform. A comparative transcriptome analysis was carried out to identify differentially expressed genes (DEGs) between the stressed and control tissues. Expression of the critical genes involved in the responses to low temperature stress were verified with a qRT-PCR analysis. 【Result】Death of wild tilapia individuals initially occurred at 11 ℃ and still 50.0% of them were alive at 8 ℃, indicating that the wild tilapia had a better tolerance to low temperature than the cultured tilapia.During the long-term low temperature stress at 14 ℃, DEGs expression in cultured tilapia brain tissue was about 10 times higher than that in wild tilapia, indicating that the stress response of cultured tilapia was much stronger. KEGG pathway enrichment analysis results showed that the up-regulated DEGs of cultured GIFT tilapia and wild tilapia maintained at 14 ℃ for 120 and 288 h were both enriched in ribosome formation, endoplasmic reticulum protein processing and spliceosome signaling pathways. The up-regulated DEGs of wild tilapia were additionally enriched in nucleotide excision repair, N-glycosylation biosynthesis and DNA replication pathways. Compared with the cultured Tilapia, there were 577 unique DEGs in wild Tilapia, which were mainly enriched in the NOD receptor signaling pathway, apoptosis, endocytosis and other pathways. The NOD receptor signaling pathway was activated, and apoptosis was inhibited. In wild tilapia, the key functional genes involved in Nod-like receptor signal pathway, such as Nemo, NFκB, P38, JNK and IL-1β, maintained a high expression under the long-term low temperature stress. Meanwhile,the key genes employed in the endocytosis pathway had the much higher expression than the cultured tilapia. 【Conclusion】Wild tilapia has stronger cold tolerance than cultured tilapia by avoiding excessive stress response and maintaining low-level metabolic cell stability to alleviate the damage of low temperature stress on the body, as well as continuing to activate NOD receptor signaling pathways and endocytosis pathways to maintain their immunity.D702AD90-53E9-41CC-98A9-65C771A49FD3
Key words: tilapia; brain tissue; low temperature stress; DEGs; transcriptome sequencing
Foundation items:National Key Research and Development Program of China“Technological Innovation of Blue Granary”(2018YFD0900101, 2018YFD0900601); Guangxi Freshwater Fish Industry Innovation Team of National Mo-dern Agricultural Industry Technology System(nycytxgxcxtd-2021-08-03)
0 引言
【研究意义】罗非鱼隶属于慈鲷科(Cichlidae)罗非鱼属(Oreochromis),是全球重要的经济养殖鱼类,具有繁殖能力强、生长速度快、抗病力强及食性杂等优点(郑雄等,2019;李柳清等,2020)。罗非鱼起源于非洲的热带亚热带地区,属于暖水性鱼类,在越冬期间会增加能量损耗,导致其免疫力下降甚至死亡(Abdel-Ghany et al.,2021),制约着其养殖区域的向北拓展。野生罗非鱼较养殖罗非鱼具有更强的低温耐受能力,因此开展野生罗非鱼低温应激分子机制研究,有助于筛选具有较强耐低温能力的野生品系作为杂交育种亲本,以拓展罗非鱼养殖区域。【前人研究进展】寒冷是水生环境中鱼类的主要应激源。近年来,已有学者运用转录组测序及其他组学研究方法探究冷应激反应下不同鱼类的基因表达模式(Mininni et al.,2014;Qian and Xue,2016)。Long等(2012)对斑马鱼(Danio rerio)幼虫的研究发现,在冷应激条件下显著差异表达的基因主要参与RNA剪接、核糖体生物合成和蛋白质分解等通路。Xu等(2018)在对冷应激存活黄姑鱼(Nibea albiflora)的研究中发现,其脑和肌肉组织中参与分子—分子相互作用、信号转导、碳水化合物代谢、脂质代谢、消化系统及内分泌系统等通路的基因表达均显著上调,可能与应激信号转导、能量代谢和应激诱导的细胞膜变化等生理过程相关。Sun等(2019)研究表明,在受低温胁迫的石斑鱼(Epinephelus coioides)肝脏转录组中,其应激反应涉及细胞黏附分子、PPAR信号通路及脂肪酸延长等途径。Wen等(2019)通过对低温胁迫后的暗纹东方鲀(Takifugu fasciatus)肝脏组织进行多组学分析,建立的mRNA—蛋白质—代谢物相互作用网络证实分子互作网络主要参与脂肪酸代谢、膜转运、信号转导及DNA损伤和修补等代谢过程。Liu等(2020)通过对低温胁迫8月龄虎皮鱼的脑、鳃、肝脏和肌肉组织进行转录组测序,发现参与昼夜节律、类固醇和脂肪酸生物合成等途径的基因在低温胁迫下其表达模式均发生改变,并证实泛素介导的蛋白质降解可能是虎皮鱼应对急性冷应激过程的关键。针对罗非鱼而言,不同罗非鱼品种的耐低温能力也存在明显差异,其致死温度范围在8.9~10.5 ℃(唐章生等,2012)。在罗非鱼耐受低温应激的分子调控机制研究中,Chen等(2002)研究认为免疫相关基因对罗非鱼的耐寒能力存在影响,当罗非鱼处于12.0 ℃时其血浆肾上腺素增加,血清皮质醇水平显著升高,血清中的高皮质醇水平对鱼体免疫产生抑制作用;Ammar等(2018)对尼罗罗非鱼在夏季和冬季的基因表达差异进行分析,发现冬季IV型防冻基因在其肠道、鳃、皮肤、脾脏、肝脏和脑组织中的表达量明显上调;Nitzan等(2019)对冷应激下的奥利亚罗非鱼鳃组织和肝脏进行转录组测序,发现基因在局部黏附和细胞—细胞外基质(ECM)相互作用通路中的表达下调,而在蛋白酶体及各种细胞内蛋白水解活性信号通路中的表达上调。可见,罗非鱼的低温应答过程主要表现在免疫、节律、信号传导、DNA修补和基础物质合成或降解等相关代谢途径的基因启动,且主要发生在免疫器官、消化器官及中枢控制器官——脑。脑是调控低温胁迫下应激反应的关键器官(Song and McDowell,2020),机体对于冷刺激的耐受机制是在神经系统的主导下进行。【本研究切入点】选择合适的亲本,采用临界非损伤性的温度适应选育方法有可能改善子代罗非鱼耐低温能力(肖俊等,2014;杜雪松等,2019),但从育种角度而言迫切需要耐低温性状能稳定遗传的亲本,并将其耐低温性状引入现有的优良家系中。野生罗非鱼是耐寒研究和育种实践的良好材料,开展野生罗非鱼低温应激反应的分子生物学研究对加速罗非鱼抗寒性育种进程具有重要意义。【拟解决的关键问题】选择野生罗非鱼进行低温胁迫试验,并以养殖的吉富罗非鱼为对照,通过Illumina HiSeq×Ten测序平台对罗非鱼的脑组织进行转录组测序,比较低温胁迫下2种罗非鱼的基因表达模式,旨在揭示野生罗非鱼低温应答的特有分子调控机制,为后续开展野生罗非鱼耐冷亲本大范围筛选打下基础。
1 材料与方法
1. 1 降温试验及样品采集
成年野生罗非鱼采自广西崇左市扶绥县左江流域(东经107°54′14.76″,北纬22°38′2.76″),选用规格相近的养殖吉富罗非鱼为对照(唐永凯等,2010)。随机挑选野生罗非鱼和吉富罗非鱼各30尾,26 ℃下暂养60 d后进行降温预试验。参考Healy等(2017)、Giordano等(2021)的降温试验方法,使水温先以1 ℃/12 h的速度从26 ℃降至8 ℃,降温过程中每2 h记录1次水温及罗非鱼死亡数量。正式取样时,使水温先以1 ℃/12 h的速度从26 ℃降至14 ℃,并保持288 h(12 d),于26 ℃、20 ℃及14 ℃保持0、120和288 h等5个时间点分别解剖罗非鱼采集脑組织样品,各时间点均取2尾罗非鱼作为平行重复。按取样水温和时间,吉富罗非鱼样品分别命名为G26、G20、G14H0、G14H120和G14H288,野生罗非鱼样品分别命名为W26、W20、W14H0、W14H120和W14H288。采集的脑组织立即冻存于液氮中备用。D702AD90-53E9-41CC-98A9-65C771A49FD3
1. 2 RNA提取与测序
采用TRIzol试剂提取罗非鱼脑组织总RNA,在安捷伦生物分析仪芯片RNA 7500 Series II上检测其质量和浓度。使用TruSeq RNA样品制备试剂盒(Illumina)从每个样品中提取3 μg总RNA,转录合成cDNA后构建平均插入片段大小为300~500 bp的测序文库,然后以Illumina HiSeq×Ten测序平台进行双端测序。
1. 3 转录组测序数据处理与分析
使用Trimomatic v0.32对测序获得的原始序列(Raw reads)进行检测和过滤(Bolger et al.,2014),过滤掉低质量序列及接头序列后得到有效序列(Clean reads)。通过HISAT v2.1.0将Clean reads比对到尼罗罗非鱼参考基因组(GenBank登录号GCA_000002035.4)上(Kim et al.,2019;Tao et al.,2021),再使用Cufflinks 2.2.1计算基因表达量(FPKM)(Trapnell et al.,2012),以R语言软件包edgeR筛选差异表达基因(Differentially expressed genes,DEGs)(Chen et al.,2016),筛选条件:[log2 Fold Change]≥1和P<0.05。通过KEGG数据库对差异表达基因进行功能注释及信号通路分析(Kanehisa et al.,2016),并用R语言软件包ggplot2对结果进行可视化处理(Ginestet,2011)。
1. 4 实时荧光定量PCR验证
采用实时荧光定量PCR对NOD受体、内吞及凋亡等信号通路上的17个差异表达基因进行表达验证。使用反转录试剂盒(TaKaRa)将各样品的RNA反轉录合成cDNA,以β-actin基因为内参基因,使用FastStart Universal SYBR Green Master(ROX)试剂在CFX96 Real-Time PCR Detection System定量PCR仪(Bio-Rad)上进行实时荧光定量PCR检测。每个样品测定3个平行,采用2-△△Ct法计算目的基因相对表达量。
2 结果与分析
2. 1 罗非鱼低温存活率测定结果
在低温预试验中,统计梯度降温过程中各时间点养殖吉富罗非鱼和野生罗非鱼的存活数量,计算存活率。结果显示,养殖吉富罗非鱼在水温降至14 ℃时开始出现个体失去平衡的现象,在水温降至11 ℃时出现个体死亡,并于8 ℃时全部死亡(图1)。相比之下,野生罗非鱼虽然在14 ℃时出现失去平衡的个体,并于11 ℃时开始出现死亡个体,但在8 ℃时仍有50.0%的存活个体,说明野生罗非鱼较养殖吉富罗非鱼具有更强的低温耐受能力。
2. 2 罗非鱼脑组织转录组测序结果
对养殖吉富罗非鱼和野生罗非鱼5个时间点的脑组织共20个样本进行转录组测序,经质控后共获得141.31 Gb的Clean reads。测序碱基达Q30(测序碱基准确率达99.9%)的比例超过92.0%(表1),即测序准确率较高。将各样品的Clean reads比对到尼罗罗非鱼参考基因组上,其比对率均在69.5%以上,最高为94.2%,满足后续分析的要求。
2. 3 低温胁迫下罗非鱼脑组织差异表达基因功能注释分析结果
为认定野生罗非鱼的种属关系,将26 ℃的野生型罗非鱼转录组测序数据比对到7种罗非鱼[奥利亚罗非鱼(NC_013750.1)、红罗非鱼(NC_014060.1)、布氏异罗非鱼(NC_023470.1)、莫桑比克罗非鱼(AY597335.1)、 尼罗罗非鱼(GU477624.1)、齐氏罗非鱼(KM658974.1)和维多利亚绿罗非鱼(NC_026109.1)]的线粒体基因组上,结果发现比对到红罗非鱼的数目最多,故推测该野生罗非鱼与红罗非鱼的亲缘关系最为接近。对G14H0 vs G26、G14H120 vs G26、G14H288 vs G26、W14H0 vs W26、W14H120 vs W26和W14H288 vs W26等6对样品的基因表达量进行比较分析,鉴定出各组差异表达基因中的上调差异表达基因如图1-D所示。其中,野生罗非鱼W14H288和W14H120共有的上调差异表达基因数量为162个,而养殖吉富罗非鱼G14H288和G14H120共有的上调差异表达基因数量为1686个,约是野生罗非鱼的10倍。野生罗非鱼W14H288特有的上调差异表达基因是200个,而养殖吉富罗非鱼G14H288特有的上调差异表达基因为2909个,也是野生罗非鱼的10倍以上,说明长期处于14 ℃下养殖吉富罗非鱼的应激反应远比野生罗非鱼强烈。
KEGG信号通路富集分析结果(图2)显示,养殖吉富罗非鱼和野生罗非鱼在14 ℃保持120和288 h的上调差异表达基因均富集到核糖体发生、内质网蛋白加工及剪接体信号等信号通路上,可能与应激状态下机体利用这些蛋白作为应激状态的供能物质有关。富集到昼夜节律信号通路上与本研究观察到低温胁迫下罗非鱼运动和摄食节奏急剧变慢的现象相符(Li et al.,2020;Zou et al.,2021);富集到蛋白酶体等信号通路上,则可能与清除细胞内冷应激下产生的异常蛋白有关(Yu et al.,2021)。与养殖吉富罗非鱼相比,野生罗非鱼的上调差异表达基因还额外富集到核苷酸切除修复、N-聚糖生物合成和DNA复制信号等通路上,可能参与冷应激过程中的损伤修复(Huang et al.,2021)。养殖吉富罗非鱼的这些差异表达基因则富集到自噬、线粒体自噬、细胞凋亡、泛素介导蛋白水解及赖氨酸降解等信号通路上,可能与长期低温胁迫下能量利用途径的改变有关,并激活自噬和细胞凋亡途径,将细胞内一些不重要的蛋白及细胞器分解以维持细胞的基本存活。可见,野生罗非鱼在长期低温胁迫下主要通过避免过度的应激反应、及时启动修复和减弱细胞调亡等机制,以获得较养殖罗非鱼更强的耐低温能力。D702AD90-53E9-41CC-98A9-65C771A49FD3
2. 4 低温胁迫下野生罗非鱼的基因表达模式
为了解野生罗非鱼特有的低温应激机制,对14 ℃胁迫下仅在野生罗非鱼中上调的差异表达基因进行分析。在计算得到野生罗非鱼在14 ℃下各时间点上调差异表达基因(W14H0 vs W26、W14H120 vs W26和W14H288 vs W26)的基础上,将这些差异表达基因中在相同时间点的养殖吉富罗非鱼脑组织也发生显著上调(G14H0 vs G26、G14H120 vs G26和G14H288 vs G26)的基因舍去,即去除养殖吉富和野生罗非鱼在相同时间点共有的上调差异表达基因后,得到在对应时间点仅在野生罗非鱼中上调的差异表达基因。通过筛选共得到这3个时间点仅在野生罗非鱼中发生表达变化的差异表达基因有577个,且主要富集在NOD受体信号通路(Nod-like receptor signal pathway,map04621)、凋亡(Apoptosis,map 04210)和内吞(Endocytosis,map04144)等通路上。对这些差异表达基因所参与的生物反应过程进行深入分析,发现在野生罗非鱼中存在NOD受体信号通路被启动而细胞凋亡受抑制的平衡(图3)。
结合参与这3条信号通路上关键差异表达基因的实时荧光定量PCR验证结果(图4),发现在NOD受体信号通路上,野生罗非鱼的B细胞编码κ轻链多肽抑制基因(Nemo)显著上调,并激活下游的核转录因子kappa B1(NFκB)、丝裂原活化蛋白激酶(p38)和C-Jun氨基末端激酶(JNK)等,促进炎症相关因子白细胞介素-1β(IL-1β)表达上升,进而启动天然免疫和炎症反应(Elabd et al.,2020),且这些基因在长期低温胁迫中均维持在一个较高的表达水平。在该过程中,养殖吉富罗非鱼的这些基因几乎没有参与全程的冷应激反应,尤其是NOD受体信号通路上的Nemo基因一直未被启动;而参与这一途径的其他基因中只观察到p38和IL-1β在个别时间点因未知原因突然剧烈升高,但随后又迅速恢复至原有水平。在细胞调亡途径中,重要抑制因子颗粒酶B(GZMB)在野生罗非魚呈显著上升,而抑制细胞调亡的发展(Yang et al.,2021)。此外,野生罗非鱼的蛋白酪氨酸磷酸酶非受体型13(ptpn13)和细胞凋亡相关半胱氨酸肽酶2(casp2)等基因参与细胞调亡调控表达量上升的倍数明显小于养殖吉富罗非鱼,细胞调亡可能在一定程度上受到抑制,而有利于避免过度应激的发生。在内吞途径中,大部分野生罗非鱼关键基因表达上升的倍数也明显高于养殖吉富罗非鱼,对及时清理细胞内的不良物质及维持细胞稳定均有利。故推测野生罗非鱼较养殖吉富罗非鱼具有更强的耐低温能力是通过避免过度应激反应及维持低水平代谢的细胞稳定来实现。
3 讨论
水温对鱼类的生长、繁殖、代谢、生理功能和行为活动均产生重要影响(Somero,2010;Ma et al.,2014;Yang and Ma,2016)。虽然鱼类可通过广泛的调节代谢及生理生化适应过程应对一定范围内的水温变化,但不适当的环境温度仍然会产生负面影响(陈子桂等,2018;Xu et al.,2018)。冷应激下,机体的稳态调节是在神经系统主导下,由下丘脑—腺垂体—甲状腺系统(HPT)、下丘脑—腺垂体—肾上腺皮质系统(HPA)和机体交感—肾上腺髓质系统(SAM)配合相关激素的作用共同实现,其原理较复杂(Oyola and Handa,2017)。脑组织对冷应激敏感,已有研究表明热带鱼的脑组织较冷水鱼的脑组织对热刺激更具稳定性,但对冷刺激不稳定(Lu et al.,2019)。在低温应激后,金鱼出现躲避游泳的现象是由一种存在鱼类后脑组织中的神经元细胞参与调控进而激活听觉传入所引起(Silva et al.,2019)。在鲤鱼中,低温会减少脑组织的血容量,防止低温血液进入大脑而影响感觉运动神经的活性(van den Burg et al.,2005)。可见,脑是鱼类冷敏感检测的理想组织。
现有关于罗非鱼对低温应答机制的研究主要是通过长时间的低温胁迫及观察基因表达变化来实现。Nitzan等(2019)对具有相同遗传背景的吉富罗非鱼进行降温试验(从24 ℃降温至12 ℃并保持),根据存活时间将吉富罗非鱼分为冷耐受鱼和冷敏感鱼,发现冷敏感鱼的差异表达基因数量多于冷耐受鱼。本研究中,冷敏感的养殖吉富罗非鱼在14 ℃保持120和288 h时表达显著上调的基因数量远多于低温耐受能力较强的野生罗非鱼,仅在冷应激早期(14 ℃保持0 h)对冷应激的响应基因稍多一些,说明野生罗非鱼对低温环境的应答启动更迅速,但在长期冷应激反应过程中对低温更敏感的养殖吉富罗非鱼中具有温度依赖性表达的基因远多于野生罗非鱼。从上调表达基因的功能注释结果来看,Nitzan等(2019)发现鳃组织中糖酵解、糖异生及肝脏中氨基酸生物合成通路上的某些基因在耐冷鱼类中呈下调表达。本研究发现在野生罗非鱼的上调差异表达基因主要富集到核苷酸切除修复和N-糖基化的生物合成等通路上,而在能量代谢途径基本上呈下调趋势,表明能量代谢的减弱有利于鱼类应对寒冷环境。
根据已有关于低温胁迫对罗非鱼免疫系统的影响研究可知,长时间冷应激反应会直接导致其免疫力下降(Ndong et al.,2007),引起血液中儿茶酚胺、皮质醇、肾上腺素、去甲肾上腺素和肾上腺素激的水平上升,从而抑制白细胞的吞噬作用,降低抗体水平(Chen et al.,2002)。此外,SGT1 和 ATG1等免疫相关基因在低温胁迫下呈下调表达的现象可能会影响过氧化物酶体、吞噬体和自噬的调节(Yang et al.,2015)。这是由于过度应激反应引发的免疫能力下降,极大增加了罗非鱼在低温环境下的病原体感染风险。本研究发现,在低温胁迫下野生罗非鱼的应激反应明显弱于养殖吉富罗非鱼,可能促使免疫反应和细胞凋亡在一定程度上受到抑制,避免对机体的损伤。然而,在对免疫相关基因的分析中发现由p38、JNK、IL-1β、ptpn13和casp2等关键功能基因共同参与的NOD受体信号通路在长期低温胁迫野生罗非鱼脑组织中始终保持着较高的水平,暗示着NOD受体信号通路可能在野生罗非鱼低温应答中发挥关键作用。在NOD受体信号通路上,Nemo基因显著上调,再激活下游的NFκB、p38和JNK基因等,促进炎症相关因子IL-1β表达上升,进而启动天然免疫和炎症反应(Velmurugan et al.,2019;Elabd et al.,2020),可能对维持野生罗非鱼的生存具有重要意义。就低温诱导的细胞凋亡或程序性死亡而言,GZMB是细胞凋亡过程中起关键作用的生物酶,其表达水平能反映细胞凋亡的程度。本研究结果表明,野生罗非鱼可能通过激活GZMB基因,抑制核纤层蛋白,达到间接减弱因核膜完整性丧失而导致的细胞凋亡,从而表现出更强的耐寒性。因此,控制免疫过度反应和细胞调亡程度,且又保持局部的免疫能力,可能是野生罗非鱼具有较强耐低温能力的原因之一。D702AD90-53E9-41CC-98A9-65C771A49FD3
来自野生群体的优良性状,始终是良种选育的重要性状来源 ,而携带有这些优良性状的野生家系是杂交育种的重要亲本材料(Li et al.,2021)。本研究对养殖吉富罗非鱼和野生罗非鱼进行耐寒能力测定,比较分析二者在低温致死极限下的差异,并通过转录组测序分析揭示野生罗非鱼低温胁迫下细胞凋亡、内吞和NOD受体等信号通路相关基因的表达模式,明确了导致罗非鱼不同低温耐受能力的分子调控机制,为今后筛选耐寒能力强的野生罗非鱼品种打下基础。
4 结论
野生罗非鱼通过避免过度应激反应和维持低水平代谢的细胞稳定以减轻低温胁迫对机体的损伤,并持续启动NOD受体信号通路及内吞途径以维持其免疫能力,即野生罗非鱼较养殖罗非鱼具有更强的耐受低温能力。
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