【作者】 胡桂林；

【导师】 陈卓； 陈晓虹；

【作者基本信息】 河南师范大学 ， 动物学， 2021， 硕士

【摘要】 约3.5亿年前,两栖动物的祖先由水登陆,并逐渐进化出一系列适应陆生生境的改变和特征,因此,两栖动物是脊椎动物进化史上具有特殊和十分重要意义的一个类群,受到进化生物学家和生态学家广泛关注。两栖动物在起源和分化过程中,面临着复杂多变的生态环境,而两栖类皮肤裸露、缺乏皮肤角质层的保护,因此,免疫适应是两栖动物适应辐射过程中面对环境病原微生物变化首先需要解决的关键问题之一。研究表明两栖动物获得性免疫系统进化尚不完善,先天性免疫系统在其适应复杂多变的栖息地环境过程中发挥着重要作用。作为先天性免疫系统的关键部分,抗菌肽被认为具有广谱抗菌活性,在两栖动物防御外界病原微生物和外界刺激中发挥重要作用。Cathelicidins家族是一类重要的抗菌肽,然而,目前我们对两栖动物cathelicidins抗菌肽家族的组成、结构、进化模式知之甚少。本研究首先以中国林蛙(Rana chensinensis)为研究对象,通过c DNA末端快速扩增技术(RACE)获得了中国林蛙cathelicidin-AL和cathelicidin-RC2抗菌肽基因的全长c DNA序列,基于序列分析、蛋白质预测及系统发育重建等生物信息学分析揭示了这两个抗菌肽基因所编码的氨基酸组成、结构域、蛋白质理化性质及进化历史。此外,通过实时荧光定量PCR实验及分析揭示了中国林蛙cathelicidin-AL和cathelicidin-RC2抗菌肽基因的时空表达模式。最后,基于本研究扩增的这两个抗菌肽基因设计了多对引物,进一步通过RACE技术克隆获得了不同生境无尾两栖类代表物种的cathelicidin-AL和cathelicidin-RC2抗菌肽基因的c DNA序列,利用贝叶斯和最大似然法分别构建了脊椎动物和两栖类cathelicidins系统发育树,并通过PAML软件分析揭示了两栖类cathelicidin-AL和cathelicidin-RC2的进化模式。结果表明:(1)首次在中国林蛙中克隆获得了cathelicidin-AL和cathelicidin-RC2基因c DNA全长编码区序列。中国林蛙cathelicidin-AL基因的c DNA序列全长为677bp,CDS长度为561bp,编码186个氨基酸,中国林蛙cathelicidin-RC2基因c DNA序列全长为779bp,CDS共有480bp,编码159个氨基酸。中国林蛙cathelicidin-AL以V132为酶切位点,成熟肽由54个氨基酸组成;cathelicidin-RC2以K123为酶切位点,成熟肽由36个氨基酸组成。其它无尾两栖类cathelicidin-AL和cathelicidin-RC2的CDS在物种间存在长度差异,cathelicidin-AL的CDS长度范围在543-552bp,cathelicidin-RC2长度范围在468-501bp。无尾两栖类cathelicidin-AL在C80-C92、C103-120形成两个二硫键;cathelicidin-RC2虽然在C75-C86、C97-C108具有4个半胱氨酸,但只形成一个二硫键。(2)组织分布表达显示cathelicidin-AL和cathelicidin-RC2基因在中国林蛙肺、皮肤、胃、肾、脾脏、脑和肝脏等组织均表达,但在心脏未见表达。cathelicidin-AL在肝脏中表达量最高,其次是脑和脾脏,而cathelicidin-RC2在脑中表达量最高,其次是肝脏和脾脏。cathelicidin-AL和cathelicidin-RC2基因在肺和皮肤中表达量均不高。(3)脊椎动物系统发育重建表明cathelicidin-AL比cathelicidin-RC2先起源。基于已知的cathelicidin系统发育树结果,我们发现中国林蛙cathelicidin-AL置于系统发育关系的基部,而中国林蛙cathelicidin-RC2与爬行类、鸟类和哺乳类聚为一支。此外,已知两栖类cathelicidins系统发育关系结果表明cathelicidin-RC2和cathelicidin-AL分别聚为一支,支持cathelicidin-AL先分化。(4)无尾两栖类cathelicidin-AL和cathelicidin-RC2选择压力分析表明free ratio model优于one ratio model,显示不同支系受选择压力不同。Branch specific model分析表明cathelicidin-AL在臭蛙属祖先支、武夷湍蛙祖先支、黑斑侧褶蛙祖先支、中华蟾蜍祖先支受到了正选择作用;cathelicidin-RC2在凹耳臭蛙、中国林蛙、黑斑侧褶蛙、树蛙科和叉舌蛙科祖先支中均受到强烈正选择作用。branch site model进一步在凹耳臭蛙支系检测到一个正选择位点(138V)。Cathelicidins基因本身由于末端成熟肽变异程度大,结合cathelicidin-AL和cathelicidin-RC2在无尾两栖类不同支系中经历了正选择作用,表明无尾两栖类cathelicidin-AL和cathelicidin-RC2在两栖类适应不同生境过程中发挥重要作用。更多还原

【Abstract】 Approximately 3.5 hundred millions years ago,the ancestors of amphibians began to transition from water to land and evolved a series of changes and characteristics adapted to the terrestrial habitat.Thus,the amphibians are special and siginificant groups in the evolutionary history of vertebrate,and they have attracted widely interests of the evolutionarists and ecologists.Due to the complex and changable ecologicial environmentsduring the origin and diversification of the amphibians as well asthe uncovered skin and lack of stratum corneum,immune adaptation was the first question for the amphibians to adapt to the pathogens changes they faced.Previous analyses suggested the acquired immune system evolved not well in amphibians and the innate immune system plays significant roles during their adaptation to the complex habitats.Antibacterial peptides,the key components of the innate immune system,was regarded to have broad spectrum antibacterial activity and plays key roles during their response to the pathogens and the exposure for amphibians.The Cathelicidins are one of the important antibacterial peptide,however,the composition,structure and evolutionary patterns of cathelicidins in amphibians are still unknown to date.In this study,the c DNA sequences of cathelicidin-AL and cathelicidin-RC2 were cloned and sequenced in Rana chensiensis by RACE.The analysis of sequence alignment,protein prediction and phylogeny reconstruction and other bioinformatic analyses were conducted to reveal the amino acids,structure domain,the proteincharacteristics and the evolutionary history.Furthermore,real-time fluorogenic quantitative PCR was used to determine the expression patterns of cathelicidin-AL and cathelicidin-RC2 in R.chensinensis.Finally,a series of primers were designed based on the newly sequences obtained in this study,and the cathelicidin-AL and cathelicidin-RC2 were sequenced in anuran representatives of different habitats.The tree of cathelicidin across vertebrate and amphibians were reconstructed based on BI and ML analysis.The PAML was also used to test the evolutionary patterns of cathelicidin-AL and cathelicidin-RC2 in amphibians.The following are the results of this study:(1)The c DNA sequence of cathelicidin-AL and cathelicidin-RC2 were first cloned and sequenced in R.chensiensis.The length of the c DNA of cathelicidin-ALin R.chensiensisis 677 bp,and the CDS is 561 bp which encodeda protein of 186 amino acids.The c DNA of cathelicidin-RC2 in R.chensiensisis 779 bp in length,and the CDS is 480 bp encoding a protein of 159 amino acids.The site V132 was used as the restriction site in cathelicidin-ALfor R.chensiensis,and the mature peptide is consited with 54 amino acids.The site K123 was found to be the the restriction site in cathelicidin-RC2,and it encoded a mature peptide of 36 amino acid.The length of both CDS of cathelicidin-AL and cathelicidin-RC2 in other anurans differed from species,the lengths of cathelicidin-AL ranged 543-552 bp,and that of cathelicidin-RC2 ranged468-501 bp.Two disulfide bonds were found at C80-C92 and C103-120 in cathelicidin-AL,whereas only one disulfide bond was found in cathelicidin-RC2 although the existence of 4 cysteines at C75-C86 and C97-C108.(2)The tissue distribution and expression analysis in R.chensinensis show that both cathelicidin-AL and cathelicidin-RC2 expressed in lung,skin,stomach,kidney,spleen,brain,and liver,but not in heart.The highest expression of cathelicidin-AL was in liver and followed by brain and spleen,whereas it reached the highest level in brain for cathelicidin-RC2 and followed by liver and spleen.The level of expression of both cathelicidin-AL and cathelicidin-RC2 in lung and skin was low.(3)The phylogenetic renconstructionof representative vertebrates suggested the earlier origin of cathelicidin-AL than cathelicidin-RC2.According to the phylogeny of reprsentative known cathelicidins,the basal postion of cathelicidin-AL of R.chensinensis was revealed,where as the cathelicidin-RC2 have a close relationship with reptiles,birds and mammals and they togther foremed a branch.In addition,representatives of cathelicidin-AL and cathelicidin-RC2 formed a group,respectively,according to the phylogeny of the known amphibian cathelicidins,and it also revealed the similar results that cathelicidin-AL appeared earlier.(4)The selectivepressure analysis suggested free ratio model of cathelicidin-AL/-RC2 is better than one ratio model,and it indicated the slective pressure was different among different lineages.Positively selection was detected along the ancestral branch of the genus Odorrana,Amolops wuyiensis,Pelophylax nigromaculatus,Bufo gargarizans in cathelicidin-AL based on the branch specific model.Significant positively selection was also detected along the branch of O.tormota,R.chensinensis,P.nigromaculatus,the common ancestor of Rhacophoridae and Dicroglossidae in cathelicidin-RC2.Interestingly,the branch site model also detected one positively selected sites(138V)in O.tormota.Due to the variable mature peptide of the end of cathelicidins,and different slective pressure detected along different branches in cathelicidin-AL and cathelicidin-RC2,they might play important roles during the adaptation to different habitats for amphibians.更多还原