俄罗斯鲟补体C7基因的克隆及表达分析
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摘要
为研究俄罗斯鲟(Acipenser gueldenstaedti)补体C7基因(AgC7)的功能,采用RACE (Rapid-amplification of cDNA ends)技术,获得AgC7的cDNA全长序列为3103 bp,包括开放阅读框(Open Read Frame, ORF) 2502 bp,编码833个氨基酸, 5′-UTR (5′untranslated region)和3′-UTR的长度分别为44和554 bp。同源性分析表明, AgC7与其他鱼类补体C7如斑点雀鳝(Lepisosteus oculatus)、斑点叉尾鲴(Ictalurus punctatus)、斑马鱼(Danio rerio)、大西洋鲑(Salmo salar)、尼罗罗非鱼(Oreochromis niloticus)氨基酸序列的一致性分别为56%、46%、49%、49%、47%,且都具有TSP1、LDLa、FIMAC和MACPF保守结构域。荧光定量qRT-PCR (quantitative Realtime PCR)结果表明, AgC7在血液、脑、鳃、性腺、心脏、头肾、肠、肝、肌肉、皮肤、脾、胃和后肾组织中均可表达,且在肠中表达水平最高;用含有壳寡糖的饲料饲喂俄罗斯鲟60d后, AgC7在这13种组织中表达均有增加,在肠中增加量最高,约为对照组的1.51倍;嗜水气单胞菌(Aeromonas hydrophila)可诱导AgC7,在血液、鳃、头肾、肠、肝和脾6种组织中瞬时上调表达,随着病原菌感染时间增加,基因表达量逐渐降低恢复至正常水平,其中,鳃中基因表达量的上调趋势最明显,最大表达量出现在感染后6h,为对照组的41.30倍, 12h后基因表达下降并恢复至正常水平,表明AgC7基因可能参与了俄罗斯鲟抗细菌感染的免疫应答。俄罗斯鲟AgC7具有典型的补体C7基因特征,且壳寡糖刺激和病原感染后均可引起AgC7基因表达变化,补体C7可能参与了俄罗斯鲟的免疫应答。
This study cloned the full-length of complement C7 cDNA by RACE(rapid-amplification of cDNA ends)from Russian sturgeon(Acipenser gueldenstaedti). It contained 3103 bp, including a 2502 bp open reading frame(ORF) encoding 833 dedueced amino acids, a 44 bp 5′-UTR and a 554 bp 3′-UTR. Multiple sequence alignment revealed that AgC7 proteins were well conserved with the typical structural motifs such as TSP1, LDLa, FIMAC,MACPF and had the sequence identity with Lepisosteus oculatus(56%), Ictalurus punctatus(46%), Danio rerio(49%),Salmo salar(49%) and Oreochromis niloticus(47%), respectively. AgC7 gene is constitutively expressed in all evaluated tissues with the highest expression in intestine and the weakest level in muscle. Chitosan oligosaccharide induced the expression of AgC7 gene to a certain degrees in all tissues with the highest increase in intestine(1.51 times). After infection with Aeromonas hydrophila, AgC7 gene exhibited distinct time-dependent response patterns in tested tissues with most obvious induction in gills and the maximum expression at 6 h after infection(41.30 times). The expression of AgC7 gene decreased rapidly and returned to normal level after 12 h. These results suggest that C7 of Russian sturgeon might play an important role in the immune defenses against bacterial infection.
This study cloned the full-length of complement C7 cDNA by RACE(rapid-amplification of cDNA ends)from Russian sturgeon(Acipenser gueldenstaedti). It contained 3103 bp, including a 2502 bp open reading frame(ORF) encoding 833 dedueced amino acids, a 44 bp 5′-UTR and a 554 bp 3′-UTR. Multiple sequence alignment revealed that AgC7 proteins were well conserved with the typical structural motifs such as TSP1, LDLa, FIMAC,MACPF and had the sequence identity with Lepisosteus oculatus(56%), Ictalurus punctatus(46%), Danio rerio(49%),Salmo salar(49%) and Oreochromis niloticus(47%), respectively. AgC7 gene is constitutively expressed in all evaluated tissues with the highest expression in intestine and the weakest level in muscle. Chitosan oligosaccharide induced the expression of AgC7 gene to a certain degrees in all tissues with the highest increase in intestine(1.51 times). After infection with Aeromonas hydrophila, AgC7 gene exhibited distinct time-dependent response patterns in tested tissues with most obvious induction in gills and the maximum expression at 6 h after infection(41.30 times). The expression of AgC7 gene decreased rapidly and returned to normal level after 12 h. These results suggest that C7 of Russian sturgeon might play an important role in the immune defenses against bacterial infection.
引文
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[18]Shen Y B,Zhang J B,Xu X Y,et al.Molecular cloning,characterization and expression analysis of the complement component C6 gene in grass carp[J].Veterinary Immunology and Immunopathology,2011,141(1-2):139-143
[19]Zarkadis I K,Duraj S,Chondrou M.Molecular cloning of the seventh component of complement in rainbow trout[J].Developmental&Comparative Immunology,2005,29(2):95-102
[20]Jiang J J,Li Y W,Chen D Q,et al.Molecular Cloning,expression and genetic diversity analysis of the seventh component of the complement in Hypophthalmichthys molitrix[J].Journal of Fishery Sciences of China,2012,19(6):994-1000[蒋菁菁,李英文,陈大庆,等.鲢补体C7基因的克隆、表达和序列分析.中国水产科学,2012,19(6):994-1000]
[21]Wang S,Gao Y,Shu C,et al.Characterization and evolutionary analysis of duplicated C7 in miiuy croaker[J].Fish&Shellfish Immunology,2015,45(2):672-679
[22]Papanastasiou AD,Zarkadis IK.Gene duplication of the seventh component of complement in rainbow trout[J].Immunogenetics,2005,(57):703-708
[23]Ye X F,Zhang S R,Li X,et al.Research progress on effects and mechanisms of immunological regulation of chitosan oligosaccharide[J].Feed Review,2014,(7):34-37[叶小飞,张石蕊,李霞,等.壳寡糖的免疫调节作用及机理研究进展.饲料博览,2014,(7):34-37]
[24]Huang R L,Deng Z Y,Yang C B,et al.Dietary oligochitosan supplementation enhances immune status of broilers[J].Journal of the Science of Food&Agriculture,2007,87(1):153-159
[25]Dou J,Xu Q,Tan C,et al.Effects of chitosan oligosaccharides on neutrophils from glycogen-induced peritonitis mice model[J].Carbohydrate Polymers,2009,75(1):119-124
[26]Wang J P,Yoo J S,Kim H J,et al.Nutrient digestibility,blood profiles and fecal microbiota are influenced by chitooligosaccharide supplementation of growing pigs[J].Livestock Science,2009,125(2):298-303
[2]Godahewa G I,Bathige S D,Herath H M,et al.Characterization of rock bream(Oplegnathus fasciatus)complement components C1r and C1s in terms of molecular aspects,genomic modulation,and immune responsive transcriptional profiles following bacterial and viral pathogen exposure[J].Fish&Shellfish Immunology,2015,46(2):656-668
[3]Berends ETM,Mohan S,Miellet W R,et al.Contribution of the complement membrane attack complex to the bactericidal activity of human serum[J].Molecular Immunology,2015,65(2):328-335
[4]Liu Q.Study on the off-flavor compounds character of sturgeon and their relationship with lipid oxidation[D].Ocean University of China.2013[刘奇.鲟鱼腥味物质特征及其与脂肪酸氧化的关系研究.中国海洋大学.2013]
[5]Wang Y J,Hu M H.Diseases and prevention of sturgeon in artificial culture[J].Beijing Aquaculture,2004,(4):32-34[王有基,胡梦红.人工养殖鲟鱼中的疾病与防治.北京水产,2004,(4):32-34]
[6]Yao H T,Luo M N,Li C C.Chitosan oligosaccharides reduce acetaminophen-induced hepatotoxicity by suppressing CYP-mediated bioactivation[J].Journal of Functional Foods,2015,12:262-270
[7]Liu X,Xia W,Jiang Q,et al.Chitosan oligosaccharide-N-chlorokojic acid mannich base 2015,polymer as a potential antibacterial material[J].Carbohydrate Polymers,2018,182:225-234
[8]Sun L W,Wen H,Jiang M,et al.Effect of dietary chitosan on growth performance,non-specific immunity and hematological parameters of juvenile Gift tilapia,Oreochromis niloticus[J].Journal of Guangdong Ocean University,2011,31(3):43-49[孙立威,文华,蒋明,等.壳寡糖对吉富罗非鱼幼鱼生长性能、非特异性免疫及血液学指标的影响.广东海洋大学学报,2011,31(3):43-49]
[9]Zhang Y,Xu W,Cui L Q,et al.Effects of chitosan oligosaccharides in diets of turbot(Scophthalmus maximus)on the growth and immune performance[J].Periodical of Ocean University of China,2012,42(10):71-74[张艺,徐玮,崔丽卿,等.壳寡糖对大菱鲆生长和免疫功能的影响.中国海洋大学学报(自然科学版),2012,42(10):71-74]
[10]Luo L,Cai X,He C,et al.Immune response,stress resistance and bacterial challenge in juvenile rainbow trouts Oncorhynchus mykiss fed diets containing chitosan-oligo saccharides[J].Current Zoology,2009,55(6):416-422
[11]Lin S,Mao S,Guan Y,et al.Effects of dietary chitosan oligosaccharides and Bacillus coagulans on the growth,innate immunity and resistance of koi(Cyprinus carpio koi)[J].Aquaculture,2012,342-343:36-41
[12]Amoah Y T,Moniruzzaman M,Lee S,et al.Evaluation of different dietary additives based on growth performance,innate immunity and disease resistance in juvenile Amur catfish,Silurus asotus[J].International Aquatic Research,2017,9(4):351-360
[13]Zante M D,Borchel A,Brunner R M,et al.Cloning and characterization of the proximal promoter region of rainbow trout(Oncorhynchus mykiss)interleukin-6 gene[J].Fish&Shellfish Immunology,2015,43(1):249-256
[14]Zhang D L,Yu D H,Chen J,et al.Characterization and expression analysis of BCA2 gene in large yellow croaker,Larimichthys crocea[J].Fish&Shellfish Immunology,2015,46(2):543-549
[15]Chen Y,Xia Y,Shao C,et al.Discovery and identification of candidate sex-related genes based on transcriptome sequencing of Russian sturgeon(Acipenser gueldenstaedtii)gonads[J].Physiological Genomics,2016,48:464-476
[16]Zhao L,Ma L G,Yang Z A,et al.Cloning and expression analysis of C4H gene from Lepidium[J].Acta Pharmaceutica Sinica,2017,(5):821-831[赵乐,马利刚,杨泽岸,等.独行菜C4H基因克隆与表达分析.药学学报,2017,(5):821-831]
[17]Ren Y,Zhao H,Su B,et al.Expression profiling analysis of immune-related genes in channel catfish(Ictalurus punctatus)skin mucus following Flavobacterium columnare challenge[J].Fish&Shellfish Immunology,2015,46(2):537-542
[18]Shen Y B,Zhang J B,Xu X Y,et al.Molecular cloning,characterization and expression analysis of the complement component C6 gene in grass carp[J].Veterinary Immunology and Immunopathology,2011,141(1-2):139-143
[19]Zarkadis I K,Duraj S,Chondrou M.Molecular cloning of the seventh component of complement in rainbow trout[J].Developmental&Comparative Immunology,2005,29(2):95-102
[20]Jiang J J,Li Y W,Chen D Q,et al.Molecular Cloning,expression and genetic diversity analysis of the seventh component of the complement in Hypophthalmichthys molitrix[J].Journal of Fishery Sciences of China,2012,19(6):994-1000[蒋菁菁,李英文,陈大庆,等.鲢补体C7基因的克隆、表达和序列分析.中国水产科学,2012,19(6):994-1000]
[21]Wang S,Gao Y,Shu C,et al.Characterization and evolutionary analysis of duplicated C7 in miiuy croaker[J].Fish&Shellfish Immunology,2015,45(2):672-679
[22]Papanastasiou AD,Zarkadis IK.Gene duplication of the seventh component of complement in rainbow trout[J].Immunogenetics,2005,(57):703-708
[23]Ye X F,Zhang S R,Li X,et al.Research progress on effects and mechanisms of immunological regulation of chitosan oligosaccharide[J].Feed Review,2014,(7):34-37[叶小飞,张石蕊,李霞,等.壳寡糖的免疫调节作用及机理研究进展.饲料博览,2014,(7):34-37]
[24]Huang R L,Deng Z Y,Yang C B,et al.Dietary oligochitosan supplementation enhances immune status of broilers[J].Journal of the Science of Food&Agriculture,2007,87(1):153-159
[25]Dou J,Xu Q,Tan C,et al.Effects of chitosan oligosaccharides on neutrophils from glycogen-induced peritonitis mice model[J].Carbohydrate Polymers,2009,75(1):119-124
[26]Wang J P,Yoo J S,Kim H J,et al.Nutrient digestibility,blood profiles and fecal microbiota are influenced by chitooligosaccharide supplementation of growing pigs[J].Livestock Science,2009,125(2):298-303