魁蚶(Scapharca broughtonii)血红蛋白Ⅰ基因克隆及表达特征
|
摘要
魁蚶(Scapharca broughtonii)的血淋巴中因含有血红蛋白而不同于其他多数双壳贝类。为明确魁蚶血红蛋白基因的结构特征、组织发生分布、免疫活性等特点,本研究以魁蚶转录组数据库中的部分序列为基础,通过cDNA末端快速扩增(Rapid amplication of cDNA ends, RACE)技术,克隆获得了一种魁蚶血红蛋白基因cDNA全长序列(命名为SbHbⅠ),并研究了不同条件下的m RNA表达。序列和结构分析显示,SbHbⅠ基因cDNA全长为867 bp,其中包括长度为483 bp的开放阅读框(Open reading frame, ORF),编码160个氨基酸,预测蛋白分子量为17.5 kDa,理论等电点为9.68;氨基酸序列具有多个血红素结合位点,序列与所选其他物种的相似度范围为57%～93%,与蚶科相似度较高。实时荧光定量PCR (qRT-PCR)结果显示,在魁蚶成体的斧足、鳃、外套膜、闭壳肌、血淋巴、肝胰腺及受精卵至发育23 d的幼虫体内,均能检测到SbHbⅠ基因;该基因在成体魁蚶血淋巴中表达量最高,与其他5个组织中的表达量差异显著(P<0.05);受精卵至23 d幼虫期的表达,基本呈现为先下降后上升的趋势,且在12d时急剧上升(P<0.05);在鳗弧菌(Vibrioanguillarum)刺激和低氧胁迫后,血淋巴中SbHbⅠ基因的表达均呈先上升后下降的趋势,表达量最高值均出现在16 h。本研究丰富了贝类血红蛋白相关研究资料。
Hemoglobin is the main component of haemolymph, which is widely distributed in vertebrates and some invertebrates. It has many biological functions, including transporting oxygen, storing energy, sustaining osmotic pressure, immunological functions, and so on. According to previous reports, the functions of hemoglobin from different organisms are varied, especially in invertebrate animals, because of less conservation in gene structure. However, the study of the immune function of hemoglobin in mollusks is limited. The ark shell, Scapharca broughtonii, is one of the important economic marine shellfishes in China, Korea, and Japan, and other places. In particular, previous studies showed that there were three types of hemocytes in S. broughtonii: red blood cells, white blood cells, and thrombus cells. To determine the structure, distribution, and immunological activity of the hemoglobin gene, the cDNA sequence of hemoglobin(named SbHbⅠ) was obtained using EST and RACE methods. Sequence analysis revealed that the cDNA was 867 bp in length, including an open reading frame(ORF) of 483 bp encoding a polypeptide of 160 amino acid residues. The predicted molecular weight was 17.5 kDa and isoelectric point(pI) was 9.68. The mRNA expression analysis of SbHbⅠin tested tissues by quantitative real-time PCR(qRT-PCR) revealed that the mRNA of SbHb I could all be detected in the foot, gill, mantle, adductor muscle, haemocytes, hepatopancreas, and the larvae aged from oosperm to 23 d. The expression level of SbHbⅠin haemocytes was more than 70 folds that in the adductor muscle. The expression of SbHbⅠin the larvae from the oosperm to 23 d was basically showed a declining tendency at the beginning and increased later, with a sharp increase at 12 d(P<0.05). After being stimulated in Vibrio anguillarum, SbHbⅠfirst showed an upgraded tendency and later descended, and the same tendency occurred during hypoxia stress, during which SbHbⅠreached the maximum(P<0.05) at 16 h at three dissolved oxygen levels(4.5 mg/L, 2.5 mg/L, and 0.5 mg/L). Basic data were provided from the study of hemoglobin in mollusks.
Hemoglobin is the main component of haemolymph, which is widely distributed in vertebrates and some invertebrates. It has many biological functions, including transporting oxygen, storing energy, sustaining osmotic pressure, immunological functions, and so on. According to previous reports, the functions of hemoglobin from different organisms are varied, especially in invertebrate animals, because of less conservation in gene structure. However, the study of the immune function of hemoglobin in mollusks is limited. The ark shell, Scapharca broughtonii, is one of the important economic marine shellfishes in China, Korea, and Japan, and other places. In particular, previous studies showed that there were three types of hemocytes in S. broughtonii: red blood cells, white blood cells, and thrombus cells. To determine the structure, distribution, and immunological activity of the hemoglobin gene, the cDNA sequence of hemoglobin(named SbHbⅠ) was obtained using EST and RACE methods. Sequence analysis revealed that the cDNA was 867 bp in length, including an open reading frame(ORF) of 483 bp encoding a polypeptide of 160 amino acid residues. The predicted molecular weight was 17.5 kDa and isoelectric point(pI) was 9.68. The mRNA expression analysis of SbHbⅠin tested tissues by quantitative real-time PCR(qRT-PCR) revealed that the mRNA of SbHb I could all be detected in the foot, gill, mantle, adductor muscle, haemocytes, hepatopancreas, and the larvae aged from oosperm to 23 d. The expression level of SbHbⅠin haemocytes was more than 70 folds that in the adductor muscle. The expression of SbHbⅠin the larvae from the oosperm to 23 d was basically showed a declining tendency at the beginning and increased later, with a sharp increase at 12 d(P<0.05). After being stimulated in Vibrio anguillarum, SbHbⅠfirst showed an upgraded tendency and later descended, and the same tendency occurred during hypoxia stress, during which SbHbⅠreached the maximum(P<0.05) at 16 h at three dissolved oxygen levels(4.5 mg/L, 2.5 mg/L, and 0.5 mg/L). Basic data were provided from the study of hemoglobin in mollusks.
引文
Bao YB,Li PF,Dong YH,et al.Polymorphism of the multiple hemoglobins in blood clam Tegillarca granosa and its association with disease resistance to Vibrio parahaemolyticus.Fish and Shellfish Immunology,2013,34(5):1320-1324
Brittain T.Molecular aspects of embryonic hemoglobin function.Molecular Aspects of Medicine,2002,23(4):293-342
Datta S,Machaal A,Thekkudan J,et al.Inhaled nitric oxide as rescue therapy for right ventricular insufficiency after orthotopic heart transplantation.Journal of Heart and Lung Transplantation,2004,23(2):158
Hang YH,Liu ZH,Wu B,et al.Gene cloning and expression analysis of catalase in Scapharca broughtonii.Journal of Fisheries of China,2016,40(6):856-866[黄永欢,刘志鸿,吴彪,等.魁蚶过氧化氢酶基因克隆及表达分析.水产学报,2016,40(6):856-866]
Jin Z.Study on phenoloxidase-like properties and antibacterial activity of hemoglobin from blood clam Scapharca kagoshimensis.Doctoral Dissertation of Ocean University of China,2011[荆昭.毛蚶(Scapharca kagoshimensis)血红蛋白的类酚氧化酶性质及其抗菌活性研究.中国海洋大学博士研究生学位论文,2011]
Kato Y,Shoji Y,Morita K,et al.Evaluation of changes in oxyhemoglobin during Shiritori task in elderly subjects including those with Alzheimers disease.Psychogeriatrics,2017,17(4):238-246
Li M,Zhu L,Zhou CY,et al.Molecular characterization and expression of a novel big defensin(Sb-BDef1)from ark shell,Scapharca broughtonii.Fish and Shellfish Immunology,2012,33(5):1167-1173
Qi XQ,Wang T.Molecular evolution of hemoglobin families.Hubei Agricultural Sciences,2014,53(18):4447-4456[齐小琼,王艇.植物血红蛋白基因家族的分子进化研究.湖北农业科学,2014,53(18):4447-4456]
Shen SF,Zhu L,Li JQ,et al.Molecular cloning and expression analysis of C-type lectin from Scapharca broughtonii.Progress in Fishery Sciences,2018,39(1):128-136[沈淑芳,朱玲,李加琦,等.魁蚶C型凝集素基因c DNA的克隆及表达分析.渔业科学进展,2018,39(1):128-136]
Sun J,Wang BJ,Sun SJ,et al.cDNA cloning and sequence analysis of hemocyaninin Fenneropenaeus chinensis.Progress in Fishery Sciences,2010,31(1):80-88[孙杰,王宝杰,孙姝娟,等.中国对虾血蓝蛋白基因c DNA的克隆与序列分析.渔业科学进展,2010,31(1):80-88]
Toulmond A.Tide-related changes of blood respiratory variables in the lugworm Arenicola marina.Respiration Physiology,1973,19(2):130-144
Wang SF,Bao YB,Shi NJ,et al.Purification and antibacterial activity of hemoglobin from Tegillarca granosa.Acta Oceanologica Sinica,2014,36(12):67-73[王素芳,包永波,施淼江,等.泥蚶血红蛋白的制备及其抗菌活性研究.海洋学报,2014,36(12):67-73]
Wang Q,Jiang C,Fang S,et al.Antihyperglycemic,antihyperlipidemic and antioxidant effects of ethanol and aqueous extracts of Cyclocarya paliurus leaves in type 2diabetic rats.Journal of Ethnopharmacology,2013,150(3):1119-1127
Wang Q,Xiang RH,Bao YB,et al.Research progress in animal hemoglobin.Journal of Ningbo University(NSEE),2011,24(2):20-24[汪青,项荣花,包永波,等.动物血红蛋白研究进展.宁波大学学报(理工版),2011,24(2):20-24]
Wang Q,Lin ZH,Bao YB,et al.Clone and analysis of hemoglobin gene(Tg-HbⅡA)and immune expression research in Tegillarca granosa.Oceanologia et Limnologia Sinica,2012,43(1):88-94[汪青,林志华,包永波,等.泥蚶(Tegillarca granosa)血红蛋白基因(Tg-HbⅡA)克隆、分析及免疫表达研究.海洋与湖沼,2012,43(1):88-94]
Wu B,Liu ZH,Zhou LQ,et al.Dynamic expression of big-defensin in different developmental stages of early-larcva of Scapharca broughtonii.Oceanologia et Limnologia Sinica,2017,48(2):351-357[吴彪,刘志鸿,周丽青,等.魁蚶母源大防御素在子代发育早期的动态变化.海洋与湖沼,2017,48(2):351-357]
Ye SY,Chen SZ.The type and function of plant hemoglobin.Anhui Agricultural Science Bulletin,2008,14(17):82-83,98[叶水英,陈素珍.植物血红蛋白的类型及其功能.安徽农学通报,2008,14(17):82-83,98]
Yue F.Study on the ontogenesis of immune system in scallop Chlamys farreri and the maternal immunity at its early life.Doctoral Dissertation of University of Chinese Academy of Science,2013[岳峰.栉孔扇贝免疫系统发生及母源免疫的初步研究.中国科学院大学博士研究生学位论文,2013]
Zhang GM,Wu B,Yang AG,et al.Influence of low salinity challenge on survival and enzyme activities in Scapharca broughtonii.Journal of Ludong University(Natural Science Edition),2017,33(2):159-163[张广明,吴彪,杨爱国,等.盐度胁迫对魁蚶耐受性及体内酶活性的影响.鲁东大学学报(自然科学版),2017,33(2):159-163]
Zhang SP.Illustrated handbook of of marine Mollusks in China.Beijing:China Ocean Press,2008,268-269[张素萍.中国海洋贝类图鉴.北京:海洋出版社,2008,268-269]
Zheng LB,Wu B,Liu ZH,et al.Cloning and expression analysis of galectin from Scapharca broughtonii(Sb Gal).Oceanologia et Limnologia Sinica,2015,46(5):1061-1070[郑利兵,吴彪,刘志鸿,等.魁蚶(Scapharca broughtonii)半乳糖凝集素(Sb Gal)基因c DNA的克隆及表达分析.海洋与湖沼,2015,46(5):1061-1070]
Zheng LB,Wu B,Liu ZH,et al.A manganese superoxide dismutase(MnSOD)from ark shell,Scapharca broughtonii:Molecular characterization,expression and immune activity analysis.Fish and Shellfish Immunology,2015,45(2):656-665
Zheng LB,Liu ZH,Wu B,et al.Ferritin has an important immune function in the ark shell Scapharca broughtonii.Developmental and Comparative Immunology,2016,59(1):15-24
Zhou LQ,Yang AG,Wang QY,et al.Studies on the hemocytes types and their immunological functions inbloody clam(Scapharca broughtonii).Journal of Fisheries of China,2013,37(4):599-606[周丽青,杨爱国,王清印,等.魁蚶血细胞分类及其免疫功能的初步分析.水产学报,2013,37(4):599-606]
Zhou LQ,Yang AG,Wang QY,et al.Effect of Vibrio anguillarum on morphology and immunological function of blood cells in Scapharca broughtonii.Oceanologia et Limnologia Sinica,2014,45(3):536-541[周丽青,杨爱国,王清印,等.鳗弧菌对魁蚶血细胞形态及免疫功能的影响.海洋与湖沼,2014,45(3):536-541]
Zhou LQ,Yang AG,Liu ZH,et al.Changes in hemolymph characteristics of ark shell Scapharaca broughtonii dealt with Vibrio anguillarum challenge in vivo and various of anticoagulants in vitro.Fish and Shellfish Immunology,2017,61(10):9-15
Brittain T.Molecular aspects of embryonic hemoglobin function.Molecular Aspects of Medicine,2002,23(4):293-342
Datta S,Machaal A,Thekkudan J,et al.Inhaled nitric oxide as rescue therapy for right ventricular insufficiency after orthotopic heart transplantation.Journal of Heart and Lung Transplantation,2004,23(2):158
Hang YH,Liu ZH,Wu B,et al.Gene cloning and expression analysis of catalase in Scapharca broughtonii.Journal of Fisheries of China,2016,40(6):856-866[黄永欢,刘志鸿,吴彪,等.魁蚶过氧化氢酶基因克隆及表达分析.水产学报,2016,40(6):856-866]
Jin Z.Study on phenoloxidase-like properties and antibacterial activity of hemoglobin from blood clam Scapharca kagoshimensis.Doctoral Dissertation of Ocean University of China,2011[荆昭.毛蚶(Scapharca kagoshimensis)血红蛋白的类酚氧化酶性质及其抗菌活性研究.中国海洋大学博士研究生学位论文,2011]
Kato Y,Shoji Y,Morita K,et al.Evaluation of changes in oxyhemoglobin during Shiritori task in elderly subjects including those with Alzheimers disease.Psychogeriatrics,2017,17(4):238-246
Li M,Zhu L,Zhou CY,et al.Molecular characterization and expression of a novel big defensin(Sb-BDef1)from ark shell,Scapharca broughtonii.Fish and Shellfish Immunology,2012,33(5):1167-1173
Qi XQ,Wang T.Molecular evolution of hemoglobin families.Hubei Agricultural Sciences,2014,53(18):4447-4456[齐小琼,王艇.植物血红蛋白基因家族的分子进化研究.湖北农业科学,2014,53(18):4447-4456]
Shen SF,Zhu L,Li JQ,et al.Molecular cloning and expression analysis of C-type lectin from Scapharca broughtonii.Progress in Fishery Sciences,2018,39(1):128-136[沈淑芳,朱玲,李加琦,等.魁蚶C型凝集素基因c DNA的克隆及表达分析.渔业科学进展,2018,39(1):128-136]
Sun J,Wang BJ,Sun SJ,et al.cDNA cloning and sequence analysis of hemocyaninin Fenneropenaeus chinensis.Progress in Fishery Sciences,2010,31(1):80-88[孙杰,王宝杰,孙姝娟,等.中国对虾血蓝蛋白基因c DNA的克隆与序列分析.渔业科学进展,2010,31(1):80-88]
Toulmond A.Tide-related changes of blood respiratory variables in the lugworm Arenicola marina.Respiration Physiology,1973,19(2):130-144
Wang SF,Bao YB,Shi NJ,et al.Purification and antibacterial activity of hemoglobin from Tegillarca granosa.Acta Oceanologica Sinica,2014,36(12):67-73[王素芳,包永波,施淼江,等.泥蚶血红蛋白的制备及其抗菌活性研究.海洋学报,2014,36(12):67-73]
Wang Q,Jiang C,Fang S,et al.Antihyperglycemic,antihyperlipidemic and antioxidant effects of ethanol and aqueous extracts of Cyclocarya paliurus leaves in type 2diabetic rats.Journal of Ethnopharmacology,2013,150(3):1119-1127
Wang Q,Xiang RH,Bao YB,et al.Research progress in animal hemoglobin.Journal of Ningbo University(NSEE),2011,24(2):20-24[汪青,项荣花,包永波,等.动物血红蛋白研究进展.宁波大学学报(理工版),2011,24(2):20-24]
Wang Q,Lin ZH,Bao YB,et al.Clone and analysis of hemoglobin gene(Tg-HbⅡA)and immune expression research in Tegillarca granosa.Oceanologia et Limnologia Sinica,2012,43(1):88-94[汪青,林志华,包永波,等.泥蚶(Tegillarca granosa)血红蛋白基因(Tg-HbⅡA)克隆、分析及免疫表达研究.海洋与湖沼,2012,43(1):88-94]
Wu B,Liu ZH,Zhou LQ,et al.Dynamic expression of big-defensin in different developmental stages of early-larcva of Scapharca broughtonii.Oceanologia et Limnologia Sinica,2017,48(2):351-357[吴彪,刘志鸿,周丽青,等.魁蚶母源大防御素在子代发育早期的动态变化.海洋与湖沼,2017,48(2):351-357]
Ye SY,Chen SZ.The type and function of plant hemoglobin.Anhui Agricultural Science Bulletin,2008,14(17):82-83,98[叶水英,陈素珍.植物血红蛋白的类型及其功能.安徽农学通报,2008,14(17):82-83,98]
Yue F.Study on the ontogenesis of immune system in scallop Chlamys farreri and the maternal immunity at its early life.Doctoral Dissertation of University of Chinese Academy of Science,2013[岳峰.栉孔扇贝免疫系统发生及母源免疫的初步研究.中国科学院大学博士研究生学位论文,2013]
Zhang GM,Wu B,Yang AG,et al.Influence of low salinity challenge on survival and enzyme activities in Scapharca broughtonii.Journal of Ludong University(Natural Science Edition),2017,33(2):159-163[张广明,吴彪,杨爱国,等.盐度胁迫对魁蚶耐受性及体内酶活性的影响.鲁东大学学报(自然科学版),2017,33(2):159-163]
Zhang SP.Illustrated handbook of of marine Mollusks in China.Beijing:China Ocean Press,2008,268-269[张素萍.中国海洋贝类图鉴.北京:海洋出版社,2008,268-269]
Zheng LB,Wu B,Liu ZH,et al.Cloning and expression analysis of galectin from Scapharca broughtonii(Sb Gal).Oceanologia et Limnologia Sinica,2015,46(5):1061-1070[郑利兵,吴彪,刘志鸿,等.魁蚶(Scapharca broughtonii)半乳糖凝集素(Sb Gal)基因c DNA的克隆及表达分析.海洋与湖沼,2015,46(5):1061-1070]
Zheng LB,Wu B,Liu ZH,et al.A manganese superoxide dismutase(MnSOD)from ark shell,Scapharca broughtonii:Molecular characterization,expression and immune activity analysis.Fish and Shellfish Immunology,2015,45(2):656-665
Zheng LB,Liu ZH,Wu B,et al.Ferritin has an important immune function in the ark shell Scapharca broughtonii.Developmental and Comparative Immunology,2016,59(1):15-24
Zhou LQ,Yang AG,Wang QY,et al.Studies on the hemocytes types and their immunological functions inbloody clam(Scapharca broughtonii).Journal of Fisheries of China,2013,37(4):599-606[周丽青,杨爱国,王清印,等.魁蚶血细胞分类及其免疫功能的初步分析.水产学报,2013,37(4):599-606]
Zhou LQ,Yang AG,Wang QY,et al.Effect of Vibrio anguillarum on morphology and immunological function of blood cells in Scapharca broughtonii.Oceanologia et Limnologia Sinica,2014,45(3):536-541[周丽青,杨爱国,王清印,等.鳗弧菌对魁蚶血细胞形态及免疫功能的影响.海洋与湖沼,2014,45(3):536-541]
Zhou LQ,Yang AG,Liu ZH,et al.Changes in hemolymph characteristics of ark shell Scapharaca broughtonii dealt with Vibrio anguillarum challenge in vivo and various of anticoagulants in vitro.Fish and Shellfish Immunology,2017,61(10):9-15