CAJ | 학술논문

利用本实验室获得的杂色鲍(Haliotis diversicolor)转录组测序数据库,筛选出杂色鲍紫色酸性磷酸酯酶(Purple acid phosphatase, PAP)同源片段,进而克隆获得PAP的cDNA全序列,命名为HdPAP,并进行了HdPAP蛋白的结构分析和功能预测,同时利用实时定量 PCR(qRT-PCR)技术分析 HdPAP 基因的组织表达谱和应激条件下的HdPAP表达变化。HdPAP基因cDNA全长1215 bp,含有1个编码322个氨基酸的969 bp的开放阅读框。经Blast比对,与无脊椎动物半索动物门囊舌虫(Saccoglossus kowalevskii)的PAP氨基酸序列一致性最高,达59%。通过实时定量PCR检测, HdPAP在检测的杂色鲍各组织中均有表达,其中在血细胞和肝胰腺的表达量显著高于其他各组织(P<0.05)。高温应激下,在检测的6个时相中, HdPAP在血细胞、肝胰腺和鳃中均出现不同程度的表达抑制。缺氧应激后,4 h血细胞中HdPAP显著低于对照组,24 h恢复到正常水平,96 h显著高于对照组(P<0.05),到192 h又恢复到正常水平。副溶血弧菌感染实验中检测到HdPAP在血细胞中同样出现表达抑制现象,3 h和6 h均检测到感染组 HdPAP表达量显著低于对照组(P<0.05)。PAP在高温、缺氧及弧菌感染下显著的表达抑制从转录水平揭示了其作为表达下调的酯酶可能参与胁迫下的生物代谢和免疫反应。
이용본실험실획득적잡색포(Haliotis diversicolor)전록조측서수거고,사선출잡색포자색산성린산지매(Purple acid phosphatase, PAP)동원편단,진이극륭획득PAP적cDNA전서렬,명명위HdPAP,병진행료HdPAP단백적결구분석화공능예측,동시이용실시정량 PCR(qRT-PCR)기술분석 HdPAP 기인적조직표체보화응격조건하적HdPAP표체변화。HdPAP기인cDNA전장1215 bp,함유1개편마322개안기산적969 bp적개방열독광。경Blast비대,여무척추동물반색동물문낭설충(Saccoglossus kowalevskii)적PAP안기산서렬일치성최고,체59%。통과실시정량PCR검측, HdPAP재검측적잡색포각조직중균유표체,기중재혈세포화간이선적표체량현저고우기타각조직(P<0.05)。고온응격하,재검측적6개시상중, HdPAP재혈세포、간이선화새중균출현불동정도적표체억제。결양응격후,4 h혈세포중HdPAP현저저우대조조,24 h회복도정상수평,96 h현저고우대조조(P<0.05),도192 h우회복도정상수평。부용혈호균감염실험중검측도HdPAP재혈세포중동양출현표체억제현상,3 h화6 h균검측도감염조 HdPAP표체량현저저우대조조(P<0.05)。PAP재고온、결양급호균감염하현저적표체억제종전록수평게시료기작위표체하조적지매가능삼여협박하적생물대사화면역반응。
The small abalone Haliotis diversicolor is one of the most commercially important cultured abalone in southern coastal areas in China. However, the frequent occurrence of infectious disease, especially during hot summers, is a major problem that has threatened the abalone aquaculture industry for a long time. As an inverte-brate, abalone lacks an adaptive immune system and relies exclusively on innate immunity to defend against bac-terial challenge. However, research on gastropod immune responses is limited. Purple acid phosphatase (PAP) belongs to a large family of dinuclear metalloenzymes and is distinguished from other acid phosphatases by its purple color, which is due to a Tyr-to-iron (III) charge transfer transition. PAPs are a group of tartrate resistant, molybdate sensitive, iron containing acid phosphatases with a molecular weight of about 35-40 kD and a high activity towards activated phosphoric acid monoesters and anhydrides. They catalyze the hydrolysis of a wide range of phosphate esters. PAPs play important roles in response to different stresses in plants and mammals. However, to date it has not been investigated in molluscs. In this study, the first molluscan PAP gene, HdPAP from H. diversicolor, was cloned by combining the expressed sequence tag (EST) and rapid amplification of cDNA end (RACE) methods. Its full length cDNA sequence is 1 215 bp, with a 969 bp open reading frame encoding a protein of 322 amino acids (GenBank:KC337074). The 5′and 3′untranslated regions (UTR) of HdPAP contain 28 bp and 218 bp, respectively. Pairwise analysis results revealed that the HdPAP amino acid sequence has the highest iden-tity, 59%, to the invertebrate Saccoglossus kowalevskii PAP. The calculated molecular mass of deduced HdPAP is 36.8 kD with a theoretical isoelectric point (PI) of 5.27. Multiple sequence alignment of the HdPAP amino acid sequence with other known vertebrate PAPs and invertebrate PAP family proteins revealed that it was conserved, while their lengths varied among species. Predicted three-dimensional modeling (3D) of HdPAP from Swiss-model identified 7α-helices and 12β-sheets. These important secondary structures contribute to the function of this en-zyme. HdPAP expression profiles in various tissues and at different time phases after thermal, hypoxia, or bacteria challenge were determined by real-time quantitative PCR. HdPAP was detected in all tissues examined, with the highest levels being in the hemocytes and hepatopancreas, suggesting that purple acid phosphatase is an important enzyme with a vital function in metabolic stress. HdPAP expression was suppressed to varying degrees in the gill, hemocytes, and hepatopancreas by thermal stress. Its expression was significantly down-regulated after 4 h and 24 h in the gill, 4 h, 24 h, 96 h, and 192 h in the hemocytes, and 24 h, 96 h, and 192 h in the hepatopancreas at 30℃. HdPAP expression levels in the hemocytes of the hypoxic group was significantly lower than that of the control group after 4 h of exposure, then rose to the same level as the control group after 24 h. After 96 h the expression level was significantly higher than that of the control group and subsequently dropped to the same level as the control group after 192 h. Expression inhibition was also observed in the hemocytes after Vibrio parahaemolyticus (isolated from diseased H. diversicolor) injection, with the level being significantly lower than that of the control group after 3 h and 6 h. These results suggest that HdPAP plays an important role in environmental stress and bacterial challenge responses in abalone.