CAJ | 학술논문

对41尾感病和22尾抗病兴国红鲤的308个有效克隆进行测序,获得171条不同的MHC Ⅱ类α基因编码序列,分属26个不同的等位基因,其中Cyca-DXA24-Cyca-DXA36为新发现的13个等位基因。MHCⅡ类α基因片段的长度为624 bp,包括第1-4个外显子,分别编码信号肽、α1和α2结构域及连接肽/跨膜区。α1结构域的变异明显大于α2结构域,表现在α1结构域中核苷酸和氨基酸变异位点比例(55.16%和79.76%)明显高于α2结构域的变异位点比例(45.96%和68.42%)。α1结构域的PBR区的非同义碱基替换率(dN)与同义碱基替换率(dS)的比值ω(ω= dN/dS)为5.742,远远高于非抗原结合位点(non-PBR)及α2结构域的0.755、0.592,揭示兴国红鲤 MHC Ⅱ类α基因的α1结构域在进化过程中受到正向选择作用。等位基因Cyca-DXA24(P<0.01)与兴国红鲤对嗜水气单胞菌的抗性相关,等位基因Cyca-DXA3(P<0.05)、Cyca-DXA4(P<0.01)、Cyca-DXA6(P<0.05)、Cyca-DXA33(P<0.05)与兴国红鲤对嗜水气单胞菌的易感性相关。荧光定量PCR结果表明, MHCⅡ类α基因在健康兴国红鲤的肾、肝、鳃等10个组织均能普遍表达。人工感染嗜水气单胞菌后,肾、肝、脾3个组织中的MHCⅡ类α基因的表达量均发生了不同程度的变化,表明MHCⅡ类α分子在兴国红鲤的免疫反应中起到重要作用。
대41미감병화22미항병흥국홍리적308개유효극륭진행측서,획득171조불동적MHC Ⅱ류α기인편마서렬,분속26개불동적등위기인,기중Cyca-DXA24-Cyca-DXA36위신발현적13개등위기인。MHCⅡ류α기인편단적장도위624 bp,포괄제1-4개외현자,분별편마신호태、α1화α2결구역급련접태/과막구。α1결구역적변이명현대우α2결구역,표현재α1결구역중핵감산화안기산변이위점비례(55.16%화79.76%)명현고우α2결구역적변이위점비례(45.96%화68.42%)。α1결구역적PBR구적비동의감기체환솔(dN)여동의감기체환솔(dS)적비치ω(ω= dN/dS)위5.742,원원고우비항원결합위점(non-PBR)급α2결구역적0.755、0.592,게시흥국홍리 MHC Ⅱ류α기인적α1결구역재진화과정중수도정향선택작용。등위기인Cyca-DXA24(P<0.01)여흥국홍리대기수기단포균적항성상관,등위기인Cyca-DXA3(P<0.05)、Cyca-DXA4(P<0.01)、Cyca-DXA6(P<0.05)、Cyca-DXA33(P<0.05)여흥국홍리대기수기단포균적역감성상관。형광정량PCR결과표명, MHCⅡ류α기인재건강흥국홍리적신、간、새등10개조직균능보편표체。인공감염기수기단포균후,신、간、비3개조직중적MHCⅡ류α기인적표체량균발생료불동정도적변화,표명MHCⅡ류α분자재흥국홍리적면역반응중기도중요작용。
We cloned 171 sequences of MHC class IIαgene were determined from 41 susceptible and 22 resistant indi-viduals of Cyprinus carpio var. singguoensis. These sequences belonged to 26 alleles, thirteen of which were novel (Cyca-DXA24-Cyca-DXA36). The total length of the sequence was 624 bp, consisting of a 33 bp exon1, a 252 bp exon2, a 285 bp exon3, and a 54 bp exon4. These exons encoded a partial signal peptide, aα1 domain, aα2 domain and a con-necting peptide (CP)/partial transmembrane (TM), respectively. The mutation-Prone positions of nucleotide or amino acid sites in α1 domain (55.16%, 79.76%) significantly out number than those in α2 domain (45.96%, 68.42%). The ωvalues (ω=dN/dS) were 5.742 for peptide-binding region (PBR), 0.755 for non-PBR in theα1 domain and 0.592 for theα2 domain, implying that the positive selection pressure probably had occurred in the PBR region of the α1 domain. Cyca-DXA24 (P<0.01) was significantly correlated with the carp’s resistance to Aeromonas hydrophila, while Cyca-DXA3 (P<0.05), Cyca-DXA4 (P<0.01), Cyca-DXA6 (P<0.05) and Cyca-DXA33 (P<0.05) alleles were signifi-cantly associated with the fish’s susceptibility to the disease. Real-time quantitative PCR demonstrated that MHC IIαgenes were ubiquitously expressed in ten tissues. The expression was up-regulated in kidney, down-regulated in liver and spleen during infection. Our results indicated that the MHC class IIα gene might play an important role in the im-mune reactions of C. carpio var. singguoensis.