ICAR-National Bureau of Fish Genetic Resources (ICAR-NBFGR), Lucknow, India.
Indian Council of Agricultural Research (ICAR), Krishi Anusandhan Bhawan-II, New Delhi, India.
PLoS One. 2019 Jul 23;14(7):e0216144. doi: 10.1371/journal.pone.0216144. eCollection 2019.
The present study, for the first time, reported twelve A2M isoforms in Tenualosa ilisha, through SMRT sequencing. Hilsa shad, T. ilisha, an anadromous fish, faces environmental stresses and is thus prone to diseases. Here, expression profiles of different A2M isoforms in four tissues were studied in T. ilisha, for the tissue specific diversity of A2M. Large scale high quality full length transcripts (>0.99% accuracy) were obtained from liver, ovary, testes and gill transcriptomes, through Iso-sequencing on PacBio RSII. A total of 12 isoforms, with complete putatative proteins, were detected in three tissues (7 isoforms in liver, 4 in ovary and 1 in testes). Complete structure of A2M mRNA was predicted from these isoforms, containing 4680 bp sequence, 35 exons and 1508 amino acids. With Homo sapiens A2M as reference, six functional domains (A2M_N,A2M_N2, A2M, Thiol-ester_cl, Complement and Receptor domain), along with a bait region, were predicted in A2M consensus protein. A total of 35 splice sites were identified in T. ilisha A2M consensus transcript, with highest frequency (55.7%) of GT-AG splice sites, as compared to that of Homo sapiens. Liver showed longest isoform (X1) consisting of all domains, while smallest (X10) was found in ovary with one Receptor domain. Present study predicted five putative markers (I-212, I-269, A-472, S-567 and Y-906) for EUS disease resistance in A2M protein, which were present in MG2 domains (A2M_N and A2M_N2), by comparing with that of resistant and susceptible/unknown response species. These markers classified fishes into two groups, resistant and susceptible response. Potential markers, predicted in T. ilisha, placed it to be EUS susceptible category. Putative markers reported in A2M protein may serve as molecular markers in diagnosis of EUS disease resistance/susceptibility in fishes and may have a potential for inclusion in the marker panel for pilot studies. Further, challenging studies are required to confirm the role of particular A2M isoforms and markers identified in immune protection against EUS disease.
本研究首次通过 SMRT 测序报道了印度洋鲱鱼中的 12 种 A2M 异构体。长吻鮠，T. ilisha，一种洄游鱼类，面临环境压力，因此容易患病。在这里，研究了 T. ilisha 中四种组织中不同 A2M 异构体的表达谱，以研究 A2M 的组织特异性多样性。通过 PacBio RSII 上的 Iso-sequencing，从肝、卵巢、睾丸和鳃转录组中获得了大量高质量全长转录本（>0.99%的准确率）。总共在三种组织中检测到 12 种具有完整假定蛋白质的异构体（7 种在肝脏，4 种在卵巢，1 种在睾丸）。从这些异构体中预测了 A2M mRNA 的完整结构，包含 4680bp 序列、35 个外显子和 1508 个氨基酸。以智人为参考，在 A2M 共识蛋白中预测了六个功能域（A2M_N、A2M_N2、A2M、Thiol-ester_cl、补体和受体域）以及一个诱饵区。在 T. ilisha A2M 共识转录本中鉴定了总共 35 个剪接位点，与智人相比，GT-AG 剪接位点的频率最高（55.7%）。肝脏表现出最长的异构体（X1），由所有结构域组成，而最小的异构体（X10）在卵巢中，只有一个受体结构域。本研究预测了 A2M 蛋白中 5 个潜在的标记物（I-212、I-269、A-472、S-567 和 Y-906），用于 EUS 疾病抗性，这些标记物存在于 MG2 结构域（A2M_N 和 A2M_N2）中，与抗性和敏感/未知反应物种进行比较。这些标记物将鱼类分为两组，即抗性和敏感反应组。在 T. ilisha 中预测的潜在标记物将其置于 EUS 易感类别中。在 A2M 蛋白中报道的假定标记物可能作为鱼类 EUS 疾病抗性/敏感性诊断的分子标记物，并有可能被纳入试点研究的标记物组。此外，还需要进行挑战性研究来确认特定 A2M 异构体和在对 EUS 疾病的免疫保护中识别出的标记物的作用。
