实验海洋生物学重点实验室知识库

文蛤（Meretrix petechialis）是我国一种重要的海洋经济贝类。然而，近年来在文蛤的养殖过程中频繁发生由细菌和病毒引起的病害问题。因此，通过分子手段研究在微生物侵染过程中文蛤体内的信号通路响应，解析文蛤天然免疫防御机制，可为培育文蛤的抗性品系提供良好的分子基础。

小眼畸形相关转录因子（microphthalmia-associated transcription factor，MITF）是MYC转录家族成员之一，其通过本身的bHLH-LZ结构域直接调控下游靶基因的表达。MITF是控制细胞增殖、存活和免疫防御的信号转导途径的关键调节因子。在本研究中，我们鉴定了文蛤体内的MITF家族基因，分析了MITF在文蛤免疫防御和壳色形成中的重要作用，并对MITF的上下游通路进行探究，完善了MITF相关通路在贝类天然免疫防御中的调控机制。本研究的主要结果如下：

1、本研究从文蛤中克隆获得了MITF基因的两个亚型，分别命名为MpMITF-1和MpMITF-2。其中MpMITF-1基因cDNA全长为3564 bp，包含1365 bp的开放阅读框。通过组织表达分析显示，MpMITF-1基因在文蛤的各个组织中广泛表达。用50ml 5×10⁶ CFU ml⁻¹副溶血弧菌（Vibrio parahaemolyticus）注射文蛤后，MpMITF-1基因的表达量在6 h和12 h出现显著上调（P<0.05）。同时，在V. parahaemolyticus浸泡刺激后，MpMITF-1基因也出现表达上调的趋势，表明MpMITF-1参与文蛤应对弧菌的免疫响应。在MpMITF-1基因中通过SNP分型获得5个SNP和2个单体型与弧菌抗性性状相关。在文蛤抗性家系（Family-R）和敏感家系（Family-S）中对得到的分子标记进行验证，最终确定4个SNP（SNP2、5、6和13）和1个单体型（Hap1）与文蛤的弧菌抗性相关。同时，克隆获得MpMITF-2基因的cDNA全长为2026 bp，包含1116 bp的开放阅读框。MpMITF-2基因的组织表达分析显示，MpMITF-2在外套膜组织表达量很高，其他组织几乎不表达。同时，MpMITF-2基因在不同壳色品系的文蛤体内具有不同的表达量，证明MpMITF-2基因与文蛤的壳色形成相关。通过RNAi干扰MpMITF-2的表达后，新长出的贝壳壳色出现了不均匀的色素分布，充分说明MpMITF-2基因参与文蛤色素的合成。Phenoloxidase（MpPO）是MITF潜在的下游靶基因，也是参与色素形成的功能基因。当MpMITF-2基因的表达被干扰后，MpPO基因的表达量也出现显著下调。该研究阐释了MpMITF-2、MpPO和壳色之间的关系。

2、本研究通过RNAi技术干扰了MpMITF-1的表达，发现其潜在的下游靶基因phenoloxidase（PO）、cathepsin K（CTSK）和BCL-2的表达量出现显著下调（P<0.05），推测在文蛤体内MpPO、MpCTSK和MpBCL-2是受到MpMITF调控的下游靶基因。分别克隆获得了MpPO、MpCTSK和MpBCL-2基因的完整的开放阅读框，其中MpCTSK基因的ORF为1041 bp，编码346个氨基酸；MpBCL-2基因的ORF为705 bp，编码234个氨基酸；MpPO的ORF长度为2019 bp，编码672个氨基酸。基因的组织表达分析显示，MpPO、MpCTSK和MpBCL-2基因在文蛤的6个组织内均广泛表达。在弧菌浸泡刺激文蛤后，MpPO、MpCTSK和MpBCL-2基因的表达量均出现显著上调，暗示它们参与文蛤应对弧菌的免疫响应。

3、基于RNAi筛选得到的MpMITF-1的下游靶基因MpPO、MpCTSK和MpBCL-2，通过启动子扩增获得它们的启动子序列并检测到能够与MITF蛋白结合的E-box基序。通过EMSA实验发现，MpPO、MpCTSK和MpBCL-2的启动子序列与MpMITF-1蛋白直接结合；通过酵母单杂交实验进一步验证发现，MpMITF-1蛋白能调控下游基因MpPO、MpCTSK和MpBCL-2的表达。通过原核重组蛋白表达获得了MpPO和MpCTSK的重组蛋白，并利用最小抑菌浓度实验和抑菌圈实验发现MpPO和MpCTSK都可以抑制副溶血弧菌的生长，说明MpPO和MpCTSK通过抑制细菌的生长参与文蛤的免疫防御。同时，当BCL-2的活性被抑制后，文蛤血细胞的凋亡率显著上调（P<0.05），推测MpBCL-2可以通过抑制血细胞的凋亡参与免疫防御。

4、通过酵母双杂交技术对MpMITF-1的上游基因进行筛选，发现p38（MpP38）和p300（MpP300）能够直接与MpMITF-1蛋白发生直接相互作用。从文蛤中克隆获得MpP38基因的cDNA全长1720 bp，包含1095 bp的ORF，编码365个氨基酸；同时，获得MpP300基因的全长为2507 bp，包含1767 bp的ORF编码588个氨基酸。在V. parahaemolyticus浸泡刺激文蛤后，MpP38基因和MpP300基因的表达量都出现显著上调，说明它们响应弧菌的刺激。通过对MpP38和MpP300的调控机制进行研究，发现V. parahaemolyticus刺激可以促进MpP38的磷酸化，而MpP38的磷酸化水平会影响MITF下游靶基因MpPO的表达量的变化，表明磷酸化的MpP38通过激活MpMITF-1蛋白从而引起下游效应基因MpPO的表达。另外，通过酵母双杂交技术分别检测了MpP300的N端和C端与MpMITF-1蛋白的结合能力，结果发现MpP300的N端，而不是C端，能够与MpMITF-1直接结合。该研究揭示了MpMITF-1在发挥转录调控功能时自身受到的调控。

The clam Meretrix petechialis is one of the most commercial species of marine bivalves. However, in recent years, clam deseases caused by bacteria and virus are becoming more and more common in the cultivation of Meretrix petechialis. Hence, it will provide molecular basis for cultivating resistant strains of M. petechialis by exploring the signaling pathway response during microbial infection and analyse the innate immune mechanism in the clam M. petechialis.

The microphthalmia-associated transcription factor （MITF）, a member of the MYC family of transcription factors, is a basic helix-loop-helix-leucine zipper （bHLH-LZ） protein which could regulate downstream genes depending on its bHLH-LZ domain. MITF has been reported to be the key regulator of signalling pathways that control cell proliferation, survival and immune defence. In this research, we identified and characterised the MITF gene family and analysed the functions of MITF in the immune defense and color formation in clams. In addition, we explored the upstream and downstream pathways of MpMITF, which revealed the function of MITF signalling pathway in the immune system. The main results were as follows.

1. In this study, two isoforms of MITF were identified from M. petechialis, named MpMITF-1 and MpMITF-2. The full length cDNA of MpMITF-1 is 3564 bp with an ORF of 1365 bp. The tissue distribution analysis showed that MpMITF-1 mRNA was widely expressed in all tissues. The expression level of MpMITF-1 was significantly up-regulated 6 h and 12 h post-Vibrio parahaemolyticus injection （P<0.05）. The mRNA expression of MpMITF-1 also increased post-V. parahaemolyticus immersion, which suggested that MpMITF-1 is involved in the immune response in clams. Genotyping in two clam groups with different resistant levels to V. parahaemolyticus （i.e., 11-R and 11-S）, five SNPs and two haplotypes were associated with Vibrio resistance. Four SNPs （SNP2, 5, 6 and 13） and one haplotype （Hap1） were further confirmed to be associated with Vibrio resistance in M. petechialis by association analysis in different clam families. In addition, the full-length cDNA of MpMITF-2 is 2026 bp with an ORF of 1116 bp. The mRNA of MpMITF-2 was more highly expressed in the mantle compared to the other four tissues. Furthermore, there was a significant difference in the expression of MpMITF-2 among three clam strains with different shell colors. These results implied that MpMITF-2 was associated with shell color formation in the clam M. petechialis. When the mRNA expression of MpMITF-2 was knocked down, the new shell showed discontinuous pigment distribution, suggesting that the reduced expression of MpMITF-2 influenced pigment synthesis. A gene encoding phenoloxidase （MpPO） was identified as related to the shell color of the clam and was also a putative downstream gene of MITF. The expression of MpPO decreased significantly when the expression of MpMITF-2 was knocked down （P<0.05）. The results indicate the close relationships among MpMITF-2, MpPO and shell color.

2. When the expression of MpMITF-1 was knocked down by RNAi, the mRNA expression of phenoloxidase （PO）, cathepsin K （CTSK） and BCL-2 was significantly decreased （P<0.05）, indicating that PO, CTSK and BCL-2 were the target genes of MpMITF in clams. The ORF of MpCTSK is 1041 bp, encoding a protein of 346 amino acids. The ORF of MpBCL-2 is 705 bp with a predicted protein of 234 amino acids. And the ORF of MpPO is 2019 bp, encoding a protein of 672 amino acids. The tissue distribution analysis showed that MpCTSK, MpBCL-2 and MpPO were widely expressed in all tissues. Meanwhile, the mRNA expressions of MpCTSK, MpBCL-2 and MpPO were all significantly up-regulated post-V. parahaemolyticus challenge （P<0.05）, indicating that they take part in the immune response against Vibrio challenge in clams.

3. Based on the downstream genes of MpMITF-1 selected by RNAi, we cloned the promoter sequences of MpCTSK, MpBCL-2 and MpPO and detected an E-box sequence in all three of these genes, which is an essential element for the MITF protein to bind to. Our EMSA results showed that MpMITF-1 directly binds to the promoter regions of MpCTSK, MpPO and MpBCL-2. Our yeast one-hybrid assay result supports the conclusion that MpMITF-1 can directly regulate the transcription of MpCTSK, MpPO and MpBCL-2. The purified recombinant proteins, MpPO and MpCTSK, inhibited the growth of Vibrio. Additionally, the apoptosis rate of clam haemocytes rose significantly when the activity of MpBCL-2 was suppressed. These results revealed that MpPO, MpCTSK and MpBCL-2 are involved in the immune defence against V. parahaemolyticus.

4. The putative upstream genes p38 （MpP38） and p300 （MpP300） were detected to directly interact with MpMITF-1 by yeast two-hybrid assay. The full-length cDNA of MpP38 is 1720 bp consisting of a 1095 bp ORF, encoding a polypeptide of 365 amino-acid residues. Meanwhile, the full-length cDNA of MpP300 is 2507 bp consisting of a 1767 bp ORF, encoding a polypeptide of 588 amino-acid residues. The expression levels of MpP38 and MpP300 were significantly up-regulated post-V. parahaemolyticus immersion （P<0.05）, suggesting that they respond to Vibrio stimulation. To explore the regulation of MpP38 and MpP300, we found that the MpP38 phosphorylation level was increased in response to V. parahaemolyticus stimulation. And there was a correlation between the MpP38 phosphorylation level and PO expression level. Our results imply that MpP38 participates in the clam immune defence via activating MpMITF-1 and ultimately regulating the expression of an immune-related gene PO. In addition, the yeast two-hybrid assay was adopted to detect the combination between MpMITF-1 and the N-/C-terminal of MpP300. Our result found that the N-terminal of MpP300 could directly bind to MpMITF-1. This result revealed the regulation on MpMITF-1 when it regulates downtream genes.