【作者】 赵建民；

【导师】 宋林生；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2006， 博士

【摘要】 扇贝养殖是我国传统的海水养殖产业,但自1997年以来,养殖扇贝陆续爆发的大规模死亡,不但造成了巨大的经济损失,而且严重影响了该产业的健康发展。扇贝病害的不断爆发以及病因的多样性迫切要求制定新的疾病防治措施和开发新型的抗菌物质。从扇贝自身的免疫防御因子入手,筛选和克隆参与免疫防御的功能基因,一方面可以研究抗病功能基因在病原感染或环境胁迫条件下的表达规律,深入探讨扇贝的免疫防御机制,并可作为抗病良种选育的分子标记,指导扇贝的遗传改良和抗病品系的培育;另一方面,可对抗菌效应物实现重组表达,开发新型的病害预防治疗制剂,取代目前普遍使用的抗生素和化学药物。抗菌效应物是机体在免疫应答过程中产生的多肽类物质,对侵入生物体内的细菌、病毒具有很强的免疫杀灭作用,对抗菌效应物的研究有助于深入了解机体先天性免疫防御的机制。本研究采用大规模EST测序方法,结合cDNA末端快速扩增(RACE)技术,从海湾扇贝血淋巴中克隆到了大防御素基因(big defensin, AiBD)的全长cDNA序列,该cDNA全长为531 bp,其中5’非编码区(UTR)为24 bp,开放阅读框(Open Reading Frame, ORF)含有369 bp,编码122个氨基酸残基;随后为138-bp的3’ UTR,包括一个多聚腺苷酸信号序列(AATAAA)和ploy A尾巴。分析表明,海湾扇贝大防御素是以前体的形式合成,前体分子包括信号肽、前域和成熟肽三部分。采用Northern blot方法,以DIG标记的DNA探针检测了AiBD mRNA在不同组织中的表达。结果发现,AiBD基因的转录本主要在血淋巴中表达,在鳃中也有微量的表达,而在外套膜、闭壳肌、性腺及肝胰腺中检测不到杂交信号。采用QRT-PCR(quantitative real time PCR)对鳗弧菌感染后海湾扇贝血淋巴中AiBD mRNA的表达量进行了检测,结果发现在感染后8 h内, AiBD mRNA的相对表达量平缓升高;随着刺激时间的增长,AiBD基因的mRNA表达量急剧增加,在刺激后16 h和32 h分别达到了空白组的72.3倍和131.1倍。为了研究海湾扇贝大防御素的抗菌活性,将其成熟肽编码区克隆到毕赤酵母表达载体更多还原

【Abstract】 Scallop aquaculture is a big industry and contributes enormously to the economicdevelopment of coastal provinces in China. Since the summer of 1997, large-scalemortality of cultured scallop has caused catastrophic losses to scallop aquaculture,which resulted in the production decreasing drastically. The durative outbreak ofdiseases has stimulated intensive efforts for the development of better healthmanagement strategies and characterization of original immune efforts for diseasecontrol.The identification and characterization of genes involved in scallop immuneresponses are now considered to be essential for the elucidation of immune defensemechanisms and disease control because of their potential use as therapeutic agentsand genetic improvement biomarkers on disease-resistant strain selection.Antimicrobial effectors constitute the first line of innate immunity for scallop exposedto various potential pathogens in the aquatic environment by exerting broad-spectrummicrobicidal activity.The first mollusk big defensin (designated AiBD) cDNA was cloned from bayscallop Argopecten irradians by expressed sequence tag (EST) and rapidamplification of cDNA ends (RACE) techniques. The full-length cDNA of AiBDconsisted of 531 nucleotides with a canonical polyadenylation signal sequenceAATAAA and a poly(A) tail, encoding a polypeptide of 122 amino acids. Consistentwith other known big defensin sequences, AiBD was firstly synthesized as aprepropeptide. After cleavage of the signal peptide, the proregion was processed byKex-2-like proteases and resulted in the mature peptide with a theoretical mass of9.22 kDa and a pI of 9.81. The expression of AiBD in various tissues was measured byusing Northern blotting analysis. mRNA transcripts of AiBD could be detected inhaemocytes of unchallenged scallops. The temporal expression of AiBD inhaemolymph after Vibrio anguilarum challenge was recorded by quantitative real timePCR. The relative expression level of AiBD in haemolymph was up-regulated evenlyin the first 8 h, followed by a drastic increase, and increased 131.1 fold at 32 h postinjection. These results indicated that AiBD could be induced by bacterial challenge,and it should participate in the immune responses of A. irradians. Biological activityassay revealed that recombinant AiBD could inhibit the growth of both Gram-positiveand Gram-negative bacteria, and also showed strong fungicidal activity towards theexpression host.The genomic structure of the first invertebrate G-type lysozyme from Chlamysfarreri (designated CFGLys) was obtained by genome walking approach. TheCFGLys gene was 8131 bp in length, coded for 829 bp and 200 deduced amino acidresidues. By comparison to the cDNA sequence and its translation product, the codingregion was found separated in six exons of 55 bp,60 bp,90 bp,113 bp,145 bp and140 bp, respectively. The introns range in size from 592 to 2161 bp, and havetraditional spliceosomal intron 5′-GT donor and 3′-AG acceptor sites for splicing.Analysis of the 5′-flanking sequence of CFGLys gene by TRANSFAC softwarerevealed several putative binding sequences for transcriptional factor found in hostdefense genes of other animals, including sequences similar to the NF-κB, OCT-1,AP-1 and NF-IL6. The expression of CFLysG mRNA in various tissues was measuredusing Northern blot analysis. mRNA transcript of CFLysG was most abundantlyexpressed in the tissues of gills, hepatopancreas and gonad, to a lesser degree in thetissues of haemocytes and mantle, while undetectable in the adductor muscle. Theseresults suggested that CFLysG could possess combined features of both the immuneand digestive adaptive lysozymes. In order to determine the in vitro lytic activities ofCFLysG, the mature peptide coding region was cloned into Pichia pastoris forheterogeneous expression. Recombinant CFLysG showed inhibitive effect on thegrowth of both Gram-positive and Gram-negative bacteria with more potent activityagainst Gram-positive bacteria, which demonstrated the involvement of CFLysG inthe innate immunity of C. farreri.Recombinant expression of AiBD and CFLysG made it possible to furthercharacterize their biological activities, three dimensional structures and immunefunctions, and also provided potential therapeutic agents for disease control inaquaculture.更多还原