Fang Jianguang

Effect of Epinephrine on the Settlement and Metamorphosis of Manila Clam Larvae

Chemical inducement and DDRT-PCR (differential display reverse transcription PCR) are adopted to investigate the effect of epinephrine (EPI) on the settlement and metamorphosis of Manila clam larvae. Chemical inducement shows that EPI has an effect to some extent on the metamorphosis of Manila clam larvae at all concentrations and in all treatments designed. The most significant result of inducement is obtained at the concentration of 10−6 mol L−1 and for 4h. DDRT-PCR using six primer pairs shows that the gene expression pattern is quite different between EPI treatment and the control. Three hundred and forty-three amplification bands are obtained in total, among which, 67 (19.53%) are differentially appeared. Therefore, EPI has an effect on the gene expression of the eye spot larval Manila clam. It can be hypothesized that EPI is a settlement and metamorphosis inducer for Manila clam. EPI may lead to larvae settlement and metamorphosis by binding to the receptors on the membrane and then changing the gene expression of larvae cells.

魁蚶 C 型凝集素基因 cDNA 的克隆及表达分析

通过cDNA末端快速扩增(Rapid amplification of cDNA ends，RACE)技术克隆得到魁蚶(Scapharca broughtonii) C型凝集素(C-type lectin，Sb-Lec1)基因，该基因全长为700 bp，其中，5′-UTR为29 bp，3′-UTR为167 bp，开放阅读框长度为504 bp，编码167个氨基酸，包括长度为23个氨基酸的信号肽序列、129个氨基酸的糖识别结构域(CRD)以及参与二硫键形成的6个半胱氨酸。预测蛋白分子量为19.11 kDa，理论等电点为4.74。多序列比对结果显示，Sb-Lec1基因CRD编码的氨基酸序列与长牡蛎(Crassostrea gigas)、紫贻贝(Mytilus galloprovincialis)和海湾扇贝(Argopecten irradians) C型凝集素的同源性分别为38%~40%、34%~35%和38%~39%，Sb-Lec1基因编码的氨基酸序列与其他物种的凝集素基因具有相似的结构，均含有形成二硫键的4个保守半胱氨酸。系统进化分析结果显示，魁蚶先与贝类聚为一支，再与脊椎动物聚在一起，表明魁蚶Sb-Lec1基因在进化树上的位置与其传统分类所处位置一致。采用荧光定量PCR技术，检测了Sb-Lec1基因在组织中的表达情况，发现其在肝胰腺、血淋巴、鳃、外套膜、闭壳肌、斧足中均有表达，其中，肝胰腺表达量最高。同时，分析了Sb-Lec1基因在鳗弧菌(Vibrio anguillarum)刺激下的mRNA表达量变化情况。结果显示，与对照组相比，菌刺激组Sb-Lec1基因mRNA在各检测组织中的表达量均显著上调(P < 0.05)，随着刺激时间的延长，表达量呈先升高后降低的趋势。本研究表明，魁蚶Sb-Lec1基因在机体免疫防御方面发挥重要功能。