马氏珠母贝（Pinctada fucata martensii）新型清道夫受体基因克隆与表达分析

Gene Cloning and Expression Analysis of a Novel Scavenger Receptor from Pinctada fucata martensii

清道夫受体（scavenger receptors,SRs）作为一类先天性免疫受体,在宿主固有免疫防御中起着重要的作用。本研究采用RACE技术首次从马氏珠母贝c DNA文库中克隆出一个新型的马氏珠母贝SR基因全长序列（pmSR）,并且运用qRT-PCR技术检测了pmSR基因在马氏珠母贝不同组织中的表达情况和哈维氏弧菌刺激后在血淋巴中的时序表达模式。结果显示,PmSR基因序列全长为954 bp,5＇UTR长为107 bp,3＇UTR长为169 bp,开放阅读框为678 bp,其编码225个氨基酸;PmSR氨基酸序列含有α卷曲螺旋结构和具有6个保守的半胱氨酸的SRCR域（scavenger receptor cystein rich domain）,具有A型SRCR结构域的典型特征。SRCR结构域氨基酸序列多序列比对显示Pm SR的SRCR结构域与老鼠和斑马鱼MARCO（macrop Hage receptor with collagenous structure,MARCO）SRCR相似度最高,分别为44%和43%;且蛋白质聚类分析表明PmSR与SR-AI（class A scavenger receptor I,SR-AI）同源性最高。qRT-PCR结果显示,PmSR基因在马氏珠母贝闭壳肌、肝胰腺、血细胞、外套膜、性腺、鳃中均有表达,在肝胰腺表达量最高;哈维式弧菌（Vibrio harveyi）刺激后,血淋巴中PmSR基因表达量在0-8 h逐渐上升,并于8 h达到最大表达量,约为对照组（0 h）的4.2倍,表达量随后下降,直至16 h后,其表达量再次出现显著性上升,结果具有显著性差异（p〈0.05）。本研究为贝类免疫防御系统的研究提供了重要资料。

Thescavenger receptor,as a kind of congenital immune receptor,plays an important role in the host innate immune defense.This researchcloned a novel scavenger receptor genomic full-length sequence（PmSR）from Pinctada fucata martensii by using RACE technology,and tested its expression condition in each tissueof Pinctada fucata martensiiand sequencial expression patterns in the hemolymph after Vibrio harveyi stimulation by Quantitative Real-Time PCR technology.Results showed that the total length of PmSR cDNA was 954 bp,including a5＇UTR of 107 bp,a 3＇UTR of 169 bp and an open reading frame（ORF）of 678 bp which encoded 225 amino acids;PmSR was possessed of the typical characteristics of A type SRCR domain structure,which contained a alpha coiled coil region and a SRCR domain with six conservative cysteine.Multiple sequence alignment with amino acid sequence of SRCR domain indicated that Pm SR SRCR structure domain had the highest similarity with the Mus musculus and Danio rerio MARCO SRCR,which were 44% and 43%,respectively;cluster analysis of Pm SR protein sequence showed that it was highest homology to SR-AI.qRT-PCR results revealed that Pm SR was expressed in each tissue,including adductor,hepatopancreas,hemocytes,mantle,gonads and gill,and it expressed with the highest proportion in hepatopancreas;after Vibrio harveyi injection,the expression of Pm SR raised gradually in 0-8 h and reached the highest expression quantity at 8 h（4.2 times compared with 0 h）,and then the expression of Pm SR declined,until after 16 h,there was another significant ascension,the result showed a significant difference（p0.05）.This study might provide reference materials to the research of shellfish immune defense system.