螯虾免疫相关基因的检出及丝氨酸蛋白酶抑制物基因分析

Detection of immune associated genes and analysis of a new serine proteinase inhibitor gene in crayfish Procambarus clarkii

通过对抑制性差减杂交和cDNA芯片技术分离的 2 0 1个阳性克隆的测序 ,得到螯虾 (Procambarusclarkii)免疫相关基因 4 8个 ,其中正向克隆中 4 2个 ,反向克隆 6个 ,除正向克隆中SNAP - 2 5 (synatosome-associatedproteinof2 5ku)已报道外 ,其余均为新基因 ,均在GenBank登录。正向克隆中的同源基因有微管蛋白、超氧化物歧化酶前体、丝氨酸蛋白酶抑制物Ⅰ、精氨酸激酶、70kD伴侣蛋白同类物等。进一步用DotNorthernblot对克隆号PCI188进行鉴定 ,结果攻毒组的杂交信号是对照组的 3.2 4倍 ,与cDNA芯片结果相符。用快速扩增cDNA末端技术扩增克隆号PCI188基因的 5’端片段和 3’端片段 ,全长共 112 8bp ,编码的蛋白质有 2 77个氨基酸 ,分子量为 30 .2 7kD。与GenBank序列号X795 12软尾太平剌蛄 (P .le niusculus)的蛋白酶抑制物Ⅰ (为丝氨酸蛋白酶抑制物 )的基因同源性为 5 8.7% ,氨基酸同源性为 6 9.7%。该蛋白质有 5个重复的结构域 。

White spot syndrome virus(WSSV) is a major pathogen in cultured penaeid shrimp. Infection of penaeid shrimp by WSSV can result in mortality of up to 90% to 100% within 3 to 7 d. But crustacean host defense mechanisms and particularly host viral defense are relatively poorly understood. Health management requires an improved understanding of the molecular response of crustaceans to pathogens. Crayfish (Procambarus clarkii) which had been developed as an animal model to culture WSSV in our lab was used in this experiment. Combining suppression subtractive hybridization(SSH) and cDNA chip, the immune genes of crayfish were studied and 201 clones including 184 forward subtracted clones and 17 reverse subtracted clones constructed by SSH and cDNA chip were sequenced using Pinpoint-T vector sequence as primer. The sequences were aligned with GenBank using the BLASTn program. Fourty-eight immune genes of crayfish were detected, of which 42 were upregulated genes and the other 6 were downregulated genes. All of them were new genes except the SNAP-25 gene. Five upregulated genes had homologous genes in the GenBank. Their sequences showed 87%, 92%, 86%, 90% and 78% identity with those in American lobster (Homarus americanus) alpha-Ⅲ tubulin mRNA (GenBank accession number HAU68764), signal crayfish (Pacifastacus leniusculus) mRNA for protease inhibitorⅠ(GenBank accession number X79512), signal crayfish mRNA for extracellular superoxide dismutase precursor (GenBank accession number AF122900), green crab (Carcinus maenas) arginine kinase mRNA (GenBank accession number AF167313) and domestic silkworm (Bombyx mori) mRNA for heat shork 70 kD protein cognate (GenBank accession number AB016836), respectively. Totally 47 GenBank accession numbers were assigned. The SSH clones PCI188, which was similar with signal crayfish mRNA for protease inhibitorⅠ, was identified by Dot Northern blot. Analyzed by Quantity One software, the density was 3.24 times higher in the experiment group than that in the control group. This result was coincident with that got from cDNA chip. According to the sequence of PCI188, two primers was designed. And the 5’ end and 3’ end of the cDNA were amplified respectively. A 1 128 bp fragment, which encodes a signal sequence and a mature protein of 277 amino acids with a predicted molecular mass of 30.27 kD, was obtained. The nucleotide identity between this protein and protease inhibitorⅠof P. leniusculus is 58.7%. And the amino acids identity between them is 69.7%.The amino acid sequence of this protein consists of five repeated stretches, indicating that the protein has five domains. The domains have significant sequence similarities to the serine protease inhibitors of Kazal family. Detection of serine protease inhibitor in the upregulated genes confirmed the hypothesis brought forward by Roux that proPO expression was not upregulated in the same virus-infected shrimp led to the speculation that WSSV infection probably activated the expression of a protease inhibitor(s) that blocked the activity of serine protease or the activity of the proPO.